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  1. Evaluation Summary:

    This manuscript describes cellular and developmental defects at late embryonic stages during Wolbachia-induced cytoplasmic incompatibility (CI), which occurs when male insects harboring the endosymbiont bacteria Wolbachia fertilize eggs of uninfected females, triggering embryonic lethality, usually at the first nuclear division. This work presents evidence that the mechanism of late embryonic defects is independent from the ones responsible for early embryonic defects. The experiments are technically superb, and the strength of evidence provided is compelling, including beautiful single-embryo PCR analyses and convincing light microscopy. While the overall significance might be limited, the knowledge will be useful to those in the fields of cytoplasmic incompatibilities and insect embryo development.

    (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 #2 agreed to share their names with the authors.)”

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  2. Reviewer #1 (Public Review):

    This manuscript investigates the cellular and developmental defects underlying Wolbachia-induced cytoplasmic incompatibility (CI), which occurs when male insects harboring the endosymbiont bacteria Wolbachia fertilize eggs of uninfected females, triggering embryonic lethality at the first nuclear division. Characterization of the mechanisms of CI has implications for pest control in insects beyond Drosophila, and thus this topic will have broad interest.

    Previous work, including by the Sullivan lab, has shown that CI is caused by a paternal effect in which the sperm from a Wolbachia+ male triggers a dramatic early failure in the first nuclear division within the newly fertilized Drosophila egg. In this work, the authors provide compelling evidence that there is an additional, later defect that is present in ~30% of the affected embryos. These defects occur at the mid-blastula transition and beyond. They go on to show that these later embryonic defects can be due to loss of the paternal genomic DNA (creating haploids) which could be due to the early fertilization defect, but also a chromosome segregation defect independent of haploidy or the initial fertilization defect. They use elegant single embryo PCR, pooled blastoderm genomic sequencing, and FISH methods to track the origins of the blastula defects; this is a compelling set of experiments! Taking all their results together, they conclude that the latter phenotype is due to a distinct molecular mechanism than that inducing first division defects. The paper is well written and easy to follow.

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  3. Reviewer #2 (Public Review):

    My first thought about this work was, "It's about time!". The complexity and wonder of Wolbachia biology never cease to amaze, and one longstanding question in the field has been where, when and for how long does CI act in the developing egg. This work goes a long way in answering this question and in doing so raises CI analysis and interpretation to a new level. The quality of the work is excellent, the methods used are appropriate, it is organized beautifully and the results are unimpeachable. The work is clearly of great interest to Wolbachia researchers and related fields (vector control, for example) and the entire scientific field will benefit from the knowledge gained in this report.

    Having said all that, there is one overarching issue that is a weakness of the work that plagues the entire field and has done so for 25+ years, and it is a peculiar fact that insect fertilization is opaque to our eyes. Drosophila internal fertilization is impossible to visualize by current means and so many of the crucial events are not observed. Even following egg deposition, the early events and first 4-6 nuclear divisions are also completely obscured to prying eyes. We are therefore left to wonder how much we have missed, and when and where it occurred. All of this is of course added to our murky understanding of the mechanisms involved. Thus, the use of rigid and uncompromising terms and conclusions is difficult and should be avoided. This is the only substantive criticism of this outstanding tour-de-force of early (and now late!) study of CI cell biology.

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  4. Reviewer #3 (Public Review):

    This study aims to determine whether the chromosome defects induced by a bacterial endosymbiont in insects in developing embryos are a direct result of paternal chromosome defects from early embryogenesis or due to a second, independent set of defects that arise later: "we addressed whether defects observed in late CI embryos such as chromosome segregation errors and nuclear fallout are the result of first division errors or a second, distinct CI-induced defect."

    Using crosses, genetics, and fluorescent microscopy, the study claims that the defects at different embryonic stages are due to independent processes, and this work thus has mechanistic relevance to how bacteria inflict developmental harm to insect embryogenesis. The claim is not well supported by the weight of the evidence in this paper and the literature.

    The work is technically sound and proficiently completed to an expert level with appropriate statistics, but it does not provide straight-line evidence to substantiate the primary claim of the paper that later-stage embryos die for different reasons than early-stage embryos. That is no fault of the experimental rigor but rather to the difficulty of directly answering this question. It appears the field has insufficient information on the reductionist, bacterial mechanism that induces embryonic death, namely what acutely is modified by the bacteria to cause embryonic death? As such, the authors hedge that by studying different developmental stages of the embryonic defects, the answer can be surmised. However, a simple explanation for how late and early-stage embryos could die to similar mechanisms is that host cellular conditions are more or less susceptible to the same bacterial-induced change of the insect chromosomes (e.g., new chemical marks on the DNA). It's just not possible to rule this out until the acute mechanism of killing is known. For instance, some embryos may vary in their transcriptomes, proteomes, physiology, etc within a single family of fly offspring, and as such these varying embryos may be more or less susceptible to the same proximal cause of the bacteria-mediated defects. The difference is just when do they take place in development. Without knowing the bacterial mechanism of death (e.g. changes in chemical marks of the fly DNA), the study here can characterize broad strokes of chromatin biology while speculating on the weight of the evidence for whether or not different mechanisms are at play.

