The serine protease homolog Skanda modulates Toll-Phenoloxidase-mediated immunity in Drosophila

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   Jun 26, 2026

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   Reply to the reviewers

   Point-by-point response to the reviewers (____blue____)

   Dear Editor,

   Thank you for taking care of our manuscript. We are pleased to see that the reviewers are positive about our manuscript. We have amended our manuscript to address nearly all the reviewer’s comments. See below our point by points answer Although we cannot fully establish the exact function of the serine protease homolog Skanda in the Drosophila immune response, our study that combines both biochemistry and genetic provides important insight on the Toll-PO cascade and its complexity

   With best regards,

   Bruno Lemaitre on the behalf of the authors

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   __Review____er #1 (Evidence, …

   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

   Point-by-point response to the reviewers (____blue____)

   Dear Editor,

   Thank you for taking care of our manuscript. We are pleased to see that the reviewers are positive about our manuscript. We have amended our manuscript to address nearly all the reviewer’s comments. See below our point by points answer Although we cannot fully establish the exact function of the serine protease homolog Skanda in the Drosophila immune response, our study that combines both biochemistry and genetic provides important insight on the Toll-PO cascade and its complexity

   With best regards,

   Bruno Lemaitre on the behalf of the authors

   ---

   __Review____er #1 (Evidence, reproducibility and clarity (Required)): __

   In the manuscript entitled "The serine protease homolog Skanda modulates Toll-phenoloxidase-mediated immunity in Drosophila," Vasanth et al characterize in detail a previously unstudied component of the insect immune response using first biochemical and then in vivo methods. Using proteins overexpressed and purified from insect cells, the authors provide evidence that Skanda could be a negative regulator of the SP cascade, impacting cleavage of proHayan and proPsh, and consequently Toll pathway and PPO1 activation. This work reaches further by transposing these findings into the D. melanogaster in vivo model. Here, however, the picture becomes more confusing as Skanda at native levels does not appear to regulate either the Toll pathway or the melanization cascade. Only one strong phenotype was identified in that decreased expression of Skanda increased susceptibility to S. aureus infection while increased expression decreased susceptibility. The mechanism for this remains unclear. To their credit, the authors carry out an in-depth analysis to rule out all the obvious possibilities. In the discussion, the authors explore the basis of discrepancies between their biochemical and genetic findings. We would suggest that an additional one to consider is differing roles or behaviors of Skanda in the microenvironments of the local site of injury (where S. aureus may be contained when it is tolerated) and the hemolymph. In summary, this is a valuable analysis of the innate immune component Skanda whose role has become somewhat clearer through these studies, but still remains obscure.

   We thank the reviewer for this general assessment of our article. We agree with his idea that discrepancies between the biochemical and genetic findings arise from differing roles or behaviors of Skanda in the microenvironments of the local site of injury and the hemolymph’. We added the following sentence in the discussion: ‘The presence of Skanda in the hemolymph (Rommelaere et al. 2025) suggests a role in the systemic immune response; however, we cannot exclude that it may be particularly important within the local microenvironments at sites of injury’.

   __Major Comments __

   • To assess bimodal distribution of bacterial ds within single flies in Fig 6E, authors should either: increase the sample size to allow for proper statistical assessment of different distributions among genotypes, specifically between w1118 and skanda_d107; or, provide a modelling framework for statistical testing. Otherwise, the present results seem insufficient to conclude that Skanda is playing a role in resistance to S. aureus. We agree with the reviewer that our bacterial count was not enough developed. In the revised version we add a new Figure 6E with two time points 13h and 16h that were chosen before flies start to die from S. aureus. We observe at 13h a significantly higher bacterial count in the Skanda mutants but not at the 16 hours although there is higher proportion of wild-type flies that have clear the bacteria. These observations suggest a role of Skanda to resist, but also tolerate S. aureus. The fast killing induced by systemic injury with a low dose S. aureus made difficult to find a condition that would allow to see a clear load difference. So we have amended our text to highlight that Skanda could also play a role in tolerance.

   • Another way to assess a role for tolerance in the Skanda mutant would be to measure BLUDs (https://doi.org/10.7554/eLife.28298 ) and/or transcription of CrebA.

   We agree with the reviewer but measuring the BLUD with S. aureus is rather challenging as flies die quickly to this bacterium. As mentioned above and following revised figure 6E, we discuss in the revised version that Skanda could be involved in both resistance and tolerance.

   • The error bars on qRT-PCR datasets are large, the data points are not shown so we do not know how many replicates were included in the graphs (Fig 5 B and C, Fig 6C, Fig 7 A and B, and Fig 8B). Bar plots are not the most faithful reproduction of biological datasets, as they can hinder significant information regarding datapoints distribution and variation (Beyond Bar and Line Graphs: Time for a New Data Presentation Paradigm | PLOS Biology). We advise that, particularly in the case of datasets such as qRT-PCR, the final values of fold change are represented with individual dots, with the mean value clearly represented, whether with or without the additional bar graph. Furthermore, no statistical tests were applied to determine significance. Data points should be shown and appropriate statistical tests should be applied. The number of biological replicates should be included in the analysis and the statistical test applied should be noted in the figure legends.

   We have changed the figures related to qRT-PCR to show the individual points and we have added statistics in the revised version.

   • Although there are claims of Skanda conferring resistance to S. aureus infection, only Drs levels are tested. These conclusions could be strengthened by assessing expression levels of additional AMPs.

   In the revised manuscript, we report the expression of BomS1 in wild-type, skanda, and spz mutants following S. aureus infection. As previously observed for Drosomycin, Skanda does not markedly affect BomS1 expression (new Supplementary Figure S3E).

   __Minor Comments __

   • Parag. 1: (data not shown) should be removed and if possible AlphaFold prediction of skanda conformation added. Alternatively, remove sentence.

   We have removed (data not shown) and indicated that the information derived from Alphafold.

   • Parg. 3: 1000 mL? why not 1L?

   Corrected.

   • Parag. 5: , in last sentence that should be .

   Corrected.

   • Parag. 6: "a role at the same position..." does not convey the correct messageWe have improved the sentence for ‘Our results indicate that Grass processes Skanda in the Toll–PO SP cascade, consistent with Skanda acting at the same level of the proteolytic cascade as Hayan and Psh’.

   • Figure axes (5D, 5E, 6D, etc...) of melanization assays are wrongly named "% melanisation", with "s"

   We have corrected for “Melanization”.

   • Parag. 21: compound mutants (if correctly interpreted as dataset presented in Fig. 8B) were tested at 6h, 24h and 48h, and not 32h, as written in the text

   Indeed, in figure 8B, we monitored expression at 6, 24 and 32h and not 48h. This has been corrected.

   • Results section "skanda is not mandatory for the activation of the Toll pathway" adopts a literal translation which would probably be better phrased as "is not essential"

   We have corrected accordingly.

   • Discussion parag. 2: "Skanda exhibits..."

   • Discussion last parag: "..., but also underlies..."

   • It has been evidenced that

   This has been corrected.

   Additional comments:

   • The sentence on page 2 beginning with "Upon binding, these PRRs..." is very long and difficult to follow. This should be rewritten.

   We have split this sentence in two shorter ones for clarity.

   • In many places in the manuscript bacterial "dose" is used in place of bacterial burden. The dose is the amount of a substance or bacterium given to the animal.

   We have changed ‘bacterial dose’ for ‘bacterial burden’ when relevant, and we have kept the term “dose” when we mentioned the OD used to infect flies.

   Page 11: Skanda is described as a placeholder when I think a (competitive) inhibitor would be more appropriate.

   We agree that Skanda functionally resembles a competitive inhibitor, but several key differences set it apart from classical small-molecule inhibitors. First, Skanda is comparable in size and structure to Persephone and Hayan, natural substrates of Grass. Second, Skanda-like SPHs, which have close SP paralogs (e.g., Psh), are common in insects (Cao and Jiang, 2019), indicating that they may constitute a distinct class of negative regulators that warrants its own terminology. Moreover, because amplification in protease cascades typically occurs at the terminal step. Negative regulation by Skanda in an intermediate step could be more stochiometric than the freely reversible inhibition expected for a typical competitive inhibitor. As Skanda’s mechanism remains unclear. the neutral term “placeholder” seems more appropriate than “competitive inhibitor”.

