Microplastics are an emerging environmental hazard on a global scale. Their detection in agricultural environments is of particular concern because microplastics may negatively impact insect detritivores and their ecosystem functioning. Dung beetles are important detritivores and are often vulnerable to anthropogenic hazards. Here, we test whether artificial contamination of cow dung with thermoplastic polyurethane (TPU) microplastics affects juvenile development and maternal behavior in the bull-headed dung beetle Onthophagus taurus. Larvae exposed to dung containing ≥0.5 mg g-1 TPU microplastics experienced high mortality, whereas exposure to 0.1 mg g-1 TPU did not significantly increase mortality risk relative to controls. Despite these strong effects on larval survival, adult females were equally likely to provision offspring with TPU-contaminated (and lethal) dung as with uncontaminated control dung. This suggests that females cannot differentiate between highly toxic microplastic-contaminated and uncontaminated resources. Together, these results indicate the potential for severe negative impacts on dung beetle populations if TPU microplastics persist and accumulate in agricultural environments. However, environmentally relevant exposure levels remain unknown. Future work should quantify microplastic concentrations in the field, test the effects of microplastic composition and size distribution, and identify the mechanisms underlying TPU-induced toxicity. These avenues will be critical for evaluating the long-term consequences of microplastic pollution for insect-mediated ecosystem functioning.

The microbiome is central to host development and adaptation, yet the balance between vertical and environmental acquisition, and how hosts shape surrounding microbial communities, remains poorly understood. Dung beetles rely on microbial symbionts to extract nutrients from vertebrate dung, with part of their microbiome vertically inherited via a maternal faecal pellet. However, the relative importance of vertical versus horizontal transmission is unclear. We examined this in the gazelle dung beetle (Digitonthophagus gazella), rearing larvae on brood balls made of dung from grass-(high-quality), hay-(low-quality) or silage-fed (a novel fermentable energy-rich diet) cattle, with or without maternal microbes. We integrated measures of gut morphology with 16S rRNA amplicon sequencing to assess host development and the gut microbiome. Diet significantly influenced overall size, hindgut area, and microbiome composition. Silage-dung fed larvae had more even and taxonomically rich microbiomes, with higher microbial diversity in individuals reared with maternal microbes. Diet explained ~26% of the variation in microbial composition, while the vertical transmission of microbes only explained 3%. Vertical transmission only slightly increases microbial species richness and relative hindgut area but did not influence overall microbial diversity. The larval brood ball contributed 40%-50% of the hindgut microbiome, while maternal microbes contributed < 0.05%. These findings demonstrate that horizontal acquisition through diet is the dominant force shaping larval gut microbiomes, while vertical inheritance plays a minor but detectable role in enhancing richness and gut development. More broadly, this work reinforces the importance of examining host-microbiome-environment interactions in ecological and evolutionary contexts.

Evolutionary theory predicts that variation in longevity persists due to trade-offs between early-life fitness traits (e.g., growth or fecundity) and long-term somatic maintenance. However, such trade-offs can be difficult to detect and may often become apparent only under certain conditions. For instance, developing in novel or atypical environments may alter the genetic architecture of traits, revealing trade-offs that are otherwise hidden under normal conditions. To test this, we compared full-sibling families of the Mexican spadefoot (Spea multiplicata) reared across two larval diets: a typical detritus diet and an atypical live shrimp diet, which they are competitively excluded from in nature. The shrimp diet significantly increased broad-sense genetic variance and heritability for larval growth rate, whereas heritability for post-metamorphic telomere length-a known longevity correlate-remained similar across diets. Moreover, only on the shrimp diet did families with faster growth exhibit shorter telomeres, consistent with a diet-dependent trade-off between growth and somatic maintenance. Overall, our study shows that developing under atypical dietary conditions exposes previously cryptic genetic variation in growth, thereby revealing a trade-off with somatic maintenance. These findings have implications for understanding how environmental change, such as rapid dietary shifts, can shape aging processes and vulnerability to age-related disease.

