Nicole Li Ying Lee
Jia Wei Tan
Tyrone Ren Hao Tan
Nalini Puniamoorthy- Department of Biological Sciences, National University of Singapore, Singapore, Singapore
Introduction: Phenotypic plasticity in critical life-history traits can buffer populations against environmental change, but its magnitude and direction may vary across geographic lineages.
Methods: Here, we investigated how two major developmental stressors, rising temperatures and food limitation, affect life-history traits. We quantified variation in juvenile survival, development time, adult body size and male reproductive investment (testis size; sperm length) in Sepsis punctum (Fabricius, 1794), a widespread dung fly with different mating strategies and contrasting sexual size dimorphism (SSD) between North America (females larger) and Europe (males larger).
Results and Discussion: Elevated temperatures reduced larval survival and accelerated development, generating smaller adults, particularly in European populations, with no changes in SSD. Food limitation also reduced adult size but had little effect on juvenile survival. Instead, resource stress differentially impacted SSD; female-biased SSD persisted in North American populations, but male-biased SSD did not persist in European populations. Male reproductive investment showed partial resilience, where absolute trait sizes declined under both stressors, but size-independent investment remained largely stable. Testis size decreased with increasing temperature, while sperm length did not, suggesting potential canalization of sperm quality and a strategic resource allocation that trades off quantity under stress. We revealed continental divergence in phenotypic plasticity in S. punctum, highlighting the importance of geography and stressor type in shaping life-history trajectories. These findings indicate that thermal stress may have stronger evolutionary consequences than resource limitations, with implications for population resilience and reproduction under global change.
Introduction
Rapid environmental changes are intensifying the frequency and severity of developmental stressors experienced by organisms (Chevin and Hoffmann, 2017; IPCC, 2023; Peters and Lovejoy, 1994). Rising temperatures and fluctuations in resource availability directly influence an organism’s early development, altering key fitness-related traits such as survival, growth rate, and reproduction (Crean and Immler, 2021; Johnson et al., 2016). Resilience to stressful conditions partly depends on phenotypic plasticity, which is a flexible response that allows individuals to alter their phenotypes in response to environmental variation (Bonamour et al., 2019; Pigliucci et al., 2006; Stearns, 1989; West-Eberhard, 2003). However, plasticity is not universally beneficial and can involve critical trade-offs between survival (e.g., thermal tolerance) and reproduction (e.g., fecundity), which vary among sexes and populations (Bogan et al., 2024; Jarrold et al., 2019; Sisodia and Singh, 2002). Thus, adaptive plasticity in resource allocation strategies is fundamental in influencing the evolution of life-history traits, and understanding developmental stress responses in different populations is relevant for predicting resilience to global change, particularly for widespread species (Chevin and Hoffmann, 2017; Kelly, 2019; Levins, 2012; Nylin and Gotthard, 1998).
Insects are powerful models for studying developmental plasticity, given their short generation times and sensitivity to environmental stress (Gillott, 1980; Moczek, 2010; Yoon et al., 2023). In holometabolous species that undergo complete metamorphosis, juveniles often experience starkly different environmental selection pressures than adults (Truman, 2019; Weaving et al., 2022). Larval developmental conditions tend to impact overall fitness more critically, as they influence both survival to the adult stage and fitness parameters such as reproduction and longevity (Blanckenhorn et al., 2014; Boggs, 2009; English and Barreaux, 2020; Nylin and Gotthard, 1998). Abiotic factors, such as temperature, influence the overall development, reproduction, and population dynamics of various ectotherms (Adamo and Lovett, 2011; Atkinson, 1994; Barton and Ives, 2014; Gourgoulianni et al., 2023), whereas biotic stressors, such as early resource or food limitation, can influence adult dispersal and reproductive capacities (Daniel et al., 2022; Chafino et al., 2019; Dillon et al., 2009; Zhang and Puniamoorthy, 2025). Studies investigating reproductive plasticity often focus on indicators vital for mate acquisition, that is, precopulatory traits such as overall body size and/or secondary sexual structures (Emlen, 1994; Harrison et al., 2023; Yap et al., 2024). The effects on traits that mediate fertilization success or post-copulatory reproductive success, such as sperm competition, mate guarding, or increased refractory periods, are less explored (Devost and Turgeon, 2016; Hagg et al., 2024; Himuro et al., 2022; Parker, 1970). Even sperm traits can be influenced by resource investments and affect reproductive fitness via gamete quantity and quality (Boggs, 1981, 2009; Macartney et al., 2018; Mehlis et al., 2015).
