Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
Insects and their relatives are commonly infected with microbes that are transmitted from mothers to their offspring. A number of these microbes have independently evolved the ability to kill the sons of infected females very early in their development; this male killing strategy has evolved because males are transmission dead-ends for the microbe. A major question in the field has been to identify the genes that cause male killing and to understand how they work. This has been especially challenging because most male-killing microbes cannot be genetically manipulated. This study focuses on a male-killing bacterium called Wolbachia. Different Wolbachia strains kill male embryos in beetles, flies, moths, and other arthropods. This is remarkable because how sex is determined differs widely in these hosts. Two Wolbachia genes have been previously implicated in male-killing by Wolbachia: oscar (in moth male-killing) and wmk (in fly male-killing). The genomes of some male-killing Wolbachia contain both of these genes, so it is a challenge to disentangle the two.
This paper provides strong evidence that oscar is responsible for male-killing in moths. Here, the authors study a strain of Wolbachia that kills males in a pest of tea, Homona magnanima. Overexpressing oscar, but not wmk, kills male moth embryos. This is because oscar interferes with masculinizer, the master gene that controls sex determination in moths and butterflies. Interfering with the masculinizer gene in this way leads the (male) embryo down a path of female development, which causes problems in regulating the expression of genes that are found on the sex chromosomes.
We would like to thank you for evaluating our manuscript.
Strengths:
The authors use a broad number of approaches to implicate oscar, and to dissect its mechanism of male lethality. These approaches include:
(1) Overexpressing oscar (and wmk) by injecting RNA into moth eggs.
(2) Determining the sex of embryos by staining female sex chromosomes.
(3) Determining the consequences of oscar expression by assaying sex-specific splice variants of doublesex, a key sex determination gene, and by quantifying gene expression and dosage of sex chromosomes, using RNASeq.
(4) Expressing oscar along with masculinizer from various moth and butterfly species, in a silkmoth cell line.
This extends recently published studies implicating oscar in male-killing by Wolbachia in Ostrinia corn borer moths, although the Homona and Ostrinia oscar proteins are quite divergent. Combined with other studies, there is now broad support for oscar as the male-killing gene in moths and butterflies (i.e. order Lepidoptera). So an outstanding question is to understand the role of wmk. Is it the master male-killing gene in insects other than Lepidoptera and if so, how does it operate?
Thank you for your comments. Wolbachia strains often carry wmk genes, but as observed in this study, the homologs in Homona showed no apparent MK ability. These showed strong male lethality in D. melanogaster, but it is still unclear whether the genes are the master male-killing gene in Diptera. It is also possible that the genes show toxicities in other lepidopteran insects as well as in other insect taxa. Further functional validation assays in different insects are warranted to clarify whether wmk shows toxicity in different insect taxa. We have also discussed the functions of wmk in the Discussion section (lines 301-306).
Weaknesses:
I found the transfection assays of oscar and masculinizer in the silkworm cell line (Figure 4) to be difficult to follow. There are also places in the text where more explanation would be helpful for non-experts (see recommendations).
Thank you for your suggestion. We have thoroughly revised the manuscript to address all the questions, comments and suggestions you raised in “recommendations”. In particular, we have revised the section on the transfection assays of Oscar and Masc in Bm-N4 cells (result section “Hm-oscar suppresses the masculinizing functions of lepidopteran masc genes” starts on line 214 and Fig. 4; materials and methods section ”Transfection assays and quantification of BmIMPM”, starts on line 483). We have also provided more detailed explanations for non-experts in some contexts (in response to your recommendation). We believe that the resulting revisions have significantly improved the quality and comprehensiveness of our manuscript.
Reviewer #2 (Public review):
Summary:
Wolbachia are maternally transmitted bacteria that can manipulate host reproduction in various ways. Some Wolbachia induce male killing (MK), where the sons of infected mothers are killed during development. Several MK-associated genes have been identified in Homona magnanima, including Hm-oscar and wmk-1-4, but the mechanistic links between these Wolbachia genes and MK in the native host are still unclear.
