Morphological and functional analyses for investigation of sexually selected legs in the frog legged beetle Sagra femorata (Coleoptera: Chrysomelidae).

Mate choice and male-male combat over successful mating often cause disproportionate exaggeration of male trait relative to body size. However, the exaggeration is often not the only trait involved with male-male combat and mate choice: suites of co-expressed traits may function together as a coordinated unit. When this occurs, dimorphism may be expected for these additional, non-exaggerated, structures. S. femorata males have disproportionately large hind-legs used in male-male combat over females. During the fights, fore- and mid-legs are used to keep males in positions where advantageous for leverage. Because use of the exaggerated hind-legs is coordinated with the other legs, they will coevolve as a functional unit. Here, we show that 1) S. femorata has sexual size differences in all three legs; 2) males show positive allometry in the relative sizes of all three legs; and 3) microstructures of tarsi on the fore- and mid-legs are also sexually dimorphic. Despite these differences in the tarsal microstructure, 4) adhesion forces of the tarsi had no sexual difference in flat surface. The microstructure would be specialized on attaching elytra surface. These results suggest that the three pairs of legs function together during fighting behavior, with hind-legs employed primarily for fighting, and the fore- and mid-legs functioning to grip females, keeping males positioned on the back of the female during combat.

Transcriptomic and functional screening of weapon formation genes implies significance of cell adhesion molecules and female-biased genes in broad-horned flour beetle.

For understanding the evolutionary mechanism of sexually selected exaggerated traits, it is essential to uncover its molecular basis. By using broad-horned flour beetle that has male-specific exaggerated structures (mandibular horn, head horn and gena enlargement), we investigated the transcriptomic and functional characters of sex-biased genes. Comparative transcriptome of male vs. female prepupal heads elucidated 673 sex-biased genes. Counter-intuitively, majority of them were female-biased (584 genes), and GO enrichment analysis showed cell-adhesion molecules were frequently female-biased. This pattern motivated us to hypothesize that female-biased transcripts (i.e. the transcripts diminished in males) may play a role in outgrowth formation. Potentially, female-biased genes may act as suppressors of weapon structure. In order to test the functionality of female-biased genes, we performed RNAi-mediated functional screening for top 20 female-biased genes and 3 genes in the most enriched GO term (cell-cell adhesion, fat1/2/3, fat4 and dachsous). Knockdown of one transcription factor, zinc finger protein 608 (zfp608) resulted in the formation of male-like gena in females, supporting the outgrowth suppression function of this gene. Similarly, knockdown of fat4 induced rudimental, abnormal mandibular horn in female. fat1/2/3RNAi, fat4RNAi and dachsousRNAi males exhibited thick and/or short mandibular horns and legs. These cell adhesion molecules are known to regulate tissue growth direction and known to be involved in the weapon formation in Scarabaeoidea beetles. Functional evidence in phylogenetically distant broad-horned flour beetle suggest that cell adhesion genes are repeatedly deployed in the acquisition of outgrowth. In conclusion, this study clarified the overlooked functions of female-biased genes in weapon development.

Natural selection increases female fitness by reversing the exaggeration of a male sexually selected trait.

Theory shows how sexual selection can exaggerate male traits beyond naturally selected optima and also how natural selection can ultimately halt trait elaboration. Empirical evidence supports this theory, but to our knowledge, there have been no experimental evolution studies directly testing this logic, and little examination of possible associated effects on female fitness. Here we use experimental evolution of replicate populations of broad-horned flour beetles to test for effects of sex-specific predation on an exaggerated sexually selected male trait (the mandibles), while also testing for effects on female lifetime reproductive success. We find that populations subjected to male-specific predation evolve smaller sexually selected mandibles and this indirectly increases female fitness, seemingly through intersexual genetic correlations we document. Predation solely on females has no effects. Our findings support fundamental theory, but also reveal unforseen outcomes-the indirect effect on females-when natural selection targets sex-limited sexually selected characters.

Individual Ants Do Not Show Activity-Rest Rhythms in Nest Conditions.