Here, we focus on Sepsis punctum (Diptera: Sepsidae), a widespread dung fly with natural populations distributed across North America and Europe (Pont and Meier, 2002). Adults are commonly found on ephemeral dung pats and exhibit sexual dimorphism. Prior studies have documented continental divergence in mating system dynamics and associated changes in mating behavior and sexual size dimorphism, with European populations expressing male-biased SSD and North American populations expressing female-biased SSD (Dmitriew and Blanckenhorn, 2012; Puniamoorthy et al., 2012a, b). These populations have likely experienced distinct evolutionary pressures, including varying climate regimes and intensities of sexual selection, making them ideal for testing whether plastic responses to stress are consistent or lineage-specific, although existing studies have mainly focused on single populations or within-continent variation (Berger et al., 2013, 2014; Lee et al., 2024). We subjected larvae from different populations to two common forms of developmental stress (elevated temperatures and reduced food availability) and quantified their effects on juvenile survival, development time, adult body size, and male postcopulatory traits: Testis size, a proxy for sperm quantity, and sperm length, a proxy for sperm quality (Simmons et al., 1999, 2003; Snook, 2005). By comparing responses across continents, sexes, and stress types, we aim to uncover the extent to which environmental stress alters fitness-related traits and whether reproductive investment is buffered against developmental perturbations.
We predicted that developmental stress would negatively impact fitness-related traits, but the magnitude would vary between continents, with larger-bodied European populations exhibiting greater sensitivity to stress-induced size reductions. Specifically, we expect increasing temperatures to reduce larval survival, shorten developmental time and reduce overall body size, consistent with the temperature-size rule, which predicts that ectotherms mature smaller at warmer temperatures owing to the differential thermal responses of growth and development rates (Atkinson, 1994; Sheridan and Bickford, 2011; Verberk et al., 2021). Food limitation may impose similar effects on survival and growth, but may lead to prolonged development owing to potential trade-offs in reproductive allocation (Gittleman and Thompson, 1988; Martin, 1987).
Methods
Fly cultures and maintenance
Here, we studied both European and North American populations of S. punctum. Laboratory stocks were established from wild-caught mixtures collected in 2018. These flies were allowed to mate freely within each container, expanding the population size to 200–400 adults per generation, and were kept for multiple (>15) generations before the experiments were conducted in 2019. All stock cultures were maintained under standardized laboratory conditions of 26°C, 60% humidity, and 14:10 L:D photoperiod with fresh untreated cow dung, sugar, and water provided ad libitum.
Experimental design
We conducted two independent experiments to evaluate the effects of (1) developmental temperature and (2) resource availability on survival, development, adult body size, and male reproductive traits of the wasps. Each experiment included four populations (two per continent) selected to span latitudinal and ecological gradients, with one overlapping European population (Italy) to facilitate cross-stressor comparisons (Figure 1). To obtain test individuals, stock cultures were provided with cattle dung ad libitum for 2 h to 3 h for females to oviposit. First-instar larvae from each population were split into replicates of 10 larvae, which were randomly distributed into different environmental treatments within 30 h of oviposition.
Figure 1. Experimental design. Locations of S. punctum populations in (A) Europe (blue) and (B) North America (red). Schematic summary of the two experiments conducted to assess the effects of environmental stress during larval development: higher temperatures and resource limitations (C). Head width measurements were taken as an indicator of body size, and male postcopulatory traits (testes size and sperm length) were scaled against body size (D). Source: Created in BioRender. https://BioRender.com/l95vat2.
Experiment 1: Thermal stress
Two European (Sweden, SW: 58.52°N, 16.84°E; Italy, IT: 42.56°N, 12.64°E) and two North American (USA, GA: 32.17°N, −82.90°W; USA, NY: 43.711°N, −76.977°E) populations were reared at four temperatures within an ecologically relevant range: 16°C, 20°C, 24°C, and 28°C (Berger et al., 2013; Dallo et al., 2024; Rohner and Berger, 2025). Temperatures above 30°C were excluded from this study because of their known detrimental effects on reproductive success (Berger et al., 2013). For each temperature × population combination, 100 larvae (10 replicates of 10 larvae each) were reared in ad libitum dung under controlled humidity and photoperiod (60% humidity, 14:10 L:D).