   **Referee cross-commenting**

   I agree with the comments of the other reviewers.

   Reviewer #1 (Significance (Required)):

   Strengths: The authors take a multi-disciplinary biochemical and in vivo approach to understand the molecular interactions among SPs and SPHs and thereby uncover the role of the protein Skanda that might otherwise not have been appreciated. They have made extensive use of novel transgenic fly lines, generated in the context of this study, and have thoroughly tested their specificity and cis-acting potential. These will provide a resource to the field. In addition to the new description of Skanda, these findings strengthen previous knowledge regarding systemic infections with different bacteria (M. luteus, S. aureus) and reproduce the known redundancies of Psh and Hayan modes of action. Moreover, this research is relevant for the expansion of basic knowledge on innate immunity, particularly in the field of insect-pathogen interactions, making use of S. frugiperda cell lines and D. melanogaster adults and larvae. Although not at the focus of this work, the evolutionary conserved nature of these aspects of innate immunity across these two distant species enhance the importance of these findings.

   Weaknesses: Some assays do not include enough biological replicates and others do not have enough information on how many biological replicates were performed. Therefore, the conclusions drawn are difficult to assess. Lack of statistical analysis on the qPCR experiments complicates the interpretation of results.

   We thank the reviewer for his assessment. We have added the number of replicates in the revised version and make visible the variability of our data.

   __Review____er #2 (Evidence, reproducibility and clarity (Required)): __

   Summary In this work the authors identify the SPH skanda as an important player in Drosophila resistance to S. aureus infections independent of Toll and classical melanization. The authors conducted rigorous in vitro assays using recombinant proteins of various SPs in the Drosophila Toll-PO cascade to show that skanda negatively regulates activation cleavage of SPs at the level of and downstream of Psh and hayan, two key SPs that converge on Toll pathway activation with the latter playing a central role in cuticular melanization. In parallel, genetic analysis using mutant flies showed that skanda does not negatively regulate Toll pathway nor melanization. Only skanda over expression in vivo led to a reduction in S. aureus melanization which, in my opinion, is most likely due to the artificial increase in the in vivo concentration of the protein rather than an indication of a potential true function. Altogether this an interesting work as it shows the discrepancies between the biochemical and genetic approaches when it comes to dissecting the insect SP cascades regulating melanization and Toll as highlighted by the authors themselves in the discussion section. All experimental work is well controlled, methodology is robust and results are adequately discussed. I have some comments concerning few experiments and interpretations that in my opinion warrant further discussion.

   We thank the reviewer for the analysis and agree that the result showing than Skanda negatively regulates melanization could be due to over-expression.

   __Major comments: __ 1- It seems that SP48 and Grass can redundantly cleave Skanda although the later cleaves more strongly. (Fig 3B) Can other downstream SPs cleave skanda? Can ModSp alone cleave skanda? (ModSP + skanda lane was absent for Fig 3B). It is important to test these possibilities as the in vitro system may be quite relaxed as to the specificity of these cleavage events and may not reflect what happens in vivo. In fact it has been shown in Anopheles gambiae that SPH can be redundantly cleaved by multiple SP in the protease cascade. Although these are cascades with certain hierarchy, information can still flow in more than one direction along the different branches of these cascades.

   We tested whether ModSP could cleave pro-Skanda and found that it did not (data not shown). This result is consistent with our expectations, as ModSP has a chymoelastase-like specificity and preferentially cleavage after Leu. In contrast, Skanda is cleaved by Grass and cSP48, both of which are trypsin-like proteases.

   At present, there is no straightforward way to assess whether downstream SPs activate pro-Skanda. Obtaining an active downstream SP would require sequential activation of all its upstream enzymes, and it is nearly impossible to completely remove these activating proteases afterward. As a result, it is difficult to distinguish the activity of a downstream SP from that of cSP48 and Grass. We are currently developing a new approach to overcome this limitation.

   2- In Fig 4B and 4C the bands of active forms should be quantified from at least 3 immunoblots for robust results especially in Fig 4C where the differences are minimal.

   As suggested by the reviewer, we quantified the band intensities from four independent blots and presented the data in Fig. 4B and 4C (lower panels).

   3- It is not clear to me why skanda should have a specific role in resisting S. aureus infections despite that S. aureus is not a natural pathogen of Drosophila? Has other Gram-positive and Gram-negative bacteria been tested?

   It is true that S. aureus is unlikely to be a natural pathogen of Drosophila. However, this bacterium has been used in several studies (notably Dudzic 2019) to uncover a specific activity associated with melanization modules that is distinct from cuticular blackening. For this reason, we believe that S. aureus provides a sensitive assay to monitor this particular immune mechanism. We further hypothesize that other bacteria related to S. aureus—possibly members of the Staphylococcus family—may infect Drosophila and could be controlled by Skanda. We chose not to elaborate on this point to avoid overextending the scope of the article.

   4- In Fig 6E more points should be collected for statistical power. It is also better to show these data that are not normally distributed in violin charts or boxes and whiskers which give a better indication as to which quartile the bulk of the data belongs.

   We have addressed this point (see answer to Reviewer 1).

   Minor comments: 5- In Figures 3 and 4, It would be easier to follow the cleavage events if a schematic drawing is provided showing the sequence of activation cleavage events of the tested SPs

   Because the order of the two cleavage events is unclear, we felt it was simpler to include the putative cleavage sites in Fig. 2B and refer interested readers to Fig. S1, Table S1, and Fig. 3 legend.

   6- The fact that PPO1/PPO2 depleted flies exhibit increased Drs expression could be due to increased bacterial proliferation in this mutant background that trigger increased Toll stimulation, rather than a negative feedback mechanism. This increased proliferation is shown in Fig 6E.

   This is a good point. The higher expression of Drs in PO1/PPO2 depleted flies could be associated to higher bacterial load in the mutant, or to negative feedback of the melanization reaction. This higher Toll pathway activation has been further characterized in Liu et al., (Plos pathogen 2025) where it was suggested that it relate to a negative feedback loop between the Toll and the melanization cascade.

   7- In Fig 6E more points should be collected for statistical power. It is also better to show these data that are not normally distributed in violin charts or boxes and whiskers which give a better indication as to which quartile the bulk of the data belongs.

   We have addressed this point. See answer to reviewer 1 for discussion.

   8- A phenotype for skanda in melanization was observed only in over-expression assays which may artificially alter molecular interactions in the cascade.

   We agree with this statement and we have added a comment in the discussion of the revised manuscript about the potential artifactual results due to over-expression.

   9- Page 10 last paragraph "peak expression at 32 hrs or 48 hrs as shown on the figure?"

   This is 32h and has been corrected.

   10- The differences in Drs expression levels in Hayan-pshDef and psh-skandaDef double mutant flies infected with M. luteus and S. aureus is surprising. I wonder whether the observed differences are due to biochemical differences in the microbial surfaces to which these cascades are recruited.

   Drs expression is markedly higher following systemic infection with M. luteus than with S. aureus, consistent with the different bacterial doses used. We deliberately employed a low dose of S. aureus because this condition reveals a pronounced susceptibility in skanda flies. Consequently, direct comparison between these two infection regimes remains challenging.

   11- There are several typos in the manuscript

   We have carefully re-read the manuscript and corrected several typos.

   Reviewer #2 (Significance (Required)):

   The main strength of this work is that it combines biochemistry and genetics in a strong genetic model to characterize the biochemical interactions between SPH and Sp in clip cascades and relate the relevant interactions observed in vitro with potential in vivo functions. This is the first time that such a rigorous combined approach was adopted to the study of these cascades. The results obtained also show the advantages and limitations of each approach. As such i believe this study will be of interest to a broad audience in the field of insect immunity.

   __Review____er #3 (Evidence, reproducibility and clarity (Required)): __

   __Summary: __

   Serine protease cascades are central for activation of immune responses in insects. In Drosophila melanogaster, Toll signaling pathway has been quite extensively studied, and several serine proteases, serpins and serine protease homologs (SPH) with functions in Toll activation have been identified. In this work, the authors characterize a new component of this system, a SPH which they name Skanda. Skanda seems to have multiple roles/points of action, on one hand participating in the regulation of Toll together with the established serine protease in the Toll activation, Psh, and on the other hand controlling the response to a systemic S. aureus infection, via not yet fully specified mechanism.

