Adhesion and shrinkage transform the rounded pupal horn into an angular adult horn in Japanese rhinoceros beetle.

Clarifying the mechanisms underlying shape alterations during insect metamorphosis is important for understanding exoskeletal morphogenesis. The large horn of the Japanese rhinoceros beetle Trypoxylus dichotomus is the result of drastic metamorphosis, wherein it appears as a rounded shape during pupation and then undergoes remodeling into an angular adult shape. However, the mechanical mechanisms underlying this remodeling process remain unknown. In this study, we investigated the remodeling mechanisms of the Japanese rhinoceros beetle horn by developing a physical simulation. We identified three factors contributing to remodeling by biological experiments - ventral adhesion, uneven shrinkage, and volume reduction - which were demonstrated to be crucial for transformation using a physical simulation. Furthermore, we corroborated our findings by applying the simulation to the mandibular remodeling of stag beetles. These results indicated that physical simulation applies to pupal remodeling in other beetles, and the morphogenic mechanism could explain various exoskeletal shapes.

Genomic and transcriptomic analyses of the subterranean termite Reticulitermes speratus: Gene duplication facilitates social evolution.

Termites are model social organisms characterized by a polyphenic caste system. Subterranean termites (Rhinotermitidae) are ecologically and economically important species, including acting as destructive pests. Rhinotermitidae occupies an important evolutionary position within the clade representing a transitional taxon between the higher (Termitidae) and lower (other families) termites. Here, we report the genome, transcriptome, and methylome of the Japanese subterranean termite Reticulitermes speratus Our analyses highlight the significance of gene duplication in social evolution in this termite. Gene duplication associated with caste-biased gene expression was prevalent in the R. speratus genome. The duplicated genes comprised diverse categories related to social functions, including lipocalins (chemical communication), cellulases (wood digestion and social interaction), lysozymes (social immunity), geranylgeranyl diphosphate synthase (social defense), and a novel class of termite lineage-specific genes with unknown functions. Paralogous genes were often observed in tandem in the genome, but their expression patterns were highly variable, exhibiting caste biases. Some of the assayed duplicated genes were expressed in caste-specific organs, such as the accessory glands of the queen ovary and the frontal glands of soldier heads. We propose that gene duplication facilitates social evolution through regulatory diversification, leading to caste-biased expression and subfunctionalization and/or neofunctionalization conferring caste-specialized functions.

Histological Observation of Helmet Development in the Treehopper Poppea capricornis (Insecta: Hemiptera: Membracidae).

The treehoppers (Hemiptera, Membracidae) are known for possessing a large three-dimensional structure called a helmet. Although some ecological functions of the helmet have already been elucidated, the developmental mechanisms underlying the complex and diverse morphology of the helmet are still largely unknown. The process of helmet formation was first described in Antianthe expansa, which possesses a simple roof-shaped helmet. However, the developmental process in species with more complex helmet morphologies remains largely unexplored. Hence, in this study, we used Poppea capricornis, which possesses a more complex helmet structure than A. expansa, to investigate the helmet development using paraffin sections, micro-CT, and scanning electronic microscopy. Our focus was on the overall helmet developmental process common to both species and formation of structures unique to Poppea and its comparison to Antianthe. As a result, we discovered that miniature structures were also formed in Poppea, similar to Antianthe, during the helmet formation. Common structures that were shared between the two species were discernible at this stage. Additionally, we observed that suprahumeral horns and posterior horns, two morphological traits specific to the Poppea helmet that are apparently similar anatomically, are formed through two distinctly different developmental mechanisms. The suprahumeral horns appeared to be formed by utilizing the nymphal suprahumeral bud as a mold, while we could not detect any nymphal structures potentially used for a mold in the posterior horns formation. Our findings suggest that the helmet formation mechanisms of Antianthe and Poppea employ a common mechanism but form species-specific structures by multiple mechanisms.

Unveiling the role of differential growth in 3D morphogenesis: An inference method to analyze area expansion rate distribution in biological systems.