Experiment 2: Resource stress
Two European (Germany, DE: 49.48°N, 8.44°E; Italy, IT: 42.56°N, 12.64°E) and two North American (Canada, ON: 45.42°N, 75.67°W; USA, ND: 46.90°N, −96.80°W) populations were reared on five amounts of dung around previously defined thresholds: 1.5 g, 5.5 g, 9.5 g, 13.5 g, and 17.5 g, with 0.3 g per larvae previously recorded to cause substantially smaller adults (Berger et al., 2013; Blanckenhorn, 1998; Rohner et al., 2016). For each resource × population combination, 50 larvae (five replicates of 10 larvae each) were reared at a constant temperature, humidity, and photoperiod (26°C, 60% humidity, 14:10 L:D).
Life history traits
Larval survival and development time
Larval survival to adulthood, development time, and adult morphological traits were recorded for both experiments. For each replicate, the survival rate was calculated as the proportion of individuals that successfully emerged as adults. Development time was recorded as the number of days from oviposition to eclosion for each adult insect. Adult flies were morphologically sexed upon emergence and freeze-killed at −40°C for two to three days after eclosion before being preserved in 70% ethanol.
Adult morphological traits (body size and male postcopulatory traits)
We assessed whether adult body size and male post-copulatory traits (i.e., testis volume and sperm length) changed in response to environmental stress during larval development. We measured head width as a standard proxy for adult body size (Blanckenhorn et al., 2004; Puniamoorthy et al., 2012a, b) for both sexes using an Olympus SZX10 microscope. The testes of each male fly were dissected and transferred onto a glass slide with phosphate-buffered saline (PBS). The length (L) and width (W) of each testis were recorded to calculate the ellipsoid volume (V = 1/6 × L × W2 × π) (Blanckenhorn and Henseler, 2005). Subsequently, sperm were extracted from one testis on a fresh glass slide and fixed with a methanol: acetic acid (3:1) solution for 1 min before staining with 4’,6’-diamidino-2-phenylindole (DAPI) solution in the dark for 5 min. DAPI binds to nucleic acids, allowing the visualization of sperm cell DNA under an Olympus BX50 fluorescent microscope (Tanious et al., 1992). The stained sperm were mounted with one drop of glycerol before sealing with a cover slip and clear nail polish and dried in the dark. Ten sperm per male were measured (from head to tail) to obtain the average sperm length of each male using ImageJ (Abrámoff et al., 2004) and Micro-manager software (Edelstein et al., 2014).
Statistical analysis
We analyzed all data in R (version 4.0.5) (R Core Team, 2021), checking for normality with Shapiro–Wilk tests and Q–Q plots. Given the deviations from normality and the hierarchical structure of our experimental design, we used generalized linear mixed models (GLMMs) with the lmerTest package (Kuznetsova et al., 2017) or linear mixed-effects models (LMEs) with lme4 (Bates et al., 2015), depending on the response variable. For all models, we used the Akaike Information Criterion with an additional term to correct for small samples (AICc) (Burnham and Anderson, 2004) using the model.sel function from the MuMin R package (Barton, 2013) (ΔAICc <2). We checked the diagnostic plots to ensure that the model assumptions were met and removed outliers based on Cook’s distance. Figures were constructed using the ggplot2 package (Wickham, 2016). We assessed significance using the Kruskal–Wallis test (kruskal.test function in base R), followed by Dunn’s test using the dunn_test function from the rstatix package (Kassambara, 2023). We calculated the size dimorphism index (SDI) of Lovich and Gibbons (1992) using the means of each sex per treatment (Supplementary Table S1.1) and Dunn’s test to statistically test for the presence or absence of sexual size dimorphism.
Larval survival and development
We constructed candidate GLMMs with larval development time (days) and survival rate as separate response variables for each treatment. For larval development time, we used sex, population, and either temperature or resources as fixed effects and replicates as random effects. For survival rate, we used only population and either temperature or resource as fixed effects and replicates as random effects. All GLMMs were checked for any overdispersion.
