A tool for repression of RNAi overcomes sterility and separates maternal from zygotic RNAi in Tribolium castaneum

A tool for repression of RNAi overcomes sterility and separates maternal from zygotic RNAi in Tribolium castaneum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A tool for repression of RNAi overcomes sterility and separates maternal from zygotic RNAi in Tribolium castaneum Muhammad Salim Hakeemi, Ann-Kathrin Müller, Julia C. Ulrich, Gregor Bucher This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7658464/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jan, 2026 Read the published version in Developmental Biology Advances → Version 1 posted 9 You are reading this latest preprint version Abstract RNA interference (RNAi) is a natural antiviral defense mechanism in plants and animals. As a counter defense strategy, most viruses have evolved viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. Here, we utilized transgenic misexpression of a VSR from Cricket Paralysis virus (CrPV1A) to dampen RNAi in a temporal and life-stage specific way in order to overcome limitations of knocking down pleiotropic genes by the strong systemic RNAi response in the red flour beetle Tribolium castaneum . We found that ubiquitously driven VSR rescued the sterility of the females injected with Tc-axin or Tc-decapentaplegic double-stranded RNA, where sterility had previously hampered analysis. By combining this tool with a heat-shock driven VSR, we were able to separate maternal from zygotic function for the Wnt pathway inhibitor Tc-axin . Thereby, we could provide evidence that maternal Wnt signalling alone is responsible for axis formation in Tribolium . Our tool opens new experimental possibilities such as studying genes by parental RNAi, which would normally lead to sterility and separating maternal from zygotic gene functions. Further improvements are required to allow for studying zygotic gene function while rescuing maternal functions and for spatially restricting the RNAi effect. RNAi Viral suppressors of RNAi (VSRs) transgenic tool Tribolium castaneum Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction In recent years, RNA interference (RNAi) has become a highly effective and widely used reverse genetic tool to elucidate gene function in diverse emerging arthropod model organisms (1-6). It has opened the possibility to ask for the evolution of gene function across clades and to study processes that are not represented in the main model system for arthropods, the fruit fly Drosophila melanogaster . The red flour beetle Tribolium castaneum has emerged as a powerful new insect model organism for the study of gene function based on a number of established techniques: It has a strong and systemic RNAi response (7-9) and the availability of a very advanced genetic toolkit for gene function analysis i.e enhancer trapping, insertional mutagenesis, heat-inducible gene expression, the GAL4/UAS system and genome editing (10-15). Recently, the iBeetle genome-wide RNAi screen revealed divergent gene sets required for a number of homologous developmental processes in Tribolium vs, Drosophila. For example, a surprisingly high number of genes are essential for muscle development in Tribolium but seem not required in Drosophila (16-18). One notable aspect that makes Tribolium a powerful model system is the environmental nature of its RNAi response, i.e. the ability of Tribolium cells to take up dsRNA from the surrounding fluid. This has for instance allowed to inject dsRNA into the hemolymph of larvae and observe the effect in all cells (19) to inject mothers to elicit knockdown in the offspring (7) and to add dsRNA to primary cell cultures for knocking-down genes (20). However, this strength can at the same time limit gene functional analyses in several ways. For example, dsRNA injection of developmental genes that are in addition required for oogenesis leads to sterility of the injected females. As consequence, the knockdown effect is difficult to be studied in the offspring as in the case of Tc-decapentaplegic ( Tc-dpp) (21) or a reduced dsRNA concentration has to be used, which may lead to an incomplete knock-down (22). Moreover, in case of pleiotropic genes, it would be advantageous to locally or temporally knock-down the gene in order to study the effect in one process independently from other functions. Finally, upon parental RNAi of genes that have both maternal and zygotic gene function such as Tc-axin , both functions are suppressed making it difficult to separate maternal versus zygotic gene function. To overcome these restrictions, there is a need for a tool to restrict RNAi to a certain time, region or life-stage . In Drosophila , localized RNAi has been achieved by Gal4 driven hairpin-loops that induce RNAi only in the expressing cells (23-24). However, in the smaller communities of the less established model systems without central stock keeping facilities it is unrealistic to maintain genome-wide transgenic line collections. Hence, a tool combining dsRNA injection with spatio-temporal inhibition of RNAi would combine the ease of RNAi with the maintenance of a limited number of transgenic lines. The RNAi pathway is a natural antiviral defence mechanism in plants and animals (25-31). As a counter defence strategy, many viruses have evolved proteins that act as viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. These VSR proteins evolved independently and use different mechanism of action to impede the RNAi machinery (32-40). In previous work, we had tested a number of known VSRs for their functionality in the red flour beetle (41). We had found that heat-shock induced expression of a VSR from Cricket Paralysis virus (CrPV1A) was effectively blocking an ongoing RNAi response (41). Here, we utilized this VSR which interacts with the endonuclease Argonaute 2 (Ago2) and blocks its cleavage activity (40). Specifically, we tested the strategy to transgenically express that VSR in certain life stages in order to render them resistant to RNAi. As consequence, only the non-expressing cells would be affected by injection-induced RNAi. We wanted to apply the tool to answer a long-standing question on the function of the Wnt pathway in anterior-posterior (AP) axis formation. It had been suggested that maternal Tc-axin mRNA localized at the anterior pole represses Wnt signalling, which is required for the specification of the anterior pole. However, the respective experiments had to be performed with reduced amounts of Tc-axin dsRNA in order to avoid sterility (22). Further, Wnt signalling has many additional functions during segmentation and other embryonic processes. Therefore, the observed phenotype was a mixture of the maternal axis-formation phenotype superimposed by the zygotic segmentation defects. Indeed, stronger knock-down of Tc-axin resulted in embryos with heavily affected morphology or even without cuticles (called the empty egg phenotype) (22). A clear signature of a complete axis-formation phenotype is the double abdomen phenotype where the head is replaced by a second abdomen. Such double abdomen phenotypes had not been observed in our initial study (22) while in a subsequent study by others, such phenotypes had been described (42). Hence, it appeared possible that repression of Wnt signalling alone would be sufficient for specifying anterior development, but it had remained unclear, why this phenotype had not been observed in our previous experiments. Our results showed that a ubiquitously driven VSR (CrPV1A) indeed reduced RNAi activity. We demonstrate the use of the new tool to overcome the issue of sterility after parental RNAi of Tc-axin and Tc-dpp . Unfortunately, this tool alone was not efficient enough enable distinguishing zygotic from maternal gene function. However, when combined with heat-shock mediated repression in embryos, we were able to do so. Several observations suggest that strong ubiquitous CrPV1A expression may interfere with viability. As consequence, only transgenic lines with a moderate VSR expression survive such that the RNAi blocking effect may not be strong enough for separating maternal from zygotic functions. Our new tool can now be used to study the embryonic function of genes, the parental RNAi of which leads to sterility. Adjustments of the design may overcome the current limitations. Methods 1. Generation of VSR plasmid constructs Several VSR plasmid constructs were generated as follows: The VSR template CrPV1A, enhanced green fluorescent protein (EGFP) fragment and different ubiquitous promoters were amplified and subsequently cloned into pSLfa1080fa shuttle vector (43). The final VSR constructs as shown in Fig. 1, were cloned into piggyBac vector by using standard restriction enzymes provided by Thermo Fisher Scientific or Gibson assembly (NEBuilder HiFi DNA Assembly Master Mix). For bicistronic VSR lines, codon optimization of the CrPV1A fragment was performed using the Integrated DNA Technologies (IDT, https://eu.idtdna.com) codon optimization tool and subsequently ordered as a gBlock fragment from IDT. The CrPV1A plasmid was kindly provided by Ronald Van Rij (Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands). The polyubiquitin and α-tubulin promoters have been described previously (44). Please find the sequences of the final constructs in supplementary file 1; the plasmids are available from the last author upon request and on Addgene (numbers: xxx) 2. Transgenesis, RNAi and heat-shock induction of VSR Embryonic injections and transgenesis in vermillion white beetles (vw) were performed as described previously (11,13,45). In order to reduce lethality by VSR expression during