In this manuscript, Arai et al. show that Hm-oscar is the gene responsible for Wolbachia-induced MK in Homona magnanima. They provide evidence that Hm-Oscar functions through interactions with the sex determination system. They also found that Hm-Oscar disrupts sex determination in male embryos by inducing female-type dsx splicing and impairing dosage compensation. Additionally, Hm-Oscar suppresses the function of Masc. The manuscript is well-written and presents intriguing findings. The results support their conclusions regarding the diversity and commonality of MK mechanisms, contributing to our understanding of the mechanisms and evolutionary aspects of Wolbachia-induced MK.
We would like to thank you for evaluating our manuscript.
Strengths/weaknesses:
(1) The authors found that transient overexpression of Hm-oscar, but not wmk-1-4, in Wolbachia-free H. magnanima embryos induces female-biased sex ratios. These results are striking and mirror the phenotype of the wHm-t infected line (WT12). However, Table 1 lists the "male ratio," while the text presents the "female ratio" with standard deviation. For consistency, the calculation term should be uniform, and the "ratio" should be listed for each replicate.
We have revised the first results section (Hm-oscar induces female-biased sex ratios, starting from line 147) accordingly to maintain the consistency in the calculation term. In the revised manuscript, the 'male ratio' is now consistently used, in alignment with Fig. 1. In addition, we have included all sex ratio information (number of males and females) in the supplementary data file for transparency and clarity.
(2) The error bars in Figure 3 are quite large, and the figure lacks statistical significance labels. The authors should perform statistical analysis to demonstrate that Hm-oscar-overexpressed male embryos have higher levels of Z-linked gene expression.
The large error bar on each chromosome (Fig.3a-d) likely reflect the overall variation in expression levels across different transcripts. Accordingly, we have included statistical data for Figure 3 based on the Steel-Dwass test for expression levels. However, displaying statistical significance directly on the whisker plots would make the figure too cluttered due to the numerous combinations. Instead, we have provided all the statistical data in the supplementary data file. To further support the claim that Z-linked genes are more highly expressed in wHm-t-infected/Hb-Oscar-injected embryos, we have included the expression data for a Z-linked gene tpi, along with its statistical data in the revised manuscript (Fig. 3e, lines 210-212).
(3) The authors demonstrated that Hm-Oscar suppresses the masculinizing functions of lepidopteran Masc in BmN-4 cells derived from the female ovaries of Bombyx mori. They should clarify why this cell line was chosen and its biological relevance. Additionally, they should explain the rationale for evaluating the expression levels of the male-specific BmIMP variant and whether it is equivalent to dsx.
Thank you for your suggestion. We selected BmN-4 cell line because previous studies have established it as a reliable model for investigating the functions of lepidopteran masc genes and the interactions between masc and Oscar genes (Katsuma et al., 2019; 2022). In addition, BmIMPM is a male-specific regulator of the male-type dsx, making it an ideal target for assessing the 'maleness' induced by transfection of the masc gene in female-derived BmN-4 cells (Suzuki et al., 2010; Katsuma et al., 2015). We have included more detailed background information in the revised manuscript and have thoroughly revised this section (Hm-oscar suppresses the masculinizing functions of lepidopteran masc genes, starting at line 214) and Figure 4 for better clarity.
(4) Although the authors show that Hm-oscar is involved in Wolbachia-induced MK in Homona magnanima and interacts with the sex determination system in lepidopteran insects, the precise molecular mechanism of Hm-oscar-induced MK remains unclear. Further studies are needed to elucidate how Hm-oscar regulates Homona magnanima genes to induce MK, though this may be beyond the scope of the current manuscript.