Adult body size and male reproductive traits
For adult body size, we constructed a set of candidate LMEs with sex, population, and either temperature or resources as fixed effects and replicates as a random effect. For male reproductive traits, we constructed candidate LMEs with testis volume and sperm length as separate response variables and included population and either temperature or resources as fixed effects, replicates as a random effect, and body size as a covariate. We also analyzed residual reproductive investment by regressing reproductive traits on body size and using the residuals as response variables in the treatment models.
Allometric analyses
To evaluate changes in the reproductive allocation of male post-copulatory traits across populations under environmental stress (temperature/resource), we performed a log–log regression of linearized testis size and sperm length against body size and fitted global regression models across all treatment levels. We compared linear, quadratic, and cubic models using AICs and conducted separate regressions to assess geographic differences in allometric scaling.
Results
Thermal stress reduces survival and accelerates larval development across populations
Thermal stress significantly impacted survival and development time across populations, with the number of eclosed adults declining as temperature increased (Figure 2A; x2 3,160 = 63.091, p <0.001). With a total of 1,348 out of 1,616 larvae surviving to adulthood, the highest mortality was observed at 28°C, where survival dropped by up to 36% relative to 16°C (Table 1; GLMM, p <0.001). Populations from higher latitudes (NY and SW) also showed a sharp decrease in survivability (Figure 2A). Development time decreased with increasing temperature (Figure 2B; x2 3,1348 = 1,045.3, p <0.01), reflecting the expected acceleration of development under warmer conditions (Table 1; GLMM, p <0.001). Overall, at 28°C, flies emerged 37%–38% faster than at 16°C. We also observed an expected cross-continental reversal in development time between sexes in accordance with the differing SSD trends between continents (Figures 2B, C; NA, x2 1,661 = 8.4107, p <0.01; EU, x2 1,687 = 10.461, p <0.01).
Figure 2. Mean ± SE trends of thermal stress on S. punctum (A) larval survivability rate, (B) larval development time, (C) head width as a proxy for body size, (D) testis size, and (E) sperm length, along with the respective size-independent residuals (F, G) across four populations from two continents.
Table 1. Summary of best fit LME and GLMM models for S. punctum development and morphological trait measurements under thermal stress.
Thermal stress reduces adult body size and even sexual dimorphism at high latitudes
Adult body size, measured as head width, declined with increasing thermal stress across populations, consistent with the temperature-size rule (Figure 2C) (x2 3,1348 = 180.59, p <0.001) (Table 1, Supplementary Table S1.5). Consistent with previous studies, we observed a male-biased SSD in European populations and a female-biased SSD in North American populations (Figure 2C, Supplementary Tables S1.1, S1.7). European flies were larger than North American flies across temperatures, and their size declined more steeply with heat stress regardless of latitude (Table 1: EU populations × treatment, p <0.01). Three-way interactions between temperature, population, and sex were only significant at 28°C for the population at the highest latitude (SW), which even records a minimization of SSD from 24°C to 28°C (Figure 2C), suggesting a stronger effect of thermal selection than sexual selection on body size at high temperatures.
Thermal stress reduces reproductive investment with partial resilience in sperm quality but not quantity
Both absolute testis size (x2 3,643 = 112.42, p <0.001) and sperm length (x2 3,398 = 18.099, p <0.001) decreased with increasing temperatures across populations (Figures 2D, E). However, this was also attributed to the effect of body size rather than temperature alone (Table 1: LME, p <0.05). Most interestingly, when controlling for body size, only residual testis size showed a significant decline at high temperatures (x2 3,16 = 8.625, p <0.05; Table 1: temperature × population, p <0.01), while residual sperm length remained stable across temperatures in all but the highest latitude population (SW), although this was not significant (x2 3,16 = 4.9191, p >0.05) (Figures 2F, G). This phenotypic difference suggests that under thermal stress, males reduce sperm quantity but maintain their quality.
Resource stress has limited effects on larval survival but influences sex- and continent-specific development across populations
Of the 1,010 larvae, 704 survived to adulthood. Larval survival generally improved with increasing dung availability (Table 2), but there were no significant differences (x2 4,101 = 8.4082, p >0.05). Instead, there were population-level differences, with DE consistently showing the highest survival and ND the lowest (Figure 3A; x2 3,101 = 19.332, p <0.001). Hence, unlike thermal stress, food limitation did not significantly alter overall survival (Figure 3A, Table 2: GLMM, p >0.05) or systematically affect overall development time across continents (Table 2: LME, p >0.05). Instead, we documented sex- (x2 1,684 = 15.435, p <0.001) and continent-specific (x2 1,684 = 233.03, p <0.001) effects on larval development (Table 2). While North American flies show similar development rates in both sexes, European males exhibited significantly prolonged development under high-resource conditions, a trend that disappeared under severe food limitation due to sharper reductions in male size compared to females (Figure 3B, Supplementary Table S1.10).