transgenesis, dsRNA targeting the VSR at a final concentration of 1000ng/μl was added to the injection mix. The maternal RNAi into pupae and cuticle preparation was performed as described previously (16,18). All tests were performed in two biological replicates. See supplementary tables for number of analysed embryos. The heat-shock activation of VSR was performed similar to (41) by separating three hours old embryos (kept at 32°C) from flour, putting them into a glass vial with flat bottom and placing the vial for 10 minutes into a water bath at 48°C. The first heat-shock was after 10 hours and second one after 13 hours of egg collection (i.e. 10-13 and 13-16 hours of development at 32°C, respectively). The heat-shocked embryos were transferred back to a plastic vial and continued development further at 32°C for the cuticle preparation. The overnight egg collection from the same injected animals was used as negative control for cuticle analysis (embryos with no heat-shock treatment). Images were adjusted for brightness and contrast using the Levels-function in Photoshop CS5 (Adobe). Tc-axin : TC006314; Tc-dpp: TC008466; Tc-prd: TC015804; Tc-eve: TC009469; Tc-hh : TC032269. The lines are kept in the lab of the last author and are freely available upon request. Note that we had attempted to localize VSR expression to a stripe along the midline by knocking-in the construct into the vnd locus. While we found no localized expression nor effect, we noted that unexpectedly, this line performed well in some of our tests. Therefore, we kept the line and documented the results in Supplementary File 1 and the supplementary tables. Results Generating viral suppressor of RNAi (VSR) transgenic lines To overcome sterility and to separate maternal versus zygotic RNAi, we first wanted to generate transgenic lines where a VSR was expressed ubiquitously. We reasoned that when injecting into such a line, the female sterility induced by RNAi targeting certain genes might be overcome. This would allow researchers to study the embryonic RNAi phenotype of such genes in the offspring. Conversely, if RNAi is applied to females and the VSR is contributed by the males, the zygotic expression could theoretically become rescued while the maternal RNAi effect would have full effect. We used the CrPV1A from the Cricket Paralysis virus because this VSR had been identified as the most efficient of several tested VSRs in our previous work (41). We generated a series of transgenic lines by piggyBac -mediated transgenesis that expressed the VSR CrPV1A. Expression was controlled by different ubiquitous promoters (α-tubulin, polyUbiquitin and GAPDH) in order to identify the most suitable one (Fig. 1A-B). To further enhance the expression of the RNAi inhibitor, we have in addition generated bicistronic VSR lines where double amounts of VSR were expected to be produced per translation initiation event. In these constructs, two copies of the VSR are separated by the 2A-peptide were cloned into one coding sequence (Fig. 1C). The 2A sequence leads to ribosomal skipping such that two separate proteins are translated from a single mRNA transcript (46). In total, we generated 12 transgenic lines from different VSR constructs and most of these transgenic lines were subsequently tested for RNAi rescue effect (see Table S1 for details). However, some of the transgenic VSR lines driven by strong ubiquitous promoters turned out to be unhealthy and infertile and could therefore not be included in the RNAi rescue effect test (Table S1). Interestingly, high embryonic lethality was observed during microinjection of the constructs for transgenesis of the bicistronic VSR constructs and the VSR constructs driven by putatively strong ubiquitous promoter such as GAPDH (Fig. 2). Based on these observations, we considered that high amounts of VSR might be lethal. In line with this assumption, the embryonic lethality rate after injection for transgenesis was largely rescued when we co-injected these plasmids along with dsRNA against the VSR-EGFP transcript (Fig. 2). See below for discussion on our assumption that strong VSR expression could interfere with viability. Sterility rescue after maternal RNAi in VSRs lines Some genes are required both, for embryonic development and for oogenesis. Hence, parental RNAi targeting such genes induces sterility in the injected mother such that no embryos can be collected to study the gene’s embryonic function (Fig. 3A). Hence, in order to obtain knock-down embryos for analysis, the RNAi effect needs to be dampened in the injected females (Fig. 3A). To test whether the ubiquitous VSR lines helped to overcome this sterility problem, we injected homozygous female pupae from several of our VSR lines with dsRNA targeting two genes that had shown to induce sterility in previous works (Fig. 3B). In previous studies, the sterility induced by injection of dsRNA targeting Tc-dpp and Tc-axin into females had prohibited the application of parental RNAi to these genes or had required strongly reducing the amounts of injected dsRNA (21,22). For our test, we first determined and then used the minimal amount of dsRNA that induced a complete sterile phenotype for Tc-dpp and Tc-axin (500ng/µL and 250ng/µL, respectively). The injected females were mated to wildtype males and the number of embryos was scored and compared to wildtype females injected with the same dsRNAs (Figure 3B). Among all the VSR lines that were tested, only the α-tubulin driven VSR line showed a strong rescue of sterility (Supplementary Table S2). In wildtype, after Tc-axin and Tc-dpp dsRNA injection, the number of embryos produced from 16 living female animals within nine days post injections were 4 and 5 respectively. However, upon Tc-axin and Tc-dpp RNAi in the α-tubulin driven VSR line, the number of eggs significantly rose to 101 and 169, respectively (Fig. 3C and Supplementary Table S2). The results confirmed that the technique could indeed be used to overcome the sterility induced by some genes upon parental RNAi. This opens the possibility to study the embryonic function of such genes by parental RNAi. Separating maternal and zygotic gene function A number of patterning genes have both, maternal and zygotic functions. For instance, during oogenesis the mother localizes a signalling molecule in the oocyte, which acts during the first hours of embryogenesis. The later zygotic expression of the same molecules can play a different role during advanced embryogenesis. However, in parental RNAi, knock-down reduces both maternal and zygotic gene functions. As consequence, it remains unclear what part of the phenotype-if any-is maternal and what part is zygotic. Similar to the sterility effect above, a strong rescue of RNAi in the injected females of VSR lines would rescue the maternal effect of the targeted gene (i.e. VSR expression blocks RNAi in the mother). When using a heterozygous mother and a wildtype male, 50% of the offspring carry no VSR. These offspring animals should show the full zygotic phenotype of the RNAi treatment (Fig. 3B). Conversely, inhibiting the RNAi in the offspring by mating injected wildtype females with homozygous VSR males could theoretically allow for maternal knock-down but zygotic rescue of a given gene (Fig. 3D). For this rescue of the zygotic function, the VSR needs to be effective even in the heterozygous state (Fig. 3D). To identify such a line, we tested the level of RNAi rescue effect in all our VSR lines in homozygous offspring animals. We injected different zygotic genes such as Tc-paired (Tc-prd), even-skipped (Tc-eve) and Tc-hedgehog (Tc-hh). These genes are known to have only zygotic functions and do not lead to sterility in the injected mother. Indeed, the strength of the phenotypic penetrance decreased dramatically for most of the genes tested in these VSR lines confirming that the constructs were working in principle (Supplementary Table S3). Unfortunately, no VSR line showed complete phenotypic rescue (i.e. wildtype L1 offspring) even though the VSR was homozygous in these embryos . Only Tc-hh RNAi seemed to be rescued to some degree (Supplementary Table S3). These data show that the idea works in principle but that the available VSR lines have not sufficient VSR activity for that application. As alternative approach to zygotic gene rescue, we sought to separate the maternal and zygotic gene function by combining the ubiquitous VSR lines with a heat-shock driven VSR line where the RNAi inhibitor CrPV1A is expressed under the control of the endogenous Tribolium heat-shock promoter. The heat-shock driven VSR system had previously been shown to very effectively rescue an ongoing RNAi response for several genes (41). Maternal Tc-axin function is sufficient for axis formation The current model for axis formation in Tribolium poses that maternal function of Tc-axin is involved in anterior-posterior axis formation in beetles. Its mRNA of this Wnt-signalling inhibitor is localized at the anterior pole during oogenesis representing a maternal aspect of Tc-axin function. The localized presence of Tc-Axin blocks the Wnt-pathway at the anterior thereby contributing to defining the anterior pole (22). Later it was found that Tc-gcl acts upstream in that process being required for anterior localization of Tc-axin . RNAi targeting Tc-gcl led to absence of Tc-axin mRNA at the anterior pole and to double abdomen phenotypes (47). Based on these results it was hypothesized that Wnt signalling was responsible for AP axis formation. However, it had remained unclear, whether Wnt signalling was sufficient for that process because Tc-axin RNAi alone had not resulted in double abdomen phenotypes in Fu