Circadian rhythms, which respond to the day-night cycle on the earth, arise from the endogenous timekeeping system within organisms, called the "biological clock." For accurate circadian rhythms, daily fluctuations in light and temperature are considered one of the important time cues. In social insects, both abiotic and biotic factors (i.e., social interactions) play a significant role in activity-rest rhythm regulation. However, it is challenging to monitor individual activity-rest rhythms in a colony because of the large group size and small body size. Therefore, it is unclear whether individuals in a colony exhibit activity-rest rhythms and how social interactions regulate their activity-rest rhythms in the colony. This study developed an image-based tracking system using 2D barcodes for Diacamma cf. indicum from Japan (a monomorphic ant) and measured the locomotor activities of all colony members under laboratory colony conditions. We also investigated the effect of broods on activity-rest rhythms by removing all broods under colony conditions. Activity-rest rhythms appeared only in isolated ants, not under colony conditions. In addition, workers showed arrhythmic activities after brood removal. These results suggested that a mixture of social interactions, and not light and temperature, induces the loss of activity-rest rhythms. These results contribute to the knowledge of a diverse pattern of circadian activity rhythms in social insects.

Octopaminergic system orchestrates combat and mating behaviors: A potential regulator of alternative male mating tactics in an armed beetle.

Male-male combats over females and territories are widespread across animal taxa. The winner of a combat gains resources, while the loser suffers significant costs (e.g. time, energy and injury) without gaining resources. Many animals have evolved behavioral flexibility, depending on their nutritional condition and experience, to avoid combat in order to reduce such costs. In these cases, male aggression often correlates with mating behavior changes, that is, the deployment of alternative reproductive tactics. Therefore, uncovering the physiological mechanism that orchestrates combat and mating behaviors is essential to understand the evolution of alternative mating tactics. However, so far, our knowledge is limited to specific behaviors (i.e., fighting or mating) of specific model species. In this study, we used an armed beetle (Gnatocerus cornutus) and hypothesized that one of the key neuromodulators of invertebrate aggression, octopamine (OA), would control male combat and other mating behaviors. Using receptor agonists (chlordimeform and benzimidazole), we showed that the octopaminergic (OAergic) system down-regulated the combat and courtship behaviors, while it up-regulated locomotor activity and sperm size. This suggests that the OAergic system orchestrates a suite of fighting and mating behaviors, thereby implying that correlated behavioral responses to OAergic signaling may have driven the evolution of alternative mating tactics in this beetle.

Bipartite network analysis of ant-task associations reveals task groups and absence of colonial daily activity.

Social insects are one of the best examples of complex self-organized systems exhibiting task allocation. How task allocation is achieved is the most fascinating question in behavioural ecology and complex systems science. However, it is difficult to comprehensively characterize task allocation patterns due to behavioural complexity, such as the individual variation, context dependency and chronological variation. Thus, it is imperative to quantify individual behaviours and integrate them into colony levels. Here, we applied bipartite network analyses to characterize individual-behaviour relationships. We recorded the behaviours of all individuals with verified age in ant colonies and analysed the individual-behaviour relationship at the individual, module and network levels. Bipartite network analysis successfully detected the module structures, illustrating that certain individuals performed a subset of behaviours (i.e. task groups). We confirmed age polyethism by comparing age between modules. Additionally, to test the daily rhythm of the executed tasks, the data were partitioned between daytime and nighttime, and a bipartite network was re-constructed. This analysis supported that there was no daily rhythm in the tasks performed. These findings suggested that bipartite network analyses could untangle complex task allocation patterns and provide insights into understanding the division of labour.

A specific type of insulin-like peptide regulates the conditional growth of a beetle weapon.

Evolutionarily conserved insulin/insulin-like growth factor (IGF) signaling (IIS) has been identified as a major physiological mechanism underlying the nutrient-dependent regulation of sexually selected weapon growth in animals. However, the molecular mechanisms that couple nutritional state with weapon growth remain largely unknown. Here, we show that one specific subtype of insulin-like peptide (ILP) responds to nutrient status and thereby regulates weapon size in the broad-horned flour beetle Gnatocerus cornutus. By using transcriptome information, we identified five G. cornutus ILP (GcorILP1-5) and two G. cornutus insulin-like receptor (GcorInR1, -2) genes in the G. cornutus genome. RNA interference (RNAi)-mediated gene silencing revealed that a certain subtype of ILP, GcorILP2, specifically regulated weapon size. Importantly, GcorILP2 was highly and specifically expressed in the fat body in a condition-dependent manner. We further found that GcorInR1 and GcorInR2 are functionally redundant but that the latter is partially specialized for regulating weapon growth. These results strongly suggest that GcorILP2 is an important component of the developmental mechanism that couples nutritional state to weapon growth in G. cornutus. We propose that the duplication and subsequent diversification of IIS genes played a pivotal role in the evolution of the complex growth regulation of secondary sexual traits.

