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  1. Author Response:

    Evaluation Summary:

    The present work aims to increase our understanding of marine epizootics caused by the dinoflagelate parasite Hematodinium sp. in crabs. The work includes a large data set of field collected specimens from a wide geographical area. The authors have evaluated presence or absence of this parasite as well as co-infections by several other groups of pathogens and model the main factors that shape crab community structure. The topic of study is very important in the context of current marine pandemics and, therefore, adequate examination of this data set may lead to significant advances in the field. Refinement of the approaches to produce quantitative data is needed in order to reach to more solid conclusions.

    We are grateful to the reviewers and editorial members of eLife for their evaluation of our submission and recognising the importance/rarity of our dataset for advancing our understanding of marine epizootics. In our revised text, we have included further quantitative data in the context of Hematodinium-parasitised crabs and re-run all our analyses.

    Reviewer #1 (Public Review):

    The study would have benefited from qPCR instead of presence or absence. It is interesting to note that differences were observed when CFUs were compared. I understand that for most organisms, in the absence of a draft genome, assigning copy numbers to cells is not yet possible; however, it would have provided a more robust dataset for the statistical analysis.

    Similarly, histology relies upon one of a hundred possible for every single specimen. With this caveat, scoring the presence of the parasites rather than (+) (-) would also have been more informative. Without knowing the intensity of the infection for each of the other pathogens makes it more difficult to reject the hypothesis. The authors discuss other papers with cellular immune data, which is lacking in this manuscript. Observing fresh hemolymph and histology are just not enough. The data strongly support that parasite community composition is affected by the location. Smaller crabs also appear to be more likely to display co-infections compared to disease-free crabs.

    We thank reviewer 1 for spending their time considering our submission.

    We used population genetic markers for Hematodinium and crabs, and PCR-based molecular diagnostics, in addition to freshly withdrawn haemolymph and multi-tissue histology in our original submission. In our revised text we have incorporated additional quantitative Hematodinium loads in haemolymph, gill and hepatopancreas in crabs.

    In our original submission, we presented data that discriminated between Hematodinium-positive crabs (n = 162) and Hematodinium-negative crabs (n = 162) based on targeted PCR. Rather than use qPCR – due to the absence of sufficient molecular information to ensure single-copy gene targets per genome for Hematodinium spp. and the diversity of ecotypes presented in our study – we have incorporated count data (actual number of parasites per mL haemolymph) from the original liquid tissue screens using haemocytometry, and grade data (0-4) from solid (gill and hepatopancreas) tissues assessed using histology. In our view, these quantitative data are more powerful than those that we could have achieved via qPCR.

    We have analysed these additional data in the context of co-infection presence. Indeed, when we restricted the Hematodinium-positive crabs to n = 111 based on individuals that were positive for the parasite in all three diagnostic methods across key tissues (haemolymph via haemocytometry and PCR, and gill/hepatopancreas via histology), and re-ran all our analyses/models, the outcomes did not yield contradictory conclusions. Hematodinium-positive crabs were no more likely to contain co-infections when compared to Hematodinium-negative crabs, AND, location remains the determining factor for pathogen diversity among crabs.

    Reviewer #2 (Public Review):

    Strength:

    1. The authors looked into the prevailing idea that parasitic infection make crab immune-compromised although evidence to support this idea is lacking except one study by where immune gene expression was found to be modulated upon Hematodinium infection in Japanese blue crab. Apparently, the authors studying gene expression in Japanese blue crab upon parasitic infection did not identify any effort molecules of parasitic origin.
    2. It was interesting to note that haplotype diversity analysis revealed higher genetic diversity in the host compared to the Hematodinium parasite in two locations examined.

    We thank Reviewer 2 for their time and insight.

    Weakness:

    1. The authors should clarify how the sample sizes were selected.

    We apologise for the unintentional ambiguity. The initial survey covered ~50 crabs per location per month for 1 year (n = 1191) - using an alpha value of 0.05 and desired power >80% indicated a minimum of 38 (1-sided test) or 48 (2-sided test) crabs were needed based on an a priori prediction of 15% Hematodinium prevalence in the C. maenas population. The 162 Hematodinium-positive crabs (PCR) were size/sex/location matched to 162 Hematodiniumnegative crabs.

    The methods section has been edited to ensure that this information is clear.

    1. It is also recommended that details on sample collections are included (for example the times in which samples from the surrounding waters of infected crabs)

    More details on sample collection have been included in our revision

    Reviewer #3 (Public Review):

    This manuscript aims to assess the main factors that drive disease in crabs across Europe. Crabs are critical members of the trophic chain in marine environments and also serve as food source for human consumption. Therefore, understanding the health of this group of organisms is very important for ecosystem health as well as human health. This is a well written manuscript that identifies important knowledge gaps in the field. The authors evaluate whether crabs are infected by an important parasitic dinoflagelate, Hematodinium, and question several current dogmas in the field. The first one is whether or not presence of Hematodinium infection is a significant factor driving co-infections by other pathogens including trematodes, bacteria or fungi. The authors, based on their data set, conclude that this is not the case, since co-infections are observed in similar proportions in Hematodinium free animals. The authors perform in depth assessment of other drivers of infection and identify geographical location as the main factor driving community structure.