Based on our findings and previous studies in Homona, Ostrinia and Bombyx (Arai et al., 2023a; Katsuma et al., 2023; Kiuchi et al., 2014), we hypothesize that the molecular mechanisms underlying _w_Hm-induced MK are likely linked to impaired dosage compensation caused by the inhibition of Masc function by the Hm-Oscar protein. While the precise mechanisms remain unclear, unbalanced Z-linked gene expression due to the impaired dosage compensation (i.e., 2-fold higher Z-linked gene expression compared to normal males) is known to be lethal for lepidopteran males (Kiuchi et al., 2014; Fukui et al., 2015; Visser et al., 2021). We have outlined this hypothesis in the Discussion section (lines 245-254).
Reviewer #3 (Public review):
Summary:
Overall, this is a clearly written manuscript with nice hypothesis testing in a non-model organism that addresses the mechanism of Wolbachia-mediated male killing. The authors aim to determine how five previously identified male-killing genes (encoded in the prophage region of the wHm Wolbachia strain) impact the native host, Homona magnanima moths. This work builds on the authors' previous studies in which:
(1) They tested the impact of these same wHm genes via heterologous expression in Drosophila melanogaster.
(2) They examined the activity of other male-killing genes (e.g., from the wFur Wolbachia strain in its native host: Ostrinia furnacalis moths).
Advances here include identifying which wHm gene most strongly recapitulates the male-killing phenotype in the native host (rather than in Drosophila), and the finding that the Hm-Oscar protein has the potential for male-killing in a diverse set of lepidopterans, as inferred by the cell-culture assays.
Strengths:
Strengths of the manuscript include the reverse genetics approaches to dissect the impact of specific male-killing loci, and the use of a "masculinization" assay in Lepidopteran cell lines to determine the impact of interactions between specific masc and oscar homologs.
We would like to thank you for evaluating our manuscript.
Weaknesses:
My major comments are related to the lack of statistics for several experiments (and the data normalization process), and opportunities to make the manuscript more broadly accessible.
Thank you for your suggestions. We have thoroughly revised the manuscript to provide clearer explanations for non-experts. In addition, we have included more detailed statistical data for Figure 3 and Figure 4 based on the Steel-Dwass tests. For Figure 3a-d, displaying statistical significance directly on the whisker plots would make the figure too cluttered due to the numerous combinations. Therefore, we have provided all the statistical data in the supplementary data file. To further support the claim that Z-linked genes are more highly expressed in _w_Hm-t-infected/Hm-Oscar-injected embryos, we have included the expression data for a Z-linked gene tpi, along with its statistical data in the revised manuscript (Fig.3e, lines 210-212). Regarding Figure 4, we have revised the Figure based on the reviewer’s suggestions, and provided more detailed information on how the expression data were analyzed (Transfection assays and quantification of BmIMPM, lines 495-520). We have also included more detailed background information on the assay system (Hm-oscar suppresses the masculinizing functions of lepidopteran masc genes, lines 215-237). Although we did not observe statistical significance based on the Steel-Dwass test, likely due to limited replicates, the observed changes in the IMP gene expression remain clearly evident.
The manuscript I think would be much improved by providing more details regarding some of the genes and cross-lineage comparisons. I know some of this is reported in previous publications, but some summary and/or additional analysis would make this current manuscript much more approachable for a broader audience, and help guide readers to specific important findings. For example, a graphic and/or more detail on how the wmk/oscar homologs (within and across Wolbachia strains) differ (e.g., domains, percent divergence, etc) would be helpful for contextualizing some of the results. I recognize the authors discuss this in parts (e.g., lines 223-227), but it does require some bouncing between sections to follow. Similarly, the experiments presented in Figure 4 indicate that Hm-oscar has broad spectrum activity: how similar are the masc proteins from these various lepidopterans? Are they highly conserved? Rapidly evolving? Do the patterns of masc protein evolution provide any hints at how Oscar might be interacting with masc?