The three-dimensional (3D) morphologies of many organs in organisms, such as the curved shapes of leaves and flowers, the branching structure of lungs, and the exoskeletal shape of insects, are formed through surface growth. Although differential growth, a mode of surface growth, has been qualitatively identified as 3D morphogenesis, a quantitative understanding of the mechanical contribution of differential growth is lacking. To address this, we developed a quantitative inference method to analyze the distribution of the area expansion rate, which governs the growth of surfaces into 3D morphology. To validate the accuracy of our method, we tested it on a basic 3D morphology that allowed for the theoretical derivation of the area expansion rate distribution, and then assessed the difference between the predicted outcome and the theoretical solution. We also applied this method to complex 3D shapes and evaluated its accuracy through numerical experiments. The findings of the study revealed a linear decrease in error on a log-log scale with an increase in the number of meshes in both evaluations. This affirmed the reliability of the predictions for meshes that are sufficiently refined. Moreover, we employed our methodology to analyze the developmental process of the Japanese rhinoceros beetle Trypoxylus dichotomus, which is characterized by differential growth regulating 3D morphogenesis. The results indicated a notably high rate of area expansion on the left and right edges of the horn primordium, which is consistent with the experimental evidence of a higher rate of cell division in these regions. Hence, these findings confirm the efficacy of the proposed method in analyzing biological systems.

Termite soldier mandibles are elongated by dachshund under hormonal and Hox gene controls.

In social insects, interactions among colony members trigger caste differentiation with morphological modifications. In termite soldier differentiation, the mandible size considerably increases through two moltings (via the presoldier stage) under the control of juvenile hormone (JH). Regulatory genes are predicted to provide patterning information that induces the mandible-specific cell proliferation. To identify factors responsible for the mandibular enlargement, expression analyses of 18 candidate genes were carried out in the termite Hodotermopsis sjostedti Among those, dachshund (dac), which identifies the intermediate domain along the proximodistal appendage axis, showed mandible-specific upregulation prior to the molt into presoldiers, which can explain the pattern of cell proliferation for the mandibular elongation. Knockdown of dac by RNAi reduced the mandibular length and distorted its morphology. Furthermore, the epistatic relationships among Methoprene tolerant, Insulin receptor, Deformed (Dfd) and dac were revealed by combined RNAi and qRT-PCR analyses, suggesting that dac is regulated by Dfd, downstream of the JH and insulin signaling pathways. Thus, caste-specific morphogenesis is controlled by interactions between the factors that provide spatial information and physiological status.

Dimorphic sperm formation by Sex-lethal.

Sex is determined by diverse mechanisms and master sex-determination genes are highly divergent, even among closely related species. Therefore, it is possible that homologs of master sex-determination genes might have alternative functions in different species. Herein, we focused on Sex-lethal (Sxl), which is the master sex-determination gene in Drosophila melanogaster and is necessary for female germline development. It has been widely shown that the sex-determination function of Sxl in Drosophilidae species is not conserved in other insects of different orders. We investigated the function of Sxl in the lepidopteran insect Bombyx mori In lepidopteran insects (moths and butterflies), spermatogenesis results in two different types of sperm: nucleated fertile eupyrene sperm and anucleate nonfertile parasperm, also known as apyrene sperm. Genetic analyses using Sxl mutants revealed that the gene is indispensable for proper morphogenesis of apyrene sperm. Similarly, our analyses using Sxl mutants clearly demonstrate that apyrene sperm are necessary for eupyrene sperm migration from the bursa copulatrix to the spermatheca. Therefore, apyrene sperm is necessary for successful fertilization of eupyrene sperm in B. mori Although Sxl is essential for oogenesis in D. melanogaster, it also plays important roles in spermatogenesis in B. mori Therefore, the ancestral function of Sxl might be related to germline development.

Evaluation of Body Size Indicators for Morphological Analyses in Two Sister Species of Genus Dorcus (Coleoptera, Lucanidae).

The relationship between trait and body size, i.e., the scaling relationship or static allometry, is an essential concept for investigating trait size. However, usage of an inappropriate body size indicator can lead to misinterpretation of morphology. In this study, we examined several possible body size indicators in two closely related stag beetle species, Dorcus rectus and Dorcus amamianus. We raised animals in captivity and used pupal weight as a measure of true, or overall body size, and then evaluated six adult morphological traits to test whether these traits could be reliably used as body size indicators in static scaling relationship comparisons. We analyzed two comparisons, between sexes in same species and between species in same sex. We showed that the most appropriate body size indicators differ depending on the comparisons. Our results indicated that the scaling relationship of focal traits could be over- or under-estimated depending on which body size indicators are used.

Rhinoceros beetle horn development reveals deep parallels with dung beetles.

