Parental social environment has no effect on offspring development in the dung beetle: A test of adult sex ratio effects

Parental social environment has no effect on offspring development in the dung beetle: A test of adult sex ratio effects | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 3 April 2026 V1 Latest version Share on Parental social environment has no effect on offspring development in the dung beetle: A test of adult sex ratio effects Authors : Lisheng Zhang 0009-0004-6463-3787 [email protected] , Sudeshna Chakraborty , Tamas Szekely , and Jan Komdeur Authors Info & Affiliations https://doi.org/10.22541/au.177521260.09198948/v1 176 views 97 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The adult sex ratio (ASR) is a key demographic parameter that shapes sexual selection and social interactions. However, whether parental experience of ASR variation influences offspring development across generations remains unclear. Such effects fall under the unifying framework of transgenerational plasticity (TGP), which encompasses two core, non-mutually exclusive non-genetic pathways: pre-zygotic germline-mediated epigenetic inheritance, and post-zygotic effects via behavioural adjustments to parental care. Using the dung beetle Onthophagus taurus, we conducted an egg-transplantation experiment to disentangle these two pathways of TGP. Parents were assigned to female-biased, unbiased, and male-biased treatments. We first quantified contest and courtship behaviours to confirm ASR-driven social effects. Eggs from naturally produced brood balls were then transplanted into standardized artificial brood balls to isolate effects via the germline-mediated TGP pathway, while the original brood mass was weighed to assess parental investment via the behavioural TGP pathway. Results revealed that ASR strongly modulated adult interactions: male-biased treatments exhibited the highest contest intensity, whereas female-biased treatments displayed the highest courtship frequency, significantly surpassing the male-biased group. Despite these pronounced behavioural responses, parental ASR experience had no significant effect on offspring development speed, emerging weight, or developmental stage duration. Moreover, parental investment did not differ among treatments. These findings demonstrate that although ASR reliably predicts contest and courtship intensity in parents, such social pressures do not necessarily propagate to offspring metamorphic development via either core pathway of TGP within a single generation. This study underscores a dissociation between adult behavioural plasticity and intergenerational outcomes, suggesting that transgenerational ASR effects may require longer timescales or stronger selective pressures to manifest. Title : Parental social environment has no effect on offspring development in the dung beetle: A test of adult sex ratio effects Short title : Parental adult sex ratio experience and offspring development in dung beetles Lisheng Zhang 1,2 * , Sudeshna Chakraborty 2 , Tamás Székely 3,4 , Jan Komdeur 2 # 1 Yunnan Key Laboratory of Forest Ecosystem Stability and Global Change, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, Yunnan, 666303, China 2 Behavioural and Physiological Ecology Group, Groningen Institute for Evolutionary Life Science, University of Groningen, PO Box 11103, 9700CC Groningen, The Netherlands 3 Milner Centre for Evolution, University of Bath, Claverton Down, Bath BA2 7AY, UK 4 HUN-REN-DE Reproductive Strategies Research Group, University of Debrecen, Debrecen H-4032, Hungary *Corresponding author : Lisheng Zhang , Address : Yunnan Key Laboratory of Forest Ecosystem Stability and Global Change, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, Yunnan, 666303, China. E-mail: [email protected] #Co-corresponding author : Jan Komdeur , Address : Behavioural and Physiological Ecology Group, Groningen Institute for Evolutionary Life Science, University of Groningen, PO Box 11103, 9700CC Groningen, The Netherlands. E-mail : [email protected] ††slugcomment: Draft version Conflicts of Interest The authors state that there are no competing or financial interests to disclose. ††slugcomment: Draft version Funding L.Z. was supported by a Ph.D. scholarship from the China Scholarship Council (CSC) and by the Dutch Science Council grant (ALW NWO Grant No. ALWOP.531 and NWO VICI 823.01.014), and NWO TOP grant (854.11.003) to J.K. T.S. was funded by NKFIH ADVANCED 150852, HU-RIZONT-2024-00109 and HUN-REN-Debrecen University Reproductive Strategies Research Group (Ref 110220). ††slugcomment: Draft version Introduction The adult sex ratio (ASR), defined as the proportion of adult males to females in a population, is a fundamental demographic parameter that varies substantially across species and ecological contexts (Székely et al., 2014). To date, research on ASR has focused primarily on its intragenerational consequences, demonstrating its powerful role in shaping mating systems, sexual selection, and reproductive strategies (Schacht et al., 2017, 2022; Székely et al., 2014). Within a single generation, variation in the ASR alters the social environment, often modifying the intensity of competition for mates and the level of parental care (Kokko and Jennions, 2008; Liker et al., 2013). However, parental responses to the social environment can also influence the phenotype of the subsequent generation via transgenerational plasticity (TGP), a broadly defined framework encompassing all non-genetic parental effects induced by the parental environment, regardless of the transmission pathway (Bonduriansky, 2021; McAndry et al., 2025). Despite this, it remains unclear whether ASR-induced TGP is driven primarily by two distinct, non-mutually exclusive mechanisms: alterations in post-zygotic parental investment (e.g., resource provisioning via brood care) or by pre-zygotic non-genetic transfer of information via the germline. Disentangling these mechanistic pathways within the TGP framework is critical for understanding the cross-generational evolutionary implications of skewed sex ratios. Parental effects shape offspring phenotypes through both genetic and non‑genetic mechanisms. While classical genetics emphasizes Mendelian and chromosomal inheritance (Alberts et al., 2022), evidence increasingly highlights the role of non‑genetic processes, most notably TGP, the unifying core construct for all environment-induced non-genetic parental effects (Bonduriansky, 2021; McAndry et al., 2025). Contemporary consensus defines TGP as any phenotypic change in offspring triggered by parental environmental exposure, independent of changes to the nuclear DNA sequence, and encompassing two core, non-mutually exclusive mechanistic pathways: pre-zygotic germline-mediated transmission, and post-zygotic transmission via environmentally induced parental behaviours (Bell and Hellmann, 2019; Bonduriansky, 2021; Donelan et al., 2020; Herman and Sultan, 2011; McAndry et al., 2025). TGP has been widely documented in response to abiotic factors like temperature, nutrition, and toxins (Joschinski and Bonte, 2020; Walsh et al., 2024). For instance, in the reef fish Acanthochromis polyacanthus , sustained parental exposure to elevated temperatures (+1.5 °C) enhanced offspring thermal tolerance via germline-mediated TGP, illustrating transgenerational acclimatization (Bonzi et al., 2025). Recent TGP research has also turned to social environmental factors (Hellmann and Sih, 2025), including population density, social rank, and social stress, with effects mediated through both germline epigenetic and post-zygotic behavioural pathways. In the desert locust ( Schistocerca gregaria ), parental crowding experience influences offspring phase traits via chemical deposition into eggs, a classic example of germline-mediated TGP (Miller et al., 2008; Simpson et al., 2011). Similarly, social stress in humans (e.g., genocide exposure: Kellermann, 2013, 2015; chronic stress: Rodgers et al., 2013) can alter offspring birth weight, hormone profiles, and psychological vulnerability via TGP (Brunton, 2013). In zebrafish ( Danio rerio ), offspring from long-term isolated parents exhibited significantly greater body length than those from group-reared parents, whereas offspring from group-reared parents showed higher feeding rates in feeding trials, indicating that parental social environment can shape offspring phenotype via TGP (Dissinger et al., 2025). In a study of social behaviour in mice, dominant males exhibited differential DNA methylation in sperm for genes related to growth and neural development, and their offspring inherited enhanced aggressive behaviour via this germline-mediated TGP pathway (Hou et al., 2023). These findings collectively suggest that parents can perceive changes in the social environment, induce corresponding epigenetic modifications, and transmit these changes via the germline to their offspring, thereby influencing offspring growth and behaviour through the pre-zygotic TGP pathway. Alongside the well-documented germline-mediated pathway, the second core mechanistic pathway within the unified TGP framework operates via direct parental behaviours, including habitat choice, food provisioning, and cultural transmission (Clutton‑Brock, 1991; Galef and Laland, 2005). Many holometabolous insects, for example, select pupation sites to improve offspring survival via this behavioural TGP pathway (Sprague and Woods, 2015; Wang et al., 2017), and in matrilineal species, high‑ranking mothers confer social status that boosts offspring fitness via environment-induced parental behavioural effects (Vullioud et al., 2024). The influence of parental ASR on offspring also operates through diverse mechanisms. ASR-driven sexual selection, typically involving male–male competition and female choice, helps identify high-quality gametes and promotes the transmission of superior genes, consistent with “good genes” theory (Andersson, 1994; Qvarnström and Price, 2001; Wedell et al., 2002). However, multi-generational ASR manipulations show that stable behavioural and morphological divergence accumulates over dozens of generations, as seen in Drosophila melanogaster (>78 generations; Bath et al., 2021) and Tribolium castaneum (>58 generations; Godwin et al., 2020). Evidence for ASR-induced TGP, encompassing both germline and behavioural mechanisms, is also growing. In the non-caregiving beetle Callosobruchus maculatus , a male-biased parental ASR triggered transgenerational adjustments: F0 (parental generation) females showed reduced longevity and reproduction, but F1 (offspring generation) offspring displayed improved mating competitiveness (e.g., higher copulation success and reduced copulation duration), with some effects persisting into the F2 (grand-offspring generation), suggesting a non-genetic inheritance mechanism priming descendants for competitive conditions (Amiri and Bandani, 2021). Within the TGP framework, ASR can also shape offspring fitness behaviourally via mating opportunities and parental care (Székely et al., 2000). In male-biased populations, males face lower mate-finding prospects and may increase care to maximise fitness (Kokko and Jennions, 2008; Liker et al., 2013), although higher paternity uncertainty can also lead to reduced care or desertion (Alonzo, 2010; Fromhage and Jennions, 2016), indirectly affecting offspring development. Consistently, in female-biased populations the females are expected to increase their care due to their reduced mating opportunities (Székely et al., 2000). Unlike classic genetic evolution, which requires multi-generational selection to drive heritable shifts in nuclear DNA sequence, TGP enables the transmission of environmental cues across generations within a single reproductive cycle, facilitating rapid adaptive phenotypic change. For our study, which manipulates ASR across only one parental generation, this means sequence-based genetic evolution cannot explain observed offspring phenotypic differences. We therefore hypothesize that over short timescales, ASR affects offspring development primarily via the dual pathways of TGP, rather than sequence-based genetic change. The dung beetle Onthophagus taurus (Coleoptera: Scarabaeidae), a holometabolous insect, progresses through distinct developmental stages (egg, larva, pupa, adult) during holometamorphosis, a process essential to larval and adult growth (Gullan and Cranston, 2014). In nature, females typically lay one egg per brood ball, which is provisioned by both parents (mainly females). Larval development is strongly influenced by nutritional conditions, since the dung contained in the brood ball constitutes the sole food source for the immobile larva (Hanski and Cambefort, 1994; Scholtz et al., 2009). Under favorable nutritional conditions, male larvae develop into larger adults with relatively larger horns, whereas under limited nutrition they become smaller adults with reduced horns (Moczek, 2010). Furthermore, previous research confirms that the ASR is an important driver of both parental behaviour and offspring development. In male-biased O. taurus populations, contest and courtship behaviours occur at higher intensity than in female-biased and unbiased populations (Zhang et al., 2024), indicating that parents experience socially mediated stress under a skewed ASR, a process potentially facilitated by TGP. Separately, females produce fewer brood masses when males were present, and those paired with horned males constructed larger brood masses than those paired with hornless males or those breeding alone (Hunt and Simmons, 1998), suggesting that the ASR directly influences parental investment in brood ball production, thereby affecting offspring development. Yet, it remains unresolved whether the effect of ASR on offspring results from an interaction between these two TGP pathways or from their independent effects. To dissect the respective contributions of the two core mechanistic pathways underpinning ASR-induced TGP, we developed an egg-transplantation protocol in O. taurus . Eggs were transferred from their original brood balls—which vary in mass due to differences in parental investment—into standardized artificial brood balls. By comparing developmental outcomes of eggs from different parental ASR treatments within this common gestational environment, we could attribute observed differences primarily to TGP. Concurrently, the mass of the original brood balls provided a direct measure of parental investment. We propose that parental ASR can influence the development of offspring in one generation through two non–mutually exclusive pathways. 1 ) Germline-mediated TGP pathway. Parental experience of ASR stress can lead to the developmental programming of offspring via non-genetic transmission (e.g. epigenetic modification). Offspring from male–biased populations may exhibit accelerated development, manifested as faster developmental speed and less developmental duration, as an adaptive response to heightened competition intensity. 