et al. (22) while it had done so with low frequency in another study (42). Hence, it had remained disputed, whether strong knock-down of Tc-axin alone would be sufficient for generating double abdomen phenotypes or whether additional components were required. Importantly, all respective experiments were complicated by the sterility induced by Tc-axin RNAi, which required lowering the dsRNA dose such that a full knock-down would not be expected. In addition, Tc-axin has later zygotic functions, which lead to strong developmental defects prohibiting the formation of L1 cuticles such that double abdomen cuticles would not be visible. We wanted to use the combination of our tools to overcome these technical issues. First, we aimed at reducing the sterility effect in the injected mother in order to be able to increase the amount of injected dsRNA, i.e. increase RNAi strength. Second, we wanted to rescue the zygotic Tc-axin phenotype in the offspring in order to allow for normal zygotic development and formation of a cuticle. With the aim of inhibiting maternal Tc-axin function, we injected Tc-axin dsRNA (concentration: 250ng/μl) in our α-tubulin driven VSR female pupae (to reduce the sterility effect of Tc-axin RNAi) and crossed them with males carrying the previously described heat-shock VSR constructs (41). Three hour egg collection were heat-shocked two times for 10 minutes at 48°C. The first heat-shock was after 10 hours and second after 13 hours of egg collection (i.e. 10-13 and 13-16 hours of development at 32°C, respectively). Upon regular parental Tc-axin RNAi, most offspring produced empty egg phenotypes (i.e. no cuticle formed; EE) or the cuticle crumbs phenotype (i.e. cuticle remnants with no recognizable morphology) (see Fig. 4 for the range of observed phenotypes). However, in contrast to non-heat-shocked embryos (control), Tc-axin RNAi in heat-shocked embryos resulted in a reduction of the portion of empty egg phenotypes and an increased number of cuticles in two independent experiments. The L1 cuticles were classified according to the phenotypes. In both experiments, head phenotypes emerged (27 embryos; 27.55%) and in one of the experiments, several cuticles showed a double abdomen phenotypes (7 embryos; 7.14% of both experiments, 25% of the single experiment with that effect; see Supplementary Table S4) (Fig. 4F). Also, the number of wildtype cuticles increased significantly in heat-shock embryos (20 out of 98 embryos; 20.4%) compared to control without heat-shock (15 out of 512 embryos; 2.92% ) (Fig. 5 and Supplementary Table S4). Discussion Toxic effect of VSR on viability and development In order to establish a system for spatio-temporal control of RNAi in Tribolium, we planned to generate strong ubiquitous expression of the VSR to reach complete phenotypic rescue after parental RNAi. Unfortunately, our data indicate that high level of VSR expression likely had a significant effect on Tribolium viability and development. First, for many constructs that were expected to exhibit strong expression—such as those containing strong promoters or employing a bicistronic design—we either failed to obtain transgenic lines, or the resulting lines were unhealthy or exhibited reduced fertility. (Table S1). Second, only VSR lines driven by α-tubulin, and polyUbiqutin promoters could be made homozygous. Third, a comparably high embryonic lethality was observed during microinjection of bicistronic VSR constructs or VSR constructs driven by strong promoters such as GAPDH. In line with this hypothesis, the lethality rate was rescued when we co-injected the transgenesis plasmids along with dsRNA against the VSR-EGFP transcript. Fifth, In previous study we validated the toxic effect of VSR expression in Tribolium by a heat-shock experiment where the RNAi inhibitor CrPV1A was expressed under the control of endogenous Tribolium heat-shock promoter. Here, upon heat-shock activation of VSR at different stages of development (adult, pupae and larvae), the survival rate was reduced significantly in heat-shock-VSR line animals compared to wildtype (41). Indeed, other studies provided evidence that CrPV1A interferes with the Ago-2-dependent miRNA silencing and miRNA regulatory network in Drosophila as well (48). Taken together, these results suggest that a high expression level of CrPV1A has a substantial effect on fitness and viability during Tribolium development. We suggest two possible workarounds for this issue: First, heat-shock mediated expression has been shown to work efficiently (this work and 41) despite increased lethality. Second, expression mediated by the binary Gal4/UAS system established in that model (12) might be able to provide strong localized expression without affecting the processes sensitive to VSR misexpression. Expanding the scope of RNAi by using viral RNAi inhibitor Recently our lab has utilized VSR from Cricket Paralysis virus (CrPV1A) to temporally restrict RNAi during segmentation process in Tribolium (41). In the present study, we adopted that tool to overcome the sterility problem and showed that α-tubulin driven ubiquitous VSR line has a notable rescue effect on sterility after Tc-axin and Tc-dpp RNAi. The sterility rescue of VSR line highlights the potential use of this newly developed tool on overcoming the limitations of studying certain pleiotropic genes by parental RNAi if their knock-down affects fertility as well. An alternative to overcome the sterility problem is embryonic injection of dsRNA. However, this is a much more tedious procedure compared to parental RNAi, the embryos may be damaged by the injection and it is very difficult to collect RNAi embryos for WMISH or similar analyses, as each embryo has to be dissected out of the eggshell. With our new tool, standard methods for downstream analyses can be applied. Some drawbacks of the tool should be noted, too. First, we cannot exclude that VSR protein is carried over from the mother to the oocytes such that the offspring has a dampened RNAi response despite the lack of the construct in their genomes (Fig. 3B). Second, the assumed lethality induced by strong ubiquitous expression of the VSR prohibits the establishment of lines, which are strong enough to realize the approaches outlined in Fig. 3B and D. Interestingly strong VSR expression by two heat-shocks does apparently not block development of embryos (this work and 41). Hence, it could be that the observed problems to generate and maintain transgenic VSR lines stems from the chronic VSR expression or the expression in certain tissues at certain times (e.g. germ cells, ovary, brain etc.). If this is true, a sufficiently high VSR level might be reached when restricting expression to certain tissues or life stages e.g. by driving them by a binary expression system, which is available in Tribolium (12). Repression of Wnt signalling is sufficient for defining the anterior pole We applied our new tool to answer a question, which had remained open because technical difficulties prohibited testing it. It had been shown before that the Wnt-inhibitor Tc-axin was involved in the specification of the anterior-posterior axis. Specifically, maternally contributed Tc-axin mRNA was localized to the anterior pole of the oocyte thereby inhibiting Wnt signalling at the anterior. In Tc-axin RNAi embryos, the anterior fates were reduced in a dose dependent manner. This led to the idea that suppression of Wnt signalling was required for anterior development similar to findings in vertebrates and other animals (22). In line with this hypothesis, RNAi targeting Tc-germ-cell less ( Tc-gcl ) abolished anterior localized Tc-axin mRNA and led to full double abdomen phenotype, which indicated a complete loss of anterior fates (47). It was suggested that Tc-gcl acts via Tc-axin to suppress the Wnt pathway in order to define the anterior pole. However, the hypothesis that Tc-gcl acted exclusively via the Tc-axin RNA remained challenged by the discrepancy of the two phenotypes: in Tc-gcl RNAi, a double abdomen phenotype develops while in Tc-axin RNAi, such a phenotype had not been observed in those studies (22, 47). Conversely, another study reported double abdomen cuticles after Tc-axin RNAi supporting the idea that indeed, Wnt-repression is the sole determinant of the anterior pole (42). The differences in phenotypes observed in these two studies have remained unexplained. The use of lower concentrations in the study by Prühs et al. (42) might have contributed while strain-specific effects (49) are unlikely because the divergence was observed even when using the same strain (San Bernardino). Two explanations could explain the discrepancy of phenotypes of Tc-gcl and Tc-axin RNAi embryos in our previous studies. Either, another component regulated by Tc-gcl is needed to produce the full phenotype. Or Tc-gcl acts exclusively via Tc-axin but this could not be seen in our Tc-axin RNAi experiments because reduced concentrations had to be used due to sterility of Tc-axin dsRNA injected mothers. Another issue with Tc-axin RNAi is that the Wnt pathway has many functions during development, all of which are affected by standard RNAi experiments. As consequence, the resulting RNAi embryos do not form recognizable morphological structures, making phenotype analysis challenging. Moreover, they usually stop development before secreting cuticle. With our system, we were now able to distinguish between those alternative explanations. First, we could increase the concentration of Tc-axin dsRNA by injecting into females of the α-tubulin VSR line, where sterility was rescued to some degree. Second, we rescued the later Wnt functions by blocking RNAi in early embryos with heat-shock mediated activation of VSR expression. This allowed us to observe cuticular morphology of embryos. Indeed, the strong empty egg and cuticle crumbs phenotypes were converted to a large degree to embryos with recognizable cuticle morphology. We observed anterior deletions (as had been shown before) but we also saw double abdomen cuticle phenotypes (Fig. 4). The occurrence of double abdomen phenotypes strongly supports the hypothesis that indeed, blocking the Wnt pathway by maternal Tc-axin is sufficient to define the anterior pole of the Tribolium embryo (22, 42, 47). Declarations Funding: DFG BU1443/14-1ad Author Contribution M.S.H: Data analysis, visualization of the data, VSR plasmids design and cloning, transgenesis and writing the original draft; A.K.M and J.U.: VSR plasmid design and transgenesis; G.B.: Conceptualization, funding acquisition, supervision and writing the original draft. Acknowledgement We thank Elke Küster for help with the beetles and Claudia Hinners for help with molecular experiments. Data Availability The transgenic lines are available from G.B.; the plasmids are distributed by AddGene References Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans . Nature. 