Thank you for your valuable suggestion. To address this, we have included a visualization of the structural differences between the Oscar and wmk homologs in Figure 1a of the revised manuscript. In addition, we have included more detailed information for these genes and revised the introduction (lines 110-114; 124-137) and discussion (lines 255-266) to provide a clearer and more comprehensive overview. We have also described the similarity of the Masc proteins and Oscar proteins that we used, which is now reflected in the revised Figure 4b and 4d. More detailed information on these proteins is available in the supplementary data. Notably, Masc proteins exhibit high sequence variability with conserved domains (Figure 4d). Previous study identified the N-terminal region of Masc as crucial for the Oscar function (Katsuma et al., 2022). The wide spectrum of the actions of Hm-Oscar likely stems from these conserved structures of Masc, but the effects might have undergone evolutionary tuning through interactions with the native host as discussed in lines 293-294.
It is clear from Figure 1 that the combinations of wmk homologs do not cause male killing on their own. Did the authors test if any of the wmk homologs impact the MK phenotype of oscar? It looks like a previous study tested this in wFur (noted in lines 250-252), but given that the authors also highlight the differences between the wFur-oscar and Hm-oscar proteins, this may be worth testing in this system. Related to this, what is the explanation for why there would be 4 copies of wmk in Hm?
Thank you for your valuable suggestion. Unfortunately, we have not yet tested the effects of co-expression of wmk and Oscar. Due to a technical issue, the mixing of multiple constructs results in a reduced amount of mRNA (i.e. mixing wmk-3 and Hm-Oscar at the same concentration results in a 2-fold lower concentration in mRNA for both genes compared to mono-injected groups). In addition, we have previously tested injecting mRNA at the twofold higher concentration (i.e. 2 ug/ul mRNA), which resulted in very low hatchability regardless of the genes. Katsuma et al (2022) tested the effect of wmk on the sex determination system, but did not test the effect of co-injection/transfection of wmk and Oscar. Considering the results of this and previous studies (Katsuma et al., 2022; Arai et al., 2023), it is likely that the targets of the wmk and oscar genes are different (as discussed in lines 267-289). Co-injection of wmk and oscar may not produce additive effects. Nevertheless, we would like to test the results in future studies using the Drosophila system as well.
As you point out, it is an interesting point that the moth-derived MK Wolbachia _w_Hm-t encodes four wmk genes, although they have no apparent effect on host survival. The exact functional relevance of these wmk homologs remains unclear. However, they may play a role in Wolbachia biology as transcriptional regulators, given that they encode HTH domains. Wolbachia generally encode several wmk homologs in their genome, regardless of whether they induce MK. This suggests that the functions of the wmk genes may be 'suppressed' in certain Wolbachia-host systems. The wmk and Hm-oscar genes are located within a prophage region, and some wmk genes are tandemly arrayed (as described in Arai et al., 2023). These wmk homologs may have increased in number by horizontal phage transfer, and the region containing wmk and adjacent sequences may act as a genomic island for virulence. So far, the function of wmk homologs has only been tested in D. melanogaster and H. magnanima, and further studies in other Wolbachia-host systems are highly warranted to test whether wmk exerts MK effects in other insect models. These points have been briefly discussed in the revised manuscript (lines 301-306; 318-320).
Why are some of the broods male-biased (2/3) rather than ~50:50? (Lines 170-175, Figure 2a). For example, there is a strong male bias in un-hatched oscar-injected and naturally infected embryos, whereas the control uninfected embryos have normal 50:50 sex ratios. It is difficult to interpret the rate of male-killing given that the sex ratios of different sets of zygotes are quite variable.
The observed male-biased sex ratios in unhatched embryos are due to the occurrence of MK during embryogenesis. In the unhatched groups, the skew towards males reflects that fact that the male embryos were targeted and killed by Wolbachia/Oscar, leading to a surplus of unhatched male embryos. Conversely, hatched individuals show a higher proportion of females because many of the males were eliminated during embryogenesis. Thus, the unhatched embryos are more male-biased, while the hatched individuals are more female-biased in the Hm-oscar/_w_Hm-t treated groups. We have revised the relevant section (Males are killed mainly at the embryonic stage, lines 179-186) and provided more detailed information to clarify this explanation.
