A gentle urea-based hybridisation chain reaction (HCR) protocol for use in hymenopteran ovaries

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Dearden This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8159582/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In situ hybridisation is essential for visualising gene expression, yet many insect tissues are sensitive to standard protocols, especially those using formamide. Here, we present a modified hybridisation chain reaction (HCR) protocol that replaces formamide with urea to improve tissue morphology while maintaining strong and specific signal detection. Using ovaries from three hymenopteran species— Nasonia vitripennis , Vespula vulgaris , and Polistes dominula —we demonstrate that this urea-based protocol enables robust detection of conserved germline markers, vasa , nanos , and oskar . This methodological advance provides a reliable and accessible alternative for in situ hybridisation in delicate tissues and expands the toolkit for gene expression studies in non-model insects. In-situ hybridisation hymenoptera ovaries imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hymenoptera may be the most speciose and diverse order of animals on earth (Forbes et al., 2018 ). Hymenoptera also have a diverse range of social structures, many of which regulate reproduction (Fletcher & Ross, 1985 ). To understand the remarkable biology of Hymenoptera, particularly how it is influenced by the social structure of some hymenopteran species, we require robust tools to examine gene expression and function. In situ hybridisation is a key technology for visualising gene expression in cells, tissues, and embryos (Young et al., 2020 ). In recent years, fluorescent in situ hybridisation has become a robust and effective way to examine the expression of RNA from multiple genes at once in a tissue (Choi et al., 2016 ). In-situ hybridisation involves using an RNA probe complementary to the mRNA of the target gene of interest and hybridising that probe to the mRNA through complementary base pairing,(Carter et al., 2010 ). This often requires the incubation of tissues at high temperatures, which can be damaging, and so compounds such as formamide are added to reduce hybridisation temperatures to reasonable levels. Formamide, while lowering the hybridisation temperature, is toxic and can also be damaging to tissue (Sinigaglia et al., 2018 ). While examining gene expression in social wasps, we found some whose ovaries were very sensitive to the formamide concentrations and temperatures that are often used for in situ hybridisation. Recent work in other taxa (Cullen et al., 2023 ; Sinigaglia et al., 2018 ) has shown that replacing formamide with urea in hybridisation buffers can improve tissue integrity while maintaining probe sensitivity. Building on these insights, we optimised a urea-based HCR protocol for hymenopteran ovaries, allowing for extended probe incubation, improved tissue structure and better images of gene expression in ovaries. Materials and methods Finding genes Orthologs of key ovary-expressed genes were identified by a reciprocal BLASTP search (Altschul et al., 1990 ) from sequences in either Drosophila or Nasonia and then analysed with Bayesian phylogenetic techniques using MrBayes 3.2.7a (Ronquist et al., 2012 ). The mixed model setting was used to find the most appropriate model, and then trees were visualised in FigTree v1.4.4 (Rambaut, 2018 ) (Supplemental Fig. 1). If the top hits in Polistes dominula and Vespula vulgaris resolved into a clade of other proteins and not the outgroup, it was considered the ortholog in that species, from which the longest mRNA isoform was used for probe generation for Hybridisation Chain Reaction (HCR). Probes for each gene were generated by Molecular Instruments Ltd, using their HCR1.0 technology. The models for each protein were as follows: nanos Jones (Jones et al., 1992 ), vasa and oskar WAG (Whelan & Goldman, 2001 ). Fixation of ovaries Nasonia vitripennis were placed on ice to immobilise them before dissection. The head was removed, and the ovaries dissected into 1 × PBS (Phosphate-buffered Saline) under a dissecting microscope. Ovaries were placed into 900 µL of PTx (PBS + 0.1% Triton X-100) on ice, to which 100 µL of 37% formaldehyde was added. They were then gently agitated on a rocking incubator at room temperature for 15 min. They were then rinsed 3 times with PTx to remove formaldehyde. Dissections for Polistes and Vespula were carried out as for Nasonia , but animals were immobilised by being placed at 4°C. Hybridisation Chain Reaction in situ hybridisation To address poor morphology associated with formamide-based hybridisation protocols in Hymenopteran tissue (Fig. 2 ), we replaced the formamide in hybridisation and wash buffers with urea. Fresh ovaries were permeabilised for 2 hours in PTx on a rocking incubator at room temperature. PTx was removed and replaced with 500 µL of a modified 30% probe hybridisation buffer (2.4 M Urea, 5 × sodium chloride sodium citrate (SSC), 9 mM citric acid (pH 6.0), 0.1% Tween 20, 50 µg/mL heparin, 1 × Denhardt's solution, 10% dextran sulphate), for 30 minutes at 37°C. The hybridisation buffer was removed and replaced with 100 µL of fresh hybridisation buffer, to which probes were added. All probes were used at a concentration of 40 nM, except that of vasa from Vespula vulgaris , which was used at 200 nm after optimisation experiments with this probe. Probes were left to incubate for 3 days and were then washed with 500 µL of a modified probe wash buffer consisting of (2.4 M Urea, 5 × SSC, 9 mM citric acid (pH 6.0), 0.1% Tween, 50 µg/mL heparin) 4 times for 15 minutes each at 37°C. Probes were then washed 3 times for 5 minutes each with 500 µL of 5 SSCT (5X SSC, 0.1% Tween 20) at room temperature. Samples were incubated in 500 µL of amplification buffer (5X SSCT with 10% dextran sulphate) for 30 minutes at room temperature, while the hairpins were snap-cooled by heating to 90°C for 90 seconds, then cooling to room temperature in the dark for 30 minutes. The amplification buffer was replaced with 100 µL of fresh amplification buffer to which the snap-cooled hairpins were added and left to incubate in the dark at room temperature for 2 days. Samples were then washed with 500 µL of 5 × SSCT twice for 5 minutes each at room temperature, then twice for 30 minutes each at room temperature and then a final 5 minutes at room temperature on a rocking incubator. The supernatant was then removed and replaced with ProLong™ Gold Antifade Mountant with DNA Stain DAPI (Thermo Fisher Scientific catalogue P36931) and placed at 4°C in the dark until imaging. Imaging The localisation of RNA and protein from in situ hybridisation and immunohistochemistry experiments was observed using one of two confocal microscopes, an upright FV1000 or an inverted FV3000, both manufactured by Olympus. All confocal images were processed in FIJI (v1.5.4) (Schindelin et al., 2012 ). Results Although structural differences were observed among the three hymenopteran species examined in this study, the ovaries of Nasonia vitripennis , Vespula vulgaris , and Polistes dominula all conform to the polytrophic meroistic type, as previously described in insects(Büning, 1994 ). Each ovary is composed of multiple ovarioles, which contain a terminal filament, germarium, and vitellarium arranged along the anterior–posterior axis. Representative images of ovariole organisation for each species are shown in Fig. 1 . Attempts to carry out HCR in-situ hybridisation using urea-based methods in Polistes dominula ovaries led to poor gene expression patterns and poor morphology (Fig. 2 ). As noted by others, Urea-based hybridisation is a gentler approach (Sinigaglia et al., 2018 ), and so we assessed the efficacy of urea-based in situ hybridisation in hymenopteran ovaries. Gene Targets. To determine the effects of urea-based hybridisation, we first targeted a range of ovary-expressed genes in the parasitic wasp Nasonia vitripennis . Nasonia ovary structure has been studied for some time (King & Richards, 1969 ; Lynch & Desplan, 2010 ), and the expression patterns of three key ovary patterning genes have been examined. These are nanos , vasa and oskar . The vasa gene encodes a DEAD-Box RNA helicase (Doren et al., 1998 ), and is essential for germ cell formation and the assembly of pole plasm (Hay et al., 1988 ; Lasko & Ashburner, 1988 ; Liang et al., 1994 ; Schüpbach & Wieschaus, 1991 ). vasa is expressed in the testes of Drosophila (Papathanos et al., 2009 ). The vasa gene is a conserved germline marker in Apis mellifera and Nasonia vitripennis (Cullen et al., 2023 ; Ferree et al., 2019 ; Lynch et al., 2011 ; Lynch & Desplan, 2010 ). The nanos gene is a maternal effect gene in Drosophila melanogaster that is deposited into the oocyte and then accumulates in the pole plasm (Rangan et al., 2009 ; Tazuke et al., 2002 ). The nos gene is necessary for posterior patterning in Drosophila embryos (Struhl, 1989 ). While nos mRNA is found throughout the embryo, Nos protein is found within the pole plasm, forming a protein gradient (Bergsten & Gavis, 1999 ). The nos gene is well conserved within Metazoa (Mochizuki et al., 2000 ) with conserved functions in the germline (Tsuda et al., 2003 ) and maternal localisation in many species (Chang et al., 2006 ; Curtis et al., 1995 ; Dearden, 2006 ; Lall et al., 2003 ; Lynch & Desplan, 2010 ). The oskar gene encodes a protein required for the formation of pole plasm in Drosophila (Ephrussi et al., 1991 ; Kim-Ha et al., 1991 ; Lehmann & Nüsslein-Volhard, 1986 ; Rongo et al., 1995 ). The oskar gene also has neuronal roles in Drosophila melanogaster and crickets (Ewen-Campen et al., 2012 ; Kulkarni et al., 2023 ; Xu et al., 2013 ), along with somatic expression in other insects (Blondel et al., 2021 ). Ectopic expression of oskar is sufficient to induce germline cells in the embryo of Drosophila melanogaster (Ephrussi et al., 1991 ; Kim-Ha et al., 1991 ). In addition to the germline expression in Drosophila, oskar expression is also found in the adult neural stem cells of Gryllus bimaculatus (Kulkarni et al., 2023 ). Assessing known gene expression patterns using urea-based in-situ hybridisation. As the expression of these genes has been reported in Nasonia (Lynch et al., 2011 ; Lynch & Desplan, 2010 ), we tested our urea-based in-situ hybridisation technique on Nasonia ovaries (Fig. 3 ) Using our urea-based method to examine the expression of our three key patterning genes indicated that the expression we detect is very similar to that reported by others Lynch et al., 2011 ; Quan et al., 2019 ). In the vitellarium, vasa expression was restricted to nurse cells, while oskar and nanos were both expressed in the most proximal nurse cells and strongly localised to the posterior of the oocyte within the oosome. The similarity to previously published expression patterns for these genes gave us confidence to use urea-based techniques to improve in-situ hybridisation in wasp species where formamide damaged the ovary structure. Gene expression in Vespula vulgaris ovaries. Both oskar and nanos exhibit strong, germline-specific expression in Nasonia vitripennis , making them ideal candidates for exploring patterns of oocyte development across Hymenoptera. To determine whether similar spatial expression profiles occur in other species, we applied our urea-based HCR protocol to Vespula vulgaris . Vespula vulgaris is an invasive eusocial species of wasp that has become a considerable pest species in Aotearoa- New Zealand. Future efforts to control and eradicate this pest will require a knowledge of reproduction in this species, hence the need for robust gene expression analysis tools. Using our urea-based in-situ hybridisation method, we examined the expression of oskar , nanos and vasa in Vespula vulgaris ovaries. In Vespula vulgaris , oskar expression appears in developing oocytes in the germarium (Fig. 8). Notably, there is no detectable enlargement of oocytes before the onset of oskar expression, suggesting that the initiation of oskar transcription coincides with, or potentially marks, the early stages of oocyte differentiation and growth. Nanos’ expression is more diffuse, with no clear indication that it is expressed in the germline in the germarium. The expression of oskar and Vasa in the vitellarium of Vespula vulgaris closely resembled their expression patterns in Nasonia vitripennis Fig. 3 . Within the vitellarium, vasa RNA was distributed throughout the cytoplasm of the nurse cells, with particularly strong accumulation surrounding nurse-cell nuclei. In contrast, oskar transcripts were concentrated in the cytoplasm of the developing oocyte and the immediately adjacent nurse cells at the posterior end of the ovariole. Tissue morphology was well preserved under urea-based conditions, allowing clear delineation of nurse cells and oocyte boundaries for these comparisons. Gene expression in Polistes dominula ovaries. Among the germline markers examined, oskar and nanos showed strong and specific expression in Nasonia vitripennis , with similar patterns of oskar expression observed in Vespula vulgaris . To assess whether this pattern of germline gene expression is conserved across a broader range of Hymenoptera, we applied our urea-based in situ hybridisation protocol to Polistes dominula . In the germarium of Polistes dominula ovaries, oskar RNA formed a discrete domain of strong signal within the cytoplasm of developing oocytes (Fig. 5 ). Rather than a broad distribution, fluorescence was concentrated in a compact region near the posterior of each oocyte. This pattern suggests that oskar transcripts begin to localise early in oogenesis, potentially marking the establishment of oocyte polarity. In contrast, vasa expression is present in the cells directly anterior to oskar expression, but these cells lacked the enlarged morphology typical of mature nurse cells, suggesting that in Polistes dominula , vasa expression occurs before nurse cell enlargement. In later germaria stages of Polistes dominula , oskar expression was again detected early, showing the same restricted pattern as in Fig. 5 , with signal concentrated in the developing oocyte (Fig. 6 A-F). In these ovarioles, nanos transcripts also became detectable. RNA from nanos appeared slightly later than vasa , and rather than being confined to a few cells adjacent to the oskar -positive oocyte, it was weakly expressed in many cells located anterior to the oocyte. This diffuse nanos signal occurred before the surrounding cells exhibited the enlarged morphology characteristic of mature nurse cells, indicating that nanos activation precedes obvious nurse cell differentiation. The timing of nanos expression relative to vasa and oskar suggests a progressive sequence of germline gene activation, beginning with vasa in early germline precursors, followed by nanos during the onset of oocyte specification. RNA from oskar is present in early oocytes marked with an asterisk, and within the vitellarium in the oocyte and most proximal nurse cells, while early nanos expression is localised to the nurse cells in the germarium, and restricted to the more distal nurse cells of the vitellarium. Interestingly, oskar RNA is more localised to the posterior of the oocyte, which could be indicative of a pole plasm. The expression patterns observed in Polistes dominula share several features with those documented in Nasonia vitripennis and Vespula vulgaris . In all three species, oskar transcripts were detected within developing oocytes. In P. dominula , oskar localisation within mature oocytes was broader and less sharply confined than in Nasonia or Vespula . In each species, oskar was also expressed in the nurse cells most proximal to the oocyte. Vasa expression in P. dominula resembled that of Vespula , with a cytoplasmic signal present in germarial cells and nurse cells of the vitellarium. RNA from vasa appeared localised to nurse cells positioned more anteriorly than those expressing oskar . Expression of nanos began later than vasa in P. dominula , appearing weakly in anterior germarial cells before nurse cell enlargement. In the vitellarium, nanos RNA was localised to the more anterior nurse cells, similar to vasa . In contrast, in Nasonia , nanos expression was strongest in the oocyte and most proximal nurse cells, resembling oskar expression rather than vasa . Overall, vasa , nanos , and oskar displayed broadly conserved expression patterns across the three hymenopteran species, with notable species-specific differences in transcript localisation. Discussion This study demonstrates that a urea-based in situ hybridisation chain reaction (HCR) protocol provides a robust, high-resolution method for visualising germline gene expression in hymenopteran tissues. By replacing formamide with urea, we achieved improved tissue integrity and extended probe incubation times without compromising signal quality. This makes the method particularly well-suited for non-model insect systems such as Hymenoptera. Although Diptera and Hymenoptera possess polytrophic meroistic ovaries (Büning, 1994 ), their divergent life histories, such as the extremely high reproductive output of honeybee queens (Avni et al., 2014 ), suggest that aspects of oogenesis may differ. These differences underscore the importance of developing effective tools for studying reproductive biology in non-model systems. Our optimised urea-based HCR protocol enabled clear visualisation of spatial expression patterns for conserved germline markers in three hymenopteran species. These expression patterns highlight the utility of molecular imaging approaches for studying oogenesis in taxa where functional studies remain limited. The expression patterns of several germline markers have been previously shown in Nasonia vitripennis using traditional versions of in situ hybridisation techniques (Lynch et al., 2011 ; Quan et al., 2019 ). Comparative analysis of germline gene expression across Nasonia vitripennis , Vespula vulgaris , and Polistes dominula revealed both conserved and divergent spatial patterns that shed light on the evolution of oogenesis in Hymenoptera. Across all three species, oskar transcripts were consistently detected within developing oocytes. However, the degree of oskar localisation differed among species; N. vitripennis exhibited a tightly confined posterior domain, V. vulgaris showed a more diffuse posterior signal, and P. dominula displayed more cytoplasmic distribution within the oocyte. These differences suggest that while oskar expression is conserved, the structure or organisation of the pole plasm may vary among hymenopteran lineages. In contrast, nanos showed greater variability in expression between species. In N. vitripennis , nanos was co-localised with oskar in the oocyte and the proximal nurse cells, consistent with its established role in posterior patterning. In P. dominula , however, nanos was excluded from the oocyte and instead expressed in the anterior nurse cells of the vitellarium, more closely resembling vasa than oskar . This shift from oocyte to nurse cell localisation may represent an evolutionary divergence in maternal mRNA localisation mechanisms or post-transcriptional regulation of nanos during oogenesis. Expression of vasa was broadly conserved across species, with a strong cytoplasmic signal in nurse cells of the germarium and vitellarium. Early expression of both vasa and oskar in the germarium across species supports their roles as germline markers. These findings suggest that while germline genes are broadly conserved across Hymenoptera, localisation of their transcripts has diversified. The consistent oocyte localisation of oskar , contrasted with the variable nanos localisation, highlights evolutionary variation in the mechanisms that control maternal RNA localisation and retention within the oocyte. With our urea-based HCR approach, we were able to preserve tissue integrity and detect these fine-scale expression differences with high spatial resolution. This demonstrates the value of urea-based hybridisation methods for studying fragile tissues in non-model organisms and provides a foundation for future comparative studies on germline development and the evolution of oogenesis in Hymenoptera. Declarations Ethics, Consent to Participate, and Consent to Publish declarations not applicable. Funding declaration This work was funded by a grant from New Zealand’s Biological Heritage National Science Challenge to P.K.D Author Contribution P.K.D. Conceived and gathered funding for this work. JG carried out the work and the imaging, prepared the figures and drafted the manuscript. PKD and JG both revised and reviewed the manuscript. Data Availability The image datasets generated during the current study are available at Zenodo, (DOI to be provided) References Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410. https://doi.org/10.1016/S0022-2836(05)80360-2 Avni D, Hendriksma HP, Dag A, Uni Z, Shafir S (2014) Nutritional aspects of honey bee-collected pollen and constraints on colony development in the eastern Mediterranean. 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J Neuroscience: Official J Soc Neurosci 33(37):14791–14800. https://doi.org/10.1523/JNEUROSCI.5864-12.2013 Young AP, Jackson DJ, Wyeth RC (2020) A technical review and guide to RNA fluorescence in situ hybridization. PeerJ 8:e8806. https://doi.org/10.7717/peerj.8806 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8159582","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":555321625,"identity":"2f426ceb-3331-412a-8fbe-291824033b8c","order_by":0,"name":"Joshua Gilligan","email":"","orcid":"","institution":"University of Otago","correspondingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"","lastName":"Gilligan","suffix":""},{"id":555321626,"identity":"f610fa8e-88f3-4130-86a2-4ea482af0b5b","order_by":1,"name":"Peter K. 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13:47:05","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113267,"visible":true,"origin":"","legend":"","description":"","filename":"d8be105a0e06471a8b6f41d342f20aed1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8159582/v1/d9ec6d962f906564b8943744.xml"},{"id":97533957,"identity":"76f66620-df1b-4399-8ae7-74e4e6192da8","added_by":"auto","created_at":"2025-12-05 13:47:05","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126155,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8159582/v1/0c6f4ae17cc67aeb8e0c7597.html"},{"id":97533943,"identity":"491dcc7d-087b-408d-a316-ecc811fa2d26","added_by":"auto","created_at":"2025-12-05 13:47:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":500097,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum intensity projection of a z-stack of ovarioles from \u003cem\u003eNasonia vitripennis\u003c/em\u003e, \u003cem\u003eVespula vulgaris\u003c/em\u003e and \u003cem\u003ePolistes dominula\u003c/em\u003e stained with DAPI. The anterior part of the ovariole, the terminal filament, is marked with an asterisk. Posterior to the terminal filament resides the anterior part of the germarium and then the posterior germarium. Posterior to the germarium is the vitellarium, where nurse cells and oocytes alternate, and then in the posterior vitellarium, oocytes begin to enlarge. All scale bars denote 100 µm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8159582/v1/4d942b27477be771e3f77582.png"},{"id":97671454,"identity":"b735df40-3543-49e3-b6a0-070bee21ee80","added_by":"auto","created_at":"2025-12-08 09:32:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":886585,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum intensity Z-stack HCR in-situ hybridisation using formamide, imaging of RNA from conserved germline markers, a \u003cem\u003ePolistes dominula\u003c/em\u003e ovariole. A) DAPI, B) \u003cem\u003eoskar\u003c/em\u003e, C) DAPI\u003cem\u003e oskar\u003c/em\u003e, D) \u003cem\u003evasa\u003c/em\u003e, E) DAPI \u003cem\u003evasa\u003c/em\u003e, F) DAPI \u003cem\u003eoskar vasa\u003c/em\u003e. Asterisks denote evident tissue damage, appearing shrunken near oocytes. Scale bar denotes 200 µm.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8159582/v1/6d352580f6a8444a02c1e53e.png"},{"id":97672486,"identity":"96963441-6f87-432b-aa2c-f067bbd9278d","added_by":"auto","created_at":"2025-12-08 09:38:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":425716,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum intensity Z-stack HCR in-situ hybridisation imaging of RNA from conserved germline markers in the early vitellarium of a single \u003cem\u003eNasonia vitripennis\u003c/em\u003e ovariole. A) DAPI, B) \u003cem\u003evasa\u003c/em\u003e, C) \u003cem\u003enanos\u003c/em\u003e, D) \u003cem\u003eoskar\u003c/em\u003e. Asterisks mark the expression of oskar and nanos RNA in the nurse cells adjacent to the oocyte. Scale bar denotes 100 µm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8159582/v1/72c607debb20ae857b48ec2f.png"},{"id":97533947,"identity":"14eacac4-40c5-4328-84dc-2eac9a2e3851","added_by":"auto","created_at":"2025-12-05 13:47:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3695525,"visible":true,"origin":"","legend":"\u003cp\u003eHCR in-situ hybridisation imaging of RNA for conserved germline markers in the germarium of the ovary of a \u003cem\u003eVespula vulgaris\u003c/em\u003e gyne. Germarium of a \u003cem\u003eVespula vulgaris\u003c/em\u003e gyne. A) DAPI, B) \u003cem\u003eoskar\u003c/em\u003e, C) \u003cem\u003enanos\u003c/em\u003e, D) DAPI\u003cem\u003e oskar\u003c/em\u003e. Scale bar denotes 100 µm. (F-H) Vitellarium of a \u003cem\u003eVespula vulgaris\u003c/em\u003e gyne., A) DAPI, B) \u003cem\u003eoskar\u003c/em\u003e, C) \u003cem\u003evasa\u003c/em\u003e, D) Merge. Scale bar denotes 200 µm.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8159582/v1/f9096ca4d746729f4671dd47.png"},{"id":97671608,"identity":"481117aa-cbc7-41d9-adb5-11c71556cce3","added_by":"auto","created_at":"2025-12-08 09:32:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":818844,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum intensity z-stack HCR in-situ hybridisation imaging of RNA from conserved germline markers in the germarium of a \u003cem\u003ePolistes dominula ovariole\u003c/em\u003e. A-C early germarium, D-I mid germarium. A) DAPI B) \u003cem\u003evasa\u003c/em\u003e, C) DAPI \u003cem\u003evasa\u003c/em\u003e D) DAPI, E) \u003cem\u003eoskar\u003c/em\u003e, F) DAPI \u003cem\u003eoskar\u003c/em\u003e G) \u003cem\u003evasa\u003c/em\u003e, H) DAPI \u003cem\u003evasa\u003c/em\u003e, I) DAPI \u003cem\u003eoskar\u003c/em\u003e \u003cem\u003evasa\u003c/em\u003e. Scale bar denotes 100 µm.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8159582/v1/a23c7b9fc6cc4685670f3d26.png"},{"id":97533946,"identity":"4d3dd92e-a497-40cb-a29f-cab577a907ab","added_by":"auto","created_at":"2025-12-05 13:47:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2299673,"visible":true,"origin":"","legend":"\u003cp\u003eHCR in-situ hybridisation imaging of RNA from conserved germline markers in a \u003cem\u003ePolistes dominula ovariole\u003c/em\u003e. A-F maximum intensity z-stack of the germarium. A) DAPI B) \u003cem\u003eoskar\u003c/em\u003e, C) DAPI \u003cem\u003eoskar\u003c/em\u003e D) \u003cem\u003enanos\u003c/em\u003e, E) \u003cem\u003enanos\u003c/em\u003e \u003cem\u003eoskar\u003c/em\u003e, F) merge. Scale bar denotes 100 µm. G-L single slice of the vitellarium. G) DAPI, H) \u003cem\u003eoskar\u003c/em\u003e, I) DAPI \u003cem\u003eoskar\u003c/em\u003e, J) \u003cem\u003enanos\u003c/em\u003e, K) \u003cem\u003enanos oskar\u003c/em\u003e, L) merge. Scale bar denotes 100 µm.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8159582/v1/35baa3f913a1d6cc9c9a567b.png"},{"id":102415542,"identity":"205e08a3-c1e4-4b9b-b270-c1ba64482bdc","added_by":"auto","created_at":"2026-02-11 12:44:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8428419,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8159582/v1/75117024-4911-41f3-a376-731d1e2d3036.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A gentle urea-based hybridisation chain reaction (HCR) protocol for use in hymenopteran ovaries","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHymenoptera may be the most speciose and diverse order of animals on earth (Forbes et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Hymenoptera also have a diverse range of social structures, many of which regulate reproduction (Fletcher \u0026amp; Ross, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). To understand the remarkable biology of Hymenoptera, particularly how it is influenced by the social structure of some hymenopteran species, we require robust tools to examine gene expression and function.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn situ\u003c/em\u003e hybridisation is a key technology for visualising gene expression in cells, tissues, and embryos (Young et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In recent years, fluorescent \u003cem\u003ein situ\u003c/em\u003e hybridisation has become a robust and effective way to examine the expression of RNA from multiple genes at once in a tissue (Choi et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eIn-situ\u003c/em\u003e hybridisation involves using an RNA probe complementary to the mRNA of the target gene of interest and hybridising that probe to the mRNA through complementary base pairing,(Carter et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This often requires the incubation of tissues at high temperatures, which can be damaging, and so compounds such as formamide are added to reduce hybridisation temperatures to reasonable levels. Formamide, while lowering the hybridisation temperature, is toxic and can also be damaging to tissue (Sinigaglia et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). While examining gene expression in social wasps, we found some whose ovaries were very sensitive to the formamide concentrations and temperatures that are often used for in situ hybridisation. Recent work in other taxa (Cullen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sinigaglia et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) has shown that replacing formamide with urea in hybridisation buffers can improve tissue integrity while maintaining probe sensitivity. Building on these insights, we optimised a urea-based HCR protocol for hymenopteran ovaries, allowing for extended probe incubation, improved tissue structure and better images of gene expression in ovaries.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eFinding genes\u003c/h2\u003e\u003cp\u003eOrthologs of key ovary-expressed genes were identified by a reciprocal BLASTP search (Altschul et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) from sequences in either \u003cem\u003eDrosophila\u003c/em\u003e or \u003cem\u003eNasonia\u003c/em\u003e and then analysed with Bayesian phylogenetic techniques using MrBayes 3.2.7a (Ronquist et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The mixed model setting was used to find the most appropriate model, and then trees were visualised in FigTree v1.4.4 (Rambaut, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) (Supplemental Fig.\u0026nbsp;1). If the top hits in \u003cem\u003ePolistes dominula\u003c/em\u003e and \u003cem\u003eVespula vulgaris\u003c/em\u003e resolved into a clade of other proteins and not the outgroup, it was considered the ortholog in that species, from which the longest mRNA isoform was used for probe generation for Hybridisation Chain Reaction (HCR). Probes for each gene were generated by Molecular Instruments Ltd, using their HCR1.0 technology. The models for each protein were as follows: \u003cem\u003enanos\u003c/em\u003e Jones (Jones et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), \u003cem\u003evasa\u003c/em\u003e and \u003cem\u003eoskar\u003c/em\u003e WAG (Whelan \u0026amp; Goldman, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eFixation of ovaries\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eNasonia vitripennis\u003c/em\u003e were placed on ice to immobilise them before dissection. The head was removed, and the ovaries dissected into 1 \u0026times; PBS (Phosphate-buffered Saline) under a dissecting microscope. Ovaries were placed into 900 \u0026micro;L of PTx (PBS\u0026thinsp;+\u0026thinsp;0.1% Triton X-100) on ice, to which 100 \u0026micro;L of 37% formaldehyde was added. They were then gently agitated on a rocking incubator at room temperature for 15 min. They were then rinsed 3 times with PTx to remove formaldehyde.\u003c/p\u003e\u003cp\u003eDissections for \u003cem\u003ePolistes\u003c/em\u003e and \u003cem\u003eVespula\u003c/em\u003e were carried out as for \u003cem\u003eNasonia\u003c/em\u003e, but animals were immobilised by being placed at 4\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eHybridisation Chain Reaction in situ hybridisation\u003c/h3\u003e\n\u003cp\u003eTo address poor morphology associated with formamide-based hybridisation protocols in Hymenopteran tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), we replaced the formamide in hybridisation and wash buffers with urea.\u003c/p\u003e\u003cp\u003eFresh ovaries were permeabilised for 2 hours in PTx on a rocking incubator at room temperature. PTx was removed and replaced with 500 \u0026micro;L of a modified 30% probe hybridisation buffer (2.4 M Urea, 5 \u0026times; sodium chloride sodium citrate (SSC), 9 mM citric acid (pH 6.0), 0.1% Tween 20, 50 \u0026micro;g/mL heparin, 1 \u0026times; Denhardt's solution, 10% dextran sulphate), for 30 minutes at 37\u0026deg;C.\u003c/p\u003e\u003cp\u003eThe hybridisation buffer was removed and replaced with 100 \u0026micro;L of fresh hybridisation buffer, to which probes were added. All probes were used at a concentration of 40 nM, except that of vasa from \u003cem\u003eVespula vulgaris\u003c/em\u003e, which was used at 200 nm after optimisation experiments with this probe. Probes were left to incubate for 3 days and were then washed with 500 \u0026micro;L of a modified probe wash buffer consisting of (2.4 M Urea, 5 \u0026times; SSC, 9 mM citric acid (pH 6.0), 0.1% Tween, 50 \u0026micro;g/mL heparin) 4 times for 15 minutes each at 37\u0026deg;C. Probes were then washed 3 times for 5 minutes each with 500 \u0026micro;L of 5 SSCT (5X SSC, 0.1% Tween 20) at room temperature. Samples were incubated in 500 \u0026micro;L of amplification buffer (5X SSCT with 10% dextran sulphate) for 30 minutes at room temperature, while the hairpins were snap-cooled by heating to 90\u0026deg;C for 90 seconds, then cooling to room temperature in the dark for 30 minutes.\u003c/p\u003e\u003cp\u003eThe amplification buffer was replaced with 100 \u0026micro;L of fresh amplification buffer to which the snap-cooled hairpins were added and left to incubate in the dark at room temperature for 2 days. Samples were then washed with 500 \u0026micro;L of 5 \u0026times; SSCT twice for 5 minutes each at room temperature, then twice for 30 minutes each at room temperature and then a final 5 minutes at room temperature on a rocking incubator. The supernatant was then removed and replaced with ProLong\u0026trade; Gold Antifade Mountant with DNA Stain DAPI (Thermo Fisher Scientific catalogue P36931) and placed at 4\u0026deg;C in the dark until imaging.\u003c/p\u003e\n\u003ch3\u003eImaging\u003c/h3\u003e\n\u003cp\u003eThe localisation of RNA and protein from \u003cem\u003ein situ\u003c/em\u003e hybridisation and immunohistochemistry experiments was observed using one of two confocal microscopes, an upright FV1000 or an inverted FV3000, both manufactured by Olympus. All confocal images were processed in FIJI (v1.5.4) (Schindelin et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAlthough structural differences were observed among the three hymenopteran species examined in this study, the ovaries of \u003cem\u003eNasonia vitripennis\u003c/em\u003e, \u003cem\u003eVespula vulgaris\u003c/em\u003e, and \u003cem\u003ePolistes dominula\u003c/em\u003e all conform to the polytrophic meroistic type, as previously described in insects(B\u0026uuml;ning, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Each ovary is composed of multiple ovarioles, which contain a terminal filament, germarium, and vitellarium arranged along the anterior\u0026ndash;posterior axis. Representative images of ovariole organisation for each species are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAttempts to carry out HCR \u003cem\u003ein-situ\u003c/em\u003e hybridisation using urea-based methods in \u003cem\u003ePolistes dominula\u003c/em\u003e ovaries led to poor gene expression patterns and poor morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAs noted by others, Urea-based hybridisation is a gentler approach (Sinigaglia et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and so we assessed the efficacy of urea-based in situ hybridisation in hymenopteran ovaries.\u003c/p\u003e\u003cp\u003e\u003cem\u003eGene Targets.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo determine the effects of urea-based hybridisation, we first targeted a range of ovary-expressed genes in the parasitic wasp \u003cem\u003eNasonia vitripennis\u003c/em\u003e. Nasonia ovary structure has been studied for some time (King \u0026amp; Richards, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Lynch \u0026amp; Desplan, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and the expression patterns of three key ovary patterning genes have been examined. These are \u003cem\u003enanos\u003c/em\u003e, \u003cem\u003evasa\u003c/em\u003e and \u003cem\u003eoskar\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003evasa\u003c/em\u003e gene encodes a DEAD-Box RNA helicase (Doren et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), and is essential for germ cell formation and the assembly of pole plasm (Hay et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Lasko \u0026amp; Ashburner, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Liang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Sch\u0026uuml;pbach \u0026amp; Wieschaus, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). \u003cem\u003evasa\u003c/em\u003e is expressed in the testes of \u003cem\u003eDrosophila\u003c/em\u003e (Papathanos et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The \u003cem\u003evasa\u003c/em\u003e gene is a conserved germline marker in \u003cem\u003eApis mellifera\u003c/em\u003e and \u003cem\u003eNasonia vitripennis\u003c/em\u003e (Cullen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Ferree et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lynch et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Lynch \u0026amp; Desplan, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003enanos\u003c/em\u003e gene is a maternal effect gene in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e that is deposited into the oocyte and then accumulates in the pole plasm (Rangan et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Tazuke et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The \u003cem\u003enos\u003c/em\u003e gene is necessary for posterior patterning in \u003cem\u003eDrosophila\u003c/em\u003e embryos (Struhl, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). While \u003cem\u003enos\u003c/em\u003e mRNA is found throughout the embryo, Nos protein is found within the pole plasm, forming a protein gradient (Bergsten \u0026amp; Gavis, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The \u003cem\u003enos\u003c/em\u003e gene is well conserved within Metazoa (Mochizuki et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) with conserved functions in the germline (Tsuda et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and maternal localisation in many species (Chang et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Curtis et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Dearden, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Lall et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Lynch \u0026amp; Desplan, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eoskar\u003c/em\u003e gene encodes a protein required for the formation of pole plasm in \u003cem\u003eDrosophila\u003c/em\u003e (Ephrussi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Kim-Ha et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Lehmann \u0026amp; N\u0026uuml;sslein-Volhard, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Rongo et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The \u003cem\u003eoskar\u003c/em\u003e gene also has neuronal roles in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e and crickets (Ewen-Campen et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kulkarni et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), along with somatic expression in other insects (Blondel et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ectopic expression of \u003cem\u003eoskar\u003c/em\u003e is sufficient to induce germline cells in the embryo of \u003cem\u003eDrosophila melanogaster\u003c/em\u003e (Ephrussi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Kim-Ha et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). In addition to the germline expression in \u003cem\u003eDrosophila, oskar\u003c/em\u003e expression is also found in the adult neural stem cells of \u003cem\u003eGryllus bimaculatus\u003c/em\u003e (Kulkarni et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eAssessing known gene expression patterns using urea-based in-situ hybridisation.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAs the expression of these genes has been reported in \u003cem\u003eNasonia\u003c/em\u003e (Lynch et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Lynch \u0026amp; Desplan, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), we tested our urea-based \u003cem\u003ein-situ\u003c/em\u003e hybridisation technique on \u003cem\u003eNasonia\u003c/em\u003e ovaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUsing our urea-based method to examine the expression of our three key patterning genes indicated that the expression we detect is very similar to that reported by others Lynch et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Quan et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the vitellarium, \u003cem\u003evasa\u003c/em\u003e expression was restricted to nurse cells, while \u003cem\u003eoskar\u003c/em\u003e and \u003cem\u003enanos\u003c/em\u003e were both expressed in the most proximal nurse cells and strongly localised to the posterior of the oocyte within the oosome. The similarity to previously published expression patterns for these genes gave us confidence to use urea-based techniques to improve in-situ hybridisation in wasp species where formamide damaged the ovary structure.\u003c/p\u003e\u003cp\u003e\u003cem\u003eGene expression in Vespula vulgaris ovaries.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eBoth \u003cem\u003eoskar\u003c/em\u003e and \u003cem\u003enanos\u003c/em\u003e exhibit strong, germline-specific expression in \u003cem\u003eNasonia vitripennis\u003c/em\u003e, making them ideal candidates for exploring patterns of oocyte development across Hymenoptera. To determine whether similar spatial expression profiles occur in other species, we applied our urea-based HCR protocol to \u003cem\u003eVespula vulgaris\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eVespula vulgaris\u003c/em\u003e is an invasive eusocial species of wasp that has become a considerable pest species in Aotearoa- New Zealand. Future efforts to control and eradicate this pest will require a knowledge of reproduction in this species, hence the need for robust gene expression analysis tools.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUsing our urea-based in-situ hybridisation method, we examined the expression of \u003cem\u003eoskar\u003c/em\u003e, \u003cem\u003enanos\u003c/em\u003e and \u003cem\u003evasa\u003c/em\u003e in \u003cem\u003eVespula vulgaris\u003c/em\u003e ovaries. In \u003cem\u003eVespula vulgaris\u003c/em\u003e, \u003cem\u003eoskar\u003c/em\u003e expression appears in developing oocytes in the germarium (Fig.\u0026nbsp;8). Notably, there is no detectable enlargement of oocytes before the onset of \u003cem\u003eoskar\u003c/em\u003e expression, suggesting that the initiation of \u003cem\u003eoskar\u003c/em\u003e transcription coincides with, or potentially marks, the early stages of oocyte differentiation and growth. Nanos\u0026rsquo; expression is more diffuse, with no clear indication that it is expressed in the germline in the germarium.\u003c/p\u003e\u003cp\u003eThe expression of \u003cem\u003eoskar\u003c/em\u003e and \u003cem\u003eVasa\u003c/em\u003e in the vitellarium of \u003cem\u003eVespula vulgaris\u003c/em\u003e closely resembled their expression patterns in \u003cem\u003eNasonia vitripennis\u003c/em\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Within the vitellarium, \u003cem\u003evasa\u003c/em\u003e RNA was distributed throughout the cytoplasm of the nurse cells, with particularly strong accumulation surrounding nurse-cell nuclei. In contrast, \u003cem\u003eoskar\u003c/em\u003e transcripts were concentrated in the cytoplasm of the developing oocyte and the immediately adjacent nurse cells at the posterior end of the ovariole. Tissue morphology was well preserved under urea-based conditions, allowing clear delineation of nurse cells and oocyte boundaries for these comparisons.\u003c/p\u003e\u003cp\u003e\u003cem\u003eGene expression in Polistes dominula ovaries.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAmong the germline markers examined, \u003cem\u003eoskar\u003c/em\u003e and \u003cem\u003enanos\u003c/em\u003e showed strong and specific expression in \u003cem\u003eNasonia vitripennis\u003c/em\u003e, with similar patterns of \u003cem\u003eoskar\u003c/em\u003e expression observed in \u003cem\u003eVespula vulgaris\u003c/em\u003e. To assess whether this pattern of germline gene expression is conserved across a broader range of Hymenoptera, we applied our urea-based \u003cem\u003ein situ\u003c/em\u003e hybridisation protocol to \u003cem\u003ePolistes dominula\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the germarium of \u003cem\u003ePolistes dominula\u003c/em\u003e ovaries, \u003cem\u003eoskar\u003c/em\u003e RNA formed a discrete domain of strong signal within the cytoplasm of developing oocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Rather than a broad distribution, fluorescence was concentrated in a compact region near the posterior of each oocyte. This pattern suggests that \u003cem\u003eoskar\u003c/em\u003e transcripts begin to localise early in oogenesis, potentially marking the establishment of oocyte polarity. In contrast, \u003cem\u003evasa\u003c/em\u003e expression is present in the cells directly anterior to \u003cem\u003eoskar\u003c/em\u003e expression, but these cells lacked the enlarged morphology typical of mature nurse cells, suggesting that in \u003cem\u003ePolistes dominula\u003c/em\u003e, \u003cem\u003evasa\u003c/em\u003e expression occurs before nurse cell enlargement.\u003c/p\u003e\u003cp\u003eIn later germaria stages of \u003cem\u003ePolistes dominula\u003c/em\u003e, \u003cem\u003eoskar\u003c/em\u003e expression was again detected early, showing the same restricted pattern as in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, with signal concentrated in the developing oocyte (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-F). In these ovarioles, \u003cem\u003enanos\u003c/em\u003e transcripts also became detectable. RNA from \u003cem\u003enanos\u003c/em\u003e appeared slightly later than \u003cem\u003evasa\u003c/em\u003e, and rather than being confined to a few cells adjacent to the \u003cem\u003eoskar\u003c/em\u003e-positive oocyte, it was weakly expressed in many cells located anterior to the oocyte. This diffuse \u003cem\u003enanos\u003c/em\u003e signal occurred before the surrounding cells exhibited the enlarged morphology characteristic of mature nurse cells, indicating that \u003cem\u003enanos\u003c/em\u003e activation precedes obvious nurse cell differentiation. The timing of \u003cem\u003enanos\u003c/em\u003e expression relative to \u003cem\u003evasa\u003c/em\u003e and \u003cem\u003eoskar\u003c/em\u003e suggests a progressive sequence of germline gene activation, beginning with \u003cem\u003evasa\u003c/em\u003e in early germline precursors, followed by \u003cem\u003enanos\u003c/em\u003e during the onset of oocyte specification.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRNA from \u003cem\u003eoskar\u003c/em\u003e is present in early oocytes marked with an asterisk, and within the vitellarium in the oocyte and most proximal nurse cells, while early \u003cem\u003enanos\u003c/em\u003e expression is localised to the nurse cells in the germarium, and restricted to the more distal nurse cells of the vitellarium. Interestingly, \u003cem\u003eoskar\u003c/em\u003e RNA is more localised to the posterior of the oocyte, which could be indicative of a pole plasm.\u003c/p\u003e\u003cp\u003eThe expression patterns observed in \u003cem\u003ePolistes dominula\u003c/em\u003e share several features with those documented in \u003cem\u003eNasonia vitripennis\u003c/em\u003e and \u003cem\u003eVespula vulgaris\u003c/em\u003e. In all three species, \u003cem\u003eoskar\u003c/em\u003e transcripts were detected within developing oocytes. In \u003cem\u003eP. dominula\u003c/em\u003e, \u003cem\u003eoskar\u003c/em\u003e localisation within mature oocytes was broader and less sharply confined than in \u003cem\u003eNasonia\u003c/em\u003e or \u003cem\u003eVespula\u003c/em\u003e. In each species, \u003cem\u003eoskar\u003c/em\u003e was also expressed in the nurse cells most proximal to the oocyte. \u003cem\u003eVasa\u003c/em\u003e expression in \u003cem\u003eP. dominula\u003c/em\u003e resembled that of \u003cem\u003eVespula\u003c/em\u003e, with a cytoplasmic signal present in germarial cells and nurse cells of the vitellarium. RNA from \u003cem\u003evasa\u003c/em\u003e appeared localised to nurse cells positioned more anteriorly than those expressing \u003cem\u003eoskar\u003c/em\u003e. Expression of \u003cem\u003enanos\u003c/em\u003e began later than \u003cem\u003evasa\u003c/em\u003e in \u003cem\u003eP. dominula\u003c/em\u003e, appearing weakly in anterior germarial cells before nurse cell enlargement. In the vitellarium, \u003cem\u003enanos\u003c/em\u003e RNA was localised to the more anterior nurse cells, similar to \u003cem\u003evasa\u003c/em\u003e. In contrast, in \u003cem\u003eNasonia\u003c/em\u003e, \u003cem\u003enanos\u003c/em\u003e expression was strongest in the oocyte and most proximal nurse cells, resembling \u003cem\u003eoskar\u003c/em\u003e expression rather than \u003cem\u003evasa\u003c/em\u003e. Overall, \u003cem\u003evasa\u003c/em\u003e, \u003cem\u003enanos\u003c/em\u003e, and \u003cem\u003eoskar\u003c/em\u003e displayed broadly conserved expression patterns across the three hymenopteran species, with notable species-specific differences in transcript localisation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates that a urea-based \u003cem\u003ein situ\u003c/em\u003e hybridisation chain reaction (HCR) protocol provides a robust, high-resolution method for visualising germline gene expression in hymenopteran tissues. By replacing formamide with urea, we achieved improved tissue integrity and extended probe incubation times without compromising signal quality. This makes the method particularly well-suited for non-model insect systems such as Hymenoptera. Although Diptera and Hymenoptera possess polytrophic meroistic ovaries (B\u0026uuml;ning, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), their divergent life histories, such as the extremely high reproductive output of honeybee queens (Avni et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), suggest that aspects of oogenesis may differ. These differences underscore the importance of developing effective tools for studying reproductive biology in non-model systems.\u003c/p\u003e\u003cp\u003eOur optimised urea-based HCR protocol enabled clear visualisation of spatial expression patterns for conserved germline markers in three hymenopteran species. These expression patterns highlight the utility of molecular imaging approaches for studying oogenesis in taxa where functional studies remain limited.\u003c/p\u003e\u003cp\u003eThe expression patterns of several germline markers have been previously shown in \u003cem\u003eNasonia vitripennis\u003c/em\u003e using traditional versions of \u003cem\u003ein situ\u003c/em\u003e hybridisation techniques (Lynch et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Quan et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Comparative analysis of germline gene expression across \u003cem\u003eNasonia vitripennis\u003c/em\u003e, \u003cem\u003eVespula vulgaris\u003c/em\u003e, and \u003cem\u003ePolistes dominula\u003c/em\u003e revealed both conserved and divergent spatial patterns that shed light on the evolution of oogenesis in Hymenoptera. Across all three species, \u003cem\u003eoskar\u003c/em\u003e transcripts were consistently detected within developing oocytes. However, the degree of \u003cem\u003eoskar\u003c/em\u003e localisation differed among species; \u003cem\u003eN. vitripennis\u003c/em\u003e exhibited a tightly confined posterior domain, \u003cem\u003eV. vulgaris\u003c/em\u003e showed a more diffuse posterior signal, and \u003cem\u003eP. dominula\u003c/em\u003e displayed more cytoplasmic distribution within the oocyte. These differences suggest that while \u003cem\u003eoskar\u003c/em\u003e expression is conserved, the structure or organisation of the pole plasm may vary among hymenopteran lineages. In contrast, \u003cem\u003enanos\u003c/em\u003e showed greater variability in expression between species. In \u003cem\u003eN. vitripennis\u003c/em\u003e, \u003cem\u003enanos\u003c/em\u003e was co-localised with \u003cem\u003eoskar\u003c/em\u003e in the oocyte and the proximal nurse cells, consistent with its established role in posterior patterning. In \u003cem\u003eP. dominula\u003c/em\u003e, however, \u003cem\u003enanos\u003c/em\u003e was excluded from the oocyte and instead expressed in the anterior nurse cells of the vitellarium, more closely resembling \u003cem\u003evasa\u003c/em\u003e than \u003cem\u003eoskar\u003c/em\u003e. This shift from oocyte to nurse cell localisation may represent an evolutionary divergence in maternal mRNA localisation mechanisms or post-transcriptional regulation of \u003cem\u003enanos\u003c/em\u003e during oogenesis.\u003c/p\u003e\u003cp\u003eExpression of \u003cem\u003evasa\u003c/em\u003e was broadly conserved across species, with a strong cytoplasmic signal in nurse cells of the germarium and vitellarium. Early expression of both \u003cem\u003evasa\u003c/em\u003e and \u003cem\u003eoskar\u003c/em\u003e in the germarium across species supports their roles as germline markers. These findings suggest that while germline genes are broadly conserved across Hymenoptera, localisation of their transcripts has diversified. The consistent oocyte localisation of \u003cem\u003eoskar\u003c/em\u003e, contrasted with the variable \u003cem\u003enanos\u003c/em\u003e localisation, highlights evolutionary variation in the mechanisms that control maternal RNA localisation and retention within the oocyte.\u003c/p\u003e\u003cp\u003eWith our urea-based HCR approach, we were able to preserve tissue integrity and detect these fine-scale expression differences with high spatial resolution. This demonstrates the value of urea-based hybridisation methods for studying fragile tissues in non-model organisms and provides a foundation for future comparative studies on germline development and the evolution of oogenesis in Hymenoptera.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics, Consent to Participate, and Consent to Publish declarations\u003c/strong\u003e\u003cp\u003enot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003cp\u003eThis work was funded by a grant from New Zealand\u0026rsquo;s Biological Heritage National Science Challenge to P.K.D\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP.K.D. Conceived and gathered funding for this work. JG carried out the work and the imaging, prepared the figures and drafted the manuscript. 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PeerJ 8:e8806. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7717/peerj.8806\u003c/span\u003e\u003cspan address=\"10.7717/peerj.8806\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"In-situ hybridisation, hymenoptera, ovaries, imaging","lastPublishedDoi":"10.21203/rs.3.rs-8159582/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8159582/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eIn situ\u003c/em\u003e hybridisation is essential for visualising gene expression, yet many insect tissues are sensitive to standard protocols, especially those using formamide. Here, we present a modified hybridisation chain reaction (HCR) protocol that replaces formamide with urea to improve tissue morphology while maintaining strong and specific signal detection. Using ovaries from three hymenopteran species\u0026mdash;\u003cem\u003eNasonia vitripennis\u003c/em\u003e, \u003cem\u003eVespula vulgaris\u003c/em\u003e, and \u003cem\u003ePolistes dominula\u003c/em\u003e\u0026mdash;we demonstrate that this urea-based protocol enables robust detection of conserved germline markers, \u003cem\u003evasa\u003c/em\u003e, \u003cem\u003enanos\u003c/em\u003e, and \u003cem\u003eoskar\u003c/em\u003e. This methodological advance provides a reliable and accessible alternative for \u003cem\u003ein situ\u003c/em\u003e hybridisation in delicate tissues and expands the toolkit for gene expression studies in non-model insects.\u003c/p\u003e","manuscriptTitle":"A gentle urea-based hybridisation chain reaction (HCR) protocol for use in hymenopteran ovaries","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-05 13:47:00","doi":"10.21203/rs.3.rs-8159582/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b663ca64-3f78-4892-aecb-9f08ca19db88","owner":[],"postedDate":"December 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-11T12:42:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-05 13:47:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8159582","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8159582","identity":"rs-8159582","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}