An association between Dnmt1 and Wnt in the production of oocytes in the whitefly Bemisia tabaci.

The function of DNA methylation in insects and the DNA methyltransferase (Dnmt) genes that influence methylation remains uncertain. We used RNA interference to reduce the gene expression of Dnmt1 within the whitefly Bemisia tabaci (Hemiptera:Aleyrodidae; Gennadius), a hemipteran species that relies on Dnmt1 for proper gametogenesis. We then used RNA-seq to test an a priori hypothesis that meiosis-related genetic pathways would be perturbed. We generally did not find an overall effect on meiosis-related pathways. However, we found that genes in the Wnt pathway, genes associated with the entry into meiosis in vertebrates, were differentially expressed. Our results are consistent with Dnmt1 knockdown influencing specific pathways and not causing general transcriptional response. This is a finding that is also seen with other insect species. We also characterised the methylome of B. tabaci and assessed the influence of Dnmt1 knockdown on cytosine methylation. This species has methylome characteristics comparable to other hemipterans regarding overall level, enrichment within gene bodies, and a bimodal distribution of methylated/non-methylated genes. Very little differential methylation was observed, and difference in methylation were not associated with differences in gene expression. The effect on Wnt presents an interesting new candidate pathway for future studies.

Offspring overcome poor parenting by being better parents.

The evolutionary repercussions of parental effects-the impact of the developmental environment provided by parents on offspring-are often discussed as static effects that can have negative influences on offspring fitness that may even persist across generations. However, individuals are not passive recipients and may mitigate the persistence of parental effects through their behaviour. Here, we tested how the burying beetle, Nicrophorus orbicollis, a species with complex parental care, responded to poor parenting. We cross-fostered young and manipulated the duration of parental care received and measured the impact on traits of both F1 and F2 offspring to experimentally extricate the effect of poor parenting from other parental effects. As expected, reducing parental care negatively affected traits that are ecologically important for burying beetles, including F1 offspring development time and body size. However, F1 parents that received reduced care as larvae spent more time feeding F2 offspring than parents that received full care as larvae. As a result, both the number and mass of F2 offspring were unaffected by the developmental experience of their parents. Our results show that flexible parental care may be able to overcome poor developmental environments and limit negative parental effects to a single generation.

Runaway evolution from male-male competition.

Wondrously elaborate weapons and displays that appear to be counter to ecological optima are widespread features of male contests for mates across the animal kingdom. To understand how such diverse traits evolve, here we develop a quantitative genetic model of sexual selection for a male signaling trait that mediates aggression in male-male contests and show that an honest indicator of aggression can generate selection on itself by altering the social environment. This can cause selection to accelerate as the trait is elaborated, leading to runaway evolution. Thus, an evolving source of selection provided by the social environment is the fundamental unifying feature of runaway sexual selection driven by either male-male competition or female mate choice. However, a key difference is that runaway driven by male-male competition requires signal honesty. Our model identifies simple conditions that provide clear, testable predictions for empirical studies using standard quantitative genetic methods.

Gene expression underlying parenting and being parented shows limited plasticity in response to different ambient temperatures.

Flexible interactions between parents and offspring are essential for buffering families against variable, unpredictable, and challenging environmental conditions. In the subsocial carrion beetle, Nicrophorus orbicollis, mid-summer temperatures impose steep fitness costs on parents and offspring but do not elicit behavioural plasticity in parents. Here, we ask if plasticity of gene expression underpins this behavioural stability or facilitates independent compensation by larvae. To test this, we characterized gene expression of parents and offspring before and during active parenting under benign (20°C) and stressful (24°C) temperatures to identify genes of parents and offspring associated with thermal response, parenting/being parented, and gene expression plasticity associated with behavioural stability of parental care. The main effects of thermal and social condition each shaped patterns of gene expression in females, males, and larvae. In addition, we implicated 79 genes in females as "buffering" parental behaviour across environments. The majority of these underwent significant changes in expression in actively parenting mothers at the benign temperature, but not at the stressful temperature. Our results suggest that neither genetic programmes for parenting nor their effects on offspring gene expression are fundamentally different under stressful conditions, and that behavioural stability is associated primarily with the maintenance of existing genetic programmes rather than replacement or supplementation. Thus, while selection for compensatory gene expression could expand the range of thermal conditions parents will tolerate, without expanding the toolkit of genes involved selection is unlikely to lead to adaptive changes of function.