Loser-effect duration evolves independently of fighting ability.

Winning or losing contests can impact subsequent competitive behaviour and the duration of these effects can be prolonged. While it is clear effects depend on social and developmental environments, the extent to which they are heritable, and hence evolvable, is less clear and remains untested. Furthermore, theory predicts that winner and loser effects should evolve independently of actual fighting ability, but again tests of this prediction are limited. Here we used artificial selection on replicated beetle populations to show that the duration of loser effects can evolve, with a realized heritability of about 17%. We also find that naive fighting ability does not co-evolve with reductions in the duration of the loser effect. We discuss the implications of these findings and how they corroborate theoretical predictions.

Ant circadian activity associated with brood care type.

In group-living animals, social interactions influence various traits including circadian activity. Maternal care, in particular, can have a strong effect on the circadian activity of parents or nurses across taxa. In social insects, nest-mates are known to have diverse activity rhythms; however, what kind of social environment is crucial in shaping an individual's rhythm is largely unknown. Here, we show that the focal brood types being taken care of (i.e. egg, larva and pupa) have significant effects on individual activity/rest rhythm, using the monomorphic ant Diacamma (putative species indicum). When isolated from a colony, nurses exhibited a clear circadian rhythm. However, when paired with eggs or larvae, they exhibited around-the-clock activity with no apparent rhythm. In contrast, a clear activity rhythm emerged when nurses were paired with a pupa, requiring little care. Such brood-type-specific changes in circadian activity are considered to arise from the difference in caretaking demands. Our finding may contribute to the understanding of the organization of a colony in the context of behavioural variability under different microenvironments.

Social dominance alters nutrition-related gene expression immediately: transcriptomic evidence from a monomorphic queenless ant.

Queen-worker differentiation in eusocial organisms may have originated from decoupling of maternal care and reproductive behaviours. Recent advances in sequencing techniques have begun to elucidate the molecular basis of queen-worker differentiation. However, current knowledge of the molecular basis of caste differentiation is limited, especially to species with morphological castes. It seems likely that at the dawn of eusociality morphologically undifferentiated, monomorphic females underwent physiological differentiation that yielded egg-laying and caretaking castes. The molecular basis of such physiological differentiation may provide evolutionary insight into the emergent state of eusociality. In this study, we identify genes associated with monomorphic caste differentiation, specifically focusing on the onset of queen-worker differentiation, using a monomorphic queenless ant, Diacamma sp., that secondarily lost morphological castes. Using individuals experimentally manipulated to become sterile or reproductive, we identified 1546 caste-biased transcripts in brain and 10 in gaster. Because caste differentiation occurs in Diacamma soon after eclosion via behavioural dominance, identified transcripts are interpreted as molecular agents responding immediately to dominance rank formation. Among identified genes, expression levels of genes involved in nutrition processing and storage, such as insulin signalling genes and hexamerins, were strongly altered soon after dominance rank formation. We conclude that the rapid modification of nutrition-related genes in response to social rank may be the fundamental mechanism underlying caste differentiation in Diacamma. Together with functional evidence from the literature, we show that a specific set of genes frequently plays a role in reproductive differentiation across systems with and without morphological castes.

Histone deacetylases control module-specific phenotypic plasticity in beetle weapons.

Nutritional conditions during early development influence the plastic expression of adult phenotypes. Among several body modules of animals, the development of sexually selected exaggerated traits exhibits striking nutrition sensitivity, resulting in positive allometry and hypervariability distinct from other traits. Using de novo RNA sequencing and comprehensive RNA interference (RNAi) for epigenetic modifying factors, we found that histone deacetylases (HDACs) and polycomb group (PcG) proteins preferentially influence the size of mandibles (exaggerated male weapon) and demonstrate nutrition-dependent hypervariability in the broad-horned flour beetle, Gnatocerus cornutus RNAi-mediated HDAC1 knockdown (KD) in G. cornutus larvae caused specific curtailment of mandibles in adults, whereas HDAC3 KD led to hypertrophy. Notably, these KDs conferred opposite effects on wing size, but little effect on the size of the core body and genital modules. PcG RNAi also reduced adult mandible size. These results suggest that the plastic development of exaggerated traits is controlled in a module-specific manner by HDACs.