Figure 2b - it appears there are both male and female bands in the HmOsc male lane. I think this makes sense (likely a partial phenotype due to the nature of the overexpression approach), but this is worth highlighting, especially in the context of trying to understand how much of the MK phenotype might be recapitulated through these methods. Related, there is no negative control for this PCR.
Thank you for your suggestion. As you noted, a faint dsx-M band is visible in the Hm-oscar treated group in Figure 2b. This is consistent with previous findings by Arai et al. (2023), which reported that male embryos with low-density _w_Hm-t showed double bands of dsx-M and dsx-F, similar to what we observed in this study. This information has been included in the revised manuscript in lines 196-198, as follows:
“Notably, male embryos expressing Hm-oscar also exhibited weak male-type dsx splicing in addition to the female-type splicing, resembling the previously observed pattern in male embryos infected with low-titer _w_Hm-t (Arai et al., 2023a).”
Also, we appreciate your comment regarding the missing of negative control. The figure has now been revised as we realised that the negative control lane had been lost during the preparation of the figure. We also included the relevant molecular marker information in both the figure legends and Figure 2b.
It appears the RNA-seq analysis (Figure 3) is based on a single biological replicate for each condition. And, there are no statistical comparisons that support the conclusions of a shift in dosage compensation. Finally, it is unclear what exactly is new data here: the authors note "The expression data of the wHm-t-infected and non-infected groups were also calculated based on the transcriptome data included in Arai et al. (2023a)" - So, are the data in Figure 3c and 3d a re-print of previous data? The level of dosage compensation inferred by visually comparing the control conditions in 3b and 3d does not appear consistent. With only one biological replicate library per condition, what looks like a re-print of previous data, and no statistical comparisons, this is a weakly supported conclusion.
Thank you for your suggestion. In this study, we generated the RNA-seq data for the Hm-oscar/GFP-injected groups, but did not sequence the _w_Hm-t-infected/NSR lines. Instead, the previously generated RNA-seq data of _w_Hm-t-infected/NSR (Arai et al., 2023) were re-analyzed (rather than simply reprinted) to evaluate whether the expression patterns of Hm-oscar-injected and _w_Hm-t-infected groups are similar. We have revised the Results section (Hm-oscar impairs dosage compensation in male embryos, lines 200-212), the Materials and methods section (Quantification of Z chromosome-linked genes, lines 454-456), and the figure legends to provide more precise information about this analysis.
Although we did not perform replicates for the RNA-seq comparisons, it is important to note that each RNA-seq sample contains 50-60 male/female individuals. We believe the results are still robust and clearly indicative of the trends we observe. This was further supported by the quantification of Hmtpi gene expression, which we have visualized in Figure 3e (and lines 210-212). As you noted, the expression patterns in Figure 3b (GFP injected) and Figure 3d (NSR) are not completely identical. This discrepancy may be due to the differences between injection treatments and natural infections. Nevertheless, both treatments are consistent in showing that gene expressions on the Z chromosome (Chr01 and Chr15) are not upregulated.
We have also added more detailed statistical data for Figure 3 based on the Steel-Dwass tests. For Figure 3a-d, however, showing the statistical significance directly on the whisker plots would create excessive clutter due to the numerous combinations of chromosomes. Instead, we have provided the full statistical data in the supplementary data file. Furthermore, to support/strengthen our conclusion that Z-linked genes are highly expressed in _w_Hm-t-infected/Hm-Oscar-injected embryos, we have included expression data for the Z-linked gene tpi, along with statistical data, in the revised manuscript (Fig. 3e, lines 210-212).
In Figure 4: There are no statistics to support the conclusions presented here. Additionally, the data have gone through a normalization process, but it is difficult to follow exactly how this was done. The control conditions appear to always be normalized to 100 ("The expression levels of BmImpM in the Masc and Hm-Oscar/Oscar co-transfected cells were normalized by setting each Masc-transfected cell as 100"). I see two problems with this approach:
(1) This has eliminated all of the natural variation in BmImpM expression, which is likely not always identical across cells/replicates.