A Synthesis of Game Theory and Quantitative Genetic Models of Social Evolution.

Two popular approaches for modeling social evolution, evolutionary game theory and quantitative genetics, ask complementary questions but are rarely integrated. Game theory focuses on evolutionary outcomes, with models solving for evolutionarily stable equilibria, whereas quantitative genetics provides insight into evolutionary processes, with models predicting short-term responses to selection. Here we draw parallels between evolutionary game theory and interacting phenotypes theory, which is a quantitative genetic framework for understanding social evolution. First, we show how any evolutionary game may be translated into two quantitative genetic selection gradients, nonsocial and social selection, which may be used to predict evolutionary change from a single round of the game. We show that synergistic fitness effects may alter predicted selection gradients, causing changes in magnitude and sign as the population mean evolves. Second, we show how evolutionary games involving plastic behavioral responses to partners can be modeled using indirect genetic effects, which describe how trait expression changes in response to genes in the social environment. We demonstrate that repeated social interactions in models of reciprocity generate indirect effects and conversely, that estimates of parameters from indirect genetic effect models may be used to predict the evolution of reciprocity. We argue that a pluralistic view incorporating both theoretical approaches will benefit empiricists and theorists studying social evolution. We advocate the measurement of social selection and indirect genetic effects in natural populations to test the predictions from game theory and, in turn, the use of game theory models to aid in the interpretation of quantitative genetic estimates.

Evolution of Personal and Social Immunity in the Context of Parental Care.

Social immunity moderates the spread of pathogens in social groups and is especially likely in groups structured by genetic relatedness. The extent to which specific immune pathways are used is unknown. Here, we investigate the expression and social role of three functionally separate immune genes (pgrp-sc2, thaumatin, and defensin) during parental care in the beetle Nicrophorus vespilloides. These genes reside in different immune pathways, allowing us to test whether specific components of the immune system are targeted for social immunity. To test for the evolution of specificity, we manipulated the influence of social context and timing on gene expression and quantified the covariance of maternal immune gene expression and offspring fitness. Larvae reduced expression of all three genes in the presence of parents. Parental pgrp-sc2 and thaumatin increased during direct parenting, while defensin was upregulated before larvae arrived. Parental expression of pgrp-sc2 and thaumatin responded similarly to experimental manipulation of timing and presence of larvae, which differed from the response of defensin. We found a positive covariance between maternal expression and offspring fitness for pgrp-sc2 and thaumatin but not defensin. We suggest that social immunity can involve specific genes and pathways, reflecting evolution as an interacting phenotype during parenting.

Changes of gene expression but not cytosine methylation are associated with male parental care reflecting behavioural state, social context and individual flexibility.

Behaviour is often a front line response to changing environments. Recent studies show behavioural changes are associated with changes of gene expression; however, these studies have primarily focused on discrete behavioural states. We build on these studies by addressing additional contexts that produce qualitatively similar behavioural changes. We measured levels of gene expression and cytosine methylation, which is hypothesized to regulate the transcriptional architecture of behavioural transitions, within the brain during male parental care of the burying beetle Nicrophorus vespilloides in a factorial design. Male parenting is a suitably plastic behaviour because although male N. vespilloides typically do not provide direct care (i.e. feed offspring) when females are present, levels of feeding by a male equivalent to the female can be induced by removing the female. We examined three different factors: behavioural state (caring versus non-caring), social context (with or without a female mate) and individual flexibility (if a male switched to direct care after his mate was removed). The greatest number of differentially expressed genes were associated with behavioural state, followed by social context and individual flexibility. Cytosine methylation was not associated with changes of gene expression in any of the factors. Our results suggest a hierarchical association between gene expression and the different factors, but that this process is not controlled by cytosine methylation. Our results further suggest that the extent a behaviour is transient plays an underappreciated role in determining its underpinning molecular mechanisms.

Evolution of DNA Methylation across Insects.