   __Major comments: __

   Key conclusions made in this work are convincing, and backed up by the data presented. The data and methods are presented in a way that allows reproduction of the experiment. The number of individuals used especially in the infection experiment (20 male flies per a replicate) is on the lower side, but the experiments are adequately replicated and the effects seen are clear.

   While this work contributes to our understanding of the regulatory mechanisms governing Toll signaling, at times the authors' reasoning is difficult to follow. I recognize that this is a complex topic, with multiple upstream branches activating Toll signaling, and the authors do consider various mechanisms that could explain their findings. However, the manuscript would benefit from additional clarification, perhaps through a schematic model illustrating the proposed effects of Skanda, to help readers position Skanda within the broader context of Toll signaling. We have done our best to explain the Toll serine protease and added a figure at the beginning of the manuscript. Since we cannot position Skanda in the Toll-Po cascade yet, we prefer to avoid drawing a model. We believe that this study highlights our ignorance of the complexity of serine protease cascades acting upstream of Spätzle and Melanization.

   Statistical analyses for the Drs expression experiments are lacking.

   The statistical analysis for Drs expression has been added in the revised version.

   __Minor comments: __

   The authors could explain what type of cells the sf9 cells are and why they decided to use them.

   Sf9 cells are an insect ovarian cell line derived from Spodoptera frugiperda and are widely used for baculovirus-mediated expression of eukaryotic proteins. They support proper protein folding, disulfide bond formation, and post-translational processing. This information is now mentioned in the Result section in addition to methods.

   Band intensities could be measured and plotted for the immunoblots. The immunoblot methods should be fully described in the Materials and methods section.

   Thanks for the suggestion. We have done this accordingly and included the results in Fig. 4B and Fig. 4C (lower panels). Brief descriptions of densitometric analyses have been added to the figure legends.

   Protein levels of Skanda in the Skanda mutant could be shown as the mRNA levels remain relatively high (Sup. Fig 3B). If this is not possible, could the authors comment on the remaining expression of Skanda in the Skanda mutants?

   We have added a comment on this point: The *skanda *mutation is a frameshift mutation that affects the coding sequence. There are still transcripts although not functional. The decreased expression of Skanda in SkandaD107 is probably due to non-sense-mediated RNA decay caused by the frameshift.

   Under the heading "Loss of skanda does not further enhance the cuticular melanization defects caused by the loss of Hayan or psh" the text should refer to figure 5D not 5B.

   We have corrected this mistake in the revised version.

   Figure 6C shows that Drs expression is higher in the Skanda mutant than in controls at 32 h post S. aureus infection (although this has not been statistically tested). The authors don't mention this result in the manuscript, but to me it fits with the idea of Skanda acting as a negative regulator (the effect of which is accumulating and seen only late after infection). Could the authors comment on this? We do not think that the higher expression of Drs in Skanda mutant upon S. aureus systemic infection is due a negative regulation the Toll pathway but rather to higher S. aureus burden. We conclude this because Drs is not higher than the wild-type upon injection of M. luteus and proteases. At this stage, we cannot exclude that there are differences between M. luteus and S. aureus.

   Under the heading "Psh and skanda redundantly regulate Toll signaling", the comparison should likely be between Figures 7A-7B and 5B-C (rather than 5A). When examining the effects of single versus double mutants on Drs expression, the Psh-Skanda double mutant clearly reduces Drs more than the Psh single mutant. However, in the context of microbial proteases, the pattern appears different: there is virtually no difference at 6 hours, while at 48 hours there may be a slight decrease in Drs expression in the double mutant compared to the Psh single mutant, although this difference would likely not reach statistical significance if tested. I don't know what this could mean, but I'd like to hear the authors' take on this. The reviewer is correct and we have revised our manuscript to mention the appropriate figure. Figures 7A-7B and 5B-C.

   The reviewer raised a good point; we believe that the additional effect of Skanda in absence of Psh is less marked upon microbial proteases because Psh already has a strong effect by itself in sensing proteases. In contrast there is higher redundancy between Psh and Hayan upon M. luteus and consequently the double mutant psh, Skanda have a stronger effect.

   __**Referee cross-commenting** __

   I also agree with the comments and points raised by the other reviewers.

   __Review____er #3 (Significance (Required)): __

   Research on the Drosophila immune response has significantly advanced our understanding of (innate) immune responses, both generally and in an evolutionary context. Despite over three decades of study, this work demonstrates that there are aspects of Toll signaling that remain unresolved. The authors identify a novel regulator of the Toll pathway and begin to elucidate its functions. Equally important, their findings underscore the complexity and context-dependency of the regulatory events that shape immune responses.

   We fully agree with the assessment of the reviewer. Our study highlights the complexity (and our ignorance) of this important facet of Drosophila immunity, as mentioned in the last sentence of the discussion.

   My fields of expertise are Drosophila melanogaster, innate immunity, cell-mediated immunity.

   __Review____er #4 (Evidence, reproducibility and clarity (Required)): __

   __Summary __

   In this study, the authors investigate the function of Skanda, a serine protease homolog (SPH) in Drosophila innate immunity using both biochemical and genetical approaches. The reason to focus on this SPH is that it lies at the same locus as two key proteases of Drosophila immune defenses, Hayan and Persephone, all of which are induced by an immune challenge. After having modeled this SPH and shown that the three amino-acid of the serine protease catalytic triad are either mutated or poorly oriented, they report that Skanda may limit the cleavage of proteases downstream of Grass, a key event for their biochemical activation. The study of an isogenized, putatively null, mutant line failed to reveal any impact of skanda on Toll pathway activation nor on melanization, albeit a strong but not moderate overexpression somewhat inhibits the formation of a melanization scab only after "clean" but not septic injury. These results are not in keeping with the biochemical analysis: the mutant would have been expected to display an enhanced immune response. Unexpectedly, skanda mutants are as highly susceptible to a low amount of Staphylococcus aureus injection as flies deleted for the adult-expressed phenoloxidases PPO1 and PPO2, melanization playing a key role in host defense in this infection paradigm. No strong impact on the bacterial load was detected at the sole investigated time point, 24h. Because the analysis of the single skanda mutant did not unambiguously reveal its role in host defense, the authors then studied double or triple mutants of the three protease genes and found a redundant role for Skanda with Persephone for Toll pathway activation after a challenge with a nonpathogenic Gram-positive bacterium or a bacterial protease. In the case of S. aureus infection, a strong induction of the Drosomycin gene, is observed at 48h of infection in the compound mutants, which was not observed with the nonpathogenic challenges. Evidence, reproducibility and clarity

   __Major comments __

   The authors state that "These results are consistent with a role of Skanda in resistance to S. aureus". This conclusion rests on a very fragile experiment that measured the bacterial burden 24h after challenge with a low dose of S. aureus: whereas wild-type control flies exhibit a dual low and high distribution of bacterial loads, skanda flies exhibit only the higher values. However, the bacterial load in skanda appears to be as high in persephone mutant flies that are much less sensitive to S. aureus than skanda flies. This makes it highly unlikely that the high susceptibility of skanda to S. aureus is due solely to resistance. The problem is compounded by the poor description of the experiment: it is not stated anywhere how many times the experiment has been performed, whether pooled data are shown, what each data point represents, pooled or single flies. A fine-grained time course with more biological samples would definitely be needed to convince the reader of a (limited) role in resistance. The authors do not consider the alternative, but not exclusive, possibility that skanda plays also a role in disease tolerance. The determination of the bacterial load upon death of single flies may provide some clues about this alternative function (Duneau et al., eLife, 2017). Another approach might be to determine whether the bacterial supernatant is toxic and whether skanda might protect from this toxicity. As Bomanins play a role in the host defense against S. aureus (this study, but see also Hanson et al., eLife 2019 in which the 55C deficiency susceptibility phenotype was stronger) and given the role of Bomanins in host defense against Gram-positive bacteria or fungal infections both in resistance and disease tolerance (e.g., Clemmons et al. PLoS Pathogens 2015, Lindsay et al., J. Innate Immun, 2018, Xu et al., EMBO Reports 2023, Lou et al., BioRxiv, 2025) and that BomS1 has an optimal Dorsal-related Immune Factor Binding site (Busse et al. EMBO J. , 2007), it may be useful to monitor the expression of several Bom genes in complement to that of the expression of Drosomycin, especially after S. aureus challenge. Furthermore, BomT1 is the only peptide that appears to play a role in resistance against Gram-positive bacteria, namely against E. faecalis. This series of qPCR experiments is rapid to make, provided the authors have kept the cDNAs of their samples.