Beetle horns are attractive models for studying the evolution of novel traits, as they display diverse shapes, sizes, and numbers among closely related species within the family Scarabaeidae. Horns radiated prolifically and independently in two distant subfamilies of scarabs, the dung beetles (Scarabaeinae), and the rhinoceros beetles (Dynastinae). However, current knowledge of the mechanisms underlying horn diversification remains limited to a single genus of dung beetles, Onthophagus. Here we unveil 11 horn formation genes in a rhinoceros beetle, Trypoxylus dichotomus. These 11 genes are mostly categorized as larval head- and appendage-patterning genes that also are involved in Onthophagus horn formation, suggesting the same suite of genes was recruited in each lineage during horn evolution. Although our RNAi analyses reveal interesting differences in the functions of a few of these genes, the overwhelming conclusion is that both head and thoracic horns develop similarly in Trypoxylus and Onthophagus, originating in the same developmental regions and deploying similar portions of appendage patterning networks during their growth. Our findings highlight deep parallels in the development of rhinoceros and dung beetle horns, suggesting either that both horn types arose in the common ancestor of all scarabs, a surprising reconstruction of horn evolution that would mean the majority of scarab species (~35,000) actively repress horn growth, or that parallel origins of these extravagant structures resulted from repeated co-option of the same underlying developmental processes.

The activin signaling transcription factor Smox is an essential regulator of appendage size during regeneration after autotomy in the crayfish.

Members of the phylum Arthropoda, comprising over 80% of total animal species, have evolved regenerative abilities, but little is known about the molecular mechanisms mediating this process. Transforming growth factor ß (TGF-ß) signaling mediates a diverse set of essential processes in animals and is a good candidate pathway for regulation of regeneration in arthropods. In this study we investigated the role of activin signaling, a TGF-ß superfamily pathway, in limb regeneration in the crayfish. We identified and cloned a downstream transcription factor in the activin pathway, Smox, and characterized its function with regard to other elements of the activin signaling pathway. Gene knockdown of Smox by RNAi induced regeneration of complete but smaller pereopods after autotomy. This indicates that activin signaling via Smox functions in regulation of pereopod growth and size. The expression levels of both Smox and the activin receptor babo were closely correlated with molting. The expression level of Smox increased when babo was knocked down by RNAi, indicating that Smox and babo transcription are linked. Our study suggests that the Babo-Smox system in activin signaling is conserved in decapods, and supports an evolutionary conservation of this aspect of molecular signaling during regeneration between protostomes and deuterostomes.

The function of appendage patterning genes in mandible development of the sexually dimorphic stag beetle.

One of the defining features of the evolutionary success of insects is the morphological diversification of their appendages, especially mouthparts. Although most insects share a common mouthpart ground plan, there is remarkable diversity in the relative size and shapes of these appendages among different insect lineages. One of the most prominent examples of mouthpart modification can be found in the enlargement of mandibles in stag beetles (Coleoptera, Insecta). In order to understand the proximate mechanisms of mouthpart modification, we investigated the function of appendage-patterning genes in mandibular enlargement during extreme growth of the sexually dimorphic mandibles of the stag beetle Cyclommatus metallifer. Based on knowledge from Drosophila and Tribolium studies, we focused on seven appendage patterning genes (Distal-less (Dll), aristaless (al), dachshund (dac), homothorax (hth), Epidermal growth factor receptor (Egfr), escargot (esg), and Keren (Krn). In order to characterize the developmental function of these genes, we performed functional analyses by using RNA interference (RNAi). Importantly, we found that RNAi knockdown of dac resulted in a significant mandible size reduction in males but not in female mandibles. In addition to reducing the size of mandibles, dac knockdown also resulted in a loss of the serrate teeth structures on the mandibles of males and females. We found that al and hth play a significant role during morphogenesis of the large male-specific inner mandibular tooth. On the other hand, knockdown of the distal selector gene Dll did not affect mandible development, supporting the hypothesis that mandibles likely do not contain the distal-most region of the ancestral appendage and therefore co-option of Dll expression is unlikely to be involved in mandible enlargement in stag beetles. In addition to mandible development, we explored possible roles of these genes in controlling the divergent antennal morphology of Coleoptera.

Sexual dimorphism and heightened conditional expression in a sexually selected weapon in the Asian rhinoceros beetle.