1998 Feb 19;391(6669):806-11. doi: 10.1038/35888. PMID: 9486653. Hughes CL, Kaufman TC. RNAi analysis of Deformed, proboscipedia and Sex combs reduced in the milkweed bug Oncopeltus fasciatus: novel roles for Hox genes in the hemipteran head . 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Supplementary Files TableS4Heatshocknew.xlsx TableS3Zygoticnew.xlsx TableS1VSRTransgenesisandLethalitynew.xlsx TableS2VSRSterilitynew.xlsx FileS1SupplementarySequenceDatarevision.pdf Cite Share Download PDF Status: Published Journal Publication published 16 Jan, 2026 Read the published version in Developmental Biology Advances → Version 1 posted Editorial decision: Revision requested 25 Nov, 2025 Reviews received at journal 25 Nov, 2025 Reviews received at journal 22 Nov, 2025 Reviewers agreed at journal 31 Oct, 2025 Reviewers agreed at journal 29 Oct, 2025 Reviewers invited by journal 29 Oct, 2025 Editor assigned by journal 20 Sep, 2025 Submission checks completed at journal 20 Sep, 2025 First submitted to journal 19 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":265973,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of constructs.\u003c/strong\u003e (A) The RNAi inhibitor CrPV1A is expressed under the control of the \u003cem\u003eTribolium\u003c/em\u003e α-tubulin promoter. (B) A bicistronic construct encoding for CrPV1A and EGFP separated by a P2A-peptide driven either by the polyUbiquitin or the GAPDH promoter or the combination of these two promoters (double promoters). (C) Two copies of CrPV1A were expressed under the control of the polyUbiqutin or GAPDH promoters. Sequences are given in Supplementary File 1 (Schematic diagram created with BioRender.com).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7658464/v1/167977c9df4f5516ed378969.jpeg"},{"id":95523116,"identity":"e1407189-21c2-4c8e-a315-fdbde04cdb7a","added_by":"auto","created_at":"2025-11-10 09:39:15","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":143979,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLarval hatch rate after embryonic injections. \u003c/strong\u003eGrey filled columns indicate percentage hatch rate after injecting the VSR constructs for transgenesis. Grid lined columns indicate percentage hatched rate after injecting the same VSR constructs along with dsRNA targeting the mRNA of VSR-EGFP. Compared to buffer injection and the VSR construct driven only by the pUbp promoter, the embryonic hatch rate of bicistronic VSR constructs or VSR constructs driven by the GAPDH promoter was reduced . The embryonic lethality rate was rescued when these constructs were co-injected along with VSR-EGFP dsRNA for most constructs. Based on these and other results, we assume that strong VSR expression may have adverse effects on embryogenesis. However, the embryonic lethality was not rescued when the presumably strongest construct (bicistronic VSR construct driven by double promoter) was co-injected along with dsRNA. See numbers in Supplementary Table S1.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7658464/v1/35f75802a239a4e84fa71591.jpeg"},{"id":95523128,"identity":"98a19a94-df48-4b8a-ba9a-9eca58eae329","added_by":"auto","created_at":"2025-11-10 09:39:16","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":503818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConcepts and results on the rescue of sterility, maternal or zygotic functions after maternal RNAi.\u003c/strong\u003e (A) Schematic diagram depicting sterility observed after injecting some genes into wildtype females (left) and the rescue of sterility expected in VSR lines (right). Only in the latter case, sufficient embryos are produced to analyse the RNAi effect in the offspring. (B) Scheme depicting the genotypes of injected females, the males used for mating and the offspring for sterility and maternal gene function rescue. The VSR allele (+) in the female rescues sterility (or maternal gene function) while 50% of the offspring carry no allele (-/-) and should therefore lack zygotic RNAi rescue, i.e. they should display the full RNAi phenotype. (C) Eggs from 16 surviving females were collected for 9 days after dsRNA injection. Compared to wildtype (dark filled column) our α-tubulin driven VSR line strongly rescued the sterility effect of \u003cem\u003eTc-axin\u003c/em\u003e RNAi (dsRNA concentration 250ng/µL) and \u003cem\u003eTc-dpp \u003c/em\u003eRNAi\u003cem\u003e (\u003c/em\u003edsRNA concentration\u003cem\u003e \u003c/em\u003e500ng/µL\u003cem\u003e)\u003c/em\u003e (grey filled column). See numbers in Supplementary Table S2. (D) Scheme for studying maternal function of a gene while rescuing its zygotic function. The maternal gene function is knocked down by RNAi (no VSR expression; -/-) while the zygotic gene function is rescued by VSR expression derived from the father (Schematic diagram in A from BioRender.com, modified).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7658464/v1/5f16122655100005c11710a4.jpeg"},{"id":95523131,"identity":"491102de-34cd-4bff-a468-2b59fcefd5c3","added_by":"auto","created_at":"2025-11-10 09:39:16","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":288799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCuticle phenotypes of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTc-axin\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e RNAi after heat-shock treatment. \u003c/strong\u003e(A) First-instar wildtype larva with the posterior urogomphi marked by an arrow and the legs by an arrowhead. (B) Weak phenotype with head defects, (C) Phenotype with large anterior deletions including the leg bearing thoracic segements. (D,E) Cuticle crumbs without clear AP polarity. Star marks the gut. (E) Double abdomen phenotype with two posterior ends (white arrows) and a median thorax with appendages (arrowheads). The rescue of the EE phenotype to cuticles was observed in two independent replicates, the double abdomen phenotype occurred in one replicate, where it had a prevalence of 25%. Pictures are not to scale. See numbers in Supplementary Table S4.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7658464/v1/0cf69032cda0fb7cf3d146e2.jpeg"},{"id":95523107,"identity":"14488a47-b39a-4804-918e-555393b46fc8","added_by":"auto","created_at":"2025-11-10 09:39:12","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":141335,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibiting the zygotic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTc-axin\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e RNAi effect in the offspring. \u003c/strong\u003eThis figure illustrates the phenotypic changes resulting from the inhibition of \u003cem\u003eTc-axin\u003c/em\u003e RNAi effect in the offspring after heat-shock treatment (numbers added from two independent replicates – see Supplementary Table S4 for detailed numbers). The first column represents the phenotypic outcomes without heat-shock treatment, while the second column represents the phenotypic outcomes after heat-shock treatment. The most penetrant phenotypes without heat-shock treatment are empty eggs (i.e. no cuticle at all) and cuticle crumbs (see Fig. 4E). These very strong phenotypes decreased upon heat-shock treatment where many more cuticles were formed. These showed a range of phenotypes from head defects to double abdomen phenotypes. In both experiments, the dsRNA was injected into females of the α-tubulin VSR-line in order to overcome the sterility while the heat-shock induced VSR expression was based on an allele crossed in from the male.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7658464/v1/e77e61db01d8c75003b331fd.jpeg"},{"id":100615641,"identity":"7d0d77c9-15ab-4fa7-a52d-5f922517a2cb","added_by":"auto","created_at":"2026-01-19 17:35:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3896693,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7658464/v1/2107195c-03a1-4600-8807-802b9ef5650d.pdf"},{"id":95523121,"identity":"18cbbea5-953c-4b57-8081-dc61e03fe9ac","added_by":"auto","created_at":"2025-11-10 09:39:15","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":20532,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4Heatshocknew.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7658464/v1/759181391094b5f53ec8054c.xlsx"},{"id":95523126,"identity":"848a2cd0-18b8-4cf6-8180-4135cf7a5f4a","added_by":"auto","created_at":"2025-11-10 09:39:16","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23374,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3Zygoticnew.