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.

One genome, multiple phenotypes: decoding the evolution and mechanisms of environmentally induced developmental plasticity in insects.

Plasticity in developmental processes gives rise to remarkable environmentally induced phenotypes. Some of the most striking and well-studied examples of developmental plasticity are seen in insects. For example, beetle horn size responds to nutritional state, butterfly eyespots are enlarged in response to temperature and humidity, and environmental cues also give rise to the queen and worker castes of eusocial insects. These phenotypes arise from essentially identical genomes in response to an environmental cue during development. Developmental plasticity is taxonomically widespread, affects individual fitness, and may act as a rapid-response mechanism allowing individuals to adapt to changing environments. Despite the importance and prevalence of developmental plasticity, there remains scant mechanistic understanding of how it works or evolves. In this review, we use key examples to discuss what is known about developmental plasticity in insects and identify fundamental gaps in the current knowledge. We highlight the importance of working towards a fully integrated understanding of developmental plasticity in a diverse range of species. Furthermore, we advocate for the use of comparative studies in an evo-devo framework to address how developmental plasticity works and how it evolves.

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.

Functional genomics of parental care of insects.

Parental care was likely the first step most lineages made towards sociality. However, the molecular mechanisms that generate parental care are not broadly characterized. Insects are important as an evolutionary independent group from classic models of parental care, such as, house mice. They provide an opportunity to test the generality of our understanding. With this review, I survey the functional genomics of parental care of insects, summarize several recent advances in the broader framework for studying and understanding parental care, and finish with suggested priorities for further research. Although there are too few studies to draw definitive conclusions, I argue that natural selection appears to be rewiring existing gene networks to produce parental care, that the epigenetic mechanisms influencing parental care are not well understood, and, as an interesting early consensus, that genes strongly associated with carer/offspring interactions appear biased towards proteins that are secreted. I summarize the studies that have functionally validate candidate genes and highlight the increasing need to perform this work. I finish with arguments for both conceptual and practical changes moving forward. I argue that future work can increase the use of predictive frameworks, broaden its definition of conservation of mechanism to gene networks rather than single genes, and increase the use of more established comparative methods. I further highlight the practical considerations of standardizing analyses and reporting, increasing the sampling of both carers and offspring, better characterizing gene regulatory networks, better characterizing taxonomically restricted genes and any consistent role they have underpinning parental care, and using factorial designs to disentangle the influence of multiple variables on the expression of parental care.

Musculoskeletal mass and shape are correlated with competitive ability in male house mice (Mus musculus).

Intense physical competition between males for mating opportunities is widespread among mammals. In such agonistic encounters, males with combinations of morphological, physiological and behavioral characters that allow them to dominate an opponent have greater fitness. However, the specific physical traits associated with competitive ability are poorly understood. Larger body size is often correlated with fitness in mammals. Interestingly, fitness is maximized at intermediate body masses in male house mice (Mus musculus), a species with a polygynous mating system in which males compete physically for access to reproductive resources. Here, we used competition trials in semi-natural, mixed-sex population enclosures to directly measure competitive ability in male house mice based on control of a preferred nesting site. We tested the hypothesis that the musculoskeletal systems of male mice demonstrating high competitive ability are more specialized for competition by comparing the masses of 10 major muscle groups and eight bones as well as a set of 12 skeletal shape indices associated with anatomical specialization for fighting performance in a set of nine winners and 20 losers. Winning males possessed several traits hypothesized to enhance performance in male-male contests: relatively greater mass in several muscle groups and bones of the forelimb and hindlimb and larger scapular surface area. Unexpectedly, no measurements of the head and neck differed significantly between winners and losers. These results identify musculoskeletal traits associated with competitive ability in male house mice and suggest that our current understanding of mammalian fighting performance is incomplete and more nuanced than previously considered.

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.

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.

Competitive ability in male house mice (Mus musculus): genetic influences.

Conspecifics of many animal species physically compete to gain reproductive resources and thus fitness. Despite the importance of competitive ability across the animal kingdom, specific traits that influence or underpin competitive ability are poorly characterized. Here, we investigate whether there are genetic influences on competitive ability within male house mice. Additionally, we examined if litter demographics (litter size and litter sex ratio) influence competitive ability. We phenotyped two generations for a male's ability to possess a reproductive resource--a prime nesting site--using semi-natural enclosures with mixed sex groupings. We used the "Animal Model" coupled with an extensive pedigree to estimate several genetic parameters. Competitive ability was found to be highly heritable, but only displayed a moderate genetic correlation to body mass. Interestingly, litter sex ratio had a weak negative influence on competitive ability. Litter size had no significant influence on competitive ability. Our study also highlights how much remains unknown about the proximal causes of competitive ability.

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.

The role of Dmnt1 during spermatogenesis of the insect Oncopeltus fasciatus.