    We thank Reviewer 3 for their time and insight.

    A second important aspect of Hematodinium biology is the previous notion that this parasite immunosuppresses the crab host (and therefore co-infections may be found). However, there is a paucity of studies to confirm or reject this hypothesis, and therefore, the current study is important to expand our current knowledge of marine invertebrate immunobiology and specifically in the mechanisms by which Hematodinium is harmful to the host. The current study provides some evidence that, in fact, this parasite is not an immune suppressor but rather evades the cellular immune response of the host. In particular, the authors claim that the host hemocytes (immune cells) fail to phagocytose (engulf) and encapsulate (surround and fend off) this parasite. These two cellular immune responses are the most common in invertebrates such as crabs.

    Overall, this study is important because very few studies evaluate wild marine diseases, especially in invertebrate hosts and because it contributes with a large data set from a wide geographical range.

    We thank Reviewer 3 for their time and insight.

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  2. Evaluation Summary:

    The present work aims to increase our understanding of marine epizootics caused by the dinoflagelate parasite Hematodinium sp. in crabs. The work includes a large data set of field collected specimens from a wide geographical area. The authors have evaluated presence or absence of this parasite as well as co-infections by several other groups of pathogens and model the main factors that shape crab community structure. The topic of study is very important in the context of current marine pandemics and, therefore, adequate examination of this data set may lead to significant advances in the field. Refinement of the approaches to produce quantitative data is needed in order to reach to more solid conclusions.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    The study would have benefited from qPCR instead of presence or absence. It is interesting to note that differences were observed when CFUs were compared. I understand that for most organisms, in the absence of a draft genome, assigning copy numbers to cells is not yet possible; however, it would have provided a more robust dataset for the statistical analysis.

    Similarly, histology relies upon one of a hundred possible for every single specimen. With this caveat, scoring the presence of the parasites rather than (+) (-) would also have been more informative. Without knowing the intensity of the infection for each of the other pathogens makes it more difficult to reject the hypothesis. The authors discuss other papers with cellular immune data, which is lacking in this manuscript. Observing fresh hemolymph and histology are just not enough. The data strongly support that parasite community composition is affected by the location. Smaller crabs also appear to be more likely to display co-infections compared to disease-free crabs.

    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    Strength:
    1. The authors looked into the prevailing idea that parasitic infection make crab immune-compromised although evidence to support this idea is lacking except one study by where immune gene expression was found to be modulated upon Hematodinium infection in Japanese blue crab. Apparently, the authors studying gene expression in Japanese blue crab upon parasitic infection did not identify any effort molecules of parasitic origin.
    2. It was interesting to note that haplotype diversity analysis revealed higher genetic diversity in the host compared to the Hematodinium parasite in two locations examined.

    Weakness:
    1. The authors should clarify how the sample sizes were selected.
    2. It is also recommended that details on sample collections are included (for example the times in which samples from the surrounding waters of infected crabs)

    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    This manuscript aims to assess the main factors that drive disease in crabs across Europe. Crabs are critical members of the trophic chain in marine environments and also serve as food source for human consumption. Therefore, understanding the health of this group of organisms is very important for ecosystem health as well as human health.

    This is a well written manuscript that identifies important knowledge gaps in the field. The authors evaluate whether crabs are infected by an important parasitic dinoflagelate, Hematodinium, and question several current dogmas in the field. The first one is whether or not presence of Hematodinium infection is a significant factor driving co-infections by other pathogens including trematodes, bacteria or fungi. The authors, based on their data set, conclude that this is not the case, since co-infections are observed in similar proportions in Hematodinium free animals. The authors perform in depth assessment of other drivers of infection and identify geographical location as the main factor driving community structure.

    A second important aspect of Hematodinium biology is the previous notion that this parasite immunosuppresses the crab host (and therefore co-infections may be found). However, there is a paucity of studies to confirm or reject this hypothesis, and therefore, the current study is important to expand our current knowledge of marine invertebrate immunobiology and specifically in the mechanisms by which Hematodinium is harmful to the host. The current study provides some evidence that, in fact, this parasite is not an immune suppressor but rather evades the cellular immune response of the host. In particular, the authors claim that the host hemocytes (immune cells) fail to phagocytose (engulf) and encapsulate (surround and fend off) this parasite. These two cellular immune responses are the most common in invertebrates such as crabs.

    Overall, this study is important because very few studies evaluate wild marine diseases, especially in invertebrate hosts and because it contributes with a large data set from a wide geographical range.

    Was this evaluation helpful?