PONE-D-20-22239

3D imaging of undissected optically cleared Anopheles stephensi mosquitoes and midguts infected with Plasmodium parasites

PLOS ONE

Dear Dr. De Niz,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Overall the reviewers were positive about your study. However, both reviewers indicated that some of the figure legends need additional detail/modification, and both felt some additions to the discussion would make this a stronger paper. Please address these and their other minor concerns and return a revised copy of the manuscript as outlined below.

==============================

Please submit your revised manuscript by Sep 16 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Photini Sinnis, M.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Table 2 in your text; if accepted, production will need this reference to link the reader to the Table.

3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Reviewer #1: Some of the major problems of preparing mosquitos for microscopy are related to the cuticle’s biological properties, including the thickness, light scattering, extent of cuticle sclerotization, and autofluorescence. These properties make difficult the acquisition of live or fixed images of insects in general, especially those vectors of diseases. Recently, these problems have been overcome by adapting chemical clearing protocols for to increase the transparency of Anopheles mosquito cuticle, allowing acceptable preservation of both mosquito and Plasmodium parasites. For morphological studies to bring a novel conceptual advance, few characteristics must be attended, including good preservation of parasites and mosquito tissues, and the capacity to study host-parasite interactions in whole organisms. In this manuscript, De Niz et al., address some of these limitations by combining the use of chemical clearance methods with the power of Optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) to successfully 1) obtain optically cleared Anopheles mosquitoes, 2) obtain 3D reconstruction of whole mosquitoes and 3) image Plasmodium parasites on infected midguts. Overall, the paper is well-executed, methodologically sound and well-written. Some figures can benefit from additional labeling to better guide the reader on the points emphasized by the authors (see comments). If further developed, this technology has the potential of establishing new protocols for studying host-parasite interactions.

Major comments:

Most of the discussion is a repeat of the results. The discussion would benefit from a better comparison, based on their results, of the benefits and advancement that this technology provides over other methods previously used to image infected and non-infected Anopheles mosquitoes.

A key question that the authors need to address in the discussion is what new information one can learn, given the amount of work that takes to process the samples through chemically clearance? It is not clear that the resolution level achieved allows a detailed study of host-parasite interactions. The distribution of oocysts in the midgut can be easily studied ex-vivo with well-established protocol that are less laborious, time-consuming and produce similar results. These should be discussed.

One point of concern is whether this protocol introduces morphological changes in the parasite? The low resolution of the images didn’t allow a proper evaluation of the issue.

Minor comments:

Line 170: “ScaleS and SeeDB were slowest to achieve optical clearance, yet fluorescence preservation with both methods was significantly higher than with either BABB or 3DISCO.”

The results describe a significant difference however neither the Figure 2B nor the statistics in the figure legend show statistical significance.

Line 209: “OPT enabled imaging of the antennae, the mandibles and maxillae lining the alimentary canal, and the maxillary palps (Figure 3C, top panels). OPT also allowed detailed visualization of the proboscis and its structures (all structures mentioned above are marked with arrowheads). ”

The mandibles, macillae, and maxillary palps are not clearly distinguished in these images. The arrows just point to the mouth parts but there is not description in the legend about the parts referenced in the results text.

Line 216: “Moreover, the thorax harbors the dorsal blood vessel, the tracheal and dorsal tubes (or heart), the foregut, and various nerve ducts (Figure 3A-3B).”

These structures are not clear in the figure. It will be helpful if the authors include additional arrows or marks pointing at each structure referenced in the results. In addition, it is not clear to what structures the three black arrows are point to in Fig. 3A.

Line 241: “In excised cleared midguts, our technique allowed for the first time, full quantification of oocyst numbers throughout parasite development.”

The claim that their technology allows for the first-time full quantifications of oocysts is not accurate. Full quantification of oocyst numbers in dissected midguts has been done by several methods.

Movie 2: Slow down the speed of rotation to allow for better appreciation of the image.

Reviewer #2: De Niz et al nicely described different microscope techniques and compared tissue clearing methods on Anopheles mosquito. The method comparison looked at time took to clear the mosquitoes, fluorescence preservation and morphological changes which are the key factors to look at in tissue clearing. The summary tables help to understand the comparisons. The 3D-reconstruction and surface rendering from OPT imaging were a beautiful work.

There are two points that I would like to point out.

Figure 2

It is not clear which microscope was used to quantify fluorescence preservation in figure legend and result section.

Did you look into detail if you could see individual oocysts in any tested methods and assess the level of autofluorescence?

Figure 4

It is not clear what yellow rendering indicates in figure legend (line 715 to 716). It seems surface rendering of mCherry signaling. From figure legend, it could be both mcherry and egg development.

Figure 4A, S2 & S3 show autofluorescence issue and it is hard to see individual oocysts in mosquito and identifying salivary gland sporozoite signals. For OPT and LSFM imaging, it is important to have distinguishable signals to identify the localization of protein and gene of interests. It would be great to discuss possibility to solve this issue in discussion. Not only further developing tissue clearing techniques but also the wavelength using for imaging, possibility to combine with immune-fluorescence staining advantage and disadvantage etc.

Further applications (looking at sensory response, other pathogens in abdomen) using OPT and LSFM are well described in discussion and it is a great method to use.

One minor comments

line 241-244:

Have you quantified number of eggs in mosquito or dissected oocysts? (If so, how did you count it?)

If this meant for possible application, it would be better in discussion section and discuss along with possible analysis strategies.

**********

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Dear Mariana et al.,

Thank-you for your thorough response to the reviewers. This is a nice study that will be very helpful to those working on mosquitoes and likely other vectors.

All the best,

Photini

3D imaging of undissected optically cleared Anopheles stephensi mosquitoes and midguts infected with Plasmodium parasites

PONE-D-20-22239R1

Dear Dr. De Niz,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Photini Sinnis, M.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: