Unlocking new understanding of Plasmodium sporozoite biology with expansion microscopy

  1. Benjamin Liffner
  2. Thiago Luiz Alves e Silva
  3. Elizabeth Glennon
  4. Veronica Primavera
  5. Elaine Hoffman
  6. Alexis Kaushansky
  7. Joel Vega-Rodriguez
  8. Sabrina Absalon

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Abstract

Transmission of malaria relies on the formation of sporozoites in the mosquito midgut and their subsequent invasion of the salivary gland. Despite their importance, our understanding of the cell biology of sporozoite formation and salivary gland invasion is limited. Here, we apply a technique called Mo squito Tiss ue U ltrastructure Ex pansion M icroscopy (MoTissU-ExM), which physically expands infected mosquito tissues while preserving both host and parasite ultrastructure. Using MoTissU-ExM we are able to observe a range of parasite structures and organelles including features previously seen only by electron microscopy as well as structure not observed before. We leverage MoTissU-ExM to investigate a number of cell biology events during sporozoite formation and salivary gland invasion. In particular we focus on the rhoptries, a secretory organelle important for host cell invasion. We establish a timeline for sporozoite rhoptry biogenesis, show that two rhoptries are used up during salivary gland invasion, and provide the first evidence that rhoptry pairs are specialized for different invasion events. Building on this new knowledge, we characterize the rhoptry protein RON11 and identify it as the first protein involved in sporozoite rhoptry biogenesis. Disruption of RON11 led to the production of sporozoites that specifically fail to invade the salivary gland epithelial cell, thereby blocking transmission of these parasites.

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  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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    Reply to the reviewers

    We sincerely appreciate the feedback, attention to detail and timeliness of the referees for our manuscript. Below, we provide a point-by-point response to all comments from the referees, detailing the changes we have already made, and those that are in progress. Referee's comments will appear in bolded text, while our responses will be unbolded. Any text quoted directly from the manuscript will be italicised and contained within "quotation marks". Additionally, we have grouped all comments into four categories (structural changes, minor text changes, experimental changes, figure changes), comments are numbered 1-n in each of these categories. Please note: this response to reviewer's comments included some images that cannot be embedded in this text-only section.

    1. General Statements

    We appreciate the overall highly positive and enthusiastic comments from all reviewers, who clearly appreciated the technical difficulty of this study, and noted amongst other things that this study represents" a major contribution to the future advancement of oocyst-sporozoite biology" and the development of the segmentation score for oocysts as a "major advance[ment]". We apologise for the omission of line numbers on the document sent to reviewers, we removed these for the bioRxiv submission without considering that this PDF would be transferred across to Review Commons.

    We have responded to all reviewers comments through a variety of text changes, experimental inclusions, or direct query response. Significant changes to the manuscript since initial submission are as follows:

    1. Refinement of rhoptry biogenesis model: Reviewers requested more detail around the content of the AORs, which we had previously suggested were a vehicle for rhoptry biogenesis as we saw they carried the rhoptry neck protein RON4. To address this, we first attempted to address this using antibodies against rhoptry bulb proteins but were unsuccessful. We then developed a * berghei* line where there rhoptry bulb protein RhopH3 was GFP-tagged. Using this parasite line, we observed that the earliest rhoptry-like structure, which we had previously interpreted as an AOR contained RhopH3. By contrast, RhopH3 was absent from AORs. Reflecting these observations we have renamed this initial structure the 'pre-rhoptry' and suggested a model for rhoptry biogenesis where rhoptry neck cargo are trafficked via the AOR but rhoptry bulb cargo are trafficked by small vesicles that move along the rootlet fibre (previously observed by EM).
    2. Measurement of rhoptry neck vs bulb: While not directly suggested by the reviewers, we have also included an analysis that estimates the proportion of the sporozoite rhoptry that represents the rhoptry neck. By contrast to merozoites, which we show are overwhelmingly represented by the rhoptry bulb, the vast majority of the sporozoite rhoptry represents the rhoptry neck.
    3. Measurement of subpellicular microtubules: One reviewer asked if we could measure the length of subpellicular microtubules where we had previously observed that they were longer on one side of the sporozoite than the other. We have now provided absolute and relative (% sporozoite length) length measurements for these subpellicular microtubules and also calculated the proportion of the microtubule that is polyglutamylated.
    4. More detailed analysis of RON11cKD rhoptries: Multiple comments suggested a more detailed analysis of the rhoptries that were formed/not formed in RON11cKD We have included an updated analysis that shows the relative position of these rhoptries in sporozoites.

    2. Point-by-point description of the revisions

    Reviewer #1

    Minor text changes (Reviewer #1)

    __Text on page 12 could be condensed to highlight the new data of ron4 staining of the AOR. __

    We agree with the reviewer that it is a reasonable suggestion. After obtaining additional data on the contents of the AOR (as described in General Statements #1), this section has been significantly rewritten to highlight these findings.

    __Add reference on page 3 after 'disrupted parasites' __

    This sentence has been rewritten slightly with some references included and now reads: "Most data on these processes comes from electron microscopy studies 6-8, with relatively few functional reports on gene deleted or disrupted parasites9-11.

    __Change 'the basal complex at the leading edge' - this seems counterintuitive __

    This change has been made.

    __Change 'mechanisms underlying SG are poorly' - what mechanisms? of invasion or infection? __

    This was supposed to read "SG invasion" and has now been fixed.

    __On page 4: 'handful of proteins' __

    This error has been corrected.

    __What are the 'three microtubule spindle structures'? __

    The three microtubule spindle structures: hemispindle, mitotic spindle, and interpolar spindle are now listed explicitly in the text.

    __On page 5: 'little is known' - please describe what is known, also in other stages. At the end of the paper I would like to know what is the key difference to rhoptry function in other stages? __

    The following sentence already detailed that we had recently used U-ExM to visualise rhoptry biogenesis in blood-stage parasites, but the following two sentences have been added to provide extra detail on these findings: "In that study, we defined the timing of rhoptry biogenesis showing that it begun prior to cytokinesis and completed approximate coincident with the final round of mitosis. Additionally, we observed that rhoptry duplication and inheritance was coupled with centriolar plaque duplication and nuclear fission."

    __change 'rhoptries golgi-derived, made de novo' __

    This has been fixed.

    __change 'new understand to' __

    This change has been made

    __'rhoptry malformations' seem to be similar in sporozoites and merozoites. Is that surprising/new? __

    We assume this is in reference to mention of "rhoptry malformations" in the abstract. In the RON11 merozoite study (PMID:39292724) the authors noted no gross rhoptry malformations, only that one was not formed/missing. The abstract sentence has been changed to the following to better reflect this nuance: "*We show that stage-specific disruption of RON11 leads to a formation of sporozoites that only contain half the number of rhoptries of controls like in merozoites, however unlike in merozoites the majority of rhoptries appear grossly malformed."

    __What is known about crossing the basal lamina. Where rhoptries thought to be involved in this process? Or is it proteins on the surface or in other secretory organelles? __

    We are unaware of any studies that specifically look at sporozoites crossing the SG basal lamina. A review, although now ~15 years old stated that "No information is available as to how the sporozoites traverse the basal lamina" (PMID:19608457) and we don't know any more information since then. To try and better define our understanding of rhoptry secretion during SG invasion, we have added the following sentence:

    "It is currently unclear precisely when during these steps of SG invasion rhoptry proteins are required, but rhoptry secretion is thought to begin before in the haemolymph before SG invasion16."

    __On page change/specify: 'wide range of parasite structures' __

    The structures observed have been listed: centriolar plaque, rhoptry, apical polar rings, rootlet fibre, basal complex, apicoplast.

    __On page 7: is Airyscan2 a particular method or a specific microscope? __

    Airyscan2 is a detector setup on Zeiss LSM microscopes, this was already detailed in the materials and methods sections, but figure legends have been clarified to read: "...imaged by an LSM900 microscopy with an Airyscan2 detector".

    __how large is RON11? __

    RON11 is 112 kDa in * berghei*, as noted in the text.

    __There is no causal link between ookinete invasion and oocyst developmental asynchrony __

    We have deleted the sentence that implied that ookinete invasion was responsible for oocyst asynchrony. This section now simply states that "Development of each oocyst within a midgut is asynchronous..."

    __First sentence of page 24 appears to contradict what is written in results____ I don't understand the first two sentences in the paragraph titled Comparison between Plasmodium spp __

    This sentence was worded confusingly, making it appear contradictory when that was not the intention. The sentence has been changed to more clearly support what is written in the discussion and now reads: "Our extensive analysis only found one additional ultrastructural difference between Plasmodium spp."

    __On page 25 or before the vast number of electron microscopy studies should be discussed and compared with the authors new data. __

    It is not entirely clear which new data should be specifically discussed based on this comment. However, we have added a new paragraph that broadly compares MoTissU-ExM and our findings with other imaging methods previously used on mosquito-stage malaria parasites:

    "*Comparison of MoTissU-ExM and other imaging modalities

    Prior to the development of MoTissU-ExM, imaging of mosquito-stage malaria parasites in situ had been performed using electron microscopy7,8,11,28, conventional immunofluorescence assays (IFA)10, and live-cell microscopy25. MoTissU-ExM offers significant advantages over electron microscopy techniques, especially volume electron microscopy, in terms of accessibility, throughput, and detection of multiple targets. While we have benchmarked many of our observations against previous electron microscopy studies, the intracellular detail that can be observed by MoTissU-ExM is not as clear as electron microscopy. For example, previous electron microscopy studies have observed Golgi-derived vesicles trafficking along the rootlet fibre8 and distinguished the apical polar rings44; both of which we could not observe using MoTissU-ExM. Compared to conventional IFA, MoTissU-ExM dramatically improves the number and detail of parasite structures/organelles that can be visualised while maintaining the flexibility of target detection. By contrast, it can be difficult or impossible to reliably quantify fluorescence intensity in samples prepared by expansion microscopy, something that is routine for conventional IFA. For studying temporally complex processes, live-cell microscopy is the 'gold-standard' and there are some processes that fundamentally cannot be studied or observed in fixed cells. We attempt to increase the utility of MoTissU-ExM in discerning temporal relationships through the development of the segmentation score but note that this cannot be applied to the majority of oocyst development. Collectively, MoTissU-ExM offers some benefits over these previously applied techniques but does not replace them and instead serves as a novel and complementary tool in studying the cell biology of mosquito-stage malaria parasites.**"

    __First sentence on page 27: there are many studies on parasite proteins involved in salivary gland invasion that could be mentioned/discussed. __

    The sentence in question is "To the best of our knowledge, the ability of sporozoites to cross the basal lamina and accumulate in the SG intercellular space has never previously been reported."

    This sentence has now been changed to read as follows: "While numerous studies have characterized proteins whose disruption inhibited SG invasion9,10,15,59-63, to the best of our knowledge the ability of sporozoites to cross the basal lamina and accumulate in the SG intercellular space has never previously been reported ."

    __On page 10 I suggest to qualify the statement 'oocyst development has typcially been inferred by'. There seem a few studies that show that size doesn't reflect maturation. __

    In our opinion, this statement is already qualified in the following sentence which reads: "Recent studies have shown that while oocysts increase in size initially, their size eventually plateaus (11 days pot infection (dpi) in P. falciparum4)."

    __On page 16 the authors state that different rhoptries might have different function. This is an interesting hypothesis/result that could be mentioned in the abstract. __

    The abstract already contains the following statement: "...and provide the first evidence that rhoptry pairs are specialised for different invasion events." We see this as an equivalent statement.

    Experimental changes (Reviewer #1)

    On page 19: do the parasites with the RON11 knockout only have the cytoplasmic or only the apical rhoptries?

    The answer to this is not completely clear. We have added the following data to Figures 6 and 8 where we quantify the proportion of rhoptries that are either apical or cytoplasmic: In both wildtype parasites and RON11ctrl parasites, oocyst spz rhoptries are roughly 50:50 apical:cytoplasmic (with a small but consistent majority apical), while almost all rhoptries are found at the apical end (>90%) in SG spz. Presumably, after the initial apical rhoptries are 'used up' during SG invasion, the rhoptries that were previously cytoplasmic take their place. In RON11cKD the ratio of apical:cytoplasmic rhoptries is fairly similar to control oocyst spz. In RON11cKD SG spz, the proportion of cytoplasmic rhoptries decreases but not to the same extent as in wildtype or RON11Ctrl. From this, we infer that the two rhoptries that are lost/not made in RON11cKD sporozoites are likely a combination of both the apical and cytoplasmic rhoptries we find in control sporozoites.

    __in panel G: Are the dense granules not micronemes? What are the dark lines? Rhoptries?? __

    We have labelled all of Figure 1 more clearly to point out that the 'dark lines' are indeed rhoptries. Additionally, we have renamed the 'protein-dense granules' to 'protein-rich granules', as it seems we are suggesting that these structures are dense granules the secretory organelle. At this stage we simply do not know what all of these granules are. The observation that some but not all of these granules contain CSP (Supplementary Figure 2) suggests that they may represent heterogenous structures. It is indeed possible that some are micronemes, however, we think it is unlikely that they are all micronemes for a number of reasons: (1) micronemes are not nearly this protein dense in other *Plasmodium *lifecycle stages, (2) some of them carry CSP which has not been demonstrated to be micronemal, (3) very few of these granules are present in SG sporozoites, which would be unexpected because microneme secretion is required for hepatocyte invasion.

    __Figure 2 seems to add little extra compared to the following figures and could in my view go to the supplement. __

    We agree that Figure 2b adds little and so have moved that to Supplementary Figure 2, but think that the relative ease at which it can be distinguished if sporozoites are in the secretory cavity or SG epithelial cell is a key observation because of the difficulty in doing this by conventional IFA.

    __On page 8 the authors mention a second layer of CSP but do not further investigate it. It is likely hard to investigate this further but to just let it stand as it is seems unsatisfactory, considering that CSP is the malaria vaccine. What happens if you add anti-CSP antibodies? I would suggest to shorten the opening paragraphs of this paper and to focus on the rhoptries. This could be done be toning down the text on all aspects that are not rhoptries and point to the open question some of the observations such as the CSP layers raise for future studies. __

    When writing the manuscript, we were unsure whether to include this data at all as it is a purely incidental finding. We had no intention of investigating CSP specifically, but anti-CSP antibodies were included in most of the salivary gland imaging experiments so we could more easily find sporozoites. Given the tremendous importance of CSP to the field, we figured that these observations were potentially important enough that they should be reported in the literature even though they are not something we have the intention or resources to investigate subsequently. Additionally, after consultation with other microscopists we think there is a reasonable chance that this double-layer effect could be a product of chemical fixation. To account for this, we have qualified the paragraph on CSP with this sentence:

    "We cannot determine if there is any functional significance of this second CSP layer and considering that it has not been observed previously it may well represent an artefact of chemical (paraformaldehyde) fixation."

    __Maybe include more detail of the differences between species on rhoptry structure into Figure 4. I would encourage to move the Data on rhoptries in Figure S6 to the main text ie to Figure 4. __

    We have moved the images of developing rhoptries in * falciparum *(previously Figure S6a and b) into figure 4, which now looks as follows:

    Figure S8 (previously S6c) now consists only of the MG spz rhoptry quantification

    Manuscript structural changes (Reviewer #1)

    Abstract: don't focus on technique but on the questions you tried to answer (ie rewrite or delete the 3rd and 4th sentence)

    'range of cell biology processes' - I understand the paper that the key discovery concerns rhoptry biogenesis and function, so focus on that, all other aspects appear rather peripheral.

    'Much of this study focuses on the secretory organelles': I would suggest to rewrite the intro to focus solely on those, which yield interesting findings.

    Page 11: I am tempted to suggest the authors start their study with Figure 3 and add panel A from Figure 2 to it. This leads directly to their nice work on rhoptries. Other features reported in Figures 1 and 2 are comparatively less exciting and could be moved to the supplement or reported in a separate study.____ Page 23: I suggest to delete the first sentence and focus on the functional aspects and the discoveries.

    __Maybe add a conclusion section rather than a future application section, which reads as if you want to promoted the use of ultrastructure expansion microscopy. To my taste the technological advance is a bit overplayed considering the many applications of this techniques over the last years, especially in parasitology, where it seems widely used. In any case, please delete 'extraordinarily' __

    Response to Reviewer#1 manuscript structural changes 1-5: This reviewer considers the findings related to rhoptry biology as the most significant aspect of the study and suggests rewriting the manuscript to emphasize these findings specifically. Doing so might make the key findings easier to interpret. However, in our view, this approach could misrepresent how the study originated and what we see as the most important outcomes. We did not develop MoTissU-ExM specifically to investigate rhoptry biology. Instead, this technique was created independently of any particular biological question, and once established, we asked what questions it could answer, using rhoptry biology as a proof of concept. Given the authors' previous work and available resources, we chose to focus on rhoptry biology. Since this was driven by basic research rather than a specific hypothesis, it's important to acknowledge this in the manuscript. While we agree that the findings related to rhoptry biology are valuable, we believe that highlighting the technique's ability to observe organelles, structures, and phenotypes with unprecedented ease and detail is more important than emphasizing the rhoptry findings alone. For these reasons, we have decided not to restructure the manuscript as suggested.

    Reviewer #2

    Minor text changes (Reviewer #2)

    __The 'image Z-depth' value indicated in the figures is ambiguous. It is not clear whether this refers to the distance from the coverslip surface or the starting point of the z-stack image acquisition. A precise definition of this parameter would be beneficial. __

    In the legend of Figure 1, the image Z-depth has been clarified as "sum distance of Z-slices in max intensity projection".

    __Paragraph 3 of the introduction - line 7, "handful or proteins" should be handful of proteins __

    This has been corrected.

    __Paragraph 5 of the introduction - line 7, "also able to observed" should be observe __

    This has been changed.

    __In the final paragraph of the introduction - line 1, "leverage this new understand" should be understanding __

    This has been fixed.

    __The first paragraph of the discussion summary contains an incomplete sentence on line 7, "PbRON11ctrl-infected SGs." __

    This has been removed.

    __The second paragraph of the discussion - line 10, "until cytokinesis beings" should be begins __

    This mistake has been corrected.

    __One minor point that author suggest that oocyst diameter is not appropriate for the development of sporozoite develop. This is not so true as oocyst diameter tells between cell division and cell growth so it is important parameter especially where the proliferation with oocyst does not take place but the growth of oocyst takes place. __

    We agree that this was not highlighted enough in the text. The final sentence of the results section about this now reads:

    "While diameter is a useful readout for oocyst development in the early stages of its growth, this suggests that diameter is a poor readout for oocyst development once sporozoite formation has begun and highlights the usefulness of the segmentation score as an alternative.", and the final sentence of the discussion section about this now reads "Considering that oocyst size does not plateau until cytokinesis begins4, measuring oocyst diameter may represent a useful biological clock specifically when investigating the early stages of oocyst development."

    __How is the apical polarity different to merozoite as some conoid genes are present in ookinete and sporozoite but not in merozoite. __

    Our hypothesis is that apical polarity is established by the positioning and attachment of the centriolar plaque to the parasite plasma membrane in both forming merozoites and sporozoites. While the apical polar ring proteins are obviously present at the apical end, and have important functions, we think that they themselves are unlikely to regulate polarity establishment directly. Additionally, it seems that the apical polar rings are visible in forming sporozoites far before the comparable stages of merozoite formation. An important note here is that at this point, this is largely inferences based on observational differences and there is relatively little functional data on proteins that regulate polarity establishment at any stage of the Plasmodium

    __Therefore, I think that electron microscopy remains essential for the observation of such ultra-fine structures __

    We have added a paragraph in the discussion that provides a more clear comparison between MoTissU-ExM and other imaging modalities previously applied on mosquito-stage parasites (see response to Reviewer#1 (Minor text changes) comment #17).

    __The author have not mentioned that sometimes the stage oocyst development is also dependent on the age of mosquito and it vary between different mosquito gut even if the blood feed is done on same day. __

    In our opinion this can be inferred through the more general statement that "development of each oocyst within a midgut is asynchronous..."

    Figure changes (Reviewer #2)

    __Fig 3B: stage 2 and 6 does not show the DNA cyan, it would-be good show the sate of DNA at that particular stage, especially at stage 2 when APR is visible. And box the segment in the parent picture whose subset is enlarged below it. __

    We completely agree with the reviewer that the stage 2 image would benefit from the addition of a DNA stain. Many of the images in Figure 3b were done on samples that did not have a DNA stain and so in these * yoelii* samples we did not find examples of all segmentation scores with the DNA stain. Examples of segmentation score 2 and 6 for P. berghei, and 6 for P. falciparum can be found with DNA stains in Figure S8.

    __For clarity, it would be helpful to add indicators for the centriolar plaques in Figure 1b, as their locations are not immediately obvious. __

    The CPs in Figure 1a and 1b have been circled on the NHS ester only panel for clarity.

    __Regarding Figure 1c, the authors state that 'the rootlet fiber is visible'. However, such a structure cannot be confirmed from the provided NHS ester image. Can the authors present a clearer image where the rootlet fibre is more distinct? Furthermore, please provide the basis for identifying this structure as a rootlet fiber based on the NHS ester observation alone. __

    The image in Figure 1c has been replaced with one that more clearly shows the rootlet fibre.

    Based on electron microscopy studies, the rootlet fibre has been defined as a protein dense structure that connects the centriolar plaque to the apical polar rings (PMID: 17908361). Through NHS ester and tubulin staining, we could identify the apical polar rings and centriolar plaque as sites on the apical end of the parasite and nucleus that microtubules are nucleated from. There is a protein dense fibre that connects these two structures. Based on the fact that the protein density of this structure was previously considered sufficient for its identification by electron microscopy, we consider its visualisation by NHS ester staining sufficient for its identification by U-ExM.

    __Fig 1B - could the tubulin image in the hemispindle panel be made brighter? __

    The tubulin staining in this panel was not saturated, and so this change has been made.

    __Fig 4A - the green text in the first image panel is not visible. Also, the cyan text in the 3rd image in Fig 1A is also difficult to see. There's a few places where this is the case __

    We have made all microscopy labels legible at least when printed in A4/Letter size.

    __Fig 6A - how do the authors know ron11 expression is reduced by 99%? Did they test this themselves or rely on data from the lab that gifted them the construct? Also please provide mention the number of oocyst and sporozoites were observed. __

    The way Figure 6a was previously designed and described was an oversight, that wrongly suggested we had quantified a >99% reduction in *ron11 * The 99% reduction has been removed from Figure 6a and the corresponding part of the figure legend has been rewritten to emphasise that this was previously established:

    "(a) Schematic showing previously established Ron11Ctrl and Ron11cKD parasite lines where ron11 expression was reduced by >99%9."

    As to the second part of the question, we did not independently test either protein or RNA level expression of RON11, but we were gifted the clonal parasite lines established by Prof. Ishino's lab in PMID: 31247198 not just the genetic constructs.

    __Fig 6E - are the data point colours the wrong way round on this graph? Just looking at the graph it looks as though the RON11cKD has more rhoptries than the control which does not match what is said in the text. __

    Thank you for pointing out this mistake, the colours have now been corrected.

    __Fig S8C, PbRON11 ctrl, pie chart shows 89.7 % spz are present in the secretory cavity while the text shows 100 %, 35/35 __

    The text saying 100% (35/35) only considered salivary glands that were infected (ie. Uninfected SGs were removed from the count. The two sentences that report this data have been clarified to reflect this better:

    "*Of **PbRON11ctrl SGs that were infected (35/39), 100% (35/35) contained sporozoites in the secretory cavity (Figure S8c). Conversely of infected PbRON11cKD SGs (59/82), only 24% (14/59) contained sporozoites within the secretory cavity (Figure S9d)."

    __Fig S9D shows that RON11 ckd contains 17.1% sporozoites in secretory cavity while the text says 24%. __

    Please see the response to Reviewer#2 Figure Changes Comment #8 where this was addressed.

    Experimental changes (Reviewer #2)

    __Why do the congruent rhoptries have similar lengths to each other, while the dimorphic rhoptries have different lengths? Is this morphological difference related to the function of these rhoptries? __

    We hypothesise that this morphological difference arises because the congruent rhoptries are 'used' during SG invasion, while the dimorphic rhoptries are utilized during hepatocyte invasion. It is not straightforward to test this functionally at this point, as no protein is known to have differential localization between the two. Additionally, RON11 is likely directly involved in both SG and hepatocyte invasion through a secreted portion of the protein (as seen in RBC invasion). Therefore, RON11cKD sporozoites may have combined defects, meaning we cannot assume any defect is solely due to the absence of two rhoptries. Determining this functionally is of high interest to our research groups and remains an area of ongoing study, but it is beyond the scope of this study.

    Would it be possible to show whether RON11 localises to the dimorphic rhoptries, the congruent rhoptries, or both, by using expansion microscopy and a parasite line that expresses RON11 tagged with GFP or a peptide tag?

    __ __We do not have access to a parasite line that expresses a tagged copy of RON11, or anti-PbRON11 antibodies. Based on previously published localisation data, however, it seems likely that RON11 localises to both sets of rhoptries. Below are excerpts from Figure 1c of PMID: 31247198, where RON11 (in green) seems to have a more basally-extended localisation in midgut (MG) sporozoites than in salivary gland (SG) sporozoites. From this we infer that in the MG sporozoite you're seeing RON11 in both pairs of rhoptries, but only the one remaining pair in the SG sporozoite.

    __The knockdown of RON11 disrupts the rhoptry structure, making the dimorphic and congruent rhoptries indistinguishable. Does this suggest that RON11 is important for the formation of both types of rhoptries? I believe that it would be crucial to confirm whether RON11 localises to all rhoptries or is restricted to specific rhoptries for a more precise discussion of RON11's function. __

    Based on our analysis, it does indeed seem that RON11 is important for both types of rhoptries as when RON11 isn't expressed sporozoites still have both apical and cytoplasmic rhoptries (ie. Not just one pair is lost; see Reviewer #1 Experimental changes comment #1).

    __The authors state that 64% of RON11cKD SG sporozoites contained no rhoptries at all. Does this mean RON11cKD SG sporozoites used up all rhoptries corresponding to the dimorphic and congruent pairs during SG invasion? If so, this contradicts your claims that sporozoites are 'leaving the dimorphic rhoptries for hepatocyte invasion' and that 'rhoptry pairs are specialized for different invasion events'. If that is not the case, does it mean that RON11cKD sporozoites failed to form the rhoptries corresponding to the dimorphic pair? A more detailed discussion would be needed on this point and, as I mentioned above, on the specific role of RON11 in the formation of each rhoptry pair. __

    We do not agree that this constitutes a contradiction; instead, more nuance is needed to fully explain the phenotype. As shown in the new graph added in response to Reviewer#1 Figure changes comment #1 in RON11cKD oocyst sporozoites, 64% of all rhoptries are located at the apical end. Our hypothesis is that these rhoptries are used for SG invasion and, therefore, would not be present in RON11cKD SG sporozoites. Consequently, the fact that 64% of RON11cKD sporozoites lack rhoptries is exactly what we would expect. Essentially, we predict three slightly different 'pathways' for RON11cKD sporozoites: If they had 2 apical rhoptries in the oocyst, we predict they would have zero rhoptries in the SG. If they had 2 cytoplasmic rhoptries in the oocyst, we predict they would have two rhoptries in the SG. If they had one apical and one cytoplasmic rhoptry in the oocyst, we predict they would have one rhoptry in the SG. In any case, we expect the apical rhoptries to be 'used up,' which appears to be supported by the data.

    __Out of pure curiosity, is it possible to measure the length and number of subpellicular microtubules in the sporozoites observed in this study using expansion microscopy? __

    We have performed an analysis of subpellicular microtubules which is now included as Supplementary Figure 2. We could not always distinguish every SPMT from each other and so have not quantified SPMT number. We have, however, quantified their absolute length on both the 'long side' and 'short side', their relative length (as % sporozoite length) and the degree to which they are polyglutamylated.

    A description of this analysis is now found in the results section as follows: "*We quantified the length and degree of polyglutamylation of SPMTs on the 'long side' and 'short side' of the sporozoite (Figure S2). 'Short side' SPMTs were on average 33% shorter (mean = 3.6 µm {plus minus}SD 1.0 µm) than 'long side' SPMTs (mean = 5.3 µm {plus minus}SD 1.5 µm) and extended 17.4% less of the total sporozoite length. While 'short side' SPMTs were significantly shorter, a greater proportion of their length (87.9% {plus minus}SD 11.2%) was polyglutamylated compared to 'long side' SPMTs (69.4% {plus minus}SD 13.8%)."

    *Supplementary Figure 2: Analysis of sporozoite subpellicular microtubules. **Isolated P. yoelii salivary gland sporozoites were prepared by U-ExM and stained with anti-tubulin (microtubules) and anti-PolyE (polyglutamylated SPMTs) antibodies. SPMTs were defined as being on either the 'long side' (nucleus distant from plasma membrane) or 'short side' (nucleus close to plasma membrane) of the sporozoite as depicted in Figure 1f. (a) SPMT length along with (b) SPMT length as a proportion of sporozoite length were both measured. (c) Additionally, the proportion of the SPMT that was polyglutamylated was measured. Analysis comprises 25 SPMTs (11 long side, 14 short side) from 6 SG sporozoites. ** = p The following section has also been added to the methods to describe this analysis:

    "*Subpellicular microtubule measurement

    •      To measure subpellicular microtubule length and polyglutamylation maximum intensity projections were made of sporozoites stained with NHS Ester, anti-tubulin and anti-PolyE antibodies, and SYTOX Deep Red. The side where the nucleus was closest to the parasite plasma membrane was defined as the 'short side', while the side where the nucleus was furthest from the parasite plasma membrane was defined as the 'long side'. Subpellicular microtubules were then measured using a spline contour from the apical end of the sporozoite to the basal-most end of the microtubule with fluorescence intensity across the contour plotted (Zeiss ZEN 3.8). Sporozoite length was defined as the distance from the sporozoite apical polar rings to the basal complex, measuring through the centre of the cytoplasm. The percentage of the subpellicular microtubule that was polyglutamylated was determined by assessing when along the subpellicular microtubule contour the anti-PolyE fluorescence intensity last dropped below a pre-defined threshold."

    __In addition to the previous point, in the text accompanying Figure 7a, the authors claim that "64% of PbRON11cKD SG sporozoites contained no rhoptries at all, while 9% contained 1 rhoptry and 27% contained 2 rhoptries". Could this data be used to infer which rhoptry pair are missing from the RON11cKD oocyst sporozoites? Can it be inferred that the 64% of salivary gland sporozoites that had no rhoptries in fact had 2 congruent rhoptries in the oocyst sporozoite stage and that these have been discharged already? __

    Please see the response to Reviewer #2 Experimental Changes Comment #4.

    __Is it possible that the dimorphic rhoptries are simply precursors to the congruent rhoptries? Could it be that after the congruent rhoptries are used for SG invasion, new congruent rhoptries are formed from the dimorphic ones and are then used for the next invasion?____ Would it be possible to investigate this by isolating sporozoites some time after they have invaded the SG and performing expansion microscopy? This would allow you to confirm whether the dimorphic rhoptries truly remain in the same form, or if new congruent rhoptries have been formed, or if there have been any other changes to the morphology of the dimorphic rhoptries. __

    In theory, it is possible that the dimorphic rhoptries are precursors to the uniform rhoptries, specifically how the larger one of the two in the dimorphic pair might be a precursor. Maybe the smaller one is, but we have no evidence to suggest that this rhoptry lengthens after SG invasion. We are interested in isolating sporozoites from SGs to add a temporal perspective, but currently, this isn't feasible. When sporozoites are isolated from SGs, they are collected at all stages of invasion. Additionally, we don't know how long each step of SG invasion takes, so a time-based method might not be effective either. We are developing an assay to better determine the timing of events during SG invasion with MoTissU-ExM, but this is beyond the scope of this study.

    __In the section titled "Presence of PbRON11cKD sporozoites in the SG intercellular space", the authors state that "the majority of PbRON11cKD-infected mosquitoes contained some sporozoites in their SGs, but these sporozoites were rarely inside either the SG epithelial cell or secretory cavity". - this is suggestive of an invasion defect as the authors suggest. Could the authors collect these sporozoites and see if liver hepatocyte infection can be established by the mutant sporozoites? They previously speculate that the two different types of rhoptries (congruent and dimorphic) may be specific to the two invasion events (salivary gland epithelial cell and liver cell infection). __

    It has already been shown that RON11cKD sporozoites fail hepatocyte invasion (PMID: 31247198), even when isolated from the haemolymph and so it seems very unlikely that they would be invasive following SG isolation. As mentioned in the discussion, RON11 in merozoites has a 'dual-function' where it is partially secreted during merozoite invasion in addition to its rhoptry biogenesis functions. Assuming this is also the case in sporozoites, using the RON11cKD parasite line we cannot differentiate these two functions and therefore cannot ascribe invasion defects purely to issues with rhoptry biogenesis. In order to answer this question functionally, we would need to identify a protein that only has roles in rhoptry biogenesis and not invasion directly.

    Reviewer #3

    Minor text changes (Reviewer #3)

    __Page 3 last paragraph: ...the molecular mechanisms underlying SG (invasion?) are poorly understood. __

    This has been corrected

    __The term "APR" does not refer to a tubulin structure per se, but rather to the proteinaceous structure to which tubulin anchors. Are there any specific APR markers that can be used in Figure 1C? If not, I recommend avoiding the use of "APR" in this context. __

    The text does not state that the APR is a tubulin structure. Given that it is a proteinaceous structure, we visualise the APRs through protein density (NHS Ester). It has been standard for decades to define APRs by protein density using electron microscopy, and it has previously been sufficient in Plasmodium using expansion microscopy (PMIDs: 41542479, 33705377) so it is unclear why it should not be done so in this study.

    __I politely disagree with the bold statements ‚ Little is known about cell biology of sporozoite formation.....from electron microscopy studies now decades old' (p.3, 2nd paragraph); ‚To date, only a handful of (instead of ‚or') proteins have been implicated in SG invasion' (p. 4, 1st paragraph). These claims may overlook existing studies; a more thorough review of the literature is recommended. __

    This study includes at least 50 references from papers broadly related to sporozoite biology, covering publications from every decade since the 1970s. The most recent review that discusses salivary gland invasion cites 11 proteins involved in SG invasion. We have replaced "handful" with a more precise term, as it is not the best adjective, but it is hardly an exaggeration.

    Figure changes (Reviewer #3)

    __The hypothesis that Plasmodium utilizes two distinct rhoptry pairs for invading the salivary gland and liver cells is intriguing but remains clearly speculative. Are the "cytoplasmic pair" and "docked pair" composed of the same secretory proteins? Are the paired rhoptries identical? How does the parasite determine which pair to use for salivary gland versus liver cell invasion? Is there any experimental evidence showing that the second pair is activated upon successful liver cell invasion? Without such data this hypothesis seems rather premature. __

    We are unaware of any direct protein localisation evidence suggesting that the rhoptry pairs may carry different cargo. However, only a few proteins have been localised in a way that would allow us to determine if they are associated with distinct rhoptry pairs, so this possibility cannot be ruled out either. It seems unlikely that the parasite 'selects' a specific pair, as rhoptries are typically always found at the apical end. What appears more plausible is that the "docked pair" forms first and immediately occupies the apical docking site, preventing the cytoplasmic pair from docking there. Regarding any evidence that the second pair is activated during liver cell invasion, it has been well documented over decades that rhoptries are involved in hepatocyte invasion. If the dimorphic rhoptries are the only ones present in the parasite during hepatocyte invasion, then they must be used for this process.

    __The quality of the "Roolet fibre" image is not good and resembles background noise from PolyE staining. Additional or alternative images should be provided to convincingly demonstrate that PolyE staining indeed visualizes the Roolet fibre. It is puzzling that the structure is visible with PolyE staining but not with tubulin staining. __

    This is a logical misinterpretation based on the image provided in Figure 1c. Our intention was not to imply that PolyE staining enables us to see the rootlet fibre but that PolyE and tubulin allow us to see the APR to which the rootlet fibre is connected. There is some PolyE staining that likely corresponds to the early SPMTs that in 1c appears to run along the rootlet fibre but this is a product of the max-intensity projection. Please see Reviwer#2 Figure Changes Comment #3 for the updated Figure 1c.

    __More arrows should be added to Figures 6b and 6c to guide readers and improve clarity. __

    We have added arrows to Figure 6b and 6c which point out what we have defined as normal and aberrant rhoptries more clearly. These panels now look like this:

    __Figure 2a zoomed image of P. yoelii infected SG is different than the highligted square. __

    We agree that the highlighted square and the zoomed area appear different, but this is due to the differing amounts of light captured by the objectives used in these two panels. The entire SG panel was captured with a 5x objective, while the zoomed panel was captured with a 63x objective. Because of this difference, the plane of focus of the zoomed area is hard to distinguish in the whole SG image. The zoomed image is on the 'top' of the SG (closest to the coverslip), while most of the signal you see in the whole SG image comes from the 'middle' of the SG. To demonstrate this more clearly, we have provided the exact region of interest shown in the 63x image alongside a 5x image and an additional 20x image, all of which are clearly superimposable.__

    __

    __Figure 3 legend: "P. yoelii infected midguts harvested on day 15" should be corrected. More general, yes, "...development of each oocyst within a single midgut is asynchronous." but it is still required to provide the dissection days. __

    We are unsure what the suggested change here is. We do not know what is wrong with the statement about day 15 post infection, that is when these midguts were dissected. __ Experimental Changes (Reviewer #3)__

    __The proposed role of AOR in rhoptry biogenesis appears highly speculative. It is unclear how the authors conclude that "AORs carry rhoptry cargo" solely based on the presence of RON4 within the structure. Inclusion of additional markers to characterize the content of AOR and rhoptries will be essential to substantiate the hypothesis that this enigmatic structure supports rhoptry biogenesis. __

    It is important to note that the hypothesis that AORs, or rhoptry anlagen, carry rhoptry cargo and serve as vehicles of rhoptry biogenesis was proposed long before this study (PMID: 17908361). In that study, it was assumed that structures now called AORs or rhoptry anlagen were developing rhoptries. Although often visualised by EM and presumed to carry rhoptry cargo (PMID: 33600048, 26565797, 25438048), it was only more recently that AORs became the subject of dedicated investigation (PMID: 31805442), where the authors stated that "...AORs could be immature rhoptr[ies]...". Our observation that AORs contain the rhoptry protein RON4, which is not known to localize to any other organelle, we therefore consider sufficient to conclude that AORs carry rhoptry cargo and are thus vehicles for rhoptry biogenesis.

    __The study of RON11 appears to be a continuation of previous work by a collaborator in the same group. However, neither this study nor the previous one adequately addresses the evolutionary context or structural characteristics of RON11. Notably, the presence of an EF-hand motif is an important feature, especially considering the critical role of calcium signaling in parasite stage conversion. Given the absence of a clear ortholog, it would be interesting to know whether other Apicomplexan parasites harbor rhoptry proteins with transmembrane domains and EF-hand motifs, and if these proteins might respond similarly to calcium stimulation. Investigating mutations within the EF-hand domain could provide valuable functional insights into RON11. __

    We are unsure what suggests that RON11 lacks a clear orthologue. RON11 is conserved across all apicomplexans and is also present in Vitrella brassicaformis (OrthoMCL orthogroup: OG7_0028843). A phylogenetic comparison of RON11 across apicomplexans has previously been performed (PMID: 31247198), and this study provides a structural prediction of PbRON11 with the dual EF-hand domains annotated (Supplementary Figure 9).

    __The study cannot directly confirm that membrane fusion occurs between rhoptries and AORs. __

    This is already stated verbatim in the results "Our data cannot directly confirm that membrane fusion occurs between rhoptries and AORs..."

    __It is unclear what leads to the formation of the aberrant rhoptries observed in RON11cKD sporozoites. Since mosquitoes were not screened for infection prior to salivary gland dissection, The defect reports and revisited of RON11 knockdown does not aid in interpreting rhoptry pair specialization, as there was no consistent trend as to which rhoptry pair was missing in RON11cKD oocyst sporozoites. The notion that RON11cKD parasites likely have ‚combinatorial defects that effect both rhoptry biogenesis and invasion' poses challenges to understand the molecular role(s) of RON11 on biogenesis versus invasion. Of note, RON11 also plays a role in merozoite invasion. __

    We are unclear about the comment or suggestion here, as the claims that RON11cKD does not help interpret rhoptry pair specialization, and that these parasites have combined defects, are both directly stated in the manuscript.

    __Do all SG PbRON11cKD sporozoites lose their reduced number of rhoptries during SG invasion as in Figure 7a (no rhoptries)? __

    Not all RON11cKD SG sporozoites 'use up' their rhoptries during SG invasion. This is quantified in both Figure 7a and the text, which states: "*64% of **PbRON11cKD SG sporozoites contained no rhoptries at all, while 9% contained 1 rhoptry and 27% contained 2 rhoptries."

    Different mosquito species/strains are used for P. yoelii, P. berghei, and P. falciparum. Does it effect oocyst sizes/stages? Is it ok to compare?

    __ __We agree that a direct comparison between for example * yoelii* and *P. berghei *oocyst size would be inappropriate, however Figure 3c and Supplementary Figure 4 are not direct comparisons between two species, but a summation of all oocysts measured in this study to indicate that the trends we observe transcend parasite/mosquito species differences. Our study was not set up with the experimental power to determine if mosquito host species alter oocyst size.

    __While I acknowledge that UExM has significantly advanced resolution capabilities in parasite studies, the value of standard microscopy technique should not be overlooked. Particularly, when discussing the function of RON11, relevant IFA and electron microscopy (EM) images should be included to support claims about RON11's role in rhoptry biogenesis. This would complement the UExM data and substantially strengthen the conclusions. Importantly, UExM can sometimes produce unexpected localization patterns due to the denaturation process, which warrants caution. __

    The purpose of this study is not to discredit, undermine, or supersede other imaging techniques. It is simply to use U-ExM to answer biological questions that cannot or have not been answered using other techniques. Please refer to Reviewer # 1 Minor text changes comment#17 to see the new paragraph "Comparison of MoTissU-ExM and other imaging modalities" that addresses this

    Both conventional IFA and immunoEM have already been performed on RON11 in sporozoites before (PMID: 31247198). When assessing defects caused by RON11 knockdown, conventional IFA isn't especially helpful because it doesn't allow visualization of individual rhoptries. Thin-section TEM also doesn't provide the whole-cell view needed to draw these kinds of conclusions. Volume EM could likely support these observations, but we don't have access to or expertise in this technique, and we believe it is beyond the scope of this study. It's also important to note that for the defect we observe-missing or abnormal rhoptries-the visualization with NHS ester isn't significantly different from what would be seen with EM-based techniques, where rhoptries are easily identified based on their protein density.

    The statement that "UExM can sometimes produce unexpected localisation patterns due to the denaturation process..." is partially correct but lacks important nuance in this context. Based on our extensive experience with U-ExM, there are two main reasons why the localisation of a single protein may look different when comparing U-ExM and traditional IFA images. First, denaturation: in conventional IFAs, antibodies need to recognize conformational epitopes to bind to their target, whereas in U-ExM, antibodies must recognize linear epitopes. This doesn't mean the target protein's localisation changes, only that the antibody's ability to recognize it does. Second, antibody complexes seem unable to freely diffuse out of the gel, which can result in highly fluorescent signals not related to the target protein appearing in the image, as we have previously reported (PMID: 36993603). Importantly, neither of these factors applies to our phenotypic analysis of RON11 knockdown. All phenotypes described are based solely on NHS Ester (total protein) staining, so the considerations about changes in the localisation of individual proteins are not relevant.

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    Referee #3

    Evidence, reproducibility and clarity

    Overall, the manuscript is well-written and -structured. However, I would like to raise several major points for consideration:

    1. While I acknowledge that UExM has significantly advanced resolution capabilities in parasite studies, the value of standard microscopy technique should not be overlooked. Particularly, when discussing the function of RON11, relevant IFA and electron microscopy (EM) images should be included to support claims about RON11's role in rhoptry biogenesis. This would complement the UExM data and substantially strengthen the conclusions. Importantly, UExM can sometimes produce unexpected localization patterns due to the denaturation process, which warrants caution.
    2. The proposed role of AOR in rhoptry biogenesis appears highly speculative. It is unclear how the authors conclude that "AORs carry rhoptry cargo" solely based on the presence of RON4 within the structure. Inclusion of additional markers to characterize the content of AOR and rhoptries will be essential to substantiate the hypothesis that this enigmatic structure supports rhoptry biogenesis.
    3. The hypothesis that Plasmodium utilizes two distinct rhoptry pairs for invading the salivary gland and liver cells is intriguing but remains clearly speculative. Are the "cytoplasmic pair" and "docked pair" composed of the same secretory proteins? Are the paired rhoptries identical? How does the parasite determine which pair to use for salivary gland versus liver cell invasion? Is there any experimental evidence showing that the second pair is activated upon successful liver cell invasion? Without such data this hypothesis seems rather premature.
    4. The study of RON11 appears to be a continuation of previous work by a collaborator in the same group. However, neither this study nor the previous one adequately addresses the evolutionary context or structural characteristics of RON11. Notably, the presence of an EF-hand motif is an important feature, especially considering the critical role of calcium signaling in parasite stage conversion. Given the absence of a clear ortholog, it would be interesting to know whether other Apicomplexan parasites harbor rhoptry proteins with transmembrane domains and EF-hand motifs, and if these proteins might respond similarly to calcium stimulation. Investigating mutations within the EF-hand domain could provide valuable functional insights into RON11.
    5. The study cannot directly confirm that membrane fusion occurs between rhoptries and AORs.
    6. It is unclear what leads to the formation of the aberrant rhoptries observed in RON11cKD sporozoites. Since mosquitoes were not screened for infection prior to salivary gland dissection, The defect reports and revisited of RON11 knockdown does not aid in interpreting rhoptry pair specialization, as there was no consistent trend as to which rhoptry pair was missing in RON11cKD oocyst sporozoites. The notion that RON11cKD parasites likely have ‚combinatorial defects that effect both rhoptry biogenesis and invasion' poses challenges to understand the molecular role(s) of RON11 on biogenesis versus invasion. Of note, RON11 also plays a role in merozoite invasion. I like the introduction of a segmentation score to Plasmodium oocyst maturation.

    Minor comments:

    1. The term "APR" does not refer to a tubulin structure per se, but rather to the proteinaceous structure to which tubulin anchors. Are there any specific APR markers that can be used in Figure 1C? If not, I recommend avoiding the use of "APR" in this context.
    2. The quality of the "Roolet fibre" image is not good and resembles background noise from PolyE staining. Additional or alternative images should be provided to convincingly demonstrate that PolyE staining indeed visualizes the Roolet fibre. It is puzzling that the structure is visible with PolyE staining but not with tubulin staining.
    3. Figure 2a zoomed image of P. yoelii infected SG is different than the highligted square.
    4. Figure 3 legend: "P. yoelii infected midguts harvested on day 15" should be corrected. More general, yes, "...development of each oocyst within a single midgut is asynchronous." but it is still required to provide the dissection days.
    5. More arrows should be added to Figures 6b and 6c to guide readers and improve clarity.
    6. Do all SG PbRON11cKD sporozoites lose their reduced number of rhoptries during SG invasion as in Figure 7a (no rhoptries)?
    7. Different mosquito species/strains are used for P. yoelii, P. berghei, and P. falciparum. Does it effect oocyst sizes/stages? Is it ok to compare?
    8. I politely disagree with the bold statements ‚ Little is known about cell biology of sporozoite formation.....from electron microscopy studies now decades old' (p.3, 2nd paragraph); ‚To date, only a handful of (instead of ‚or') proteins have been implicated in SG invasion' (p. 4, 1st paragraph). These claims may overlook existing studies; a more thorough review of the literature is recommended.
    9. Page 3 last paragraph: ...the molecular mechanisms underlying SG (invasion?) are poorly understood.