Environmental Factors Affecting Pupation Decision in the Horned Flour Beetle Gnatocerus cornutus.

Social environments often affect the development of organisms. In Tenebrionidae beetles, larval development can be arrested at the final instar stage in the presence of conspecific larvae. This developmental plasticity is considered to be an anti-cannibalistic strategy but the critical environmental determinants and actual effects remain to be elucidated. In this study, we examined the effects of the heterospecific environment, conspecific sexual environment (i.e., presence of conspecific male or female), and abiotic physical stimulation on the pupation decision of the sexually dimorphic horned-flour beetle Gnatocerus cornutus. Additionally, actual anti-cannibalistic or antipredatory effects of developmental arrest were evaluated by analyzing stage-dependent vulnerabilities. When G. cornutus larvae were maintained with a G. cornutus larva, a G. cornutus adult, or T. castaneum adult, the developmental period up to the prepupal stage was significantly elongated, suggesting that the cue is not species-specific. Sexual environment did not affect the timing of pupation in G. cornutus; however, we found that abiotic tactile stimulations by glass beads could repress pupation. We also discovered that prepupal and pupal stages were more vulnerable to cannibalism and predation than the larval stage. These data suggest that G. cornutus larvae use non-species specific tactile stimulation as a decision cue for pupation and it has broader defensive effects against heterospecific predation as well as conspecific cannibalism.

Social dominance and reproductive differentiation mediated by dopaminergic signaling in a queenless ant.

In social Hymenoptera with no morphological caste, a dominant female becomes an egg layer, whereas subordinates become sterile helpers. The physiological mechanism that links dominance rank and fecundity is an essential part of the emergence of sterile females, which reflects the primitive phase of eusociality. Recent studies suggest that brain biogenic amines are correlated with the ranks in dominance hierarchy. However, the actual causality between aminergic systems and phenotype (i.e. fecundity and aggressiveness) is largely unknown due to the pleiotropic functions of amines (e.g. age-dependent polyethism) and the scarcity of manipulation experiments. To clarify the causality among dominance ranks, amine levels and phenotypes, we examined the dynamics of the aminergic system during the ontogeny of dominance hierarchy in the queenless ant Diacamma sp., which undergoes rapid physiological differentiation based on dominance interactions. Brain dopamine levels differed between dominants and subordinates at day 7 after eclosion, although they did not differ at day 1, reflecting fecundity but not aggressiveness. Topical applications of dopamine to the subordinate workers induced oocyte growth but did not induce aggressiveness, suggesting the gonadotropic effect of dopamine. Additionally, dopamine receptor transcripts (dopr1 and dopr2) were elevated in the gaster fat body of dominant females, suggesting that the fat body is a potential target of neurohormonal dopamine. Based on this evidence, we suggest that brain dopamine levels are elevated in dominants as a result of hierarchy formation, and differences in dopamine levels cause the reproductive differentiation, probably via stimulation of the fat body.

Sexually dimorphic body color is regulated by sex-specific expression of yellow gene in ponerine ant, Diacamma sp.

Most hymenopteran species exhibit conspicuous sexual dimorphism due to ecological differences between the sexes. As hymenopteran genomes, under the haplodiploid genetic system, exhibit quantitative differences between sexes while remaining qualitatively identical, sexual phenotypes are assumed to be expressed through sex-specific gene usage. In the present study, the molecular basis for expression of sexual dimorphism in a queenless ant, Diacamma sp., which exhibits a distinct color dimorphism, was examined. Worker females of the species appear bluish-black, while winged males exhibit a yellowish-brown body color. Initially, observations of the pigmentation processes during pupal development revealed that black pigmentation was present in female pupae but not in males, suggesting that sex-specific melanin synthesis was responsible for the observed color dimorphism. Therefore, five orthologs of the genes involved in the insect melanin synthesis (yellow, ebony, tan, pale and dopa decarboxylase) were subcloned and their spatiotemporal expression patterns were examined using real-time quantitative RT-PCR. Of the genes examined, yellow, which plays a role in black melanin synthesis in insects, was expressed at higher levels in females than in males throughout the entire body during the pupal stage. RNA interference of yellow was then carried out in order to determine the gene function, and produced females with a more yellowish, brighter body color similar to that of males. It was concluded that transcriptional regulation of yellow was responsible for the sexual color dimorphism observed in this species.