DNA methylation contributes to gene and transcriptional regulation in eukaryotes, and therefore has been hypothesized to facilitate the evolution of plastic traits such as sociality in insects. However, DNA methylation is sparsely studied in insects. Therefore, we documented patterns of DNA methylation across a wide diversity of insects. We predicted that underlying enzymatic machinery is concordant with patterns of DNA methylation. Finally, given the suggestion that DNA methylation facilitated social evolution in Hymenoptera, we tested the hypothesis that the DNA methylation system will be associated with presence/absence of sociality among other insect orders. We found DNA methylation to be widespread, detected in all orders examined except Diptera (flies). Whole genome bisulfite sequencing showed that orders differed in levels of DNA methylation. Hymenopteran (ants, bees, wasps and sawflies) had some of the lowest levels, including several potential losses. Blattodea (cockroaches and termites) show all possible patterns, including a potential loss of DNA methylation in a eusocial species whereas solitary species had the highest levels. Species with DNA methylation do not always possess the typical enzymatic machinery. We identified a gene duplication event in the maintenance DNA methyltransferase 1 (DNMT1) that is shared by some Hymenoptera, and paralogs have experienced divergent, nonneutral evolution. This diversity and nonneutral evolution of underlying machinery suggests alternative DNA methylation pathways may exist. Phylogenetically corrected comparisons revealed no evidence that supports evolutionary association between sociality and DNA methylation. Future functional studies will be required to advance our understanding of DNA methylation in insects.

The role of neuropeptide F in a transition to parental care.

The genetics of complex social behaviour can be dissected by examining the genetic influences of component pathways, which can be predicted based on expected evolutionary precursors. Here, we examine how gene expression in a pathway that influences the motivation to eat is altered during parental care that involves direct feeding of larvae. We examine the expression of neuropeptide F, and its receptor, in the burying beetle Nicrophorus vespilloides, which feeds pre-digested carrion to its begging larvae. We found that the npf receptor was greatly reduced during active care. Our research provides evidence that feeding behaviour was a likely target during the evolution of parental care in N. vespilloides Moreover, dissecting complex behaviours into ethologically distinct sub-behaviours is a productive way to begin to target the genetic mechanisms involved in the evolution of complex behaviours.

Vitellogenin and vitellogenin receptor gene expression is associated with male and female parenting in a subsocial insect.

Complex social behaviour in Hymenoptera has been hypothesized to evolve by co-opting reproductive pathways (the ovarian ground plan hypothesis, OGPH) and gene networks (the reproductive ground plan hypothesis, RGPH). In support of these hypotheses, in eusocial Hymenoptera where there is reproductive division of labour, the yolk precursor protein vitellogenin (Vg) influences the expression of worker social behaviour. We suggest that co-opting genes involved in reproduction may occur more generally than just in the evolution of eusociality; i.e. underlie earlier stages of social evolution such as the evolution of parental care, given that reproduction and parental care rarely overlap. We therefore examined vitellogenin (vg) gene expression associated with parental care in the subsocial beetle Nicrophorus vespilloides. We found a significant reduction in the expression of vg and its receptor, vgr, in head tissue during active parental care, and confirmed that the receptor is expressed in the brains of both sexes. Ours is the first study to show that vgr is expressed in the brain of a non-eusocial insect. Given the association between behaviour and gene expression in both sexes, and the presence of vitellogenin receptors in the brain, we suggest that Vg was co-opted early in the evolution of sociality to have a regulatory function. This extends the association of Vg in parenting to subsocial species and outside of the Hymenoptera, and supports the hypothesis that the OGPH is general and that heterochrony in gene expression is important in the evolution of social behaviour and precedes subsequent evolutionary specialization of social roles.

Correlated evolution in parental care in females but not males in response to selection on paternity assurance behaviour.

According to classical parental care theory males are expected to provide less parental care when offspring in a brood are less likely to be their own, but empirical evidence in support of this relationship is equivocal. Recent work predicts that social interactions between the sexes can modify co-evolution between traits involved in mating and parental care as a result of costs associated with these social interactions (i.e. sexual conflict). In burying beetles (Nicrophorus vespilloides), we use artificial selection on a paternity assurance trait, and crosses within and between selection lines, to show that selection acting on females, not males, can drive the co-evolution of paternity assurance traits and parental care. Males do not care more in response to selection on mating rate. Instead, patterns of parental care change as an indirect response to costs of mating for females.