   To address the reviewer’s comment, we extended the bacterial load analysis of S. aureus in skanda mutants (new figure 6E). Our results support a role for Skanda in both resistance and disease tolerance. This point is now briefly discussed in the Results section, and we have added references highlighting a role of the Toll pathway in disease tolerance. We did not elaborate further, as accurately monitoring S. aureus burden following low-dose infection remains technically challenging given the high pathogenicity of this bacterium.

   In the Discussion, the authors speculate "that Skanda acts at the level of Persephone-Hayan to allow Hayan to activate the Toll pathway. Skanda would skew the activity of the Persephone-Hayan platform to induce Toll signaling and resistance to S. aureus rather than cuticular melanization". This model does not fit with the fact that SPE is only moderately susceptible to S. aureus (Dudzic et al., 2019) and that spätzle mutant flies are either not sensitive at all (Dudzic et al., 2019) or moderately sensitive to it (Hanson et al., eLife, 2019) (see also below). Whether it may apply to host defense against other pathogens remains to be determined. To better understand the function of skanda, considering only S. aureus may be limiting as this bacterium is fundamentally not susceptible to the canonical Toll intracellular signaling cascade (e.g., Bischoff et al, Nat Immunol, 2004, Dudzic et al, Cell Reports, 2019) and to the final part of the Toll-activation proteolytic cascade as discussed above with SPE and Spätzle. The authors appear to have chosen not to display the results they have gained with Enterococcus faecalis (but forgot to remove their mention at two places in the Material and Methods): it would definitely be interesting to know what the outcome of these experiments was and also to investigate the susceptibility and microbial burden of skanda mutants to representative yeast and filamentous fungal pathogens, Aspergillus fumigatus being of special interest since its proliferation is limited through melanization whereas the Toll pathway protects against secreted virulence factors (Xu et al., EMBO Reports, 2023). This series of experiments would likely take some three months and might give additional insights into Skanda function(s).

   We agree with the reviewer that examining the role of Skanda in response to additional bacterial species could further help elucidate its function. However, the most robust phenotype we identified is a strong acute susceptibility to S. aureus, which is dependent on the Psh–Hayan–Skanda axis but independent of the SPE–Spätzle pathway. Because the bacterial strains suggested by the reviewers are primarily controlled by the SPE–Spätzle–Toll pathway, we did not pursue this direction further. However, in the revised version we have added survival analysis with Skanda to Candida albicans and *Enterococcus faecalis *(new supplement Figure 3F and G). Notably, we also observed an intermediate susceptibility to both Candida albicans and E. faecalis (see below). This indicates that Skanda is not a classical regulator of the Toll-PO cascade such as Grass, ModSP, SPE or Hayan/SPE.

   In general, figure legends are not highly informative and fail to provide key information such as the number of independent experiments, whether the data are representative or pooled, which statistical test was used, e.g., qPCR experiments (the descriptions are available for the analysis of survival and melanization experiments at the end of the Mat. and Meth section). As noted above, critical information is lacking to understand microbial load graphs. It is also difficult to check statements such as: ", while psh[sk1] flies showed a reduced Toll pathway reponse". Indeed, no statistical analysis has been performed to analyze any RTqPCR data. Given the low number of experimental data points, each data point ought to be displayed and not bar graphs, for which in addition the error bars are not defined. The Material and Methods section is incomplete. It does not include a description of all the in vitro synthesized proteins used in this study nor indicate the different tags. The primary and secondary antibodies used for Western blot analysis are not reported, e.g., those that detect cleaved spätzle. This would need to be included in the Table at the beginning of this section.

   In the revised version, we have addressed these points by adding statistical tests to the RT–qPCR analyses, displaying all data points, and improving the microbial load measurement. As discussed in the Material and Methods section, Table S2 provides information for all in vitro synthesized proteins used in this study, including affinity tags and the primary and secondary antibodies. On a more personal note, we first identified the striking susceptibility of Skanda/CG15046 flies more than 10 years ago, and the skanda project subsequently experienced a long period of discontinuation before we decided to reassemble and consolidate the most important findings. Unfortunately, this study did not result in a straightforward narrative with a “happy ending.” Nevertheless, we still consider this work an important step toward a better characterization of this aspect of fly immunity.

   __Minor points __ Introduction:

   1. The authors may want to cite Stein, Cho&Stevens, FLY, 2013 when referring to the proteolytic cascade regulating the establishment of dorso-ventral patterning.

   This reference has been added

   The statement "The Toll-PO SP cascade can be DIRECTLY activated at the level of Psh-Hayan, through direct cleavage of the Psh protease bait region by microbial proteases" may be slightly misleading as only subtilisin is able to do this, the other tested proteases producing an inactive cleaved Psh that needed to be secondarily activated by a couple of specific cathepsins (Issa et al., Molecular Cell, 2018).

   Good point. This point has been corrected with the Issa reference added.

   Results

   1. The reasoning of the second paragraph is difficult to follow as the reader does not understand how the cleavage sites can be computed. It would be important to state that the recombinant proteins are tagged. It would actually be very helpful to provide a scheme of the various recombinant proteins used in the study as had been done in the Shan et al., Science Advances article.

   We followed the reviewer’s good suggestions, modified the text accordingly, and added Table S2.

   With respect to Western blots, many of the bands are faint, e.g., SPE after the addition of Skanda cannot be detected on a printed version of the figure. It is also difficult to determine whether the reduction in band amount is reproducible as no indications are given in this respect. It is important that the images be quantified in several independent blots so that the observed reduction can be statistically assessed. With respect to PPO1 cleavage, it would be important to also check its cleavage in vivo, which would yield higher confidence on the relevance of in vitro study to the in vivo situation.

   In response to the reviewer’s suggestions, we repeated SDS-PAGE and immunoblot analysis, quantified band intensities, and performed statistical analyses for the samples shown in Fig. 3B and 3C (lower panels). The total number of blots for each representative is 3 to 4. For practical reasons, we are unable to assess PPO1 cleavage in vivo.

   First sentence of the paragraph "skanda mutants are highly susceptible": the authors might also want to cite Hanson et al, eLife 2019.

   We have added the Hanson reference and Ryckebusch et al 2025, which is more appropriate.

   In Dudzic et al., Cell Reports, 2019, the authors did not observe any susceptibility to S. aureus with Hayan[sk3] whereas here they find an intermediate sensitivity phenotype with Hayan[sk6]. Was the former not a null allele of Hayan? With respect to the 55C Bomanin deficiency, Hanson et al., 2019 had reported a stronger phenotype than that shown in Fig. 8A, with some 75% of flies dead within three days. Which study should we trust or does this reflect variations between experiments (hence the question about the representation of survival data: are these pooled data from thre independent experiments; how much variation was there between independent experiments?).

   Both Hayan mutant flies were null. We observed differences along the years with different experimenters; although the main results stand. We also tend to observe a stronger impact of psh than initially reported in response to M. luteus (Figure 5B), although this is consistent with its role in the PRR-Grass-SPE pathway. Considering all the parameters that influence survival experiments (temperature, humidity, time to form the bacterial pellet and sometimes bacterial strains) and possible cryptic infections (Nora infection), we consider these variations as expectable.

   It would be interesting to measure the S. aureus bacterial load upon skanda overexpression to confirm a putative role in resistance.

   This is an interesting suggestion but we did not do it because of the technical challenge that monitoring S. aureus burden represents. We have preferred to focus our attention on monitoring S. aureus in Skanda loss-of-function mutants.

   UAS-skanda: besides Fig. 6B, the authors should also refer the reader to Fig. S4A.

   The link to Fig S4A has been added.

   Genetic dissection of the skanda-psh-hayan gene cluster: the last sentence of the paragraph does not reflect what Fig. S7B is showing: one of the double mutants and the triple mutant displayed a significant intermediate susceptibility to S. aureus.

   This is in fact Ecc15 that we discussed. The reviewer is correct as the triple mutants and hayan,psh double have increased susceptibility to Ecc15.