(2) How then was the percentage of BmImpM calculated for each of the experimental conditions? Was each replicate sample arbitrarily paired with a control sample? This can lead to very different outcomes depending on which samples are paired with each other. The most appropriate way to calculate the change between experimental and control would be to take the difference between every single sample (6 total, 3 control, 3 experimental) and the mean of the control group. The mean of the control can then be set at 100 as the authors like, but this also maintains the variability in the dataset and then eliminates the issue of arbitrary pairings. This approach would also then facilitate statistical comparisons which is currently missing.
Thank you for your suggestion. As you pointed out in (1), the previous analysis did indeed eliminate the natural variation in BmIMP-M expression. In the revised manuscript and Figure 4, we have reanalyzed the data following your suggestion and have described the variation across replicates.
For (2), the data shown in the previous manuscript were normalized to 100 for each Masc-treated group. In doing so, each replicate sample was arbitrarily paired with a control sample from the same cell lot to account for variations that might occur due to differences in cell lots. However, following your recommendation, we have revised the figure to set the average of the Hm-masc treated group to 100, rather than using arbitrary pairings. More detailed normalization procedures have been provided in the section 'Transfection assays and quantification of BmIMP' (lines 483-520). Additionally, we have provided more detailed background information on the assay system in lines 218-223. Although we did not observe statistical significance based on the Steel-Dwass test, likely due to the limited number of replicates, the differences in IMP gene expression between the Masc-treated and Masc&Hm-oscar-treated groups remain evident.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
Line 38: change to: 'Wolbachia are maternally transmitted'.
Revised accordingly (line 38).
Line 69: remove 'seemingly'.
Revised accordingly (line 69).
Paragraph starting line 123: I don't think this is so clear to a reader who is not familiar with the work and system. It would be helpful to more clearly explain that candidate male-killing genes from Wolbachia that infect Homona were inserted into Drosophila melanogaster, and that their expression was then induced, with interesting patterns (and that it can be a bit difficult to interpret the transgenic expression of genes from a moth male-killer that are inserted into a fly). Also, the sentence about the combined action of cifA and cifB in Drosophila cytoplasmic incompatibility is also confusing to a non-expert. I would suggest removing it.
Thank you for your suggestion. We have revised the paragraph (lines 124-139) to provide clearer background information, making it easier for non-experts to follow. We have also removed the sentence regarding the combined effect of cifA and cifB to improve the flow and overall clarity.
Line 170: what is the explanation for the male-biased sex ratio instead of 50-50?
The male-biased sex ratio occurs because MK happens during embryogenesis. Unhatched embryos include males that were killed by Wolbachia/Oscar, resulting in a higher proportion of unhatched male embryos. Conversely, the hatched individuals display a female bias, as most of the males were eliminated during embryogenesis. Thus, the unhatched embryos are more male-biased, while the hatched individuals are more female-biased in the Hm-oscar/_w_Hm-t treated groups. We have revised the section “Males are killed mainly at the embryonic stage” (lines 170-186) to include more detailed information explaining this phenomenon.
Line 190: please explain what are the Z chromosomes in Bombyx and Homona and Lepidoptera in general (chromosomes 1 and 15?), as this is not so clear for a non-expert.
Thank you for your suggestion. I have revised the section (lines 200-212) to include more precise background information about the chromosome constitutions in lines 202-204 as follows:
“Unlike other lepidopteran species, Tortricidae, including H. magnanima, generally possess a large Z chromosome that is homologous to B. mori chromosomes 1 (Z) and 15 (autosome).”
Line 222: please explain oscar diversity and classification in more detail, as this is not so clear for a non-expert.
Thank you for your suggestion. We have revised the sentences to provide clearer background information on the diversity of oscar genes (lines 255-264).