Among the most dramatic examples of sexual selection are the weapons used in battles between rival males over access to females. As with ornaments of female choice, the most "exaggerated" sexually selected weapons vary from male to male more widely than other body parts (hypervariability), and their growth tends to be more sensitive to nutritional state or physiological condition compared with growth of other body parts ("heightened" conditional expression). Here, we use RNAseq analysis to build on recent work exploring these mechanisms in the exaggerated weapons of beetles, by examining patterns of differential gene expression in exaggerated (head and thorax horns) and non-exaggerated (wings, genitalia) traits in the Asian rhinoceros beetle, Trypoxylus dichotomus. Our results suggest that sexually dimorphic expression of weaponry involves large-scale changes in gene expression, relative to other traits, while nutrition-driven changes in gene expression in these same weapons are less pronounced. However, although fewer genes overall were differentially expressed in high- vs. low-nutrition individuals, the number of differentially expressed genes varied predictably according to a trait's degree of condition dependence (head horn > thorax horn > wings > genitalia). Finally, we observed a high degree of similarity in direction of effects (vectors) for subsets of differentially expressed genes across both sexually dimorphic and nutritionally responsive growth. Our results are consistent with a common set of mechanisms governing sexual size dimorphism and condition dependence.

Molecular Characterization of Eye Pigmentation-Related ABC Transporter Genes in the Ladybird Beetle Harmonia axyridis Reveals Striking Gene Duplication of the white Gene.

Many species of ladybird beetles (Coccinellidae) possess vivid body colors. These colors and patterns show diversity between coccinellid species, or even within species. However, the molecular underpinnings of these striking body colors are scarcely understood. One of the candidate pigmentation molecules responsible for ladybird body color is ommochrome pigment, which is well known as the red pigment molecule responsible for the red eyes of Drosophila. Various insects also use ommochrome in body coloration. It is known that ommochrome pigment precursors are imported into appropriate cells by the ATP binding cassette (ABC) transporter proteins White and Scarlet. Thus, these ABC transporter genes are potentially involved in various color and pattern expressions seen in ladybird beetle species. In this study, in order to identify the repertory of ABC transporter genes responsible for such body colors, we performed molecular characterization of pigment-related ABC transporter genes, especially white and scarlet, in the coccinellid Harmonia axyridis. By using whole genome data for H. axyridis and subsequent RACE-PCR, six white orthologs and one scarlet ortholog were successfully identified. According to the results of functional analyses via RNA interference (RNAi), only one of these genes had a major function in eye pigmentation. Specific effects on body color and pattern were not detected by our RNAi experiments of any of these genes. This is the first report of this striking duplication of white genes and their functional analyses in insects.

Developmental link between sex and nutrition; doublesex regulates sex-specific mandible growth via juvenile hormone signaling in stag beetles.

Sexual dimorphisms in trait expression are widespread among animals and are especially pronounced in ornaments and weapons of sexual selection, which can attain exaggerated sizes. Expression of exaggerated traits is usually male-specific and nutrition sensitive. Consequently, the developmental mechanisms generating sexually dimorphic growth and nutrition-dependent phenotypic plasticity are each likely to regulate the expression of extreme structures. Yet we know little about how either of these mechanisms work, much less how they might interact with each other. We investigated the developmental mechanisms of sex-specific mandible growth in the stag beetle Cyclommatus metallifer, focusing on doublesex gene function and its interaction with juvenile hormone (JH) signaling. doublesex genes encode transcription factors that orchestrate male and female specific trait development, and JH acts as a mediator between nutrition and mandible growth. We found that the Cmdsx gene regulates sex differentiation in the stag beetle. Knockdown of Cmdsx by RNA-interference in both males and females produced intersex phenotypes, indicating a role for Cmdsx in sex-specific trait growth. By combining knockdown of Cmdsx with JH treatment, we showed that female-specific splice variants of Cmdsx contribute to the insensitivity of female mandibles to JH: knockdown of Cmdsx reversed this pattern, so that mandibles in knockdown females were stimulated to grow by JH treatment. In contrast, mandibles in knockdown males retained some sensitivity to JH, though mandibles in these individuals did not attain the full sizes of wild type males. We suggest that moderate JH sensitivity of mandibular cells may be the default developmental state for both sexes, with sex-specific Dsx protein decreasing sensitivity in females, and increasing it in males. This study is the first to demonstrate a causal link between the sex determination and JH signaling pathways, which clearly interact to determine the developmental fates and final sizes of nutrition-dependent secondary-sexual characters.