   Paragraphs Compound mutants are EXTREMELY susceptible to S. aureus. The wording is likely too ...extreme: they do not seem to die much faster than skanda simple mutants, which were HIGHLY susceptible to S. aureus, like PPO1-PPO2 double mutants.

   The reviewer is correct and we have avoided to use the term ‘extremely’ in the revised version (replaced by ‘highly’ or removed).

   Last paragraph: psh mutants should be compared side-by-side with psh-skanda double mutants in the same RTqPCR experiment: it is difficult to judge whether the statement of equivalent Drosomycin expression after S. aureus challenge is true given the low resolution of the figures (Fig. 6C vs. Fig. 7B). Last sentence: it would be more appropriate to mention "host defense" rather than "resistance" since the authors did not check the bacterial burdens of the compound mutants.

   Experiments were done simultaneously on single and double/triple mutant but this represents kinetic with 4 times in 10 different backgrounds! We have preferred to separate the data to simplify the reading. We believe that the reader can compare the data despite display in two different panels. We have changed in all the manuscript host defense instead of resistance as following bacterial counting, we suspect that Skanda may play both in resistance and disease tolerance.

   Fig. 1: the scheme is not up to date and oversimplified. It should take into account the complexity revealed in the Shan et al. Science Advances article.

   We disagree on this point. This schema reflect inference done by genetics. An up-to-date figure is shown in Westlake, Hanson Lemaitre Handbook but would require a broad introduction. In the revised version, we have highlighted that this is simplified model based on genetics.

   Fig. S1: numbering the amino-acids in the sequence would help follow the text from Document S1. What are the residues written in light blue? It may be worth highlighting residue E194. Of note, there is a difference between the sequence for peptide 4 as found in the sequence displayed on Fig. S1: KTDRD YV and the sequence of peptide 4 in Table S1: KTDRE YV; the presence of a potential SNP should be indicated, even though it is not making a major change in terms of charge of the peptide.

   We included an asterisk at every tenth position and a numerical indicator near the end of each line to facilitate counting. Residues highlighted in cyan may represent cleavage sites of cSP48, Grass, or a trypsin-like protease released by Sf9 cells. The peptide (E194… R212) appears to undergo cleavage to generate P204LNLPLQP__R212__, which is detected in the secondary MS. The reviewer is correct on peptide 4 that we attribute to a potential SNP. This is now indicated in the legend of Figure S1.

   Document S1: trypsin digestion (just before second call to Fig. S1); should it not be purified proteases instead? The text should be somewhat reworded as it is currently slightly misleading.

   "In lane 8, peptide-1 through -19 were nearly undetectable". Table S1 shows that even though peptides 1, 2, 6, , 7 , and 11 are not expressed to strong enough a level to be displayed Fig. S1 lane 8 given the chosen scale, peptides 1, 2, 6, and 7 are expressed in the same range for slices 8B and 8C, whereas peptide 1 is found with just a two-fold difference in slices 8A and 8C.

   Points taken. To better illustrate the differences in band intensities in the top right panel of Fig. S1, we kept the same scale for bands A and B in line 8 (as well as for bands A-C in the top left and middle panels) and used the second y-axis for band C.

   Fig. S2: the effect of skanda on SP7 cleavage is not detectable when Hayan isoforms are co-incubated. The main text should be modified to take this into account. How do the authors explain that pro-MP1 levels are not different upon co-incubation with Psh or Hayan-PB with or without adding Skanda, even though the active MP1 form is detected only in the absence of Skanda? In contrast, the pro-MP1 band can be detected upon co-incubation with Skanda and Hayan-PA.

   Thanks for the comments. We repeated the experiments and obtained four independent blots for each. After scanning, integrated band densities for all paired bands (i.e., with and with Skanda) were quantified using ImageJ (Fig. S2 and data not shown). In the representative blots, Skanda had little effect on SP7 activation by Hayan-PA (507/527; 96%) or Hayan-PB (15,763/15,828; ~100%), in contrast to Psh (937/7,917; 12%). However, when ratios from all blots were considered, the mean reductions were 56 ± 14% for Psh, 49 ± 19% for Hayan-PA, and 65 ± 18% for Hayan-PB. For MP1, comparison of precursor bands is less reliable because small decreases in precursor intensity are difficult to quantify; therefore, we focused on the MP1 product. MP1 levels were reduced to 58 ± 8% (Psh), 44 ± 3% (Hayan-PA), and 90 ± 30% (Hayan-PB). SPE intensity was reduced to 38 ± 12% (Psh), 43 ± 5% (Hayan-PA), and 23 ± 4% (Hayan-PB). Ser7 intensity was reduced to 9 ± 4% (Psh), 35 ± 1% (Hayan-PA), and 27 ± 13% (Hayan-PB). In general, Skanda suppressed the activation of SP7, SPE, MP1, and Ser7 by Psh, Hayan-PA, or Hayan-PB. We included the information in Fig. S2 legend.

   Fig. S3B, S7A: the three genes of the locus are inducible upon immune challenge. Have any NF-kappaB binding sites been detected at the locus. It might be relevant to repeat the experiment shown in S3B and especially S7A after a challenge with M. luteus. These experiments are definitely not essential.

   We did not look to the presence of NF-kB sites in their promoters but they have been shown to be induced and regulated by the Toll pathway (De Gregorio 2002). We did not extend our manuscript in this direction.

   The mention 'Data not shown" is used twice. Not allReview Commons-affiliated journals accept it.

   These mentions have been removed.

   Reviewer #4 (Significance (Required)): A strength of this work is the dual biochemical and genetic characterization of a SPH, an endeavor that is important to understand further the function of this class of protease-like family of secreted proteins that have been so far imperfectly studied from both perspectives (Kambris et al., CB, 2006, but see Westlake Reproducibility study on BioRxiv, Jin et al. Frontiers Immunol. 2023). Unfortunately, the two approaches fail to provide an integrated view of Skanda's function(s). A weakness is that this study does not unambiguously reveal at this stage what are the functions of Skanda in the host defense against S. aureus, let alone against other pathogens controlled to some extent by the Toll pathway or melanization. The authors have not considered a possible role in disease tolerance to S. aureus. These limitations decrease the conceptual advance of this article.

   In the revised version, we have considered a role of Skanda in resilience. This article will be of interest to investigators working on the innate immunity of insects. This reviewer is an expert in the Drosophila innate immunity field.

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   Referee #4

   Evidence, reproducibility and clarity

   Summary

   In this study, the authors investigate the function of Skanda, a serine protease homolog (SPH) in Drosophila innate immunity using both biochemical and genetical approaches. The reason to focus on this SPH is that it lies at the same locus as two key proteases of Drosophila immune defenses, Hayan and Persephone, all of which are induced by an immune challenge. After having modeled this SPH and shown that the three amino-acid of the serine protease catalytic triad are either mutated or poorly oriented, they report that Skanda may limit the cleavage of proteases downstream of Grass, a key event for their biochemical …

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   Referee #4

   Evidence, reproducibility and clarity

   Summary

   In this study, the authors investigate the function of Skanda, a serine protease homolog (SPH) in Drosophila innate immunity using both biochemical and genetical approaches. The reason to focus on this SPH is that it lies at the same locus as two key proteases of Drosophila immune defenses, Hayan and Persephone, all of which are induced by an immune challenge. After having modeled this SPH and shown that the three amino-acid of the serine protease catalytic triad are either mutated or poorly oriented, they report that Skanda may limit the cleavage of proteases downstream of Grass, a key event for their biochemical activation. The study of an isogenized, putatively null, mutant line failed to reveal any impact of skanda on Toll pathway activation nor on melanization, albeit a strong but not moderate overexpression somewhat inhibits the formation of a melanization scab only after "clean" but not septic injury. These results are not in keeping with the biochemical analysis: the mutant would have been expected to display an enhanced immune response. Unexpectedly, skanda mutants are as highly susceptible to a low amount of Staphylococcus aureus injection as flies deleted for the adult-expressed phenoloxidases PPO1 and PPO2, melanization playing a key role in host defense in this infection paradigm. No strong impact on the bacterial load was detected at the sole investigated time point, 24h. Because the analysis of the single skanda mutant did not unambiguously reveal its role in host defense, the authors then studied double or triple mutants of the three protease genes and found a redundant role for Skanda with Persephone for Toll pathway activation after a challenge with a nonpathogenic Gram-positive bacterium or a bacterial protease. In the case of S. aureus infection, a strong induction of the Drosomycin gene, is observed at 48h of infection in the compound mutants, which was not observed with the nonpathogenic challenges. Evidence, reproducibility and clarity