    Significance

    In this study, the authors explore Ultrastructure Expansion Microscopy (U-ExM) in Plasmodium-infected mosquito tissue with the aim to enhance the visualization of parasite ultrastructure. For this purpose, they revisit sporogony, the maturation of sporozoites inside oocysts, and sporozoite invasion of salivary glands, which has been studied both by cell biological methods and experimental genetics over four decades. They focus their analysis on the biogenesis and function of key secretory organelles, termed rhoptries, which are central to parasite invasion and, again, have been studied extensively.

    This study is a follow-up of a previous study by the same authors (Ref. 19). In the former study the authors showed that U-ExM allows to visualize subcellular structures in sporozoites, including the nucleus, rhoptries, Golgi, apical polar rings (APR), and basal complex, as well as midgut-associated oocysts with developing sporozoites. Here, the authors claim a new finding by stating that sporozoites possess two distinct rhoptry pairs. Supposedly, only one pair is utilized during salivary gland invasion. The authors suggest specialization of rhoptries for different cell invasion events. The authors also revisit a RON11 knock-down parasite line, which was previously shown to be deficient in salivary gland invasion, host cell attachment, gliding locomotion, and liver invasion (Ref. 14).

    I find it difficult to estimate the significance. Obviously, attention will be limited to Plasmodium researchers only, as this study is descriptive and revisits a well-studied aspect of the Plasmodium life cycle in the Anopheles vector.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Liffner et al have used the modified expansion microscopy as they term Mosquito Tissue Ultrastructure Expansion Microscopy (MoTissU-ExM) to study a cell biology of temporal development of malaria parasite sporozoite biogenesis within mosquito host. They employed three different malaria parasite models Plasmodium yoelii, P.beghei and P falciparum and infected them in mosquito host.

    The application of MoTissU-ExM to infected mosquito tissues is a significant technical advance, enabling visualizations previously only achievable with electron microscopy.

    The major conclusion and advances are as following

    • The establishment of a "segmentation score" as a great tool for staging asynchronous oocyst development.
    • The location of Centriolar plaques, rootlet and other structures which are difficult to analyse
    • The first detailed timeline for sporozoite rhoptry biogenesis.
    • Clear quantification showing that sporozoites possess four rhoptries and utilise two during salivary gland (SG) invasion.
    • A characterization of the RON11 knockout phenotype, linking it to defects in rhoptry biogenesis and a specific block in SG epithelial cell invasion. The following points are intended to further strengthen the paper for publication.

    Points for Revision

    1. For clarity, it would be helpful to add indicators for the centriolar plaques in Figure 1b, as their locations are not immediately obvious.
    2. The 'image Z-depth' value indicated in the figures is ambiguous. It is not clear whether this refers to the distance from the coverslip surface or the starting point of the z-stack image acquisition. A precise definition of this parameter would be beneficial.
    3. Regarding Figure 1c, the authors state that 'the rootlet fiber is visible'. However, such a structure cannot be confirmed from the provided NHS ester image. Can the authors present a clearer image where the rootlet fibre is more distinct? Furthermore, please provide the basis for identifying this structure as a rootlet fiber based on the NHS ester observation alone.
    4. Why do the congruent rhoptries have similar lengths to each other, while the dimorphic rhoptries have different lengths? Is this morphological difference related to the function of these rhoptries?
    5. Would it be possible to show whether RON11 localises to the dimorphic rhoptries, the congruent rhoptries, or both, by using expansion microscopy and a parasite line that expresses RON11 tagged with GFP or a peptide tag?
    6. The knockdown of RON11 disrupts the rhoptry structure, making the dimorphic and congruent rhoptries indistinguishable. Does this suggest that RON11 is important for the formation of both types of rhoptries? I believe that it would be crucial to confirm whether RON11 localises to all rhoptries or is restricted to specific rhoptries for a more precise discussion of RON11's function.
    7. The authors state that 64% of RON11cKD SG sporozoites contained no rhoptries at all. Does this mean RON11cKD SG sporozoites used up all rhoptries corresponding to the dimorphic and congruent pairs during SG invasion? If so, this contradicts your claims that sporozoites are 'leaving the dimorphic rhoptries for hepatocyte invasion' and that 'rhoptry pairs are specialized for different invasion events'. If that is not the case, does it mean that RON11cKD sporozoites failed to form the rhoptries corresponding to the dimorphic pair? A more detailed discussion would be needed on this point and, as I mentioned above, on the specific role of RON11 in the formation of each rhoptry pair.
    8. Out of pure curiosity, is it possible to measure the length and number of subpellicular microtubules in the sporozoites observed in this study using expansion microscopy?
    9. Is it possible that the dimorphic rhoptries are simply precursors to the congruent rhoptries? Could it be that after the congruent rhoptries are used for SG invasion, new congruent rhoptries are formed from the dimorphic ones and are then used for the next invasion? Would it be possible to investigate this by isolating sporozoites some time after they have invaded the SG and performing expansion microscopy? This would allow you to confirm whether the dimorphic rhoptries truly remain in the same form, or if new congruent rhoptries have been formed, or if there have been any other changes to the morphology of the dimorphic rhoptries.
    10. In addition to the previous point, in the text accompanying Figure 7a, the authors claim that "64% of PbRON11cKD SG sporozoites contained no rhoptries at all, while 9% contained 1 rhoptry and 27% contained 2 rhoptries". Could this data be used to infer which rhoptry pair are missing from the RON11cKD oocyst sporozoites? Can it be inferred that the 64% of salivary gland sporozoites that had no rhoptries in fact had 2 congruent rhoptries in the oocyst sporozoite stage and that these have been discharged already?
    11. In the section titled "Presence of PbRON11cKD sporozoites in the SG intercellular space", the authors state that "the majority of PbRON11cKD-infected mosquitoes contained some sporozoites in their SGs, but these sporozoites were rarely inside either the SG epithelial cell or secretory cavity". - this is suggestive of an invasion defect as the authors suggest. Could the authors collect these sporozoites and see if liver hepatocyte infection can be established by the mutant sporozoites? They previously speculate that the two different types of rhoptries (congruent and dimorphic) may be specific to the two invasion events (salivary gland epithelial cell and liver cell infection).

    There are a few typing errors in the document:

    1. Paragraph 3 of the introduction - line 7, "handful or proteins" should be handful of proteins
    2. Paragraph 5 of the introduction - line 7, "also able to observed" should be observe
    3. In the final paragraph of the introduction - line 1, "leverage this new understand" should be understanding
    4. The first paragraph of the discussion summary contains an incomplete sentence on line 7, "PbRON11ctrl-infected SGs."
    5. The second paragraph of the discussion - line 10, "until cytokinesis beings" should be begins

    Some suggestions for figures

    Fig 1B - could the tubulin image in the hemispindle panel be made brighter?

    Fig 3B: stage 2 and 6 does not show the DNA cyan, it would-be good show the sate of DNA at that particular stage, especially at stage 2 when APR is visible. And box the segment in the parent picture whose subset is enlarged below it.

    Fig 4A - the green text in the first image panel is not visible. Also, the cyan text in the 3rd image in Fig 1A is also difficult to see. There's a few places where this is the case

    Fig 6A - how do the authors know ron11 expression is reduced by 99%? Did they test this themselves or rely on data from the lab that gifted them the construct? Also please provide mention the number of oocyst and sporozoites were observed.

    Fig 6E - are the data point colours the wrong way round on this graph? Just looking at the graph it looks as though the RON11cKD has more rhoptries than the control which does not match what is said in the text.

    Fig S8C, PbRON11 ctrl, pie chart shows 89.7 % spz are present in the secretory cavity while the text shows 100 %, 35/35

    Fig S9D shows that RON11 ckd contains 17.1% sporozoites in secretory cavity while the text says 24%.

    Some point to discuss

    1.One minor point that author suggest that oocyst diameter is not appropriate for the development of sporozoite develop. This is not so true as oocyst diameter tells between cell division and cell growth so it is important parameter especially where the proliferation with oocyst does not take place but the growth of oocyst takes place.