    To evaluate the primary question of whether or not there are completely separate defects across development, the study shows several pieces of data that offer a finer resolution of the broad defects of embryos that were previously characterized by the literature. The new follow-up details are robustly supported and include percentages of embryos experiencing a defect, nuclear fallout, determination of haploidy/diploid, sequencing depths, Y chromosome tracking, and developmental-staged characterizations of the chromatin defects. However, according to the text, there is effectively a single type of data that speaks to the main question of the paper - whether or not viable embryos that escaped the first mitosis had increased mitotic errors during later developmental stages.

    "Therefore, the significant increase in mitotic errors observed in diploid CI-derived embryos relative to wild-type derived embryos demonstrates the existence of a second, CI-induced defect, completely separate from the first division defect." This was already known; later-stage, chromatin defects do occur in a variety of insect species cited in the paper. In effect, the question answers itself because, in order to traverse an early lethal state that does not occur, there must be defects that ensue later in development, several of which have already been characterized, though to a lesser resolution than this study.

    Moreover, the study does not link the staged chromatin errors to the CI genes using transgenic tools that are now customary in this field. That work is quite relevant to the conclusion of the paper because the authors speculate in the discussion that additional CI genes may be necessary to explain the later defects in embryogenesis versus the initial defects. This work has been completed to a degree by the papers reporting the initial discovery of the CI genes. CI transgene expression in males causes both 1st mitosis and later chromatin defects, suggesting additional genes are not necessary to explain lethality after the first mitosis. This to me is perhaps the most significant counterpoint of the narrative of the paper's claim because the acute genetic cause of CI can lead to differently timed chromatin errors.

    This is solid work and a strong effort to refine the stages and types of embryonic lethality induced by bacteria, however, the claim that there are different acute mechanisms of death during embryogenesis is not well supported.

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  5. Author Response:

    We thank the reviewers for their thoughtful critiques and helpful suggestions for how to improve our manuscript. Described below is our response clarifying a number of issues raised by the reviewers.

    We agree with the reviewer that we cannot definitively conclude that the first division chromosome segregation defects and the later mid-blastula transition CI-induced defects are the result of distinct mechanisms. In fact, we raise this possibility in the discussion. However, our finding that the CI phenotype induces a temporally and developmentally deferred chromosome segregation defects in the late blastoderm divisions (in addition to the well-studied first division defect) alters the established view of the CI phenotype and must be taken into account when considering mechanisms of CI. Our current view is that the distinct early and late defects could be caused by either 1) a common mechanism (possibly a chromosome mark/defect inherited through the early blastoderm divisions causing segregation defects in the late blastoderm divisions) or 2) distinct early and late mechanisms that do not strictly “depend” upon one another. We have clarified this point in the revised manuscript.

    We disagree with the reviewer that this result is to be expected given previous studies. In D. simulans, a small percentage of embryos derived from the CI cross hatch. These embryos are thought to have bypassed the first division defect. It is not obvious why there must be late defects in these embryos that “escape” early CI-induced defects and subsequently hatch. Previous studies interpreted embryos that exhibit late division errors as those that have lost their entire paternal complement of chromosomes as a result of strong CI-induced defects during the first mitotic division and develop as maternal haploids. These studies, including transgene- induced CI, have focused primarily on embryos that have undergone the first mitotic division embryonic defects. To the best of our knowledge, no group has thoroughly examined embryos that progress normally through the pre-cortical cycles 2-9 as performed in this manuscript. Thus, it was entirely unexpected that these embryos would exhibit the mitotic defects during the late blastoderm divisions and the MBT. We discuss how this finding requires modified current models for the mechanisms of CI.

    Regarding the comment that “the primary claim of the paper that later-stage embryos die for different reasons than early-stage embryos,” we make no such claim. In fact, we provide evidence that the failure to hatch (late embryonic lethality) is, at least in part, due to haploid development—a direct result of the first division CI defect. The focus of our studies are those CI-derived embryos that progress normally, maintain the normal complement of chromosomes through the first division, and exhibit chromosome segregation errors during the late blastoderm divisions. We do not know the fate of these embryos, and previous studies have demonstrated that embryos suffering extensive late blastoderm segregation errors are able to hatch (Sullivan, 1990, Development 110:311-323). We have clarified these points in the discussion.

    While we agree that transgenic tools have proven invaluable in the study of CI, they are not appropriate for these studies. The purpose of our study was to undertake an unbiased re-examination of the CI phenotype. Of necessity, the transgenic studies rely on exogenous host promoters rather than the natural endogenous Wolbachia/Prophage promoters. Thus, while informative, it is unlikely the that the transgenic alleles would capture all of the complexities and nuance of the CI phenotype. In addition, the transgenic studies, of which we are aware, have only interrogated a single pair of the CI-inducing genes, while the Wolbachia genome contains both Cid and Cin CI-associated gene pairs and possibly other yet-to-be-identified CI/Rescue genes.

    Our unbiased re-examination of the CI phenotype induced by W. riverside in D. simulans identified a previously unsuspected temporally and developmentally distinct set of CI-induced defects that occur during and after the mid-blastula transition. This finding must be taken into account when considering the mechanisms that cause CI. In our revisions, we clarify the above points and qualify our statements to appropriately interpret our results in context of the nuances and uncertainties of CI and early Drosophila embryogenesis.

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