   Major comments

   The authors state that "These results are consistent with a role of Skanda in resistance to S. aureus". This conclusion rests on a very fragile experiment that measured the bacterial burden 24h after challenge with a low dose of S. aureus: whereas wild-type control flies exhibit a dual low and high distribution of bacterial loads, skanda flies exhibit only the higher values. However, the bacterial load in skanda appears to be as high in persephone mutant flies that are much less sensitive to S. aureus than skanda flies. This makes it highly unlikely that the high susceptibility of skanda to S. aureus is due solely to resistance. The problem is compounded by the poor description of the experiment: it is not stated anywhere how many times the experiment has been performed, whether pooled data are shown, what each data point represents, pooled or single flies. A fine-grained time course with more biological samples would definitely be needed to convince the reader of a (limited) role in resistance. The authors do not consider the alternative, but not exclusive, possibility that skanda plays also a role in disease tolerance. The determination of the bacterial load upon death of single flies may provide some clues about this alternative function (Duneau et al., eLife, 2017). Another approach might be to determine whether the bacterial supernatant is toxic and whether skanda might protect from this toxicity. As Bomanins play a role in the host defense against S. aureus (this study, but see also Hanson et al., eLife 2019 in which the 55C deficiency susceptibility phenotype was stronger) and given the role of Bomanins in host defense against Gram-positive bacteria or fungal infections both in resistance and disease tolerance (e.g., Clemmons et al. PLoS Pathogens 2015, Lindsay et al., J. Innate Immun, 2018, Xu et al., EMBO Reports 2023, Lou et al., BioRxiv, 2025) and that BomS1 has an optimal Dorsal-related Immune Factor Binding site (Busse et al. EMBO J. , 2007), it may be useful to monitor the expression of several Bom genes in complement to that of the expression of Drosomycin, especially after S. aureus challenge. Furthermore, BomT1 is the only peptide that appears to play a role in resistance against Gram-positive bacteria, namely against E. faecalis. This series of qPCR experiments is rapid to make, provided the authors have kept the cDNAs of their samples. In the Discussion, the authors speculate "that Skanda acts at the level of Persephone-Hayan to allow Hayan to activate the Toll pathway. Skanda would skew the activity of the Persephone-Hayan platform to induce Toll signaling and resistance to S. aureus rather than cuticular melanization". This model does not fit with the fact that SPE is only moderately susceptible to S. aureus (Dudzic et al., 2019) and that spätzle mutant flies are either not sensitive at all (Dudzic et al., 2019) or moderately sensitive to it (Hanson et al., eLife, 2019) (see also below). Whether it may apply to host defense against other pathogens remains to be determined. To better understand the function of skanda, considering only S. aureus may be limiting as this bacterium is fundamentally not susceptible to the canonical Toll intracellular signaling cascade (e.g., Bischoff et al, Nat Immunol, 2004, Dudzic et al, Cell Reports, 2019) and to the final part of the Toll-activation proteolytic cascade as discussed above with SPE and Spätzle. The authors appear to have chosen not to display the results they have gained with Enterococcus faecalis (but forgot to remove their mention at two places in the Material and Methods): it would definitely be interesting to know what the outcome of these experiments was and also to investigate the susceptibility and microbial burden of skanda mutants to representative yeast and filamentous fungal pathogens, Aspergillus fumigatus being of special interest since its proliferation is limited through melanization whereas the Toll pathway protects against secreted virulence factors (Xu et al., EMBO Reports, 2023). This series of experiments would likely take some three months and might give additional insights into Skanda function(s). In general, figure legends are not highly informative and fail to provide key information such as the number of independent experiments, whether the data are representative or pooled, which statistical test was used, e.g., qPCR experiments (the descriptions are available for the analysis of survival and melanization experiments at the end of the Mat. and Meth section). As noted above, critical information is lacking to understand microbial load graphs. It is also difficult to check statements such as: ", while psh[sk1] flies showed a reduced Toll pathway reponse". Indeed, no statistical analysis has been performed to analyze any RTqPCR data. Given the low number of experimental data points, each data point ought to be displayed and not bar graphs, for which in addition the error bars are not defined. The Material and Methods section is incomplete. It does not include a description of all the in vitro synthesized proteins used in this study nor indicate the different tags. The primary and secondary antibodies used for Western blot analysis are not reported, e.g., those that detect cleaved spätzle. This would need to be included in the Table at the beginning of this section.

   Minor points

   Introduction:

   1. The authors may want to cite Stein, Cho&Stevens, FLY, 2013 when referring to the proteolytic cascade regulating the establishment of dorso-ventral patterning.
   2. The statement "The Toll-PO SP cascade can be DIRECTLY activated at the level of Psh-Hayan, through direct cleavage of the Psh protease bait region by microbial proteases" may be slightly misleading as only subtilisin is able to do this, the other tested proteases producing an inactive cleaved Psh that needed to be secondarily activated by a couple of specific cathepsins (Issa et al., Molecular Cell, 2018). Results
   3. The reasoning of the second paragraph is difficult to follow as the reader does not understand how the cleavage sites can be computed. It would be important to state that the recombinant proteins are tagged. It would actually be very helpful to provide a scheme of the various recombinant proteins used in the study as had been done in the Shan et al., Science Advances article.
   4. With respect to Western blots, many of the bands are faint, e.g., SPE after the addition of Skanda cannot be detected on a printed version of the figure. It is also difficult to determine whether the reduction in band amount is reproducible as no indications are given in this respect. It is important that the images be quantified in several independent blots so that the observed reduction can be statistically assessed. With respect to PPO1 cleavage, it would be important to also check its cleavage in vivo, which would yield higher confidence on the relevance of in vitro study to the in vivo situation.
   5. First sentence of the paragraph "skanda mutants are highly susceptible": the authors might also want to cite Hanson et al, eLife 2019.
   6. In Dudzic et al., Cell Reports, 2019, the authors did not observe any susceptibility to S. aureus with Hayan[sk3] whereas here they find an intermediate sensitivity phenotype with Hayan[sk6]. Was the former not a null allele of Hayan? With respect to the 55C Bomanin deficiency, Hanson et al., 2019 had reported a stronger phenotype than that shown in Fig. 8A, with some 75% of flies dead within three days. Which study should we trust or does this reflect variations between experiments (hence the question about the representation of survival data: are these pooled data from thre independent experiments; how much variation was there between independent experiments?).
   7. It would be interesting to measure the S. aureus bacterial load upon skanda overexpression to confirm a putative role in resistance.
   8. UAS-skanda: besides Fig. 6B, the authors should also refer the reader to Fig. S4A.
   9. Genetic dissection of the skanda-psh-hayan gene cluster: the last sentence of the paragraph does not reflect what Fig. S7B is showing: one of the double mutants and the triple mutant displayed a significant intermediate susceptibility to S. aureus.
   10. Paragraphs Compound mutants are EXTREMELY susceptible to S. aureus. The wording is likely too ...extreme: they do not seem to die much faster than skanda simple mutants, which were HIGHLY susceptible to S. aureus, like PPO1-PPO2 double mutants.
   11. Last paragraph: psh mutants should be compared side-by-side with psh-skanda double mutants in the same RTqPCR experiment: it is difficult to judge whether the statement of equivalent Drosomycin expression after S. aureus challenge is true given the low resolution of the figures (Fig. 6C vs. Fig. 7B). Last sentence: it would be more appropriate to mention "host defense" rather than "resistance" since the authors did not check the bacterial burdens of the compound mutants.
   12. Fig. 1: the scheme is not up to date and oversimplified. It should take into account the complexity revealed in the Shan et al. Science Advances article.
   13. Fig. S1: numbering the amino-acids in the sequence would help follow the text from Document S1. What are the residues written in light blue? It may be worth highlighting residue E 194. Of note, there is a difference between the sequence for peptide 4 as found in the sequence displayed on Fig. S1: KTDRD YV and the sequence of peptide 4 in Table S1: KTDRE YV; the presence of a potential SNP should be indicated, even though it is not making a major change in terms of charge of the peptide.
   14. Document S1: trypsin digestion (just before second call to Fig. S1); should it not be purified proteases instead? The text should be somewhat reworded as it is currently slightly misleading " In lane 8, peptide-1 through -19 were nearly undetectable". Table S1 shows that even though peptides 1, 2, 6, , 7 , and 11 are not expressed to strong enough a level to be displayed Fig. S1 lane 8 given the chosen scale, peptides 1, 2, 6, and 7 are expressed in the same range for slices 8B and 8C, whereas peptide 1 is found with just a two-fold difference in slices 8A and 8C.
   15. Fig. S2: the effect of skanda on SP7 cleavage is not detectable when Hayan isoforms are co-incubated. The main text should be modified to take this into account. How do the authors explain that pro-MP1 levels are not different upon co-incubation with Psh or Hayan-PB with or without adding Skanda, even though the active MP1 form is detected only in the absence of Skanda? In contrast, the pro-MP1 band can be detected upon co-incubation with Skanda and Hayan-PA.
   16. Fig. S3B, S7A: the three genes of the locus are inducible upon immune challenge. Have any NF-kappaB binding sites been detected at the locus. It might be relevant to repeat the experiment shown in S3B and especially S7A after a challenge with M. luteus. These experiments are definitely not essential.
   17. The mention 'Data not shown" is used twice. Not all Review Commons-affiliated journals accept it.