2) Parental investment-mediated TGP pathway. P arents may alter their parental investment (e.g., food provision) in larval development in response to external ASR contexts. In male-biased populations, males may increase their contribution to brood–ball provisioning to ensure paternity thereby resulting in heavier brood balls, leading to larger offspring. Materials and Methods Establishment of the laboratory colony The laboratory colony of Onthophagus taurus was established from a founder population collected in March 2018 from [Location, Germany] and transported to [Name of Institution, Netherland]. The founder population, consisting of 50 males and 50 females, was transported in a plastic bucket (~30cm × 40 cm, Diameter × Height) filled with moist sand to serve as a substrate, with no dung added. To prevent substrate leakage and minimize air exchange, the containers were sealed with plastic film. The shipment was transported via ground courier (DHL, Germany) over a period of 2 days under moderate ambient temperatures. Upon arrival, the container was opened in a climate-controlled room. We observed zero mortality, indicating that the transport process induced minimal physical stress. Although mating activity during transit could not be excluded, spontaneous oviposition was observed in a subset of females shortly after arrival. To ensure genetic control, these eggs were euthanized by freezing at -20 °C to prevent the inclusion of offspring with unknown paternity in the stock colony. The founder beetles were maintained separately in boxes (10 cm × 7 cm × 8 cm, Length (L) × Width (W) × Height (H)) in a climate-controlled room at 22 ± 3 °C under a 16h : 8h (L : D) photoperiod within a substrate of a 1:1 soil-sand mixture. To ensure optimal physiological and reproductive condition, they were provided with 60 g of fresh dung every five days for a minimum of six weeks prior to breeding. To produce subsequent generations, female and male founder beetles were paired and placed in standard breeding chambers (16 cm × 9 cm × 10 cm, L ×W ×H). Each chamber was filled three-quarters full with the moistened soil-sand mixture and provisioned with 200 g of fresh dung on the surface. The breeding period was set at five days. After egg-laying, the brood balls from each chamber were collected, cleaned, and individually reburied in boxes with the same substrate until offspring emergence. Upon reaching adulthood, a portion of the new-generation beetles was allocated for experiments, while the remainder was used to maintain the colony under the same pairwise breeding protocol. Maintenance of experimental parents The virgin adult beetles used in this study were the offspring from matings of the third laboratory generation. From emergence, these experimental beetles were maintained under identical housing, feeding, and environmental conditions as described above. Each beetle was used for a single trial only. All beetles used were sexually mature and in good physical condition. Fresh cow dung was obtained from an organic, pesticide-free dairy farm in [Location Anonymized for Review] ([Farm Name and Coordinates Redacted]) and stored at -20 °C in 10 L buckets. For use, the dung was thawed at room temperature for 24 hours and thoroughly homogenized before being provided for feeding or used in experimental procedures. Further methodological details are described in a previous study [Reference X, redacted for double-blind review]. Transplantation system ††slugcomment: Draft version Basic structure of artificial brood balls Artificial brood balls were fabricated by modifying traditional Chinese herbal pills (GB 13731, China), which were purchased online from a commercial supplier (Taobao, www.taobao.com). Each pill comprises two empty detachable hemispherical shells featuring an interlocking structure that forms a complete hollow sphere. The artificial brood balls had an internal diameter of ~30 mm. To ensure adequate oxygen diffusion into the ball interior for offspring development, 6 auxiliary ventilation holes (~2 mm diameter) were drilled evenly over each hemispherical shell using a mini electric drill (DZTZ02, Gerbala), resulting in a total of 12 auxiliary ventilation holes per artificial brood ball. Additionally, 15 g of preprocessed dung was placed inside each ball to serve as food throughout larval development (Fig. 1, Fig. 3d, e). The internal space and food quantity provided were sufficient to support complete offspring development. Consequently, each larva experienced a consistent and uniform nutritional environment throughout the developmental process. Preparation and pre-loading of preprocessed dung Preprocessed dung inside the artificial brood balls was prepared by mixing and compacting fresh and dried dung. The fresh dung was thawed for 24 hours and equilibrated to room temperature before use. Dried dung was prepared by spreading fresh dung (<1 cm thickness) onto rigid acrylic plates and drying it in a drying oven at 60 °C for 48 hours until a constant weight was achieved. Then dried dung was then scraped off the plates using a plastic spatula, transferred to a clean, dry container, and ground into a fine powder for later use. Humidity levels in the artificial brood balls were maintained to approximate natural conditions by controlling the fresh-to-dried dung ratio. During formulation, fresh dung was first placed into a stainless-steel bowl (20 cm diameter, 2 L capacity), followed by gradual addition of dried dung in small portions. The mixture was continuously stirred with steel tweezers until a homogeneous mixture resembling natural brood ball composition was achieved. Suitability of the dung mixture was assessed through visual and tactile evaluation. After preparation, preprocessed dung was distributed into artificial brood balls. Initially, each hemispherical shell was completely filled to form a solid, compact half-sphere. Subsequently, dung was removed from the central area of each hemisphere using forceps to create a cavity. The cavity surface was then carefully smoothed and compressed with blunt-tipped forceps to ensure dryness and cleanliness. When joined, the hemispheres formed a central chamber (~1 cm diameter) for fertilized egg transplantation. A fine sewing needle (0.5 mm in diameter) was then inserted through one ventilation hole into the central cavity, establishing an air channel connecting the cavity to the external environment. This main ventilation hole, together with the auxiliary ventilation holes, facilitated air exchange while simulating natural developmental conditions (Fig. 1). During experiments, the external opening of this channel was sealed with soil to prevent light penetration while restricting airflow. On the morning preceding surgical transplantation, preprocessed dung was prepared and dispensed into ~ 250 artificial brood balls following the aforementioned protocol. The artificial brood balls were then placed in a large container under static conditions until transplantation. Prior to transplantation initiation, the dung substrate and internal air in all artificial brood balls had equilibrated to ambient room temperature. ††slugcomment: Draft version Transferring fertilized eggs from original brood balls to artificial brood balls During transplantation, the sealing dung plug at each original brood ball’s aperture was carefully dislodged using fine-pointed forceps. Surrounding debris and soil fragments were subsequently cleared. Following debris removal, the brood ball was opened and expanded with forceps to fully expose its contents (Fig. 3a). Dung fragments dislodged during dissection were recompacted into the original brood balls after egg transplantation to avoid interference with subsequent parental care measurement. Blunt-tipped forceps sterilized with 75% ethanol were then used to gently apply pressure to the mid-region of the egg. The egg was gently detached from its basal attachment and transferred onto the inner surface of the cavity within the preformed artificial brood ball. All operations were performed with precision and while wearing gloves to prevent external microbial contamination and avoid mechanical damage. Throughout transplantation, the egg’s basal region adhered to the dung substrate surface inside the cavity, while the remainder remained fully air-exposed. This methodology precisely replicates natural brood ball developmental conditions under controlled artificial settings. Assessment of the transplantation system To assess system feasibility, a pilot experiment was conducted one week before the main experiment commenced. In summary, ten male-female pairs were individually housed in simplified breeding boxes (16 cm × 9 cm × 10 cm, L × W × H) containing fresh dung (100 g) atop soil for a three-day mating and breeding period. Following this, brood balls were collected from each box, yielding 37 brood balls. Eggs from each naturally produced brood ball were individually transplanted into corresponding artificial brood balls using the described method. After a 5-day incubation period, all artificial brood balls were opened to evaluate embryonic development. Successful larval development occurred in 35 of 37 transplantation attempts, yielding a 94.59% success rate. These findings demonstrate that the designed transplantation system is safe, efficient, scalable, and easily implementable. Experimental procedure Alignment of ASR and OSR in the experimental system Importantly, while it is critical to distinguish the demographic adult sex ratio (ASR) from the operational sex ratio (OSR), the ratio of sexually active males to receptive females at any given time (Emlen and Oring, 1977), we consider the two metrics to be functionally equivalent within our experimental framework. In natural environments, OSR often fluctuates independently of ASR. However, our experimental design strictly utilized fully sexually mature individuals in peak physiological condition, ensuring that all beetles were continuously capable of, and receptive to, mating. Furthermore, the experiments were conducted in spatially confined arenas, which maximizes inter-individual encounter rates and prevents temporary withdrawal from the mating pool. Under these highly controlled conditions, the manipulated ASR directly dictates the immediate competitive pressure, effectively generating stark and consistent gradients of OSR and socially mediated stress across the treatments. ASR treatments design The experiment was conducted from November to December 2021. A total of 180 adult parental beetles were randomly assigned to one of three ASR treatments: female‑biased ( FB ), unbiased ( UB ), and male‑biased ( MB ) (Table 1). Previous results indicated that ASR did not significantly affect parental investment—measured as the weight of dried brood balls—under a narrower ASR range ( FB : 2 M : 4 F, 33%; UB : 3 M : 3 F, 50%; MB : 4 M : 2 F, 66%). To better elucidate the effects of ASR, and to achieve a steeper ASR gradient under the constraint of a fixed total number of experimental beetles, we varied the group size per replicate between the biased and unbiased treatments. Specifically, in the unbiased treatment, each replicate contained 4 individuals ( UB : 2 M:2 F, 50%), whereas replicates in the female‑biased and male‑biased treatments contained 8 individuals each ( FB : 2 M : 6 F, 25%; MB : 6 M : 2 F, 75%). To equalize population density across treatments, the size of the experimental arena was scaled proportionally to group size, ensuring that the available space per individual remained constant (details see below). We acknowledge that, despite standardized per capita density, group size itself may act as an independent social variable influencing competitive interactions, mating dynamics and TGP effects. To mitigate this confounding effect, our core between-treatment comparisons focused predominantly on the FB and MB groups as these two treatment groups were fully matched for replicate group size, per capita arena density, and all other non-focal experimental variables, eliminating the confounding effect of group size variation and providing a highly robust, directly comparable framework for our hypothesis testing. The UB treatment was fully included in all statistical models as a neutral baseline control for supplementary pairwise comparisons and to contextualize the magnitude of ASR effects across the full gradient. Furthermore, this experimental design also ensured a baseline level of intrasexual competition (at least two same‑sex individuals per replicate) across all treatments. All beetles were allowed to mate freely within their assigned replicate populations for the entire breeding period, to ensure individuals experienced the full natural social context of the manipulated ASR, including unconstrained mating interactions and intrasexual competition dynamics. O. taurus is an obligately polyandrous species in natural populations, with intense pre- and post-copulatory sexual selection and sperm competition (McCullough et al., 2017; Simmons and Holley, 2011); This mating system can alter the genetic composition of offspring, and in turn, shape their developmental trajectories during metamorphosis. A well-established experimental protocol to control for offspring genetic background is to ensure females mate exclusively with males from the same full-sib family line, thus standardizing genetic similarity between parents (Sato et al., 2023). However, in accordance with our established rearing protocol, all experimental males were derived from the random mating of individuals from the preceding generation, precluding the definitive characterization of their individual genetic backgrounds. To address this limitation, we utilized a fully randomized assignment strategy for all parental beetles across the treatments. This randomization homogenizes the genetic variance of sires across the ASR groups, effectively mitigating potential biases driven by intense sexual selection or sperm competition. All replicates within a given treatment were run simultaneously. We individually marked beetles with distinct color dots on their pronotum and wings using a permanent marker (Edding 751, Japan) to facilitate identification and tracking, with no observed impairment to movement (Fig. 2a). Pronotum length, measured using a digital caliper (± 0.01 mm, Mahr, Germany), served as a proxy for body size. No significant differences in parental body size occurred between sexes or among ASR treatments (Sex: F = 0.076, DF = 1, p = 0.784; ASR treatment: F = 0.918, DF = 2, p = 0.401) (Table 1). Pre-breeding behavioural observation To ensure that individuals from different ASR treatments received the corresponding ASR-induced stimuli, individuals from each group were placed into treatment-specific observation boxes for monitoring. Through the observational experiment, we investigated the impact of ASR on competition intensity, which is widely recognized as a fundamental source of social pressure within populations. Beetle density was kept consistent across treatments using different box sizes. Female-biased and male-biased treatments used transparent plastic boxes measuring 16 cm × 9 cm × 10 cm (L × W × H, ~17 cm²/ind), while the control treatment used boxes measuring 10 cm × 7 cm × 8 cm (L × W × H, frequency of contest and courtship behaviours across various ASR treatments (Fig. 2a). Three plastic boxes, each containing one ASR treatment, were arranged side by side at a 50 cm distance on a horizontal desk under uniform dim lighting and a constant temperature of 22 ± 3 ℃. Contest and courtship behaviour were recorded for 90 minutes using a video camera (JVC, GZ-R405BEU, Japan) positioned above each box to track individual beetle movements (Fig. 2a). This observation period was selected to ensure adequate exposure to ASR-induced stress while minimizing risks of physical injury. During pre-breeding behavioural observation, detailed definitions and quantitative protocols for contest and courtship behaviours were established in Zhang et al. (2024). Summarized as: Contest behaviour was defined as agonistic interactions involving physical combat, predominantly head butting and pushing (Beckers et al., 2017). These interactions occurred primarily between males, though instances involving females or intersexual contests were also observed (Fig. 2a). To evaluate the impact of ASR on contest intensity, we quantified the total number of contest events per box; Courtship behaviour was characterized by a male repeatedly drumming the tarsi of its forelegs on the pronotum or elytra of a female (Fig. 2a, Beckers et al., 2017). To assess the effect of ASR on courtship intensity, we recorded the number of courtship initiations per observation box. Breeding protocol and brood balls collection Following the recording, all individuals from each observation box were correspondingly transferred to new breeding chambers identical in size and structure to their original observation boxes. These chambers contained three-quarters of a mixture of moist sand and soil (1:1 ratio) and were topped with adequate dung (200 g for FB and MB , 100 g for UB ) for breeding (Fig. 2b). After a five–day breeding period, all brood balls were gently removed and subsequently sieved using a 0.5 cm aperture mesh (Fig. 2c, Fig. 3b). Any adhering soil or surface debris was meticulously picked off with fine forceps. Additionally, impurities lodged within the fecal matter were meticulously extracted using a micro-dissecting needle (Hunt and Simmons, 2002a). Assessment of offspring development Somatic growth The intact brood balls were subjected to meticulous dissection (details were provided above), resulting in the separation of two distinct components: eggs and the remaining brood balls. In parallel, the eggs were transplanted from the original brood balls into artificial brood balls for developmental assessment. Eggs were individually transferred into the artificial brood balls following the previously established transplantation protocol (see above). Each artificial brood ball was marked on the external surface using a permanent marker (Edding 751, Japan). These artificial brood balls from different ASR treatments were randomly buried in soil identical to that used in the breeding chambers (Fig. 3f, temperature: 20 ± 3 °C; volumetric water content: ~ 28–35%, measured using a WET-2 sensor, Delta-T Devices Ltd., UK). To assess larval development stage and body mass during development, the following non-invasive weighing procedure was performed at regular intervals. During each inspection, the artificial brood balls buried in the soil were gently excavated, and any adhering soil and gravel on their surfaces were carefully removed using a soft brush. The artificial brood ball was then carefully separated into two halves using a utility knife inserted along the gap of the interlocking seam at its midline. The developing larva was then located and gently removed with blunt-tipped forceps for weighing. Prior to each measurement, the electronic balance (± 0.0001 g, Mettler Toledo, AG204, Switzerland) was zeroed and a clean sheet of white paper was placed on its surface. The larva was positioned on the paper, and a new sheet was used for each individual weighing to prevent cross-contamination. After weighing, the larva was returned to its original position within the artificial brood ball (Fig. 3g, h, i). In some cases, larvae excreted a small amount of dark fluid from the mouth for rumination during handling, which was not accounted for in the weight measurement. Additionally, any dung fragments adhering to the larval body were gently removed using forceps before weighing. All procedures were conducted at ambient room temperature and did not cause any observable adverse effects on larval development. All experimental procedures were conducted under dim lighting conditions to avoid direct exposure to artificial light. To ensure consistency across measurements, all weighing procedures were required to be completed within a 3 hour time window. To meet this criterion, we intentionally reduced the number of transplants in the female-biased treatment group by selecting 60 out of 107 artificial brood balls for subsequent developmental assessments. In the unbiased and male-biased treatments, all fertilized eggs from the original brood balls were transplanted into artificial ones to permit full tracking of offspring development, as the number of brood balls obtained was inherently limited. Developmental stages testing At each sampling point, the developmental stage of the offspring was recorded. Upon completion of the experiment, we calculated the duration of larval, pupal, and emerging stages. The larval stage refers to the period in the offspring’s life cycle from hatching to the pupal stage. During this stage, larvae possess a mobile body and are capable of feeding using mandibular mouthparts (Fig. 3g). The pupal stage refers to the period in the life cycle of the offspring, starting from the conclusion of the larval stage and extending until the emergence of the adult (Fig. 3h). During this process, larvae cease feeding, become immobile, undergo significant morphological changes, and assume a body shape that closely resembles that of the adult, preparing for the emergence of the adult form. In addition, the point at which newborn beetles could stretch their limbs and move freely was defined as the emergence of offspring (Fig. 3i, Hanski and Cambefort, 1994; Scholtz et al., 2009). Due to the inability to determine the precise timing of original brood ball production, the duration of the egg stage remained uncertain and was thus not considered in the present study. We recorded the peak weight of offspring and the time taken to reach peak weight during development. Developmental speed, an indicator of beetle growth rate, was represented by the ratio of peak weight to the time taken to reach peak weight (peak weight (g) / time reaching peak weight (days)). Upon emergence of newborn beetles, we recorded their sex, body weight (emerging weight), body size and horn size (for males) of offspring in each breeding box. We observed an extremely positive correlation between the weight of emerging beetles and both their body size (Pearson correlation coefficient, r = 0.902, p < 0.001) and the size of their horns in males (r = 0.873, p < 0.001). As a result, we decided to include only the emerging weight variable in our results analyses. Measurement of parental care After transplantation, the remaining original brood balls without eggs were dried for 48 hours at 60 ℃ to constant weight. After removal of adherent sand with micro-probes, mass measurements were performed using an electronic balance (± 0.0001 g, Mettler Toledo, AG204, Switzerland) (Fig. 3c). The weight of brood balls served as indicators of parental investment from parental dung beetles. Statistical analyses All statistical analyses were conducted using R version 4.3.1 (R Core Team, 2023). We fitted linear mixed-effects models (LMMs) using the ‘lme4’ package (Bates et al., 2015), with P-values for fixed effects calculated via the ‘lmerTest’ package (Kuznetsova et al., 2017). Model assumptions, specifically residual normality and homoscedasticity, were verified using simulation-based diagnostics in the ‘DHARMa’ package (Hartig, 2022). Initial models included ASR treatment, sex (where applicable), and their relevant interactions (e.g. with developmental stage) as fixed effects. Model selection was based on the Akaike Information Criterion (AIC); nonsignificant interactions were removed to simplify the models, while main effects were always retained. To account for the hierarchical structure of the data, experimental group (i.e. box) and/or offspring ID were included as random intercepts. Post hoc pairwise comparisons were performed using the ‘emmeans’ package (Lenth, 2023), with adjustments for multiple comparisons applied using the ‘multcomp’ package (Hothorn et al., 2008). Intensity of contest and courtship behaviour Intensity of contest and courtship behaviour We analysed the frequency of contest and courtship behaviours using an LMM with ASR treatment, behaviour type, and their interaction as fixed factors. Experimental group was included as a random intercept to account for the non-independence of observations within the same group. Assessment of offspring’s development We first conducted paired t-tests to examine differences between developmental stages. To assess the effects of ASR treatment and sex on development speed and emergence weight, we fitted separate LMMs. Initial models included the interaction between ASR treatment and sex; however, as the interaction was non-significant for both variables, it was removed, and final models contained only the main effects. Experimental group was included as a random factor. To analyse developmental duration across stages, we structured the data in a long format and fitted an LMM with ASR treatment, sex, and developmental stage as fixed factors, following the approach by Snijders and Bosker (2012). We tested two-way and three-way interactions; the full three-way interaction model provided the best fit based on AIC and was retained. Both experimental group and individual ID were included as random factors to account for repeated measures on the same individuals across stages. Parental care testing Parental care was evaluated by comparing the dried brood-ball mass among ASR treatments using an LMM. ASR treatment was specified as the fixed effect, and experimental group was included as a random factor. Intensity of contest and courtship behaviours among different ASR treatments The effects of ASR on the intensity of contest and courtship behaviour of parents were observed to differ. A significant interaction effect was found between ASR treatments and types of behaviour (Table 2). In terms of contest behaviour, the frequency of contests in the male-biased treatment was higher compared to both unbiased (t = 5.956, p < 0.001) and female-biased treatments (t = 2.990, p = 0.014), while the frequency of contests in the female-biased treatment surpassed that of the unbiased treatment (t = 2.789, p = 0.023) (Fig. 4a). For courtship behaviour, both male-biased (t = 2.767, p = 0.025) and female-biased (t = 5.759, p < 0.001) treatments exhibited significantly higher frequency than the unbiased treatment, additionally, the courtship frequency in female-biased treatment was significantly higher than that in male-biased treatment (t = 2.971, p = 0.015) (Fig. 4b). Development of larva A total of 60 fully developed females (24 in FB , 23 in UB , and 13 in MB ) and 63 males (32 in FB , 19 in UB , and 12 in MB ) offspring were collected. ASR had no effect on larval development, with no significant differences observed in development speed among the three treatments and sexes (Table 3, Fig. 5a, b), A detailed visualization of the growth trajectories separated by both ASR treatment and offspring sex confirms this consistent pattern (Appendixes, Fig. S1). In addition, no significant differences in emerging weight were detected among treatments and sexes (Table 3). The total developmental time until emergence consisted of two distinct phases: the larval stage and the pupal stage. Specifically, the duration of the larval stage was significantly longer than that of the pupal stage (paired t-test: t = 33.80, p < 0.001, mean difference = 12.07 days). In contrast, the emerging time was significantly longer than both larval stage (t = -155.13, p < 0.001, mean difference = -18.18 days) and pupal stage (t = -103.28, p < 0.001, mean difference = -30.24 days). These results indicate that the larval phase contributed the majority of the overall developmental period, while the pupal stage was considerably shorter in duration (Fig. 5c, d). Similarly, ASR was not a significant factor in the duration of developmental stages (Table 4). Although no significant interaction was observed between sex and stages in our model, post hoc analyses revealed that males exhibited longer durations in both the larval (t = 2.596, p =0.009) and emerging time (t = 3.625, p < 0.001) stages compared to females. However, no significant difference was detected between males and females in the pupal stage (t = 1.192, p = 0.234) (Table 4, Fig. 5c, d). A detailed breakdown confirms that these sex-specific differences in developmental duration remained consistent across all three ASR treatments (Appendixes, Fig. S2). Parental care A total of 205 dried to constant weight brood balls were collected (107 in FB , 64 in UB , and 34 in MB ). ASR had no impact on parental care, as evidenced by the lack of significant differences in dried brood ball weight among the three treatments (Table 5, Fig. 6). ††slugcomment: Draft version Discussion Our study successfully disentangled the independent effects of two core pathways of non-genetic TGP in response to ASR variation: behavioural parental investment-mediated effects and germline-mediated effects. The results yield two distinct conclusions. First, at the parental level, ASR serves as a potent driver of behavioural plasticity: male-biased populations exhibited intensified contest competition, while female-biased populations unexpectedly showed the highest levels of courtship intensity. Second, despite this intense social stress and behavioural modification in the parents, we found no evidence that this environmental information is transmitted to offspring via either germline-mediated TGP or altered parental investment (brood ball mass). Offspring developmental trajectories remained robust regardless of the parental social environment when reared in standardized conditions. These findings suggest that while Onthophagus taurus adults are highly sensitive to social demography, their offspring’s development may be canalized against such social fluctuations, potentially due to the buffering nature of the brood ball system. ASR’s influence on contest and courtship behaviour in parents We demonstrated that the ASR significantly influenced contest and courtship behaviours. Contest frequency was highest in the male-biased treatment, surpassing both the unbiased and female-biased treatments; the female-biased treatment also exhibited higher contest frequency than the unbiased treatment. These results are consistent with previous study (Zhang et al., 2024) for the male-biased treatment, though the elevated contest frequency in the female-biased treatment represents a new finding, as it was not significant in earlier work. This pattern partially aligns with Weir et al. (2011), who reported a nonlinear relationship between OSR and contest intensity—initially increasing then decreasing with increasing male bias. For courtship behaviour, frequency was significantly lower in the unbiased treatment than in either biased treatment, and was significantly higher in the female-biased treatment than in the male-biased treatment. This outcome partially corresponds to Weir et al. (2011), who found decreased male courtship with increasing ASR. While our finding that courtship intensity in both female-biased and male-biased treatments was significantly higher than in the unbiased treatment aligns with the observations of Zhang et al. (2024), a key divergence emerged in the comparison between female-biased and male-biased treatments. Contrary to Zhang et al. (2024), who reported peak courtship in male-biased ASR—likely driven by intense intrasexual competition—our results demonstrated significantly higher intensity in the female-biased treatment. This suggests that in our study species, males may shift their strategy from competitive scramble to enhanced individual courtship when female availability is high, potentially reflecting a mechanism of male mate choice or a strategic allocation of reproductive effort when the risk of male interference is minimized. Such discrepancies highlight that ASR-mediated modulation is not merely a response to competition, but a complex trade-off influenced by species-specific reproductive traits and social density. These behavioural shifts align with sexual selection theory. As suggested by Székely et al. (2014), male-biased ASR intensifies male-male competition and aggression, while female-biased conditions predict reduced aggression and increased courtship (Clark and Grant, 2010; Forsgren et al., 2004; Grant et al., 2000). Such differences may reflect trade-offs in time and energy between competition and courtship under varying mate encounter rates (Grant et al., 2000). In summary, our results clearly demonstrated that male-biased ASR can intensify both contest and courtship behaviour, creating conditions for individuals to accumulate experience with ASR-driven social consequences. ASR’s influence on the development of offspring It is well-established that parental experience can shape offspring phenotype and fitness via non-genetic mechanisms, including transgenerational plasticity (TGP). For example, human studies show that major adversities, such as the Holocaust (Kellermann, 2013), famine (Roseboom et al., 2006), and chronic stress (Brunton, 2013), are associated with offspring alterations including reduced birth weight, hormonal changes, and psychological vulnerability. In zebrafish, offspring from long-term isolated parents were larger, while those from group-reared parents had higher feeding rates (Dissinger et al., 2025). In mice, dominant males showed sperm DNA methylation changes in genes related to growth and neural development, and their offspring displayed enhanced aggression (Hou et al., 2023). These findings indicate that parents can perceive social environments and transmit experiential information to offspring via TGP, encompassing both germline-mediated epigenetic inheritance and parental behavioural effects, with effects typically persisting across one to several generations. ASR regulates intra- and inter-sexual relationships within populations (Schacht et al., 2017, 2022; Székely et al., 2014). Under male-biased conditions, male competition intensifies (Zhang et al., 2024), often alongside increased sexual harassment of females (Hailey and Willemsen, 2000; Le Galliard et al., 2005), indicating that both sexes experience selective pressure from skewed ASRs. Previous work suggests such social environmental information can be transmitted via non-genetic inheritance mechanisms in gametes, influencing offspring development. A comparative analysis revealed distinct genome-wide DNA methylation patterns in sex ratio-biased lines, with male-biased lines showing more testicular methylation sites in genes involved in reproduction and epigenetics (Hosseini et al., 2022). While this work identifies a correlative link between ASR and epigenetic variation, causality remains unclear—these marks could be either cause or consequence of sex ratio bias. We therefore proposed that parental ASR exposure could induce epigenetic changes that alter offspring phenotype, with ASR acting via the germline-mediated TGP pathway documented in other social environmental contexts. However, in our study, offspring metamorphic development traits (i.e. developing speed, emerging weight, duration of developmental stages and total development time) did not differ among ASR treatments when reared in our standardized common garden environment. This result provides no evidence that parental ASR exposure influences offspring development via germline-mediated TGP in our study system. This aligns with findings in Callosobruchus maculatus , where multi-generational male-biased ASR did not affect larval development duration (Amiri and Bandani, 2021), indicating that parental ASR exposure does not consistently trigger transgenerational effects across insect species. One explanation is that the ASR gradient or treatment duration was insufficient to induce stable, transgenerational epigenetic changes in the parental germline. We also found no significant difference in brood ball weight across the three treatments, indicating that ASR did not drive shifts in parental investment via brood provisioning, the second core pathway of TGP in this system. Studies on the dung beetle Onthophagus taurus have reported varied effects of ASR on parental care. For instance, compared to females without male exposure, those exposed to males produced fewer brood masses, and females paired with horned males produced larger brood masses than those paired with hornless males or breeding alone (Hunt and Simmons, 1998). This suggests that males, particularly horned males, may contribute to parental care during brood ball construction, often resulting in heavier brood balls. Conversely, a higher number of males—typical in male-biased conditions—could disrupt females during brood ball production, such as by blocking tunnels. However, the absence of significant differences in brood ball weight across treatments in our study implies that males did not provide additional assistance to females, regardless of ASR. In previous study, parents breeding under different ASR conditions also showed no significant change in the dry weight of brood balls (Zhang et al., 2024), which aligns with the current results. One possible explanation for this null result involves compensatory behaviour by females. In biparental species, compensatory responses in offspring investment can significantly influence parental care outcomes (Hunt and Simmons, 2002b). Research on O. taurus has shown that unassisted females spend more time on care and display more parental behaviours, though this compensation is often incomplete, leading to lighter brood masses compared to biparental pairs (Hunt and Simmons, 2002b). In our experiment, although males in different ASR treatments may have differed in the level of care provided, females may have buffered or compensated for these differences, thereby mitigating the observable effects of male care on brood ball weight. In summary, we found no evidence that ASR influences offspring metamorphic development via either of the two core pathways underpinning TGP: pre-zygotic germline-mediated transmission, or post-zygotic parental investment via brood ball provisioning. The absence of transgenerational effects is likely driven by the lack of ASR-induced shifts in parental care behaviour, or by female compensatory care that offset any potential variation in male provisioning across treatments. Recommendations for future research Although our findings offer significant insights, several limitations highlight critical avenues for future investigation. First, a key limitation of our experimental design lies in potential confounding variables that constrained our ability to rigorously isolate ASR-induced TGP effects. Our ASR treatments included variable group sizes across treatments, and while we standardized population density, group size itself can act as an independent driver of social interactions, competitive dynamics, and TGP, introducing unaccounted variation in parental social experience. Additionally, our ad libitum mating design allowed for variable mating frequencies and paternity skew across ASR treatments: given the high polyandry of Onthophagus taurus (McCullough et al., 2017; Simmons and Holley, 2011), this may have led to systematic differences in the genetic composition of offspring across treatments, preventing us from fully excluding genetic effects on offspring phenotypic variation. To address these limitations, future studies testing ASR-induced TGP should strictly standardize group size across all sex ratio treatments, manipulating only ASR as the focal variable while holding all other demographic parameters constant. Furthermore, future work should implement a controlled mating design, in which parental individuals are exposed to different ASR social environments but females are restricted to mating with males of consistent genetic background, as outlined in Sato et al. (2023). This design will ensure consistent genetic background of offspring across all treatments, enabling the unambiguous attribution of offspring phenotypic differences to TGP rather than genetic variation. Second, while we employed a range of ASR treatments, the resolution may have been insufficient to fully characterize the precise thresholds of parental sensitivity to population-level sex ratio fluctuations. Future studies should