Abstract Rapid adaptation in complex environments depends not only on the amount of genetic variation, but also on patterns of covariation among traits targeted by selection. Anthropogenic stressors create rapidly changing and multifaceted environments and provide powerful systems in which to investigate the potential for adaptation to multiple coinciding stressors. We investigate the combined effects of heat and chemical stress on survival in the black scavenger fly, Sepsis neocynipsea , and determine the genetic basis for resistance. In a fully factorial experiment, we expose isofemale lines to combinations of heat stress and ivermectin, a veterinary antiparasitic to which these flies are naturally exposed in agricultural landscapes. Using a Bayesian quantitative genetic approach, we estimate broad-sense genetic variation and cross-environmental genetic correlations. First, we show that these two stressors have synergistic effects on survival, with heat stress exacerbating the lethal effects of ivermectin. Second, we find that the largest component of genetic variation is the response to heat and ivermectin in combination (genotype-by-environment-by-environment; G×E×E). Third, cross-environmental genetic correlations are weak, implying that relative genetic performance is dependent on the specific combination of stressors. Together, these results suggest that incorporating G×E×E is essential for understanding adaptive potential in multi-stressor environments.

Coprophagous insects frequently encounter veterinary medication residues that are excreted unmetabolized in livestock dung. These residues often negatively affect insect survival, reproduction, and ecosystem services and may contribute to the rapid decline in insect populations. Ivermectin is an antiparasitic drug widely used to treat parasites in livestock. While it has long been recognized that ivermectin residues affect insect survival, the potential interactive effects between ivermectin exposure and other ecologically relevant abiotic stressors remain poorly understood. Here, we study these effects in the black scavenger fly Sepsis neocynipsea, which depends on cow dung for reproduction. Using a fully factorial experimental design, we test whether the effects of ivermectin exposure on adult survival interact with heat and desiccation stress and whether the severity of these effects depends on an individual's size and sex. We found that ivermectin exposure had strong negative impacts on adult survival overall, but that mortality was approximately three times higher in females compared to males. The combination of ivermectin exposure, heat, and desiccation stress drastically reduced survival. Interestingly, individuals simultaneously exposed to heat and ivermectin stress survived better-on average- than individuals only exposed to ivermectin, suggesting potential hormetic effects. Taken together, our findings highlight how the complex interactions between veterinary pharmaceuticals and abiotic stressors could drive changes in coprophagous insect populations and their ecological functions.

Microplastics are an emerging environmental hazard on a global scale. Their detection in agricultural environments is of particular concern not only for food contamination, but also because microplastics negatively impact detritivores and their ecosystem functioning. Dung beetles in particular provide vital ecosystem services in agricultural environments and are often vulnerable to anthropogenic hazards, but whether they are affected by microplastics remains unclear. Here, we test whether artificial contamination of cow dung with thermoplastic polyurethane (TPU) has the potential to affect the juvenile development and maternal behavior of the bull-headed dung beetle Onthophagus taurus . Dung beetles exhibited high mortality when exposed to elevated concentrations of TPU. In addition, females were equally likely to provision offspring with TPU-spiked (and lethal) cow dung as with control dung, suggesting that females cannot differentiate between highly toxic microplastic-contaminated and uncontaminated cow dung. Our findings highlight potentially severe consequences for dung beetles if microplastics persist and accumulate, although the levels of exposure in the field are unknown. Although the direct environmental hazards and the mechanisms mediating the negative impacts of TPU microplastics remain to be assessed, this study suggests that microplastics may negatively impact dung beetles and their ecosystem services. Future work assessing exposure levels in the field as well as dung beetles’ potential to evolve resistance against microplastic pollution will be necessary to assess the long-term impact of microplastic presence on dung beetle ecosystem functioning. ### Competing Interest Statement The authors have declared no competing interest.