Identification and functional analyses of sex determination genes in the sexually dimorphic stag beetle Cyclommatus metallifer.

BACKGROUND: Genes in the sex determination pathway are important regulators of sexually dimorphic animal traits, including the elaborate and exaggerated male ornaments and weapons of sexual selection. In this study, we identified and functionally analyzed members of the sex determination gene family in the golden metallic stag beetle Cyclommatus metallifer, which exhibits extreme differences in mandible size between males and females. RESULTS: We constructed a C. metallifer transcriptomic database from larval and prepupal developmental stages and tissues of both males and females. Using Roche 454 pyrosequencing, we generated a de novo assembled database from a total of 1,223,516 raw reads, which resulted in 14,565 isotigs (putative transcript isoforms) contained in 10,794 isogroups (putative identified genes). We queried this database for C. metallifer conserved sex determination genes and identified 14 candidate sex determination pathway genes. We then characterized the roles of several of these genes in development of extreme sexual dimorphic traits in this species. We performed molecular expression analyses with RT-PCR and functional analyses using RNAi on three C. metallifer candidate genes--Sex-lethal (CmSxl), transformer-2 (Cmtra2), and intersex (Cmix). No differences in expression pattern were found between the sexes for any of these three genes. In the RNAi gene-knockdown experiments, we found that only the Cmix had any effect on sexually dimorphic morphology, and these mimicked the effects of Cmdsx knockdown in females. Knockdown of CmSxl had no measurable effects on stag beetle phenotype, while knockdown of Cmtra2 resulted in complete lethality at the prepupal period. These results indicate that the roles of CmSxl and Cmtra2 in the sex determination cascade are likely to have diverged in stag beetles when compared to Drosophila. Our results also suggest that Cmix has a conserved role in this pathway. In addition to those three genes, we also performed a more complete functional analysis of the C. metallifer dsx gene (Cmdsx) to identify the isoforms that regulate dimorphism more fully using exon-specific RNAi. We identified a total of 16 alternative splice variants of the Cmdsx gene that code for up to 14 separate exons. Despite the variation in RNA splice products of the Cmdsx gene, only four protein isoforms are predicted. The results of our exon-specific RNAi indicated that the essential CmDsx isoform for postembryonic male differentiation is CmDsxB, whereas postembryonic female specific differentiation is mainly regulated by CmDsxD. CONCLUSIONS: Taken together, our results highlight the importance of studying the function of highly conserved sex determination pathways in numerous insect species, especially those with dramatic and exaggerated sexual dimorphism, because conservation in protein structure does not always translate into conservation in downstream function.

The Fat/Hippo signaling pathway links within-disc morphogen patterning to whole-animal signals during phenotypically plastic growth in insects.

BACKGROUND: Insects exhibit a diversity of environmentally sensitive phenotypes that allow them to be an extraordinarily successful group. For example, mandible size in male stag beetles is exquisitely sensitive to the larval nutritional environment and is a reliable signal of male condition. RESULTS: To date, studies of how such phenotypically plastic traits develop have focused on two types of mechanistic processes. Local, tissue-specific genetic mechanisms specify the shape and approximate final size of structures, whereas whole-animal hormonal signaling mechanisms modulate trait growth in response to environmental circumstance, including the body size and nutritional state of each individual. Hormones such as juvenile hormone, ecdysteroids, and/or ligands of the insulin-signaling pathway specify whether traits grow and regulate how much growth occurs across a diversity of insect groups. What remains to be shown is how the local, tissue-specific developmental genetic pathways interact with these whole animal hormonal signaling pathways during development to yield phenotypically plastic patterns of trait growth. CONCLUSIONS: Because the Fat/Hippo signaling pathway coordinates trait growth and development through its interactions with morphogens and hormonal pathways, we propose that Fat/Hippo signaling is a missing mechanistic link coordinating environmentally sensitive trait development in insects. Developmental Dynamics 244:1039-1045, 2015. © 2015 Wiley Periodicals, Inc.

Exaggerated trait growth in insects.