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7658464/v1/778a0d80b515451de05d01b5.xlsx"},{"id":95523119,"identity":"f86cff98-7c8a-4e8f-ae94-ae19a8ef69d9","added_by":"auto","created_at":"2025-11-10 09:39:15","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":22766,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1VSRTransgenesisandLethalitynew.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7658464/v1/1126ffbd92c4b924482efcfb.xlsx"},{"id":95523117,"identity":"bb7211bb-38cc-4af7-bdfd-67e6fdf13e5d","added_by":"auto","created_at":"2025-11-10 09:39:15","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18039,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2VSRSterilitynew.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7658464/v1/97376242db6ee0ad304d00d6.xlsx"},{"id":95523133,"identity":"7842491c-91df-499a-bcd2-d883073520ce","added_by":"auto","created_at":"2025-11-10 09:39:17","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1109467,"visible":true,"origin":"","legend":"","description":"","filename":"FileS1SupplementarySequenceDatarevision.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7658464/v1/10f10db4c9ea1900f15d661e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A tool for repression of RNAi overcomes sterility and separates maternal from zygotic RNAi in Tribolium castaneum","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, RNA interference (RNAi) has become a highly effective and widely used reverse genetic tool to elucidate gene function in diverse emerging arthropod model organisms (1-6). It has opened the possibility to ask for the evolution of gene function across clades and to study processes that are not represented in the main model system for arthropods, the fruit fly \u003cem\u003eDrosophila melanogaster\u003c/em\u003e. The red flour beetle \u003cem\u003eTribolium castaneum\u003c/em\u003e has emerged as a powerful new insect model organism for the study of gene function based on a number of established techniques: It has a strong and systemic RNAi response (7-9) and the availability of a very advanced genetic toolkit for gene function analysis i.e enhancer trapping, insertional mutagenesis, heat-inducible gene expression, the GAL4/UAS system and genome editing (10-15). Recently, the \u003cem\u003eiBeetle\u003c/em\u003e genome-wide RNAi screen revealed divergent gene sets required for a number of homologous developmental processes in \u003cem\u003eTribolium\u003c/em\u003e vs, \u003cem\u003eDrosophila.\u003c/em\u003e For example, a surprisingly high number of genes are essential for muscle development in \u003cem\u003eTribolium\u003c/em\u003e but seem not required in \u003cem\u003eDrosophila\u0026nbsp;\u003c/em\u003e(16-18). One notable aspect that makes \u003cem\u003eTribolium\u003c/em\u003e a powerful model system is the environmental nature of its RNAi response, i.e. the ability of \u003cem\u003eTribolium\u003c/em\u003e cells to take up dsRNA from the surrounding fluid. This has for instance allowed to inject dsRNA into the hemolymph of larvae and observe the effect in all cells (19) to inject mothers to elicit knockdown in the offspring (7) and to add dsRNA to primary cell cultures for knocking-down genes (20).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, this strength can at the same time limit gene functional analyses in several ways. For example, dsRNA injection of developmental genes that are in addition required for oogenesis leads to sterility of the injected females. As consequence, the knockdown effect is difficult to be studied in the offspring as in the case of \u003cem\u003eTc-decapentaplegic\u003c/em\u003e (\u003cem\u003eTc-dpp)\u0026nbsp;\u003c/em\u003e(21) or a reduced dsRNA concentration has to be used, which may lead to an incomplete knock-down (22). Moreover, in case of pleiotropic genes, it would be advantageous to locally or temporally knock-down the gene in order to study the effect in one process independently from other functions. Finally, upon parental RNAi of genes that have both maternal and zygotic gene function such as \u003cem\u003eTc-axin\u003c/em\u003e, both functions are suppressed making it difficult to separate maternal versus zygotic gene function. To overcome these restrictions, there is a need for a tool to restrict RNAi to a certain time, region or life-stage\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eDrosophila\u003c/em\u003e, localized RNAi has been achieved by Gal4 driven hairpin-loops that induce RNAi only in the expressing cells (23-24). However, in the smaller communities of the less established model systems without central stock keeping facilities it is unrealistic to maintain genome-wide transgenic line collections. Hence, a tool combining dsRNA injection with spatio-temporal inhibition of RNAi would combine the ease of RNAi with the maintenance of a limited number of transgenic lines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe RNAi pathway is a natural antiviral defence mechanism in plants and animals (25-31). As a counter defence strategy, many viruses have evolved proteins that act as viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. These VSR proteins evolved independently and use different mechanism of action to impede the RNAi machinery (32-40). In previous work, we had tested a number of known VSRs for their functionality in the red flour beetle (41). We had found that heat-shock induced expression of a VSR from \u003cem\u003eCricket Paralysis virus\u003c/em\u003e (CrPV1A) was effectively blocking an ongoing RNAi response (41). Here, we utilized this VSR which interacts with the endonuclease Argonaute 2 (Ago2) and blocks its cleavage activity (40). Specifically, we tested the strategy to transgenically express that VSR in certain life stages in order to render them resistant to RNAi. As consequence, only the non-expressing cells would be affected by injection-induced RNAi.\u003c/p\u003e\n\u003cp\u003eWe wanted to apply the tool to answer a long-standing question on the function of the Wnt pathway in anterior-posterior (AP) axis formation. It had been suggested that maternal \u003cem\u003eTc-axin\u003c/em\u003e mRNA localized at the anterior pole represses Wnt signalling, which is required for the specification of the anterior pole. However, the respective experiments had to be performed with reduced amounts of \u003cem\u003eTc-axin\u003c/em\u003e dsRNA in order to avoid sterility (22). Further, Wnt signalling has many additional functions during segmentation and other embryonic processes. Therefore, the observed phenotype was a mixture of the maternal axis-formation phenotype superimposed by the zygotic segmentation defects. Indeed, stronger knock-down of \u003cem\u003eTc-axin\u003c/em\u003e resulted in embryos with heavily affected morphology or even without cuticles (called the empty egg phenotype) (22). A clear signature of a complete axis-formation phenotype is the double abdomen phenotype where the head is replaced by a second abdomen. Such double abdomen phenotypes had not been observed in our initial study (22) while in a subsequent study by others, such phenotypes had been described (42). Hence, it appeared possible that repression of Wnt signalling alone would be sufficient for specifying anterior development, but it had remained unclear, why this phenotype had not been observed in our previous experiments.\u003c/p\u003e\n\u003cp\u003eOur results showed that a ubiquitously driven VSR (CrPV1A) indeed reduced RNAi activity. We demonstrate the use of the new tool to overcome the issue of sterility after parental RNAi of \u003cem\u003eTc-axin\u003c/em\u003e and \u003cem\u003eTc-dpp\u003c/em\u003e. Unfortunately, this tool alone was not efficient enough enable distinguishing zygotic from maternal gene function. However, when combined with heat-shock mediated repression in embryos, we were able to do so. Several observations suggest that strong ubiquitous CrPV1A expression may interfere with viability. As consequence, only transgenic lines with a moderate VSR expression survive such that the RNAi blocking effect may not be strong enough for separating maternal from zygotic functions. Our new tool can now be used to study the embryonic function of genes, the parental RNAi of which leads to sterility. Adjustments of the design may overcome the current limitations.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e1. Generation of VSR plasmid constructs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeveral VSR plasmid constructs were generated as follows: The VSR template CrPV1A, enhanced green fluorescent protein (EGFP) fragment and different ubiquitous promoters were amplified and subsequently cloned into \u003cem\u003epSLfa1080fa\u0026nbsp;\u003c/em\u003eshuttle vector (43). The final VSR constructs as shown in Fig. 1, were cloned into \u003cem\u003epiggyBac\u003c/em\u003e vector by using standard restriction enzymes provided by Thermo Fisher Scientific or Gibson assembly (NEBuilder HiFi DNA Assembly Master Mix). For bicistronic VSR lines, codon optimization of the CrPV1A fragment was performed using the Integrated DNA Technologies (IDT, https://eu.idtdna.com) codon optimization tool and subsequently ordered as a gBlock fragment from IDT. The CrPV1A plasmid was kindly provided by Ronald Van Rij (Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands). The polyubiquitin and \u0026alpha;-tubulin promoters have been described previously (44). Please find the sequences of the final constructs in supplementary file 1; the plasmids are available from the last author upon request and on Addgene (numbers: xxx)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Transgenesis, RNAi and heat-shock induction of VSR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEmbryonic injections and transgenesis in \u003cem\u003evermillion white\u003c/em\u003e beetles (vw) were performed as described previously (11,13,45). In order to reduce lethality by VSR expression during transgenesis, dsRNA targeting the VSR at a final concentration of 1000ng/\u0026mu;l was added to the injection mix. The maternal RNAi into pupae and cuticle preparation was performed as described previously (16,18).\u003c/p\u003e\n\u003cp\u003eAll tests were performed in two biological replicates. See supplementary tables for number of analysed embryos.\u003c/p\u003e\n\u003cp\u003eThe heat-shock activation of VSR was performed similar to (41) by separating three hours old embryos (kept at 32\u0026deg;C) from flour, putting them into a glass vial with flat bottom and placing the vial for 10 minutes into a water bath at 48\u0026deg;C. The first heat-shock was after 10 hours and second one after 13 hours of egg collection (i.e. 10-13 and 13-16 hours of development at 32\u0026deg;C, respectively). The heat-shocked embryos were transferred back to a plastic vial and continued development further at 32\u0026deg;C for the cuticle preparation. The overnight egg collection from the same injected animals was used as negative control for cuticle analysis (embryos with no heat-shock treatment). Images were adjusted for brightness and contrast using the Levels-function in Photoshop CS5 (Adobe). \u003cem\u003eTc-axin\u003c/em\u003e: TC006314; Tc-dpp: TC008466; Tc-prd: TC015804; Tc-eve: TC009469; \u003cem\u003eTc-hh\u003c/em\u003e: TC032269. The lines are kept in the lab of the last author and are freely available upon request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNote that we had attempted to localize VSR expression to a stripe along the midline by knocking-in the construct into the vnd locus. While we found no localized expression nor effect, we noted that unexpectedly, this line performed well in some of our tests. Therefore, we kept the line and documented the results in Supplementary File 1 and the supplementary tables.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eGenerating viral suppressor of RNAi (VSR) transgenic lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo overcome sterility and to separate maternal versus zygotic RNAi, we first wanted to generate transgenic lines where a VSR was expressed ubiquitously. We reasoned that when injecting into such a line, the female sterility induced by RNAi targeting certain genes might be overcome. This would allow researchers to \u0026nbsp;study the embryonic RNAi phenotype of such genes in the offspring. Conversely, if RNAi is applied to females and the VSR is contributed by the males, the zygotic expression could theoretically become rescued while the maternal RNAi effect would have full effect. We used the CrPV1A from the \u003cem\u003eCricket Paralysis virus\u003c/em\u003e because this VSR had been identified as the most efficient of several tested VSRs in our previous work (41).\u003c/p\u003e\n\u003cp\u003eWe generated a series of transgenic lines by \u003cem\u003epiggyBac\u003c/em\u003e-mediated transgenesis that expressed the VSR CrPV1A. Expression was controlled by different ubiquitous promoters (\u0026alpha;-tubulin, polyUbiquitin and GAPDH) in order to identify the most suitable one (Fig. 1A-B). To further enhance the expression of the RNAi inhibitor, we have in addition generated bicistronic VSR lines where double amounts of VSR were expected to be produced per translation initiation event. In these constructs, two copies of the VSR are separated by the 2A-peptide were cloned into one coding sequence (Fig. 1C). The 2A sequence leads to ribosomal skipping such that two separate proteins are translated from a single mRNA transcript (46).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn total, we generated 12 transgenic lines from different VSR constructs and most of these transgenic lines were subsequently tested for RNAi rescue effect (see Table S1 for details). However, some of the transgenic VSR lines driven by strong ubiquitous promoters turned out to be unhealthy and infertile and could therefore not be included in the RNAi rescue effect test (Table S1). Interestingly, high embryonic lethality was observed during microinjection of the constructs for transgenesis of the bicistronic VSR constructs and the VSR constructs driven by putatively strong ubiquitous promoter such as GAPDH (Fig. 2). Based on these observations, we considered that high amounts of VSR might be lethal. In line with this assumption, the embryonic lethality rate after injection for transgenesis was largely rescued when we co-injected these plasmids along with dsRNA against the VSR-EGFP transcript (Fig. 2). See below for discussion on our assumption that strong VSR expression could interfere with viability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSterility rescue after maternal RNAi in VSRs lines\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSome genes are required both, for embryonic development and for oogenesis. Hence, parental RNAi targeting such genes induces sterility in the injected mother such that no embryos can be collected to study the gene\u0026rsquo;s embryonic function (Fig. 3A). Hence, in order to obtain knock-down embryos for analysis, the RNAi effect needs to be dampened in the injected females (Fig. 3A). To test whether the ubiquitous VSR lines helped to overcome this sterility problem, we injected homozygous female pupae from several of our VSR lines with dsRNA targeting two genes that had shown to induce sterility in previous works (Fig. 3B). In previous studies, the sterility induced by injection of dsRNA targeting \u003cem\u003eTc-dpp\u003c/em\u003e and \u003cem\u003eTc-axin\u0026nbsp;\u003c/em\u003einto females had prohibited the application of parental RNAi to these genes or had required strongly reducing the amounts of injected dsRNA (21,22). For our test, we first determined and then used the minimal amount of dsRNA that induced a complete sterile phenotype for \u003cem\u003eTc-dpp\u003c/em\u003e and \u003cem\u003eTc-axin\u003c/em\u003e (500ng/\u0026micro;L and 250ng/\u0026micro;L, respectively). The injected females were mated to wildtype males and the number of embryos was scored and compared to wildtype females injected with the same dsRNAs (Figure 3B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong all the VSR lines that were tested, only the \u0026alpha;-tubulin driven VSR line showed a strong rescue of sterility (Supplementary Table S2). In wildtype, after \u003cem\u003eTc-axin\u003c/em\u003e and \u003cem\u003eTc-dpp\u003c/em\u003e dsRNA injection, the number of embryos produced from 16 living female animals within nine days post injections were 4 and 5 respectively. However, upon \u003cem\u003eTc-axin\u003c/em\u003e and \u003cem\u003eTc-dpp\u003c/em\u003e RNAi in the \u0026alpha;-tubulin driven VSR line, the number of eggs significantly rose to 101 and 169, respectively (Fig. 3C and Supplementary Table S2). The results confirmed that the technique could indeed be used to overcome the sterility induced by some genes upon parental RNAi. This opens the possibility to study the embryonic function of such genes by parental RNAi.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSeparating maternal and zygotic gene function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA number of patterning genes have both, maternal and zygotic functions. For instance, during oogenesis the mother localizes a signalling molecule in the oocyte, which acts during the first hours of embryogenesis. The later zygotic expression of the same molecules can play a different role during advanced embryogenesis. However, in parental RNAi, knock-down reduces both maternal and zygotic gene functions. As consequence, it remains unclear what part of the phenotype-if any-is maternal and what part is zygotic. Similar to the sterility effect above, a strong rescue of RNAi in the injected females of VSR lines would rescue the maternal effect of the targeted gene (i.e. VSR expression blocks RNAi in the mother). When using a heterozygous mother and a wildtype male, 50% of the offspring carry no VSR. These offspring animals should show the full zygotic phenotype of the RNAi treatment (Fig. 3B). Conversely, inhibiting the RNAi in the offspring by mating injected wildtype females with homozygous VSR males could theoretically allow for maternal knock-down but zygotic rescue of a given gene (Fig. 3D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor this rescue of the zygotic function, the VSR needs to be effective even in the heterozygous state (Fig. 3D). To identify such a line, we tested the level of RNAi rescue effect in all our VSR lines in homozygous offspring animals. We injected different zygotic genes such as \u003cem\u003eTc-paired (Tc-prd), even-skipped (Tc-eve)\u003c/em\u003e and \u003cem\u003eTc-hedgehog (Tc-hh).\u0026nbsp;\u003c/em\u003eThese genes are known to have only zygotic functions and do not lead to sterility in the injected mother. Indeed, the strength of the phenotypic penetrance decreased dramatically for most of the genes tested in these VSR lines confirming that the constructs were working in principle (Supplementary Table S3). Unfortunately, no VSR line showed complete phenotypic rescue (i.e. wildtype L1 offspring) even though the VSR was homozygous in these embryos . Only \u003cem\u003eTc-hh\u0026nbsp;\u003c/em\u003eRNAi seemed to be rescued to some degree (Supplementary Table S3). These data show that the idea works in principle but that the available VSR lines have not sufficient VSR activity for that application.