Evolutionary change necessitates genetic variation, and a reigning paradigm in biology is that rates of microevolution can be predicted from estimates of available genetic variation within populations. However, the accuracy of such predictions should decay on longer evolutionary timescales, as the influence of genetic constraints diminishes. Here, we show that intrinsic developmental variability and standing genetic variation in wing shape in two distantly related flies, Drosophila melanogaster and Sepsis punctum, are aligned and predict deep divergence in the dipteran phylogeny, spanning >900 taxa and 185 My. This alignment cannot easily be explained by constraint hypotheses unless most of the quantified standing genetic variation is associated with deleterious side-effects and effectively unusable for evolution. However, phenotyping of 71 genetic lines of S. punctum revealed no covariation between wing shape and fitness, lending no support to this hypothesis. We also find little evidence for genetic constraints on the pace of wing shape evolution along the dipteran phylogeny. Instead, correlational selection related to allometric scaling, simultaneously shaping developmental bias and deep divergence in fly wings, emerges as a potential explanation for the observed alignment. This suggests that pervasive natural selection has the potential to shape developmental architectures of some morphological characters such that their intrinsic variability predicts their long-term evolution. ### Competing Interest Statement The authors have declared no competing interest.

Climate adaptation in insects can proceed via responses in life-history traits and their thermal plasticity and through phenological shifts mediated by responses to photoperiodic cues (photoperiodism). While experimental studies demonstrate evolutionary potential for both modes of adaptation, it remains unclear how evolution will unfold in natural populations, limiting our ability to predict how insects will respond to climate change. Here, we review the literature and analyze published studies revealing that photoperiodism for diapause induction evolves predictably along latitude, with high-latitude populations entering diapause earlier. In contrast, although a few species showed clinal variation in life history and thermal plasticity, the direction of these clines was not consistent across taxa. These findings suggest that while insect life history and physiological adaptation to temperature can evolve, phenological shifts via evolution of photoperiodism are likely to be more common and predictable responses to future climate change.

Evolutionary change requires genetic variation, and a reigning paradigm in biology is that rates of microevolution can be predicted from estimates of available genetic variation within populations. However, the accuracy of such predictions should decay on longer evolutionary timescales, as the influence of genetic constraints diminishes. Here we show that intrinsic developmental variability and standing genetic variation in wing shape in two distantly related flies, Drosophila melanogaster and Sepsis punctum , are aligned and predict deep divergence in the dipteran phylogeny, spanning >900 taxa and 185 million years. This alignment cannot be easily explained by constraint hypotheses unless most of the quantified standing genetic variation is associated with deleterious side effects and is effectively unusable for evolution. However, phenotyping of 71 genetic lines of S. punctum revealed no covariation between wing shape and fitness, lending no support to this hypothesis. We also find little evidence for genetic constraints on the pace of wing shape evolution along the dipteran phylogeny. Instead, correlational selection related to allometric scaling, simultaneously shaping developmental variability and deep divergence in fly wings, emerges as a potential explanation for the observed alignment. This suggests that pervasive natural selection has the potential to shape developmental architectures of some morphological characters such that their intrinsic variability predicts their long-term evolution.

Abstract Selection on standing heritable variation and novel mutations can fuel adaptive evolutionary responses to climate change in insect morphology, behavior, physiology and life history. Although adaptation has been regarded as a slow process, more recent studies highlight that insects have the potential to adapt rapidly to climatic challenges. However, most insects also show environment-dependent trait expression (i.e., phenotypic plasticity), which can affect phenotypes within an individual’s lifetime. While such responses can be adaptive, they may also hamper evolutionary responses to environmental change. In addition, plasticity itself can evolve and bias evolution via genetic accommodation. The interactions between plastic and genetic responses are therefore manifold and complex. Integrating quantitative genetic and developmental perspectives, this chapter illustrates plastic and genetic responses of insects to climate change. Special focus is placed on the role of developmental plasticity and genetic accommodation in evolution.