BACKGROUND: The function of DNA methyltransferase genes of insects is a puzzle, because an association between gene expression and methylation is not universal for insects. If the genes normally involved in cytosine methylation are not influencing gene expression, what might be their role? We previously demonstrated that gametogenesis of Oncopeltus fasciatus is interrupted at meiosis following knockdown of DNA methyltransferase 1 (Dnmt1) and this is unrelated to changes in levels of cytosine methylation. Here, using transcriptomics, we tested the hypothesis that Dmnt1 is a part of the meiotic gene pathway. Testes, which almost exclusively contain gametes at varying stages of development, were sampled at 7 days and 14 days following knockdown of Dmnt1 using RNAi. RESULTS: Using microscopy, we found actively dividing spermatocysts were reduced at both timepoints. However, as with other studies, we saw Dnmt1 knockdown resulted in condensed nuclei after mitosis-meiosis transition, and then cellular arrest. We found limited support for a functional role for Dnmt1 in our predicted cell cycle and meiotic pathways. An examination of a priori Gene Ontology terms showed no enrichment for meiosis. We then used the full data set to reveal further candidate pathways influenced by Dnmt1 for further hypotheses. Very few genes were differentially expressed at 7 days, but nearly half of all transcribed genes were differentially expressed at 14 days. We found no strong candidate pathways for how Dnmt1 knockdown was achieving its effect through Gene Ontology term overrepresentation analysis. CONCLUSIONS: We, therefore, suggest that Dmnt1 plays a role in chromosome dynamics based on our observations of condensed nuclei and cellular arrest with no specific molecular pathways disrupted.

Phenotypic Plasticity: What Has DNA Methylation Got to Do with It?

How does one genome give rise to multiple, often markedly different, phenotypes in response to an environmental cue? This phenomenon, known as phenotypic plasticity, is common amongst plants and animals, but arguably the most striking examples are seen in insects. Well-known insect examples include seasonal morphs of butterfly wing patterns, sexual and asexual reproduction in aphids, and queen and worker castes of eusocial insects. Ultimately, we need to understand how phenotypic plasticity works at a mechanistic level; how do environmental signals alter gene expression, and how are changes in gene expression translated into novel morphology, physiology and behaviour? Understanding how plasticity works is of major interest in evolutionary-developmental biology and may have implications for understanding how insects respond to global change. It has been proposed that epigenetic mechanisms, specifically DNA methylation, are the key link between environmental cues and changes in gene expression. Here, we review the available evidence on the function of DNA methylation of insects, the possible role(s) for DNA methylation in phenotypic plasticity and also highlight key outstanding questions in this field as well as new experimental approaches to address these questions.

Survey of neurotransmitter receptor gene expression into and out of parental care in the burying beetle Nicrophorus vespilloides.

Understanding the genetic influences of traits of nonmodel organisms is crucial to understanding how novel traits arise. Do new traits require new genes or are old genes repurposed? How predictable is this process? Here, we examine this question for gene expression influencing parenting behavior in a beetle, Nicrophorus vespilloides. Parental care, produced from many individual behaviors, should be influenced by changes of expression of multiple genes, and one suggestion is that the genes can be predicted based on knowledge of behavior expected to be precursors to parental care, such as aggression, resource defense, and mating on a resource. Thus, testing gene expression during parental care allows us to test expectations of this "precursor hypothesis" for multiple genes and traits. We tested for changes of the expression of serotonin, octopamine/tyramine, and dopamine receptors, as well as one glutamate receptor, predicting that these gene families would be differentially expressed during social interactions with offspring and associated resource defense. We found that serotonin receptors were strongly associated with social and aggression behavioral transitions. Octopamine receptors produced a complex picture of gene expression over a reproductive cycle. Dopamine was not associated with the behavioral transitions sampled here, while the glutamate receptor was most consistent with a behavioral change of resource defense/aggression. Our results generate new hypotheses, refine candidate lists for further studies, and inform the genetic mechanisms that are co-opted during the evolution of parent-offspring interactions, a likely evolutionary path for many lineages that become fully social. The precursor hypothesis, while not perfect, does provide a starting point for identifying candidate genes.

Sexual dimorphism in postcranial skeletal shape suggests male-biased specialization for physical competition in anthropoid primates.

Sexual dimorphism often arises as a response to selection on traits that improve a male's ability to physically compete for access to mates. In primates, sexual dimorphism in body mass and canine size is more common in species with intense male-male competition. However, in addition to these traits, other musculoskeletal adaptations may improve male fighting performance. Postcranial traits that increase strength, agility, and maneuverability may also be under selection. To test the hypothesis that males, as compared to females, are more specialized for physical competition in their postcranial anatomy, we compared sex-specific skeletal shape using a set of functional indices predicted to improve fighting performance. Across species, we found significant sexual dimorphism in a subset of these indices, indicating the presence of skeletal shape sexual dimorphism in our sample of anthropoid primates. Mean skeletal shape sexual dimorphism was positively correlated with sexual dimorphism in body size, an indicator of the intensity of male-male competition, even when controlling for both body mass and phylogenetic relatedness. These results suggest that selection on male fighting ability has played a role in the evolution of postcranial sexual dimorphism in primates.