Animal structures occasionally attain extreme proportions, eclipsing in size the surrounding body parts. We review insect examples of exaggerated traits, such as the mandibles of stag beetles (Lucanidae), the claspers of praying mantids (Mantidae), the elongated hindlimbs of grasshoppers (Orthoptera: Caelifera), and the giant heads of soldier ants (Formicidae) and termites (Isoptera). Developmentally, disproportionate growth can arise through trait-specific modifications to the activity of at least four pathways: the sex determination pathway, the appendage patterning pathway, the insulin/IGF signaling pathway, and the juvenile hormone/ecdysteroid pathway. Although most exaggerated traits have not been studied mechanistically, it is already apparent that distinct developmental mechanisms underlie the evolution of the different types of exaggerated traits. We suggest this reflects the nature of selection in each instance, revealing an exciting link between mechanism, form, and function. We use this information to make explicit predictions for the types of regulatory pathways likely to underlie each type of exaggerated trait.

A general mechanism for conditional expression of exaggerated sexually-selected traits.

Sexually-selected exaggerated traits tend to be unusually reliable signals of individual condition, as their expression tends to be more sensitive to nutritional history and physiological circumstance than that of other phenotypes. As such, these traits are the foundation for many models of sexual selection and animal communication, such as "handicap" and "good genes" models. Exactly how expression of these traits is linked to the bearer's condition has been a central yet unresolved question, in part because the underlying physiological mechanisms regulating their development have remained largely unknown. Recent discoveries across animals as diverse as deer, beetles, and flies now implicate the widely conserved insulin-like signaling pathway, as a common physiological mechanism regulating condition-sensitive structures with extreme growth. This raises the exciting possibility that one highly conserved pathway may underlie the evolution of trait exaggeration in a multitude of sexually-selected signal traits across the animal kingdom.

The draft genome sequence of the Japanese rhinoceros beetle Trypoxylus dichotomus septentrionalis towards an understanding of horn formation.

The Japanese rhinoceros beetle Trypoxylus dichotomus is a giant beetle with distinctive exaggerated horns present on the head and prothoracic regions of the male. T. dichotomus has been used as a research model in various fields such as evolutionary developmental biology, ecology, ethology, biomimetics, and drug discovery. In this study, de novo assembly of 615 Mb, representing 80% of the genome estimated by flow cytometry, was obtained using the 10 × Chromium platform. The scaffold N50 length of the genome assembly was 8.02 Mb, with repetitive elements predicted to comprise 49.5% of the assembly. In total, 23,987 protein-coding genes were predicted in the genome. In addition, de novo assembly of the mitochondrial genome yielded a contig of 20,217 bp. We also analyzed the transcriptome by generating 16 RNA-seq libraries from a variety of tissues of both sexes and developmental stages, which allowed us to identify 13 co-expressed gene modules. We focused on the genes related to horn formation and obtained new insights into the evolution of the gene repertoire and sexual dimorphism as exemplified by the sex-specific splicing pattern of the doublesex gene. This genomic information will be an excellent resource for further functional and evolutionary analyses, including the evolutionary origin and genetic regulation of beetle horns and the molecular mechanisms underlying sexual dimorphism.

Evolution of horn length and lifting strength in the Japanese rhinoceros beetle Trypoxylus dichotomus.

What limits the size of nature's most extreme structures? For weapons like beetle horns, one possibility is a tradeoff associated with mechanical levers: as the output arm of the lever system-the beetle horn-gets longer, it also gets weaker. This "paradox of the weakening combatant" could offset reproductive advantages of additional increases in weapon size. However, in contemporary populations of most heavily weaponed species, males with the longest weapons also tend to be the strongest, presumably because selection drove the evolution of compensatory changes to these lever systems that ameliorated the force reductions of increased weapon size. Therefore, we test for biomechanical limits by reconstructing the stages of weapon evolution, exploring whether initial increases in weapon length first led to reductions in weapon force generation that were later ameliorated through the evolution of mechanisms of mechanical compensation. We describe phylogeographic relationships among populations of a rhinoceros beetle and show that the "pitchfork" shaped head horn likely increased in length independently in the northern and southern radiations of beetles. Both increases in horn length were associated with dramatic reductions to horn lifting strength-compelling evidence for the paradox of the weakening combatant-and these initial reductions to horn strength were later ameliorated in some populations through reductions to horn length or through increases in head height (the input arm for the horn lever system). Our results reveal an exciting geographic mosaic of weapon size, weapon force, and mechanical compensation, shedding light on larger questions pertaining to the evolution of extreme structures.

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.