Developmental plasticity is an important product of evolutionary processes, allowing organisms to maintain high fitness in the face of environmental perturbations. Once evolved, plasticity also has the potential to influence subsequent evolutionary outcomes, for example, by shaping phenotypic variation visible to selection and facilitating the emergence of novel trait variants. Furthermore, organisms may not just respond to environmental conditions through plasticity but may also actively modify the abiotic and (sym)biotic environments to which they themselves respond, causing plasticity to interact in complex ways with niche construction. Here, we explore developmental mechanisms and evolutionary consequences of plasticity in horned dung beetles. First, we discuss how post-invasion evolution of plasticity in an introduced Onthophagus species facilitated rapid range expansion and concurrent local adaptation of life history and morphology to novel climatic conditions. Second, we discuss how, in addition to plastically responding to variation in nutritional conditions, dung beetles engage in behaviors that modify the environment that they themselves respond to during later development. We document that these environment-modifying behaviors mask heritable variation for life history traits within populations, thereby shielding genetic variants from selection. Such cryptic genetic variation may be released and become selectable when these behaviors are compromised. Together, this work documents the complex interactions between plasticity, symbionts and niche construction, and highlights the usefulness of an integrative Eco-Evo-Devo framework to study the varied mechanisms and consequences of plasticity in development and evolution.

Diverse organisms actively manipulate their (sym)biotic and physical environment in ways that feed back on their own development. However, the degree to which these processes affect microevolution remains poorly understood. The gazelle dung beetle both physically modifies its ontogenetic environment and structures its biotic interactions through vertical symbiont transmission. By experimentally eliminating (i) physical environmental modifications and (ii) the vertical inheritance of microbes, we assess how environment modifying behaviour and microbiome transmission shape heritable variation and evolutionary potential. We found that depriving larvae of symbionts and environment modifying behaviours increased additive genetic variance and heritability for development time but not body size. This suggests that larvae's ability to manipulate their environment has the potential to modify heritable variation and to facilitate the accumulation of cryptic genetic variation. This cryptic variation may become released and selectable when organisms encounter environments that are less amenable to organismal manipulation or restructuring. Our findings also suggest that intact microbiomes, which are commonly thought to increase genetic variation of their hosts, may instead reduce and conceal heritable variation. More broadly, our findings highlight that the ability of organisms to actively manipulate their environment may affect the potential of populations to evolve when encountering novel, stressful conditions.

Abstract Many symbionts are sexually transmitted and impact their host's development, ecology, and evolution. While the significance of symbionts that cause sexually transmitted diseases (STDs) is relatively well understood, the prevalence and potential significance of the sexual transmission of mutualists remain elusive. Here, we study the effects of sexually transmitted mutualist nematodes on their dung beetle hosts. Symbiotic Diplogastrellus monhysteroides nematodes are present on the genitalia of male and female Onthophagus beetles and are horizontally transmitted during mating and vertically passed on to offspring during oviposition. A previous study indicates that the presence of nematodes benefits larval development and life history in a single host species, Onthophagus taurus. However, Diplogastrellus nematodes can be found in association with a variety of beetle species. Here, we replicate these previous experiments, assess whether the beneficial effects extend to other host species, and test whether nematode‐mediated effects differ between male and female host beetles. Rearing three relatively distantly related dung beetle species with and without nematodes, we find that the presence of nematodes benefits body size, but not development time or survival across all three species. Likewise, we found no difference in the benefit of nematodes to male compared to female beetles. These findings highlight the role of sexually transmitted mutualists in the evolution and ecology of dung beetles.