Soldier morphogenesis in the damp-wood termite is regulated by the insulin signaling pathway.

Eusocial insects exhibit various morphological castes associated with the division of labor within a colony. Termite soldiers possess defensive traits including mandibles that are greatly exaggerated and enlarged, as compared to termite reproductives and workers. The enlarged mandibles of soldiers are known to result from dynamic morphogenesis during soldier differentiation that can be induced by juvenile hormone and its analogs. However, the detailed developmental mechanisms still remain unresolved. Because the insulin/insulin-like growth factor signaling (IIS) pathway has been shown to regulate the relative sizes of organs (i.e., allometry) in other insects, we examined the expression profiles of major IIS factors in the damp-wood termite Hodotermopsis sjostedti, during soldier differentiation. The relative expression patterns of orthologs for termite InR (HsjInR), PKB/Akt (HsjPKB/Akt), and FOXO (HsjFOXO) suggest that HsjInR and HsjPKB/Akt were up-regulated in the period of elongation of mandibles during soldier development. In situ hybridization showed that HsjInR was strongly expressed in the mandibular epithelial tissues, and RNA interference (RNAi) for HsjInR disrupted soldier-specific morphogenesis including mandibular elongation. These results suggest that signaling through the IIS pathway is required for soldier-specific morphogenesis. In addition, up-regulation of the IIS pathway in other body tissues occurred at earlier stages of development, indicating that there is tissue-specific IIS regulation. Because the IIS pathway is generally thought to act upstream of JH in insects, our results suggest the damp-wood termite may have evolved a novel feedback loop between JH and IIS that enables social interactions, rather than nutrition, to regulate caste determination.

Male courtship behavior and weapon trait as indicators of indirect benefit in the bean bug, Riptortus pedestris.

Females prefer male traits that are associated with direct and/or indirect benefits to themselves. Male-male competition also drives evolution of male traits that represent competitive ability. Because female choice and male-male competition rarely act independently, exploring how these two mechanisms interact is necessary for integrative understanding of the evolution of sexually selected traits. Here, we focused on direct and indirect benefits to females from male attractiveness, courtship, and weapon characters in the armed bug Riptortus pedestris. The males use their hind legs to fight other males over territory and perform courtship displays for successful copulation. Females of R. pedestris receive no direct benefit from mating with attractive males. On the other hand, we found that male attractiveness, courtship rate, and weapon size were significantly heritable and that male attractiveness had positive genetic covariances with both courtship rate and weapon traits. Thus, females obtain indirect benefits from mating with attractive males by producing sons with high courtship success rates and high competitive ability. Moreover, it is evident that courtship rate and hind leg length act as evaluative cues of female choice. Therefore, female mate choice and male-male competition may facilitate each other in R. pedestris. This is consistent with current basic concepts of sexual selection.

Deficiency screening for genomic regions with effects on environmental sensitivity of the sensory bristles of Drosophila melanogaster.

Environmental canalization is defined as a reduction in the effect of external environmental perturbations on a phenotype, while phenotypic plasticity is defined as the production of different phenotypes in alternative environments. These terms describe different aspects of the same phenomenon, that is, the sensitivity of the phenotype to the environment. Genetic regulation of the environmental sensitivity has been a central topic in the field of evolutionary biology. In this study, we performed deficiency screening to detect genomic regions with effects on the environmental sensitivity of Drosophila melanogaster sensory bristles. We used a collection of isogenic deficiency strains established by the DrosDel Project for screening. We screened 423 genomic deficiencies that encompassed approximately 63.6% of the entire D. melanogaster genome. We identified 29 genomic deficiencies showing significant effects on environmental sensitivity, suggesting that multiple genomic regions may influence phenotypic variation. We also found significant correlations among the effects of deficiencies on environmental sensitivity for different bristle traits, suggesting that the same genetic mechanism can regulate environmental sensitivity of multiple traits. Current high-resolution mapping will facilitate the examination of individual candidate genes using mutations or RNAi approaches in future studies.