Table 2. Summary of best fit LME and GLMM models for S. punctum development and morphological trait measurements under resource stress.
Figure 3. Mean ± SE trends of resource availability on S. punctum (A) larval survivability rate, (B) larval development time, (C) head width as a proxy for body size, (D) testis size, and (E) sperm length, along with the respective size-independent residuals (F, G) across four populations from two continents.
Resource stress reduces body size and affects size dimorphism across continents, but has limited effects on male postcopulatory traits
Similar to thermal stress, resource limitation also reduced adult body size across populations (Figure 3C) (Table 2, Supplementary Table S1.8; x2 4,684 = 274.83, p <0.001). However, the magnitude and direction of size reduction varied according to sex and continent. European males were generally larger than females (x2 1,684 = 236.42, p <0.001), although this difference disappeared under extreme food limitation (Figure 3C, Supplementary Tables S1.2, S1.10). In contrast, North American populations consistently exhibited female-biased SSD, regardless of food level (Figure 3C, Supplementary Tables S1.2, S1.10).
Similar to the temperature experiment, European populations maintained a larger testis size (x2 1,329 = 176.64, p <0.001) and sperm length (x2 1,208 = 154.89, p <0.001) than North American populations (Supplementary Table S1.9). Dunn’s tests indicated significant differences in testis size, mainly between the lowest (1.5 g) and higher dung availabilities (Supplementary Table S1.8, Figure 3D), whereas sperm length variation fluctuated. However, the best-fit models suggest that neither post-copulatory trait was significantly influenced by resource availability alone. Testis size was positively associated with body size (Table 2: LME, p <0.01), and sperm length was only significantly influenced by body size and resources for the IT population (Table 2). Unlike thermal stress, resource stress had no significant effect on size-independent investment in male postcopulatory traits, as evidenced by the residual variation in testis size (x2 4,20 = 2.5713, p >0.05) and sperm length (x2 4,20 = 2.2571, p >0.05) across treatments (Figures 3F, G).
Continental divergence in allometric scaling of reproductive traits under environmental stress
Under thermal stress, testis size and sperm length remained positively correlated with male body size, but allometric relationships diverged between continents, possibly reflecting the differing mating systems (Figure 4). For instance, best-fit models suggest that testis size scales non-linearly with body size in Europe, while North American populations maintained a linear trend (Figure 4A, Table 3). Even after accounting for body size, temperature still showed an independent negative influence on residual (i.e., size-controlled) testis size (Figure 2F; x2 3,16 = 8.625, p <0.05). In contrast, sperm length relationships did not differ between continents (Figure 4B, Table 3), although low R2 values suggest that body size did not fully explain the variability in sperm length. Indeed, residual sperm length showed similar trends to absolute sperm length across temperatures (Figures 2E, G; x2 3,16 = 4.919, p >0.05).
Figure 4. Allometric relationships between S. punctum body size and testis size (A, C) or sperm length (B, D) when exposed to thermal (A, B) or resource stress (C, D). Cross-stressor comparisons using Italy as the focal population showed diverging allometric trajectories in the testes (E), and particularly in sperm (F). The dotted gray lines indicate an isometric slope of 1.
Table 3. Allometric equations for male postcopulatory traits under environmental stress.
In contrast, resource stress induced population-specific scaling in testis size instead of a clear continental effect, indicating local adaptations. Resource availability showed no independent effect on residual testis size (Figure 3F, x2 4,20 = 2.257, p >0.05). Sperm length showed a positive hypo-allometric relationship with body size, similar to the temperature treatments (Figure 4D, Table 3), but the residual variation indicated no clear trend with resource availability (Figures 2E, G, x2 3,20 = 0.703, p >0.05). Overall, North American flies displayed more variable scaling relationships without a clear pattern across treatments, unlike the significantly stronger allometries observed in European flies, suggesting that European populations exhibit greater plasticity and potentially greater constraints on reproductive investment under environmental stress.