Figure 4: I found this difficult to follow. Why are there 2 rows (HmOscar and Oscar)? Does oscar here refer to oscar from Ostrinia? I am also a bit confused about the baseline control of Masc in these cell lines. If I understand Lepidoptera sex determination, then these cell lines are expressing high levels of female-specific piRNAs that suppress Masc. How specific are these piRNAs (i.e. do Bombyx piRNAs suppress Mascs from other Lepidoptera)? How much extra Masc will override endogenous piRNA? Information is lost by setting Masc expression to 100% in each separate comparison.
Yes, the Oscar indicates the _w_Fur-encoded oscar (Oscar from Ostrinia) that was tested to compare function with the Homona-derived Hm-oscar gene. In addition, following the reviewer's suggestions, we have revised the figure and included more detailed information on how we adjusted the expressions in the M&M section.
A previous study (Shoji et al., 2017, RNA 23:86–97) demonstrated that the Fem piRNA (29 bp) in Bombyx mori requires a 17 bp complementary sequence from its 5' region for its function. However, in species other than B. mori, no significant homology (i.e., over 17 bp matches) was found between the B. mori Fem piRNA and the masc genes analyzed in this study. Therefore, it is likely that the Fem piRNA expressed in BmN-4 cells is unable to suppress the masculinizing function driven by masc genes in other lepidopteran species. In addition, we did not quantify the levels of piRNA in this system, but the expression levels of masc are probably too high to be suppressed.
Figure 4 legend: spelling of Spodoptera.
Revised accordingly.
Reviewer #2 (Recommendations for the authors):
In Figure 2, what is the dsx splicing type for the hatched male in the Hm-oscar-injected group and the wHm-t infected line? Dsx-F or dsx-M?
Thank you for your suggestion. Unfortunately, we have not tested splicing in the hatched male neonates (1st instar larvae), partly due to difficulties in obtaining sufficient material for RNA extraction. Based on the previous publication in the Ostrinia system, where Oscar-bearing _w_Sca induces MK, the hatched males (ZZ) exhibit female type dsx as observed in the male embryos (Herran et al., 2022). The hatched Homona males may show double bands for dsx-M and dsx-F as observed in this study.
The size of the markers (in kilobase pairs) should be indicated in Figure 2.
We have accordingly included the marker information in the revised Figure 2b and the figure legends.
In Figure 3, could the authors identify which genes exhibit higher expression levels in the Hm-oscar-injected group and the wHm-t infected line? Could they provide hints for the possible mechanism of male-killing?
In the RNA-seq data shown in Figure 3a-d, we observed that both the Hm-oscar-injected and _w_Hm-infected groups generally exhibited upregulated expression of Z-linked genes. Rather than the upregulation or downregulation of a specific gene, we consider that global upregulation of Z-linked genes, caused by improper dosage compensation, is lethal for males. The Z chromosome contains various genes involved in key biological processes such as endocrine function and detoxification, and disruption of these processes may contribute to male lethality. Additionally, in this revised manuscript, we have provided more detailed information on the expression level of the Z-linked gene tpi. We have also discussed the potential mechanisms of MK in the Discussion section (lines 245-254).
The format of the references should be consistent. Gene and species names should be italicized.
We have accordingly formatted.
Reviewer #3 (Recommendations for the authors):
The authors use the term "upstream" (e.g., Oscar suppressed the function of masculinizer, the upstream male sex determinant...), which was sometimes confusing. In many cases, it reads as though the masculinizer was upstream of oscar, but what I think the authors are trying to convey is that masculinizer is a primary sex-determining factor.
Thank you for your suggestion. We have accordingly revised the term.
Line 101: which insect is wFur from?
It is from Ostrinia furnacalis - line 104 has been revised.
Figure 1: it would be helpful to indicate the statistical results on the figure.
Accordingly, we have added statistical data (binominal test) for Figure 1. The data for the Steel-Dwass test have been included in the supplementary data.
Figure 2b: please label the ladder on the gel.
Thank you for your suggestion. We have accordingly labeled the DNA ladder on the gel.