Juvenile hormone mediates developmental integration between exaggerated traits and supportive traits in the horned flour beetle Gnatocerus cornutus.

Sexually selected exaggerated traits are often coupled with modifications in other nontarget traits. In insects with weapons, enlargements of nontarget characters that functionally support the weapon often occur (i.e. supportive traits). The support of sexual traits requires developmental coordination among functionally related multiple traits-an explicit example of morphological integration. The genetic theory predicts that developmental integration among different body modules, for which development is regulated via different sets of genes, is likely to be coordinated by pleiotropic factors. However, the developmental backgrounds of morphological integrations are largely unknown. We tested the hypothesis that the juvenile hormone (JH), as a pleiotropic factor, mediates the integration between exaggerated and supportive traits in an armed beetle Gnatocerus cornutus. During combat, males of this beetle use exaggerated mandibles to lift up their opponents with the supportive traits, that is, the head and prothoracic body parts. Application of methoprene, a JH analog (JHA), during the larval to prepupal period, induced the formation of large mandibles relative to the body sizes in males. Morphometric examination of nontarget traits elucidated an increase in the relative sizes of supportive traits, including the head and prothoracic body parts. In addition, reductions in the hind wing area and elytra length, which correspond to flight and reproductive abilities, were detected. Our findings are consistent with the genetic theory and support the idea that JH is a key pleiotropic factor that coordinates the developmental integration of exaggerated traits and supportive characters, as well as resource allocation trade-offs.

Screening of upregulated genes induced by high density in the vetch aphid Megoura crassicauda.

Aphids exhibit several polyphenisms in which discontinuous, alternative phenotypes are produced depending on environmental conditions. One representative example is the wing polyphenism, where winged and wingless females are produced through parthenogenesis. Previous work has shown that, in some aphid species, the density condition sensed by the mother aphid determines the developmental fate of embryos in her ovary, with high densities leading to winged progeny and low densities to wingless progeny. However, little is known about the molecular and physiological mechanisms underlying the wing polyphenism. To identify genes involved in the wing-morph determination in the vetch aphid, Megoura crassicauda, we compared maternal and embryonic transcripts between high- and low-density conditions using differential display, followed by quantitative real-time PCR (qRT-PCR). Under the high-density condition, two genes (Uba1 and Naca) were found to be upregulated in maternal tissues without ovaries, while one gene (ClpP) was upregulated in ovaries containing embryos. Uba1 and Naca encode factors that function in protein modification or transcriptional/translational regulation, respectively. In addition to differential display, candidate gene approaches focusing on morphogenetic and endocrine genes, i.e., wg, dpp, ap, hh, InR, IRS, Foxo, EcR, and USP, were also carried out. We found that wg was upregulated in maternal tissues under the high-density condition. The identified genes from both approaches are candidates for further study of their involvement in the transduction of density signals in mother aphids and/or the initial process of wing differentiation in embryos.

Deficiency mapping of the genomic regions associated with effects on developmental stability in Drosophila melanogaster.

Developmental stability is the tendency of morphological traits to resist the effects of developmental noise, and is commonly evaluated by examining fluctuating asymmetry (FA)-random deviations from perfect bilateral symmetry. Molecular mechanisms that control FA have been a long-standing topic of debate in the field of evolutionary biology and quantitative genetics. In this study, we mapped genomic regions associated with effects on the mean and FA of morphological traits, and characterized the trait specificity of those regions. A collection of isogenic deficiency strains established by the DrosDel project was used for deficiency mapping of genome regions associated with effects on FA. We screened 435 genome deficiencies or approximately 64.9% of the entire genome of Drosophila melanogaster to map the region that demonstrated a significant effect on FA of morphological traits. We found that 406 deficiencies significantly affected the mean of morphological traits, and 92 deficiencies increased FA. These results suggest that several genomic regions have the potential to affect developmental stability. They also suggest the possibility of the existence of trait-specific and trait-nonspecific mechanisms for stabilizing developmental processes. The new findings in this study could provide insight into the understanding of the genetic architecture underlying developmental stability.