Reproductive investments may be less dependent on body size under increased stress to maintain reproductive advantages, and cross-stressor comparisons suggest that increased temperatures are more stressful, particularly for sperm length (Figures 4E, F). S. punctum sperm length scales hypo-allometrically with body size under optimal circumstances (Lee et al., 2024); however, b was much shallower under warmer temperatures than under dung restriction (Table 3).
Discussion
Here, we examined the effects of environmental stress, specifically high larval developmental temperature and food limitation, on S. punctum populations from North America and Europe. We show that developmental stress during the juvenile stage can have profound consequences on adult reproductive traits and highlight geographic and stress-specific patterns of plasticity.
Developmental stress alters fitness-related traits, with temperature exerting stronger effects than resource
We demonstrated that developmental stress from elevated temperatures and resource limitation significantly alters S. punctum life-history traits, but with divergent effects depending on the type of stress. Thermal stress imposed the strongest fitness costs, reducing larval survival, accelerating development, and leading to smaller adult body sizes. This aligns with the predictions of thermal reaction norms and the temperature-size rule (Atkinson, 1994), mirroring the stress responses observed in other holometabolous insects (Sheridan and Bickford, 2011; Verberk et al., 2021). In contrast, food limitation had minimal impact on larval survival, suggesting that S. punctum larvae may be buffered against short-term nutrient deficits, which is a possible adaptation to ephemeral and patchy dung substrates in their natural habitat (Blanckenhorn, 1998).
Interestingly, while thermal stress consistently shortened the development time, food limitation did not produce uniform effects. In contrast, larger European males exhibited stark reductions in development time under severe resource scarcity compared to their smaller female counterparts, suggesting sex-specific trade-offs in growth and reproduction. Emerging earlier and smaller with increasing resource limitation is a generally rare life-history response (Stearns and Koella, 1986), but is common for species inhabiting heterogeneous environments, such as dung pats, where quantity and quality vary unpredictably in time and space (Blanckenhorn, 1998). While prolonged development increases body size and reproductive fitness, the benefits of reducing time in stressful larval environments and early breeding may outweigh the disadvantages of being small (Reim et al., 2006; Roff, 1992).
Geographic divergence in plasticity may influence climate resilience
Plastic responses to environmental stressors appear to be mediated by population background. Across the latitudinal gradient, we did not find resource-induced patterns in life history traits, similar to Berger et al. (2014), although lower latitude populations showed higher thermal tolerance, particularly with regard to survivability and sperm length. However, previous studies have also highlighted that these patterns in thermal tolerance may be broadly explained by the extreme temperatures experienced within different localities (Berger et al., 2014; Sunday et al., 2019).
Instead of a longitudinal gradient, we observed a sharp divergence in size-based sexual selection between North American and European populations, consistent with previous studies. Differential investment in pre- vs. postcopulatory sexual selection led to male-biased SSD in Europe, with stronger sexual selection on male size and sperm production, as reflected here, and female-biased SSD in North America, with males investing more in precopulatory mate acquisition (Dmitriew and Blanckenhorn, 2012; Puniamoorthy et al., 2012a, b; Rohner et al., 2016). European flies are also generally larger than North American flies (Puniamoorthy et al., 2012a, b), which in turn influenced the strength of plasticity responses between continents, consistent with previous invertebrate studies (Tseng et al., 2018; Wonglersak et al., 2021), with larger European flies showing sharper decreases in body and testis sizes in response to environmental stress.
Interestingly, while continent-specific SSD trends were maintained across extreme temperatures, this was not the case under resource limitations. The loss of male-biased SSD in European populations under low food conditions indicates that developmental constraints can override sexually selected growth advantages when resources are limited (Miller et al., 2016) and that the larger sex tends to be more sensitive to resource stress (Teder and Kaasik, 2023).
Partial resilience in male post-copulatory trait investment against environmental stress
Sperm quantity and quality are vital for male reproductive fitness (Crean and Immler, 2021; Pattarini et al., 2006; Pitnick, 1996). These traits play crucial roles in post-copulatory courtship, where either intrasexual sperm competition or cryptic female choice occurs (Macartney et al., 2019; Snook, 2005). After accounting for the effects of body size, only the effect of temperature on testis size (a proxy for sperm quantity) remained significant, suggesting that these dung flies are better adapted to compensate for resource constraints over extreme temperatures (Gourgoulianni et al., 2025; Hellriegel and Blanckenhorn, 2002). Investment in male post-copulatory traits may be buffered or canalized against resource variation to maintain reproductive advantages (Lee et al., 2024; Macartney et al., 2019; Waddington, 1942; Wagner et al., 1997).