Effects of resource variation during early life and adult social environment on contest outcomes in burying beetles: a context-dependent silver spoon strategy?

Good early nutritional conditions may confer a lasting fitness advantage over individuals suffering poor early conditions (a 'silver spoon' effect). Alternatively, if early conditions predict the likely adult environment, adaptive plastic responses might maximize individual performance when developmental and adult conditions match (environmental-matching effect). Here, we test for silver spoon and environmental-matching effects by manipulating the early nutritional environment of Nicrophorus vespilloides burying beetles. We manipulated nutrition during two specific early developmental windows: the larval environment and the post-eclosion environment. We then tested contest success in relation to variation in adult social environmental quality experienced (defined according to whether contest opponents were smaller (good environment) or larger (poor environment) than the focal individual). Variation in the larval environment influenced adult body size but not contest success per se for a given adult social environment experienced (an 'indirect' silver spoon effect). Variation in post-eclosion environment affected contest success dependent on the quality of the adult environment experienced (a context-dependent 'direct' silver spoon effect). By contrast, there was no evidence for environmental-matching. The results demonstrate the importance of social environmental context in determining how variation in nutrition in early life affects success as an adult.

Sexual selection: When crickets go quiet.

Mutations that change male cricket song should be at a disadvantage because the song is used by females to choose amongst males. A new study caught evolution in action and showed that females may have flexible preferences, and new songs may even be preferred so that the mutations spread.

Integrated and independent evolution of heteromorphic sperm types.

Sperm are a simple cell type with few components, yet they exhibit tremendous between-species morphological variation in those components thought to reflect selection in different fertilization environments. However, within a species, sperm components are expected to be selected to be functionally integrated for optimal fertilization of eggs. Here, we take advantage of within-species variation in sperm form and function to test whether sperm components are functionally and genetically integrated both within and between sperm morphologies using a quantitative genetics approach. Drosophila pseudoobscura males produce two sperm types with different functions but which positively interact together in the same fertilization environment; the long eusperm fertilizes eggs and the short parasperm appear to protect eusperm from a hostile female reproductive tract. Our analysis found that all sperm traits were heritable, but short sperm components exhibited evolvabilities 10 times that of long sperm components. Genetic correlations indicated functional integration within, but not between, sperm morphs. These results suggest that sperm, despite sharing a common developmental process, can become developmentally and functionally non-integrated, evolving into separate modules with the potential for rapid and independent responses to selection.

Male age mediates reproductive investment and response to paternity assurance.

Theory predicts that male response to reduced paternity will depend on male state and interactions between the sexes. If there is little chance of reproducing again, then males should invest heavily in current offspring, regardless of their share in paternity. We tested this by manipulating male age and paternity assurance in the burying beetle Nicrophorus vespilloides. We found older males invested more in both mating effort and parental effort than younger males. Furthermore, male age, a component of male state, mediated male response to perceived paternity. Older males provided more prenatal care, whereas younger males provided less prenatal care, when perceived paternity was low. Adjustments in male care, however, did not influence selection acting indirectly on parents, through offspring performance. This is because females adjusted their care in response to the age of their partner, providing less care when paired with older males than younger males. As a result offspring, performance did not differ between treatments. Our study shows, for the first time, that a male state variable is an important modifier of paternity-parental care trade-offs and highlights the importance of social interactions between males and females during care in determining male response to perceived paternity.

takeout gene expression is associated with temporal kin recognition.

A key component of parental care is avoiding killing and eating one's own offspring. Many organisms commit infanticide but switch to parental care when their own offspring are expected, known as temporal kin recognition. It is unclear why such types of indirect kin recognition are so common across taxa. One possibility is that temporal kin recognition may evolve through alteration of simple mechanisms, such as co-opting mechanisms that influence the regulation of timing and feeding in other contexts. Here, we determine whether takeout, a gene implicated in coordinating feeding, influences temporal kin recognition in Nicrophorus orbicollis. We found that takeout expression was not associated with non-parental feeding changes resulting from hunger, or a general transition to the full parental care repertoire. However, beetles that accepted and provided care to their offspring had a higher takeout expression than beetles that committed infanticide. Together, these data support the idea that the evolution of temporal kin recognition may be enabled by co-option of mechanisms that integrate feeding behaviour in other contexts.