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs alternative approach to zygotic gene rescue, we sought to separate the maternal and zygotic gene function by combining the ubiquitous VSR lines with a heat-shock driven VSR line where the RNAi inhibitor CrPV1A is expressed under the control of the endogenous \u003cem\u003eTribolium\u003c/em\u003e heat-shock promoter. The heat-shock driven VSR system had previously been shown to very effectively rescue an ongoing RNAi response for several genes (41).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMaternal \u003cem\u003eTc-axin\u003c/em\u003e function is sufficient for axis formation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe current model for axis formation in \u003cem\u003eTribolium\u003c/em\u003e poses that maternal function of\u003cem\u003e\u0026nbsp;Tc-axin\u0026nbsp;\u003c/em\u003eis involved in anterior-posterior axis formation in beetles. Its mRNA of this Wnt-signalling inhibitor is localized at the anterior pole during oogenesis representing a maternal aspect of \u003cem\u003eTc-axin\u003c/em\u003e function. The localized presence of Tc-Axin blocks the Wnt-pathway at the anterior thereby contributing to defining the anterior pole (22). Later it was found that \u003cem\u003eTc-gcl\u003c/em\u003e acts upstream in that process being required for anterior localization of \u003cem\u003eTc-axin\u003c/em\u003e. RNAi targeting \u003cem\u003eTc-gcl\u003c/em\u003e led to absence of \u003cem\u003eTc-axin\u003c/em\u003e mRNA at the anterior pole and to double abdomen phenotypes (47). Based on these results it was hypothesized that Wnt signalling was responsible for AP axis formation. However, it had remained unclear, whether Wnt signalling was sufficient for that process because \u003cem\u003eTc-axin\u003c/em\u003e RNAi alone had not resulted in double abdomen phenotypes in Fu et al. (22) while it had done so with low frequency in another study (42). Hence, it had remained disputed, whether strong knock-down of \u003cem\u003eTc-axin\u003c/em\u003e alone would be sufficient for generating double abdomen phenotypes or whether additional components were required. Importantly, all respective experiments were complicated by the sterility induced by \u003cem\u003eTc-axin\u003c/em\u003e RNAi, which required lowering the dsRNA dose such that a full knock-down would not be expected. In addition, \u003cem\u003eTc-axin\u003c/em\u003e has later zygotic functions, which lead to strong developmental defects prohibiting the formation of L1 cuticles such that double abdomen cuticles would not be visible. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe wanted to use the combination of our tools to overcome these technical issues. First, we aimed at reducing the sterility effect in the injected mother in order to be able to increase the amount of injected dsRNA, i.e. increase RNAi strength. Second, we wanted to rescue the zygotic \u003cem\u003eTc-axin\u003c/em\u003e phenotype in the offspring in order to allow for normal zygotic development and formation of a cuticle.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWith the aim of inhibiting maternal \u003cem\u003eTc-axin\u003c/em\u003e function, we injected \u003cem\u003eTc-axin\u003c/em\u003e dsRNA (concentration: 250ng/\u0026mu;l) in our \u0026alpha;-tubulin driven VSR female pupae (to reduce the sterility effect of \u003cem\u003eTc-axin\u003c/em\u003e RNAi) and crossed them with males carrying the previously described heat-shock VSR constructs (41). Three hour egg collection were heat-shocked two times for 10 minutes at 48\u0026deg;C. The first heat-shock was after 10 hours and second after 13 hours of egg collection (i.e. 10-13 and 13-16 hours of development at 32\u0026deg;C, respectively). Upon regular parental \u003cem\u003eTc-axin\u003c/em\u003e RNAi, most offspring produced empty egg phenotypes (i.e. no cuticle formed; EE) or the cuticle crumbs phenotype (i.e. cuticle remnants with no recognizable morphology) (see Fig. 4 for \u0026nbsp;the range of observed phenotypes). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, in contrast to non-heat-shocked embryos (control), \u003cem\u003eTc-axin\u0026nbsp;\u003c/em\u003eRNAi in heat-shocked embryos resulted in a reduction of the portion of empty egg phenotypes and an increased number of cuticles in two independent experiments. The L1 cuticles were classified according to the phenotypes. In both experiments, head phenotypes emerged (27 embryos; 27.55%) and in one of the experiments, several cuticles showed a double abdomen phenotypes (7 embryos; 7.14% of both experiments, 25% of the single experiment with that effect; see Supplementary Table S4) (Fig. 4F). Also, the number of wildtype cuticles increased significantly in heat-shock embryos (20 out of 98 embryos; 20.4%) compared to control without heat-shock (15 out of 512 embryos; 2.92% ) (Fig. 5 and Supplementary Table S4).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eToxic effect of VSR on viability and development\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to establish a system for spatio-temporal control of RNAi in \u003cem\u003eTribolium,\u003c/em\u003e we planned to generate strong ubiquitous expression of the VSR to reach complete phenotypic rescue after parental RNAi. Unfortunately, our data indicate that high level of VSR expression likely had a significant effect on \u003cem\u003eTribolium\u003c/em\u003e viability and development. First, for many constructs that were expected to exhibit strong expression\u0026mdash;such as those containing strong promoters or employing a bicistronic design\u0026mdash;we either failed to obtain transgenic lines, or the resulting lines were unhealthy or exhibited reduced fertility. (Table S1). Second, only VSR lines driven by \u0026alpha;-tubulin, and polyUbiqutin promoters could be made homozygous. Third, a comparably high embryonic lethality was observed during microinjection of bicistronic VSR constructs or VSR constructs driven by strong promoters such as GAPDH. In line with this hypothesis, the lethality rate was rescued when we co-injected the transgenesis plasmids along with dsRNA against the VSR-EGFP transcript. Fifth, In previous study we validated the toxic effect of VSR expression in \u003cem\u003eTribolium\u0026nbsp;\u003c/em\u003eby a heat-shock experiment where the RNAi inhibitor CrPV1A was expressed under the control of endogenous Tribolium heat-shock promoter. Here, upon heat-shock activation of VSR at different stages of development (adult, pupae and larvae), the survival rate was reduced significantly in heat-shock-VSR line animals compared to wildtype (41).\u003c/p\u003e\n\u003cp\u003eIndeed, other studies provided evidence that CrPV1A interferes with the Ago-2-dependent miRNA silencing and miRNA regulatory network in Drosophila as well (48). Taken together, these results suggest that a high expression level of CrPV1A has a substantial effect on fitness and viability during \u003cem\u003eTribolium\u003c/em\u003e development. We suggest two possible workarounds for this issue: First, heat-shock mediated expression has been shown to work efficiently (this work and 41) despite increased lethality. Second, expression mediated by the binary Gal4/UAS system established in that model (12) might be able to provide strong localized expression without affecting the processes sensitive to VSR misexpression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpanding the scope of RNAi by using viral RNAi inhibitor\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRecently our lab has utilized VSR from \u003cem\u003eCricket Paralysis virus\u003c/em\u003e (CrPV1A) to temporally restrict RNAi during segmentation process in \u003cem\u003eTribolium\u003c/em\u003e (41). In the present study, we adopted that tool to overcome the sterility problem and showed that \u0026alpha;-tubulin driven ubiquitous VSR line has a notable rescue effect on sterility after \u003cem\u003eTc-axin\u003c/em\u003e and \u003cem\u003eTc-dpp\u003c/em\u003e RNAi. The sterility rescue of VSR line highlights the potential use of this newly developed tool on overcoming the limitations of studying certain pleiotropic genes by parental RNAi if their knock-down affects fertility as well. An alternative to overcome the sterility problem is embryonic injection of dsRNA. However, this is a much more tedious procedure compared to parental RNAi, the embryos may be damaged by the injection and it is very difficult to collect RNAi embryos for WMISH or similar analyses, as each embryo has to be dissected out of the eggshell. With our new tool, standard methods for downstream analyses can be applied.