Testis size investment was reduced with increasing temperatures in accordance with Bergmann’s rule extended to ectotherms (Atkinson and Sibly, 1997; Blanckenhorn and Henseler, 2005; Hellriegel and Blanckenhorn, 2002). While both sperm quantity and quality contribute to fertilization success, it is theorized trade-offs may occur under physical or energetic constraints (Parker, 1970; Pattarini et al., 2006; Pitnick, 1996). Testis size, a common proxy for sperm quantity, may be more condition-dependent owing to body size constraints under temperature stress. Although classic sperm competition theory focuses on sperm quantity advantages, sperm quality also plays an important role in post-copulatory sexual selection (Crean and Immler, 2021; Macartney et al., 2019; Pattarini et al., 2006). Our results suggest the potential canalization of sperm quality and strategic resource allocation trading off quantity under stress.
Conclusion: implications for climate change resilience
In widespread species exposed to varying environments, responses to environmental stress vary owing to local adaptations (Berger et al., 2013, 2014; Chakraborty et al., 2023), with implications for species persistence with global change. Here, we documented phenotypic plasticity in S. punctum life-history traits in response to temperature and resource stress, the strength of which varied between the type of stress and geographic lineage. Reproductive investment in males may be less sensitive to environmental stress than developmental investment, as body size-independent investment in male post-copulatory traits was not affected by environmental stress, except for testis size at higher temperatures. This suggests that warmer temperatures could be more stressful than resource limitations for dung flies accustomed to ephemeral dung pats. While we tested development under constant environmental parameters, this may not reflect field conditions, as wild populations experience frequent temperature variations across their lifetimes. Future studies should explore fluctuating stress regimes that are more ecologically relevant, with possible consequences for trait expression (Hoffmann et al., 2003; Kjærsgaard et al., 2013). We also acknowledge that we only studied reproductive post-copulatory investments in males, and analyzing the impacts of environmental stress on females is crucial for the persistence of the species as a whole because of sex-specific ecological repertoires (Kjærsgaard et al., 2013; Lande, 1980). Additionally, investigating transgenerational variation in a quantitative genetic framework would improve our understanding of how natural populations respond to changing environments.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.
Ethics statement
The manuscript presents research on animals that do not require ethical approval for their study.
Author contributions
NLYL: Methodology, Data curation, Formal analysis, Project administration, Visualization, Writing – original draft. JWT: Investigation, Data curation, Formal analysis, Writing – original draft. YW: Investigation, Formal analysis, Writing – original draft. TRHT: Formal analysis, Project administration, Validation, Visualization, Writing – review & editing. NP: Conceptualization, Funding acquisition, Resources, Methodology, Supervision, Visualization, Writing – review & editing.
Funding
The author(s) declare that financial support was received for the research and/or publication of this article. This paper was supported by undergraduate research funding (UROPs) from the Department of Biological Sciences, National University of Singapore as well as a Ministry of Education Tier 1 grant (R-154-000-A75-114) awarded to Nalini Puniamoorthy.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declare that no Generative AI was used in the creation of this manuscript.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fevo.2025.1711105/full#supplementary-material
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Keywords: condition dependence, phenotypic plasticity, thermal stress, resource limitation, postcopulatory traits, reproduction, population, Diptera
Citation: Lee NLY, Tan JW, Wang YB, Tan TRH and Puniamoorthy N (2025) Continental divergence in male reproductive plasticity to thermal and resource stress in a widespread dung fly. Front. Ecol. Evol. 13:1711105. doi: 10.3389/fevo.2025.1711105
Received: 23 September 2025; Accepted: 20 November 2025; Revised: 12 November 2025;
Published: 12 December 2025.
Edited by:
Carolina Bravo, UMR7372 Centre d’études Biologiques de Chizé (CEBC), FranceReviewed by:
Pedro Simões, University of Lisbon, PortugalJorge Ari Noriega, University of Los Andes, Colombia
Copyright © 2025 Lee, Tan, Wang, Tan and Puniamoorthy. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Nalini Puniamoorthy, bmFsaW5pQG51cy5lZHUuc2c=