   Significance

   A strength of this work is the dual biochemical and genetic characterization of a SPH, an endeavor that is important to understand further the function of this class of protease-like family of secreted proteins that have been so far imperfectly studied from both perspectives (Kambris et al., CB, 2006, but see Westlake Reproducibility study on BioRxiv, Jin et al. Frontiers Immunol. 2023). Unfortunately, the two approaches fail to provide an integrated view of Skanda's function(s). A weakness is that this study does not unambiguously reveal at this stage what are the functions of Skanda in the host defense against S. aureus, let alone against other pathogens controlled to some extent by the Toll pathway or melanization. The authors have not considered a possible role in disease tolerance to S. aureus. These limitations decrease the conceptual advance of this article.

   This article will be of interest to investigators working on the innate immunity of insects. This reviewer is an expert in the Drosophila innate immunity field.

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   Referee #3

   Evidence, reproducibility and clarity

   Summary:

   Serine protease cascades are central for activation of immune responses in insects. In Drosophila melanogaster, Toll signaling pathway has been quite extensively studied, and several serine proteases, serpins and serine protease homologs (SPH) with functions in Toll activation have been identified. In this work, the authors characterize a new component of this system, a SPH which they name Skanda. Skanda seems to have multiple roles/points of action, on one hand participating in the regulation of Toll together with the established serine protease in the Toll activation, Psh, and on the other hand controlling the response to …

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   Referee #3

   Evidence, reproducibility and clarity

   Summary:

   Serine protease cascades are central for activation of immune responses in insects. In Drosophila melanogaster, Toll signaling pathway has been quite extensively studied, and several serine proteases, serpins and serine protease homologs (SPH) with functions in Toll activation have been identified. In this work, the authors characterize a new component of this system, a SPH which they name Skanda. Skanda seems to have multiple roles/points of action, on one hand participating in the regulation of Toll together with the established serine protease in the Toll activation, Psh, and on the other hand controlling the response to a systemic S. aureus infection, via not yet fully specified mechanism.

   Major comments:

   Key conclusions made in this work are convincing, and backed up by the data presented. The data and methods are presented in a way that allows reproduction of the experiment. The number of individuals used especially in the infection experiment (20 male flies per a replicate) is on the lower side, but the experiments are adequately replicated and the effects seen are clear.

   While this work contributes to our understanding of the regulatory mechanisms governing Toll signaling, at times the authors' reasoning is difficult to follow. I recognize that this is a complex topic, with multiple upstream branches activating Toll signaling, and the authors do consider various mechanisms that could explain their findings. However, the manuscript would benefit from additional clarification, perhaps through a schematic model illustrating the proposed effects of Skanda, to help readers position Skanda within the broader context of Toll signaling.

   Statistical analyses for the Drs expression experiments are lacking.

   Minor comments:

   The authors could explain what type of cells the sf9 cells are and why they decided to use them.

   Band intensities could be measured and plotted for the immunoblots. The immunoblot methods should be fully described in the Materials and methods section.

   Protein levels of Skanda in the Skanda mutant could be shown as the mRNA levels remain relatively high (Sup. Fig 3B). If this is not possible, could the authors comment on the remaining expression of Skanda in the Skanda mutants?

   Under the heading "Loss of skanda does not further enhance the cuticular melanization defects caused by the loss of Hayan or psh" the text should refer to figure 5D not 5B.

   Figure 6C shows that Drs expression is higher in the Skanda mutant than in controls at 32 h post S. aureus infection (although this has not been statistically tested). The authors don't mention this result in the manuscript, but to me it fits with the idea of Skanda acting as a negative regulator (the effect of which is accumulating and seen only late after infection). Could the authors comment on this?

   Under the heading "Psh and skanda redundantly regulate Toll signaling", the comparison should likely be between Figures 7A-7B and 5B-C (rather than 5A). When examining the effects of single versus double mutants on Drs expression, the Psh-Skanda double mutant clearly reduces Drs more than the Psh single mutant. However, in the context of microbial proteases, the pattern appears different: there is virtually no difference at 6 hours, while at 48 hours there may be a slight decrease in Drs expression in the double mutant compared to the Psh single mutant, although this difference would likely not reach statistical significance if tested. I don't know what this could mean, but I'd like to hear the authors' take on this.

   Referee cross-commenting

   I also agree with the comments and points raised by the other reviewers.

   Significance

   Research on the Drosophila immune response has significantly advanced our understanding of (innate) immune responses, both generally and in an evolutionary context. Despite over three decades of study, this work demonstrates that there are aspects of Toll signaling that remain unresolved. The authors identify a novel regulator of the Toll pathway and begin to elucidate its functions. Equally important, their findings underscore the complexity and context-dependency of the regulatory events that shape immune responses.

   My fields of expertise are Drosophila melanogaster, innate immunity, cell-mediated immunity.

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   Referee #2

   Evidence, reproducibility and clarity

   Summary

   In this work the authors identify the SPH skanda as an important player in Drosophila resistance to S. aureus infections independent of Toll and classical melanization. The authors conducted rigorous in vitro assays using recombinant proteins of various SPs in the Drosophila Toll-PO cascade to show that skanda negatively regulates activation cleavage of SPs at the level of and downstream of Psh and hayan, two key SPs that converge on Toll pathway activation with the latter playing a central role in cuticular melanization. In parallel, genetic analysis using mutant flies showed that skanda does not negatively regulate Toll …

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   Referee #2

   Evidence, reproducibility and clarity

   Summary

   In this work the authors identify the SPH skanda as an important player in Drosophila resistance to S. aureus infections independent of Toll and classical melanization. The authors conducted rigorous in vitro assays using recombinant proteins of various SPs in the Drosophila Toll-PO cascade to show that skanda negatively regulates activation cleavage of SPs at the level of and downstream of Psh and hayan, two key SPs that converge on Toll pathway activation with the latter playing a central role in cuticular melanization. In parallel, genetic analysis using mutant flies showed that skanda does not negatively regulate Toll pathway nor melanization. Only skanda over expression in vivo led to a reduction in S. aureus melanization which, in my opinion, is most likely due to the artificial increase in the in vivo concentration of the protein rather than an indication of a potential true function. Altogether this an interesting work as it shows the discrepancies between the biochemical and genetic approaches when it comes to dissecting the insect SP cascades regulating melanization and Toll as highlighted by the authors themselves in the discussion section. All experimental work is well controlled, methodology is robust and results are adequately discussed. I have some comments concerning few experiments and interpretations that in my opinion warrant further discussion.