Insects associated with livestock dung frequently encounter veterinary medication residues. These residues often have negative effects on insect survival, reproduction, and ecosystem functioning and may contribute to the rapid decline in temperate insect populations. Ivermectin is an antiparasitic drug widely used to treat parasites in livestock. While it has long been recognized that ivermectin effects insect survival, the potential interactive effects between ivermectin exposure and other ecologically relevant abiotic stressors remain poorly understood. Here, we study these effects in the black scavenger fly Sepsis neocynipsea, which depends on cow dung for reproduction. Using a fully factorial design, we test whether the effects of ivermectin exposure on adult survival interact with heat and desiccation stress, and whether these effects depend on size and sex. Ivermectin exposure had strong negative impacts on adult survival, but its effects were stronger in females and large individuals. While heat stress also had a strong effect on adult survival, the combined effects of heat and ivermectin exposure were less severe than the expected additive effects of both stressors applied independently, suggesting some cross-resistance. We did not find an interaction between ivermectin and desiccation stress. Taken together, our findings highlight how the complex interactions between insecticides and abiotic stressors could drive changes in coprophagous insect populations and their ecological functions across different ecosystems and climates. ### Competing Interest Statement The authors have declared no competing interest.

Many organisms actively manipulate the environment in ways that feed back on their own development, a process referred to as developmental niche construction. Yet, the role that constructed biotic and abiotic environments play in shaping phenotypic variation and its evolution is insufficiently understood. Here, we assess whether environmental modifications made by developing dung beetles impact the environment-sensitive expression of secondary sexual traits. Gazelle dung beetles both physically modify their ontogenetic environment and structure their biotic interactions through the vertical inheritance of microbial symbionts. By experimentally eliminating (i) physical environmental modifications and (ii) the vertical inheritance of microbes, we assess the degree to which (sym)biotic and physical environmental modifications shape the exaggeration of several traits varying in their degree and direction of sexual dimorphism. We expected the experimental reduction of a larva's ability to shape its environment to affect trait size and scaling, especially for traits that are sexually dimorphic and environmentally plastic. We find that compromised developmental niche construction indeed shapes sexual dimorphism in overall body size and the absolute sizes of male-limited exaggerated head horns, the strongly sexually dimorphic fore tibia length and width, as well as the weakly dimorphic elytron length and width. This suggests that environmental modifications affect sex-specific phenotypic variation in functional traits. However, most of these effects can be attributed to nutrition-dependent plasticity in size and non-isometric trait scaling rather than body-size-independent effects on the developmental regulation of trait size. Our findings suggest that the reciprocal relationship between developing organisms, their symbionts, and their environment can have considerable impacts on sexual dimorphism and functional morphology.

Plastic responses to environmental conditions may themselves depend on other environmental conditions, but how such environment-by-environment (E×E) interactions may impact evolution remains unclear. We investigate how temperature shapes the nutritional polyphenism in horn length in a beetle and test whether "allometric plasticity" (a form of E×E) predicts latitudinal differentiation during a rapid range expansion. Rearing populations under common garden conditions demonstrates that increased temperatures reduce the body size threshold separating two male morphs in all populations but also that the magnitude of temperature-dependent changes in allometry diverged across recently established populations. Furthermore, we found a latitudinal increase in the threshold in the species' exotic range at one of the temperatures, suggesting that allometric plasticity in response to temperature may predict evolved clinal differences. Our findings demonstrate that E×E interactions can be similar in magnitude to G×E interactions and that allometric plasticity and its evolution may impact population's responses to environmental changes.