    1. The author have not mentioned that sometimes the stage oocyst development is also dependent on the age of mosquito and it vary between different mosquito gut even if the blood feed is done on same day.
    2. How is the apical polarity different to merozoite as some conoid genes are present in ookinete and sporozoite but not in merozoite.

    Significance

    The following aspects are important:

    This is novel and more cell biology approach to study the challenging stage of malaria parasite within mosquito. By using MoTissU-ExM, the authors have enabled the three-dimensional observation of ultrastructures of oocyst-sporozoite development that were previously difficult to observe with conventional electron microscopy alone. This includes the developmental process and entire ultrastructure of oocysts and sporozoites, and even the tissue architecture of the mosquito salivary gland and its epithelia cells.

    Advances:

    By observing sporozoites formation within the oocyst and the overall ultrastructure of the sporozoite with MoTissU-ExM, the authors have provided detailed descriptions of the complete structure and three-dimensional spatial relationships of the rhoptries, rootlet fibre, nucleus, and other organelles. Furthermore, their detailed localisation analysis of sporozoites within the salivary gland is also a great achievement. Considering that such observations were technically and laboriously very difficult with conventional electron microscopy, enabling these analyses with higher efficiency and relatively lower difficulty represents a major contribution to the future advancement of oocyst-sporozoite biology. The development of the 'segmentation score' for sporozoite formation within the oocyst is another major advance. I think this will enable detailed descriptions of structural changes at each developmental stage and of the molecular mechanisms involved in the development of oocysts-sporozoites This has its advantages if antibodies can be used and somewhat reduces the need for immuno-EM. Secondly, in terms of sporozoite rhoptry biology, the Schrevel et al Parasitology 2007 seems to only focus on oocyst sporozoite rhoptries as they say that the sporozoites have 4 rhoptries. This study on the other hand also looks at salivary gland sporozoites and shows that there are potentially important differences between the two - namely the reduction from 4 rhoptries to two. This also leads to further questions about the different types of rhoptries in oocyst sporozoites and whether they're adapted to invasion of different cell types (sal gland epithelial cells or liver hepatocytes)

    Limitation

    It would be that expansion microscopy alone still has its limits when it comes to observing ultra-fine structures. For example, visualising the small vesicular structures that Schrevel et al. observed in detail with electron microscopy, or seeing ultra-high resolution details such as the fusion of membrane structures and their interactions with structures like the rootlet fibre and microtubules. Therefore, I think that electron microscopy remains essential for the observation of such ultra-fine structures The real impact of this work is mostly cell biologist working with malaria parasite and more in mosquito stages. But the approaches can be applied to any material from any species where temporal dynamics need to be studied with tissue related structures and where UExM can be applied. I am parasite cell biologist working with parasites stages within mosquito vector host.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    In this paper the authors use ultrastructure expansion microscopy to investigate the mosquito stages of the malaria parasite, specifically the stage called oocyst and the process of sporozoite development. They report a number of observations of which the ones concerning rhoptries are the most interesting. There are four of these organelles in the first form of sporozoites in the oocyst and only two in the mature form in the salivary gland. Using a gene knockout of a protein that was reported to be important for rhoprty formation in merozoites, the parasites invading into human red blood cells, they found that fewer rhoptries are formed also in sporozoites and that these cannot enter into the salivary gland cells any more. The presented data are in my view conclusive and no additional experiments are needed for this work to be published. The described experiments should be readily reproducible and have a high statistical power. The text is mostly clearly written but could be improved to make it more concise and more precise and to avoid overstatements. Some references could be added. It would have helped to have line numbers in the manuscript. My suggestions are as following:

    Abstract: don't focus on technique but on the questions you tried to answer (ie rewrite or delete the 3rd and 4th sentence)

    Add reference on page 3 after 'disrupted parasites' Change 'the basal compelx at the leading edge' - this seems counterintuitive Change 'mechanisms underlying SG are poorly' - what mechanisms? of invasion or infection? On page 4: 'handful of proteins' 'range of cell biology processes' - I understand the paper that the key discovery concerns rhoptry biogenesis and function, so focus on that, all other aspects appear rather peripheral. what are the 'three microtubule spindle structures'? 'Much of this study focuses on the secretory organelles': I would suggest to rewrite the intro to focus solely on those, which yield interesting findings. On page 5: 'little is known' - please describe what is known, also in other stages. At the end of the paper I would like to know what is the key difference to rhoptry function in other stages? change 'rhoptries golgi-derived, made de novo' change 'new understand to' 'rhoptry malformations' seem to be similar in sporozoites and merozoites. Is that surprising/new? What is known about crossing the basal lamina. Where rhoptries thought to be involved in this process? Or is it proteins on the surface or in other secretory organelles? On page change/specify: 'wide range of parasite structures' On page 7: is Airyscan2 a particular method or a specific microscope? what are the dark lines in panel E? in panel G: Are the dense granules not micronemes? What are the dark lines? Rhoptries?? On page 8 the authors mention a second layer of CSP but do not further investigate it. It is likely hard to investigate this further but to just let it stand as it is seems unsatisfactory, considering that CSP is the malaria vaccine. What happens if you add anti-CSP antibodies? I would suggest to shorten the opening paragraphs of this paper and to focus on the rhoptries. This could be done be toning down the text on all aspects that are not rhoptries and point to the open question some of the observations such as the CSP layers raise for future studies. Figure 2 seems to add little extra compared to the following figures and could in my view go to the supplement. On page 10 I suggest to qualify the statement 'oocyst development has typcially been inferred by'. There seem a few studies that show that size doesn't reflect maturation. Page 11: I am tempted to suggest the authors start their study with Figure 3 and add panel A from Figure 2 to it. This leads directly to their nice work on rhoptries. Other features reported in Figures 1 and 2 are comparatively less exciting and could be moved to the supplement or reported in a separate study. Text on page 12 could be condensed to highlight the new data of ron4 staining of the AOR. Maybe include more detail of the differences between species on rhoptry structure into Figure 4. I would encourage to move the Data on rhoptries in Figure S6 to the main text ie to Figure 4. On page 16 the authors state that different rhoptries might have different function. This is an interesting hyopthesis/result that could be mentioned in the abstract. how large is RON11? On page 19: do the parasites with the RON11 knockout only have the cytoplasmic or only the apical rhoptries? Page 23: I suggest to delete the first sentence and focus on the functional aspects and the discoveries. There is no causal link between ookinete invasion and oocyst developmental asynchrony First sentence of page 24 appears to contradict what is written in results I don't understand the first two sentences in the paragraph titled Comparison between Plasmodium spp On page 25 or before the vast number of electron microscopy studies should be discussed and compared with the authors new data. First sentence on page 27: there are many studies on parasite proteins involved in salivary gland invasion that could be mentioned/discussed. Maybe add a conclusion section rather than a future application section, which reads as if you want to promoted the use of ultrastructure expansion microscopy. To my taste the technological advance is a bit overplayed considering the many applications of this techniques over the last years, especially in parasitology, where it seems widely used. In any case, please delete 'extraordinarily'

    Significance

    This interesting study investigates the development of malaria parasites in the mosquito using ultrastructure expansion microscopy adapted to mosquito tissue. It provides new and beautiful views of the process of sporozoite formation. The authors discovered that four secretory vesicles called rhoptires are formed in the sporozoites with two pairs being important for distinct functions, one pair functions during invasion of the salivary glands of the mosquito and the other in liver infection, although the latter is not shown but inferred from prior data.

    This study will thus be of interest to scientists investigating malaria parasites in the mosquito as well as to scientist working on vesicle secretion and invasion in these parasites.

    The authors use a previously generated parasite line that lack a protein to investigate its function in rhoptry biogenesis and find that its absence leads to fewer rhoptries which impacts the capacity of the parasite to enter into salivary gland cells. This is a nice functional addition to an otherwise largely descriptive study, but mimics largely the previously reported results from the blood stages. It is not clear to this reviewer how much the study advances the field over the many previous electron microscopy studies. This could be better elaborated in the text.

    Strength of the study: beautiful microscopy, new insights into rhoptry formation and function, new technique to study malaria parasites in the mosquito

    Weakness of the study: Some loose ends in the description of spindles and CSP layers, text could be more focussed on the key advancements reported

  5. Version published to 10.1101/2025.04.09.648058 on bioRxiv