Relating quantitative variation within a behavior to variation in transcription.

Many studies have shown that variation in transcription is associated with changes in behavioral state, or with variation within a state, but little has been done to address if the same genes are involved in both. Here, we investigate the transcriptional basis of variation in parental provisioning using two species of burying beetle, Nicrophorus orbicollis and Nicrophorus vespilloides. We used RNA-seq to compare transcription in parents that provided high amounts of provisioning behavior versus low amounts in males and females of each species. We found no overarching transcriptional patterns distinguishing high from low caring parents, and no informative transcripts that displayed particularly large expression differences in either sex. However, we did find subtler gene expression differences between high and low provisioning parents that are consistent across both sexes and species. Furthermore, we show that transcripts previously implicated in transitioning into parental care in N. vespilloides had high variance in the levels of transcription and were unusually likely to display differential expression between high and low provisioning parents. Thus, quantitative behavioral variation appears to reflect many transcriptional differences of small effect. Furthermore, the same transcripts required for the transition between behavioral states are also related to variation within a behavioral state.

Ethological principles predict the neuropeptides co-opted to influence parenting.

Ethologists predicted that parental care evolves by modifying behavioural precursors in the asocial ancestor. As a corollary, we predict that the evolved mechanistic changes reside in genetic pathways underlying these traits. Here we test our hypothesis in female burying beetles, Nicrophorus vespilloides, an insect where caring adults regurgitate food to begging, dependent offspring. We quantify neuropeptide abundance in brains collected from three behavioural states: solitary virgins, individuals actively parenting or post-parenting solitary adults and quantify 133 peptides belonging to 18 neuropeptides. Eight neuropeptides differ in abundance in one or more states, with increased abundance during parenting in seven. None of these eight neuropeptides have been associated with parental care previously, but all have roles in predicted behavioural precursors for parenting. Our study supports the hypothesis that predictable traits and pathways are targets of selection during the evolution of parenting and suggests additional candidate neuropeptides to study in the context of parenting.

Duplication and Sub/Neofunctionalization of Malvolio, an Insect Homolog of Nramp, in the Subsocial Beetle Nicrophorus vespilloides.

With growing numbers of sequenced genomes, increasing numbers of duplicate genes are being uncovered. Here we examine Malvolio, a gene in the natural resistance-associated macrophage protein (Nramp) family, that has been duplicated in the subsocial beetle, Nicrophorus vespilloides, which exhibits advanced parental behavior. There is only one copy of Mvl in honey bees and Drosophila, whereas in vertebrates there are two copies that are subfunctionalized. We first compared amino acid sequences for Drosophila, beetles, mice, and humans. We found a high level of conservation between the different species, although there was greater variation in the C-terminal regions. A phylogenetic analysis across multiple insect orders suggested that Mvl has undergone several independent duplications. To examine the potential for different functions where it has been duplicated, we quantified expression levels of Mvl1 and Mvl2 in eight tissues in N. vespilloides We found that while Mvl1 was expressed ubiquitously, albeit at varying levels, expression of Mvl2 was limited to brain and midgut. Because Mvl has been implicated in behavior, we examined expression during different behavioral states that reflected differences in opportunity for social interactions and expression of parental care behaviors. We found differing expression patterns for the two copies, with Mvl1 increasing in expression during resource preparation and feeding offspring, and Mvl2 decreasing in these same states. Given these patterns of expression, along with the protein analysis, we suggest that Mvl in N. vespilloides has experienced sub/neofunctionalization following its duplication, and may be evolving differing and tissue-specific roles in behavior and physiology.

Expression of octopaminergic receptor genes in 4 nonneural tissues in female Nicrophorus vespilloides beetles.

Octopamine regulates the function of many tissues and physiological processes in invertebrates. The expression of octopamine receptor genes has been examined in multiple tissue types in several different insect orders. However, little work has addressed this issue in Coleoptera. Most studies characterize individual genes in different tissue types, but here we describe the expression of 6 octopamine receptor genes in thoracic musculature, oviducts, Malpighian tubules, and fat body of female Nicrophorus vespilloides beetles to characterize both different genes and different tissues within a single study. We then compare the gene expression profiles found in this beetle to other insects to examine the extent to which expression profiles are conserved across insects. We also examine the relative involvement of octopamine verses octopamine/tyramine receptors based on receptor gene expression in each tissue to help elucidate if tyramine plays a role in the regulation of these tissues. We find a high degree of overlap in the expression profile of the 6 genes examined in the thoracic musculature, a moderate amount for the oviducts, and divergent profiles for Malpighian tubules and fat body. Based on expression difference in receptor subtypes, our results also support the suggestion that tyramine is a biogenic amine with physiological actions separate from octopamine.