Static allometry is a major component of morphological variation. Much of the literature on the development of allometry investigates how functional perturbations of diverse pathways affect the relationship between trait size and body size. Often, this is done with the explicit objective to identify developmental mechanisms that enable the sensing of organ size and the regulation of relative growth. However, changes in relative trait size can also be brought about by a range of other distinctly different developmental processes, such as changes in patterning or tissue folding, yet standard univariate biometric approaches are usually unable to distinguish among alternative explanations. Here, we utilize geometric morphometrics to investigate the degree to which functional genetic manipulations known to affect the size of dung beetle horns also recapitulate the effect of horn shape allometry. We reasoned that the knockdown phenotypes of pathways governing relative growth should closely resemble shape variation induced by natural allometric variation. In contrast, we predicted that if genes primarily affect alternative developmental processes, knockdown effects should align poorly with shape allometry. We find that the knockdown effects of several genes (e.g., doublesex, Foxo) indeed closely aligned with shape allometry, indicating that their corresponding pathways may indeed function primarily in the regulation of relative trait growth. In contrast, other knockdown effects (e.g., Distal-less, dachs) failed to align with allometry, implicating these pathways in potentially scaling-independent processes. Our findings moderate the interpretation of studies focusing on trait length and highlight the usefulness of multivariate approaches to study allometry and phenotypic plasticity.

Male sexual ornaments often evolve rapidly and are thought to be costly, thus contributing to sexual size dimorphism. However, little is known about their developmental costs, and even less about costs associated with structural complexity. Here, we quantified the size and complexity of three morphologically elaborate sexually dimorphic male ornaments that starkly differ across sepsid fly species (Diptera: Sepsidae): (i) male forelegs range from being unmodified, like in most females, to being adorned with spines and large cuticular protrusions; (ii) the fourth abdominal sternites are either unmodified or are converted into complex de novo appendages; and (iii) male genital claspers range from small and simple to large and complex (e.g. bifurcated). We tracked the development of 18 sepsid species from egg to adult to determine larval feeding and pupal metamorphosis times of both sexes. We then statistically explored whether pupal and adult body size, ornament size and/or ornament complexity are correlated with sex-specific development times. Larval growth and foraging periods of male and female larvae did not differ, but the time spent in the pupal stage was ca 5% longer for sepsid males despite emerging 9% smaller than females on average. Surprisingly, we found no evidence that sexual trait complexity prolongs pupal development beyond some effects of trait size. Evolving more complex traits thus does not incur developmental costs at least in this system.

The degree to which developmental biases affect trait evolution is subject to much debate. Here, we first quantify fluctuating asymmetry as a measure of developmental variability, i.e., the propensity of developmental systems to create some phenotypic variants more often than others, and show that it predicts phenotypic and standing genetic variation as well as deep macroevolutionary divergence in wing shape in sepsid flies. Comparing our data to the findings of a previous study demonstrates that developmental variability in the sepsid fly Sepsis punctum strongly aligns with mutational, standing genetic, and macroevolutionary variation in the Drosophilidae--a group that diverged from the sepsid lineage ca. 64 My ago. We also find that developmental bias in S. punctum wing shape aligns with the effects of allometry, but less so with putatively adaptive thermal plasticity and population differentiation along latitude. Our findings demonstrate that developmental bias in fly wings predicts evolvability and macroevolutionary trajectories on a much greater scale than previously appreciated but also suggest that causal explanations for such alignments may go beyond simple constraint hypotheses.

Directional sexual selection drives the evolution of traits that are most closely linked to reproductive success, giving rise to trait exaggeration and sexual dimorphism. Exaggerated structures are often costly and, therefore, thought to be expressed in a condition-dependent manner. Sexual selection theory thus predicts a direct link between directional sexual selection, sexual dimorphism, and sex-specific condition dependence. However, only a handful of studies investigate the relationship between sexual dimorphism and condition dependence. Using 21 genetic lines of Drosophila prolongata , we here compared the degree of sexual dimorphism and sex-specific condition dependence, measured as allometric slopes, in sexually selected and non-sexual traits. Our data revealed male-biased sexual dimorphism in all traits examined, most prominently in the sexually selected forelegs. However, there was no relationship between the degree of sex-specific condition dependence and sexual dimorphism across traits and genetic lines. Our results contradict theoretical predictions and highlight the importance of understanding the role of exaggerated traits in the context of both sexual and natural selection.