   Major comments:

   1. It seems that SP48 and Grass can redundantly cleave Skanda although the later cleaves more strongly. (Fig 3B) Can other downstream SPs cleave skanda? Can ModSp alone cleave skanda? (ModSP + skanda lane was absent for Fig 3B). It is important to test these possibilities as the in vitro system may be quite relaxed as to the specificity of these cleavage events and may not reflect what happens in vivo. In fact it has been shown in Anopheles gambiae that SPH can be redundantly cleaved by multiple SP in the protease cascade. Although these are cascades with certain hierarchy, information can still flow in more than one direction along the different branches of these cascades.
   2. In Fig 4B and 4C the bands of active forms should be quantified from at least 3 immunoblots for robust results especially in Fig 4C where the differences are minimal.
   3. It is not clear to me why skanda should have a specific role in resisting S. aureus infections despite that S. aureus is not a natural pathogen of Drosophila? Has other Gram-positive and Gram-negative bacteria been tested?
   4. In Fig 6E more points should be collected for statistical power. It is also better to show these data that are not normally distributed in violin charts or boxes and whiskers which give a better indication as to which quartile the bulk of the data belongs.

   Minor comments:

   1. In Figures 3 and 4, It would be easier to follow the cleavage events if a schematic drawing is provided showing the sequence of activation cleavage events of the tested SPs
   2. The fact that PPO1/PPO2 depleted flies exhibit increased Drs expression could be due to increased bacterial proliferation in this mutant background that trigger increased Toll stimulation, rather than a negative feedback mechanism. This increased proliferation is shown in Fig 6E.
   3. In Fig 6E more points should be collected for statistical power. It is also better to show these data that are not normally distributed in violin charts or boxes and whiskers which give a better indication as to which quartile the bulk of the data belongs.
   4. A phenotype for skanda in melanization was observed only in over-expression assays which may artificially alter molecular interactions in the cascade.
   5. Page 10 last paragraph "peak expression at 32 hrs or 48 hrs as shown on the figure?"
   6. The differences in Drs expression levels in Hayan-pshDef and psh-skandaDef double mutant flies infected with M. luteus and S. aureus is surprising. I wonder whether the observed differences are due to biochemical differences in the microbial surfaces to which these cascades are recruited.
   7. There are several typos in the manuscript

   Significance

   The main strength of this work is that it combines biochemistry and genetics in a strong genetic model to characterize the biochemical interactions between SPH and Sp in clip cascades and relate the relevant interactions observed in vitro with potential in vivo functions. This is the first time that such a rigorous combined approach was adopted to the study of these cascades. The results obtained also show the advantages and limitations of each approach. As such i believe this study will be of interest to a broad audience in the field of insect immunity.

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5. 

   Review Commons

   Jun 26, 2026

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #1

   Evidence, reproducibility and clarity

   In the manuscript entitled "The serine protease homolog Skanda modulates Toll-phenoloxidase-mediated immunity in Drosophila," Vasanth et al characterize in detail a previously unstudied component of the insect immune response using first biochemical and then in vivo methods. Using proteins overexpressed and purified from insect cells, the authors provide evidence that Skanda could be a negative regulator of the SP cascade, impacting cleavage of proHayan and proPsh, and consequently Toll pathway and PPO1 activation. This work reaches further by transposing these findings into the D. melanogaster in vivo model. Here, however, the …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   In the manuscript entitled "The serine protease homolog Skanda modulates Toll-phenoloxidase-mediated immunity in Drosophila," Vasanth et al characterize in detail a previously unstudied component of the insect immune response using first biochemical and then in vivo methods. Using proteins overexpressed and purified from insect cells, the authors provide evidence that Skanda could be a negative regulator of the SP cascade, impacting cleavage of proHayan and proPsh, and consequently Toll pathway and PPO1 activation. This work reaches further by transposing these findings into the D. melanogaster in vivo model. Here, however, the picture becomes more confusing as Skanda at native levels does not appear to regulate either the Toll pathway or the melanization cascade. Only one strong phenotype was identified in that decreased expression of Skanda increased susceptibility to S. aureus infection while increased expression decreased susceptibility. The mechanism for this remains unclear. To their credit, the authors carry out an in-depth analysis to rule out all the obvious possibilities. In the discussion, the authors explore the basis of discrepancies between their biochemical and genetic findings. We would suggest that an additional one to consider is differing roles or behaviors of Skanda in the microenvironments of the local site of injury (where S. aureus may be contained when it is tolerated) and the hemolymph. In summary, this is a valuable analysis of the innate immune component Skanda whose role has become somewhat clearer through these studies, but still remains obscure.

   Major Comments

   • To assess bimodal distribution of bacterial loads within single flies in Fig 6E, authors should either: increase the sample size to allow for proper statistical assessment of different distributions among genotypes, specifically between w1118 and skanda_d107; or, provide a modelling framework for statistical testing. Otherwise, the present results seem insufficient to conclude that Skanda is playing a role in resistance to S. aureus.
   • Another way to assess a role for tolerance in the Skanda mutant would be to measure BLUDs (https://doi.org/10.7554/eLife.28298 ) and/or transcription of CrebA (https://doi.org/10.1371/journal.ppat.1006847).
   • The error bars on qRT-PCR datasets are large, the data points are not shown so we do not know how many replicates were included in the graphs (Fig 5 B and C, Fig 6C, Fig 7 A and B, and Fig 8B). Bar plots are not the most faithful reproduction of biological datasets, as they can hinder significant information regarding datapoints distribution and variation (Beyond Bar and Line Graphs: Time for a New Data Presentation Paradigm | PLOS Biology). We advise that, particularly in the case of datasets such as qRT-PCR, the final values of fold change are represented with individual dots, with the mean value clearly represented, whether with or without the additional bar graph. Furthermore, no statistical tests were applied to determine significance. Data points should be shown and appropriate statistical tests should be applied. The number of biological replicates should be included in the analysis and the statistical test applied should be noted in the figure legends.
   • Although there are claims of Skanda conferring resistance to S. aureus infection, only Drs levels are tested. These conclusions could be strengthened by assessing expression levels of additional AMPs.

   Minor Comments

   • Parag. 1: (data not shown) should be removed and if possible AlphaFold prediction of skanda conformation added. Alternatively, remove sentence.
   • Parg. 3: 1000 mL? why not 1L?
   • Parag. 5: , in last sentence that should be .
   • Parag. 6: "a role at the same position..." does not convey the correct message< replace with equivalent?
   • Figure axes (5D, 5E, 6D, etc...) of melanization assays are wrongly named "% melanisation", with "s"
   • Parag. 21: compound mutants (if correctly interpreted as dataset presented in Fig. 8B) were tested at 6h, 24h and 48h, and not 32h, as written in the text
   • Results section "skanda is not mandatory for the activation of the Toll pathway" adopts a literal translation which would probably be better phrased as "is not essential"
   • Discussion parag. 2: "Skanda exhibits..."
   • Discussion last parag: "..., but also underlies..."
   • It has been evidenced that

   Additional comments:

   • The sentence on page 2 beginning with "Upon binding, these PRRs..." is very long and difficult to follow. This should be rewritten.
   • In many places in the manuscript bacterial "dose" is used in place of bacterial burden. The dose is the amount of a substance or bacterium given to the animal.
   • Page 11: Skanda is described as a placeholder when I think a (competitive) inhibitor would be more appropriate.

   Referee cross-commenting

   I agree with the comments of the other reviewers.

   Significance

   Strengths: The authors take a multi-disciplinary biochemical and in vivo approach to understand the molecular interactions among SPs and SPHs and thereby uncover the role of the protein Skanda that might otherwise not have been appreciated. They have made extensive use of novel transgenic fly lines, generated in the context of this study, and have thoroughly tested their specificity and cis-acting potential. These will provide a resource to the field. In addition to the new description of Skanda, these findings strengthen previous knowledge regarding systemic infections with different bacteria (M. luteus, S. aureus) and reproduce the known redundancies of Psh and Hayan modes of action. Moreover, this research is relevant for the expansion of basic knowledge on innate immunity, particularly in the field of insect-pathogen interactions, making use of S. frugiperda cell lines and D. melanogaster adults and larvae. Although not at the focus of this work, the evolutionary conserved nature of these aspects of innate immunity across these two distant species enhance the importance of these findings.

   Weaknesses: Some assays do not include enough biological replicates and others do not have enough information on how many biological replicates were performed. Therefore, the conclusions drawn are difficult to assess. Lack of statistical analysis on the qPCR experiments complicates the interpretation of results.

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6. 

   Version published to 10.1101/2025.09.30.679548 on bioRxiv

   Oct 1, 2025