implement a finer gradient of both ASR and OSR to map the sensitivity landscape of parental responses, and disentangle the independent and interactive effects of ASR and OSR on parental behaviour and offspring development. Third, our focus on the F1 generation within a single lifecycle precludes an assessment of long-term evolutionary consequences. We therefore recommend examining outcomes over multiple consecutive generations to elucidate the persistence and cumulative impacts of ASR-induced transgenerational effects. Fourth, we focused on morphological and developmental traits (body size at emergence, developmental rate). It is possible that TGP manifests in behavioural traits (e.g., offspring aggression or mating competitiveness) rather than developmental morphology, as seen in the Callosobruchus maculatus study (Amiri and Bandani, 2021). Future studies should incorporate a suite of offspring behavioural assays to fully characterize the scope of ASR-induced TGP in this system. A further limitation is the absence of targeted epigenetic data, which constrains our understanding of the mechanistic underpinnings of offspring development. Subsequent research should incorporate molecular analyses to disentangle the physiological pathways of transgenerational inheritance. Given that ASR effects are likely mediated through gametic alterations, future work should specifically investigate how social environments influence gamete quality and composition. Particular emphasis should be placed on sperm-mediated epigenetic changes resulting from male–male competition, as well as maternal cytoplasmic inheritance—beyond oocyte epigenetics—as a potential vector of maternal effects (Buzatto et al., 2012). Finally, although short-term ASR manipulation is unlikely to drive rapid shifts in allele frequencies, the relative contributions and potential interactions of “good genes” effects, the two core pathways of TGP, and within-generation behavioural modifications remain to be disentangled. These pathways may operate synergistically or antagonistically. We encourage further empirical and theoretical work to develop an integrative framework that unifies these mechanisms within the context of offspring fitness, aligned with contemporary consensus definitions of TGP (Bonduriansky, 2021; McAndry et al., 2025). To facilitate such mechanistic and ecological inquiries, we developed a novel egg–brood ball transplantation system. This method offers distinct advantages over existing protocols. Unlike previous open systems that exposed eggs to external environmental fluctuations (e.g., light, uncontrolled humidity) and potential interference from conspecifics (Buzatto et al., 2012), our system isolates eggs within artificial brood balls. This encapsulation ensures a stable internal microenvironment while allowing for the precise manipulation of experimental variables. For example, the substrate composition can be standardized or modified—such as through the inoculation of specific microbial communities—to experimentally test the functional roles of the microbiome in dung beetle development. Furthermore, this system significantly enhances ecological validity by allowing artificial brood balls to be buried in soil, thereby simulating natural subterranean developmental conditions. The system is also cost-effective and structurally robust, facilitating handling during large-scale cross-breeding or translocation experiments. For instance, this method enables reciprocal transplant designs to investigate altitudinal adaptation: brood balls collected from high-elevation sites can be reared in artificial chambers and transferred to low-elevation environments (and vice versa). We suggest that this methodological approach will be a valuable tool for future research into the developmental ecology of dung beetles. Conclusion In conclusion, our results establish that Onthophagus taurus exhibits distinct behavioural plasticity in response to ASR variation. While male-biased social environments maximized contest competition, female-biased social environments triggered peak courtship intensity, suggesting a strategic shift in male reproductive investment based on mate availability. Crucially, despite these profound behavioural adjustments in parents, we found no evidence of ASR-induced TGP via either of its two core mechanistic pathways. Using our egg-transplant protocol, we demonstrated that offspring development is robust to parental social history when nutritional investment is standardized, providing no support for germline-mediated TGP. Furthermore, the constancy of brood ball mass across treatments indicates ASR did not alter parental investment via the post-zygotic behavioural care pathway. Collectively, these findings highlight a decoupling between adult behavioural responses and intergenerational outcomes over short timescales. While adults rapidly adjust their social interactions to the prevailing sex ratio, offspring development appears canalized, likely ensuring survival stability regardless of the parental social environment. Future research should incorporate standardized experimental designs to isolate TGP effects, alongside assays of gametic epigenetic markers (e.g., sperm methylation profiles) to determine whether social experience leaves molecular traces that might manifest under different environmental contexts or over longer evolutionary timescales. Acknowledgements Sincere gratitude is extended to members of the Komdeur and Székely groups for their pivotal role in guiding the experimental design, implementation, and manuscript development. The diligent work of the bachelor student, who was instrumental in completing demanding experimental procedures, is also acknowledged. We thank the Terrestrial Ecotoxicology Laboratory for providing the founding dung beetle colony and Mr. Jan Hendrik (manager of Martinizicht farm) for the essential supply of high-quality cow dung. We also thank the Faculty Department of Animal Care, especially Dr. H. Martijn Salomons for use of lab and climate rooms for this study. We are grateful to Dr. Joshua Jones and Dr. Patrick Rohner of the Moczek group at Indiana University for the provision of several life history images of dung beetles used in this study. Funding L.Z. was supported by a Ph.D. scholarship from the China Scholarship Council (CSC) and by the Dutch Science Council grant (ALW NWO Grant No. ALWOP.531 and NWO VICI 823.01.014), and NWO TOP grant (854.11.003) to J.K. T.S. was funded by NKFIH ADVANCED 150852, HU-RIZONT-2024-00109 and HUN-REN-Debrecen University Reproductive Strategies Research Group (Ref 110220). Conflicts of Interest The authors state that there are no competing or financial interests to disclose. Data Availability Statement The data that support the findings of this study are openly available in Dryad at http://datadryad.org/share/LINK_NOT_FOR_PUBLICATION/gAE6NjQWPOgJDQDR3GdpLz-74MOMMmL-mwhE9NlSWic (accessed on 01.04.2026). 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Keywords behavioral ecology description experimental evolution invertebrate laboratory method development theoretical theory Authors Affiliations Lisheng Zhang 0009-0004-6463-3787 [email protected] XTBG View all articles by this author Sudeshna Chakraborty GELIFES View all articles by this author Tamas Szekely University of Bath Milner Centre for Evolution View all articles by this author Jan Komdeur GELIFES View all articles by this author Metrics & Citations Metrics Article Usage 176 views 97 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Lisheng Zhang, Sudeshna Chakraborty, Tamas Szekely, et al. Parental social environment has no effect on offspring development in the dung beetle: A test of adult sex ratio effects. Authorea . 03 April 2026. DOI: https://doi.org/10.22541/au.177521260.09198948/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. 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