Spatiotemporal variation in the competitive environment, with implications for how climate change may affect a species with parental care.

Burying beetles of the genus Nicrophorus have become a model for studying the evolution of complex parental care in laboratory studies. Nicrophorus species depend on small vertebrate carcasses to breed, which they process and provision to their begging offspring. However, vertebrate carcasses are highly sought after by a wide variety of species and so competition is expected to be critical to the evolution of parental care. Despite this, the competitive environment for Nicrophorus is rarely characterized in the wild and remains a missing factor in laboratory studies. Here, we performed a systematic sampling of Nicrophorus orbicollis living near the southern extent of their range at Whitehall Forest in Clarke County, Georgia, USA. We determined the density of N. orbicollis and other necrophilous species that may affect the availability of this breeding resource through interference or exploitation competition. In addition, we characterize body size, a key trait involved in competitive ability, for all Nicrophorus species at Whitehall Forest throughout the season. Finally, we compare our findings to other published natural history data for Nicrophorines. We document a significantly longer active season than was observed 20 years previously at Whitehall Forest for both N. orbicollis and Nicrophorus tomentosus, potentially due to climate change. As expected, the adult body size of N. orbicollis was larger than N. tomentosus, the only other Nicrophorus species that was captured in 2022 at Whitehall Forest. The other most prevalent insects captured included species in the families Staphylinidae, Histeridae, Scarabaeidae, and Elateridae, which may act as competitors or predators of Nicrophorus young. Together, our results indicate significant variation in intra- and interspecific competition relative to populations within the N. orbicollis range. These findings suggest that the competitive environment shows extensive spatiotemporal variation, providing the basis to make predictions for how ecology may influence parenting in this species.

Insect homolog of oxytocin/vasopressin associated with parenting of males but not females in a subsocial beetle.

Parental care is thought to evolve through modification of behavioral precursors, which predicts that mechanistic changes occur in the genes underlying those traits. The duplicated gene system of oxytocin/vasopressin has been broadly co-opted across vertebrates to influence parenting, from a preduplication ancestral role in water balance. It remains unclear whether co-option of these genes for parenting is limited to vertebrates. Here, we experimentally tested for associations between inotocin gene expression and water balance, parental acceptance of offspring, and active parenting in the subsocial beetle Nicrophorus orbicollis, to test whether this single-copy homolog of the oxytocin/vasopressin system has similarly been co-opted for parental care in a species with elaborate parenting. As expected, inotocin was associated with water balance in both sexes. Inotocin expression increased around sexual maturation in both males and females, although more clearly in males. Finally, inotocin expression was not associated with acceptance of larvae, but was associated with a transition to male but not female parenting. Moreover, level of offspring provisioning behavior and gene expression were positively correlated in males but uncorrelated in females. Our results suggest a broad co-option of this system for parenting that may have existed prior to gene duplication.

The role of Dnmt1 in oocyte development.

The whitefly Bemisia tabaci is a globally important crop pest that is difficult to manage through current commercially available methods. While RNA interference (RNAi) is a promising strategy for managing this pest, effective target genes remain unclear. We suggest DNA methyltransferase 1 (Dnmt1) as a potential target gene due to its effect on fecundity in females in other taxa of insects. We investigated the role of Dnmt1 in B. tabaci using RNAi and immunohistochemistry to confirm its potential conserved function in insect reproduction, which will define its usefulness as a target gene. Using RNAi to downregulate Dnmt1 in female B. tabaci, we show that Dnmt1 indeed has a conserved role in reproduction, as knockdown interfered with oocyte development. Females in which Dnmt1 was knocked down had greatly reduced fecundity and fertility; this supports Dnmt1 as a suitable target gene for RNAi-mediated pest management of B. tabaci.