\u003c/p\u003e\n\u003cp\u003eSome drawbacks of the tool should be noted, too. First, we cannot exclude that VSR protein is carried over from the mother to the oocytes such that the offspring has a dampened RNAi response despite the lack of the construct in their genomes (Fig. 3B). Second, the assumed lethality induced by strong ubiquitous expression of the VSR prohibits the establishment of lines, which are strong enough to realize the approaches outlined in Fig. 3B and D. Interestingly strong VSR expression by two heat-shocks does apparently not block development of embryos (this work and 41). Hence, it could be that the observed problems to generate and maintain transgenic VSR lines stems from the chronic VSR expression or the expression in certain tissues at certain times (e.g. germ cells, ovary, brain etc.). If this is true, a sufficiently high VSR level might be reached when restricting expression to certain tissues or life stages e.g. by driving them by a binary expression system, which is available in \u003cem\u003eTribolium\u003c/em\u003e (12). \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRepression of Wnt signalling is sufficient for defining the anterior pole\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe applied our new tool to answer a question, which had remained open because technical difficulties prohibited testing it. It had been shown before that the Wnt-inhibitor \u003cem\u003eTc-axin\u003c/em\u003e was involved in the specification of the anterior-posterior axis. Specifically, maternally contributed \u003cem\u003eTc-axin\u003c/em\u003e mRNA was localized to the anterior pole of the oocyte thereby inhibiting Wnt signalling at the anterior. In \u003cem\u003eTc-axin\u003c/em\u003e RNAi embryos, the anterior fates were reduced in a dose dependent manner. This led to the idea that suppression of Wnt signalling was required for anterior development similar to findings in vertebrates and other animals (22). In line with this hypothesis, RNAi targeting \u003cem\u003eTc-germ-cell less\u003c/em\u003e (\u003cem\u003eTc-gcl\u003c/em\u003e) abolished anterior localized \u003cem\u003eTc-axin\u003c/em\u003e mRNA and led to full double abdomen phenotype, which indicated a complete loss of anterior fates (47). It was suggested that \u003cem\u003eTc-gcl\u003c/em\u003e acts via \u003cem\u003eTc-axin\u003c/em\u003e to suppress the Wnt pathway in order to define the anterior pole. However, the hypothesis that \u003cem\u003eTc-gcl\u003c/em\u003e acted exclusively via the \u003cem\u003eTc-axin\u003c/em\u003e RNA remained challenged by the discrepancy of the two phenotypes: in \u003cem\u003eTc-gcl\u003c/em\u003e RNAi, a double abdomen phenotype develops while in \u003cem\u003eTc-axin\u003c/em\u003e RNAi, such a phenotype had not been observed in those studies (22, 47). Conversely, another study reported double abdomen cuticles after \u003cem\u003eTc-axin\u003c/em\u003e RNAi supporting the idea that indeed, Wnt-repression is the sole determinant of the anterior pole (42). The differences in phenotypes observed in these two studies have remained unexplained. The use of lower concentrations in the study by Pr\u0026uuml;hs et al. (42) might have contributed while strain-specific effects (49) are unlikely because the divergence was observed even when using the same strain (San Bernardino).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTwo explanations could explain the discrepancy of phenotypes of \u003cem\u003eTc-gcl\u003c/em\u003e and \u003cem\u003eTc-axin\u003c/em\u003e RNAi embryos in our previous studies. Either, another component regulated by \u003cem\u003eTc-gcl\u003c/em\u003e is needed to produce the full phenotype. Or \u003cem\u003eTc-gcl\u003c/em\u003e acts exclusively via \u003cem\u003eTc-axin\u003c/em\u003e but this could not be seen in our \u003cem\u003eTc-axin\u003c/em\u003e RNAi experiments because reduced concentrations had to be used due to sterility of \u003cem\u003eTc-axin\u003c/em\u003e dsRNA injected mothers. Another issue with \u003cem\u003eTc-axin\u003c/em\u003e RNAi is that the Wnt pathway has many functions during development, all of which are affected by standard RNAi experiments. As consequence, the resulting RNAi embryos do not form recognizable morphological structures, making phenotype analysis challenging. Moreover, they usually stop development before secreting cuticle.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWith our system, we were now able to distinguish between those alternative explanations. First, we could increase the concentration of \u003cem\u003eTc-axin\u003c/em\u003e dsRNA by injecting into females of the \u0026alpha;-tubulin VSR line, where sterility was rescued to some degree. Second, we rescued the later Wnt functions by blocking RNAi in early embryos with heat-shock mediated activation of VSR expression. This allowed us to observe cuticular morphology of embryos.\u003c/p\u003e\n\u003cp\u003eIndeed, the strong empty egg and cuticle crumbs phenotypes were converted to a large degree to embryos with recognizable cuticle morphology. We observed anterior deletions (as had been shown before) but we also saw double abdomen cuticle phenotypes (Fig. 4). The occurrence of double abdomen phenotypes strongly supports the hypothesis that indeed, blocking the Wnt pathway by maternal \u003cem\u003eTc-axin\u003c/em\u003e is sufficient to define the anterior pole of the \u003cem\u003eTribolium\u003c/em\u003e embryo (22, 42, 47). \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eDFG BU1443/14-1ad\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.S.H: Data analysis, visualization of the data, VSR plasmids design and cloning, transgenesis and writing the original draft; A.K.M and J.U.: VSR plasmid design and transgenesis; G.B.: Conceptualization, funding acquisition, supervision and writing the original draft.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Elke K\u0026uuml;ster for help with the beetles and Claudia Hinners for help with molecular experiments.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe transgenic lines are available from G.B.; the plasmids are distributed by AddGene\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eFire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. \u003cstrong\u003ePotent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans\u003c/strong\u003e. 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PMID: 23324472; PMCID: PMC3574008.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"developmental-biology-advances","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"evod","sideBox":"Learn more about [EvoDevo](http://evodevojournal.biomedcentral.com/)","snPcode":"13227","submissionUrl":"https://submission.nature.com/new-submission/13227/3","title":"Developmental Biology Advances","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"RNAi, Viral suppressors of RNAi (VSRs), transgenic tool, Tribolium castaneum","lastPublishedDoi":"10.21203/rs.3.rs-7658464/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7658464/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRNA interference (RNAi) is a natural antiviral defense mechanism in plants and animals. As a counter defense strategy, most viruses have evolved viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. Here, we utilized transgenic misexpression of a VSR from \u003cem\u003eCricket Paralysis virus\u003c/em\u003e (CrPV1A) to dampen RNAi in a temporal and life-stage specific way in order to overcome limitations of knocking down pleiotropic genes by the strong systemic RNAi response in the red flour beetle \u003cem\u003eTribolium castaneum\u003c/em\u003e. We found that ubiquitously driven VSR rescued the sterility of the females injected with \u003cem\u003eTc-axin\u003c/em\u003e or \u003cem\u003eTc-decapentaplegic\u003c/em\u003e double-stranded RNA, where sterility had previously hampered analysis. By combining this tool with a heat-shock driven VSR, we were able to separate maternal from zygotic function for the Wnt pathway inhibitor \u003cem\u003eTc-axin\u003c/em\u003e. Thereby, we could provide evidence that maternal Wnt signalling alone is responsible for axis formation in \u003cem\u003eTribolium\u003c/em\u003e. Our tool opens new experimental possibilities such as studying genes by parental RNAi, which would normally lead to sterility and separating maternal from zygotic gene functions. Further improvements are required to allow for studying zygotic gene function while rescuing maternal functions and for spatially restricting the RNAi effect.\u003c/p\u003e","manuscriptTitle":"A tool for repression of RNAi overcomes sterility and separates maternal from zygotic RNAi in Tribolium castaneum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-10 09:38:42","doi":"10.21203/rs.3.rs-7658464/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-26T01:20:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-25T22:14:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-22T11:18:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"254046682901067155012150429978152159476","date":"2025-10-31T18:54:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2454425997162130577602678356002617962","date":"2025-10-29T16:51:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-29T06:03:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-20T13:34:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-20T13:30:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"EvoDevo","date":"2025-09-19T11:52:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"developmental-biology-advances","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"evod","sideBox":"Learn more about [EvoDevo](http://evodevojournal.biomedcentral.com/)","snPcode":"13227","submissionUrl":"https://submission.nature.com/new-submission/13227/3","title":"Developmental Biology Advances","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b70ed966-898a-4e23-9487-376ef1f97bd9","owner":[],"postedDate":"November 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T17:02:05+00:00","versionOfRecord":{"articleIdentity":"rs-7658464","link":"https://doi.org/10.1186/s13227-025-00258-2","journal":{"identity":"developmental-biology-advances","isVorOnly":false,"title":"Developmental Biology Advances"},"publishedOn":"2026-01-16 16:28:30","publishedOnDateReadable":"January 16th, 2026"},"versionCreatedAt":"2025-11-10 09:38:42","video":"","vorDoi":"10.1186/s13227-025-00258-2","vorDoiUrl":"https://doi.org/10.1186/s13227-025-00258-2","workflowStages":[]},"version":"v1","identity":"rs-7658464","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7658464","identity":"rs-7658464","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}