Search

The elaborate ornaments and weapons of sexual selection, such as the vast array of horns observed in scarab beetles, are some of the most striking outcomes of evolution. How these novel traits have arisen, develop, and respond to condition is governed by a complex suite of interactions that require coordination between the environment, whole-animal signals, cell-cell signals, and within-cell signals. Endocrine factors, developmental patterning genes, and sex-specific gene expression have been shown to regulate beetle horn size, shape, and location, yet no overarching mechanism of horn shape has been described. Recent advances in microscopy and computational analyses combined with a functional genetic approach have revealed that patterning genes combined with intricate epithelial folding and movement are responsible for the final shape of a beetle head horn.

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.

The sex determination gene doublesex (dsx) encodes a transcription factor with two domains, oligomerization domain 1 (OD1) and OD2, and is present throughout insects. Sex-specific Dsx splicing isoforms regulate the transcription of target genes and trigger sex differentiation in all Holometabola examined to date. However, in some hemimetabolous insects, dsx is not spliced sexually and its sequence is less conserved. Here, to elucidate evolutionary changes in dsx in domain organisation and regulation in termites, we searched genome and/or transcriptome databases for the dsx OD1 and OD2 in seven termite species and their sister group (Cryptocercus woodroaches). Molecular phylogenetic and synteny analyses identified OD1 sequences of termites and C. punctulatus that clustered with dsx of Holometabola and regarded them as dsx orthologues. The Cryptocercus dsx orthologue containing OD2 was spliced sexually, as previously shown in other insects. However, OD2 was not found in all termite dsx orthologues. These orthologues were encoded by a single exon in three termites for which genome information is available; they were not alternatively spliced but transcribed in a male-specific manner in two examined species. Evolution of dsx regulation from sex-specific splicing to male-specific transcription may have occurred at an early stage of social evolution in termites.

Significance Gene duplication is a major source of evolutionary innovation and is associated with the increases in biological complexity and adaptive radiation. Termites are model social organisms characterized by a sophisticated caste system. We analyzed the genome of the Japanese subterranean termite, an ecologically and economically important insect acting as a destructive pest. The analyses revealed the significance of gene duplication in social evolution. Gene duplication associated with caste-biased gene expression was prevalent in the termite genome. Many of the duplicated genes were related to social functions, such as chemical communication, social immunity, and defense, and they were often expressed in caste-specific organs. We propose that gene duplication facilitates social evolution through regulatory diversification leading to caste-biased expression and functional specialization., 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.

The exaggerated horns of beetles are attractive models for studying the origin of novel traits and morphological evolution. Closely related species often differ profoundly in the size, number, and shape of their horns, and in the body region from which they extend. In addition, beetle horns exhibit exquisite nutrition-dependent phenotypic plasticity, leading to disproportionate growth of the horns in the largest, best-condition individuals and much smaller - even stunted - horn sizes in poor-condition individuals. These exciting phenomena in beetle horns have recently been revealed at the molecular level with the advent of next-generation sequencing. This section reviews the latest research on a horned beetle, the Japanese rhinoceros beetle Trypoxylus dichotomus, whose genome was recently sequenced.

Significance Sperm exhibit dramatic evolutionarily divergent morphologies in almost all taxa. Some sexually reproductive species show polymorphisms in the sperm produced by single males. Here, we focused on Sex-lethal (Sxl), which is the master sex-determination gene in Drosophila melanogaster, and investigated its function in the lepidopteran insect Bombyx mori. Our genetic analyses revealed that Sxl is essential for the formation of anucleate nonfertile parasperm. It is not expected that Sxl would be involved in sperm polymorphisms. Yet, whereas many morphological observations and ecological surveys have been conducted on sperm polymorphisms, this paper identifies the gene involved in sperm polymorphisms. Moreover, we clearly demonstrate that parasperm of B. mori is necessary for sperm migration in female organs., 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.

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.

The beetle horn primordium is a complex and compactly folded epithelial sheet located beneath the larval cuticle. Only by unfolding the primordium the complete 3D shape of the horn appears, suggesting that the morphology of beetle horns is coded in the primordial folding pattern. To decipher the folding pattern, we have developed a method to manipulate the primordial local folding, reproduced it on a computer, and clarified the contribution of the folding of each primordium region to transformation. We found that the three major morphological changes (branching of distal tips, proximodistal elongation, and angular change) were caused by the folding of different regions, and that the folding mechanism was also different depending on the region. The computational methods we used are applicable to the morphological study of other exoskeletal animals.

The head horn of the Asian rhinoceros beetle develops as an extensively folded primordium before unfurling into its final 3D shape at the pupal molt. The information of the final 3D structure of the beetle horn is prefigured in the folding pattern of the developing primordium. However, the developmental mechanism underlying epithelial folding of the primordium is unknown. In this study, we addressed this gap in our understanding of the developmental patterning of the 3D horn shape of beetles by focusing on the formation of furrows at the surface of the primordium that become the bifurcated 3D shape of the horn. By gene knockdown analysis via RNAi, we found that knockdown of the gene Notch disturbed overall horn primordial furrow depth without affecting the 2D furrow pattern. In contrast, knockdown of CyclinE altered 2D horn primordial furrow pattern without affecting furrow depth. Our results show how the depth and 2D pattern of primordial surface furrows are regulated at least partially independently during beetle horn development, and how both can alter the final 3D shape of the horn.

Insects can dramatically change their outer morphology at molting. To prepare for this drastic transformation, insects generate new external organs as folded primordia under the old cuticle. At molting, these folded primordia are physically extended to form their final outer shape in a very short time. Beetle horns are a typical example. Horn primordia are derived from a flat head epithelial sheet, on which deep furrows are densely added to construct the complex folded structure. Because the 3D structure of the pupa horn is coded in the complex furrow pattern, it is indispensable to know how and where the furrows are set. Here, we studied the mechanism of furrow formation using dachsous (ds) gene knocked down beetles that have shorter and fatter adult horns. The global shape of the beetle horn primordia is mushroom like, with dense local furrows across its surface. Knockdown of ds by RNAi changed the global shape of the primordia, causing the stalk region become apparently thicker. The direction of cell division is biased in wildtype horns to make the stalk shape thin and tall. However, in ds knocked down beetles, it became random, resulting in the short and thick stalk shape. On the other hand, a fine and dense local furrow was not significantly affected by the ds knockdown. In developing wildtype horn primordia, we observed that, before the local furrow is formed, the apical constriction signal emerged at the position of the future furrow, suggesting the pre-pattern for the fine furrow pattern. According to the results, we propose that development of complex horn primordia can be roughly divided to two distinct processes, 1) development of global primordia shape by anisotropic cell division, and 2) local furrow formation via actin-myosin dependent apical constriction of specific cells.

Males of the Asian rhinoceros beetle, Trypoxylus dichotomus, possess exaggerated head and thoracic horns that scale dramatically out of proportion to body size. While studies of insulin signaling suggest that this pathway regulates nutrition-dependent growth including exaggerated horns, what regulates disproportionate growth has yet to be identified. The Fat signaling pathway is a potential candidate for regulating disproportionate growth of sexually-selected traits, a hypothesis we advanced in a previous paper (Gotoh et al., 2015). To investigate the role of Fat signaling in the growth and scaling of the sexually dimorphic, condition-dependent traits of the in the Asian rhinoceros beetle T. dichotomus, we used RNA interference to knock down expression of fat and its co-receptor dachsous. Knockdown of fat, and to a lesser degree dachsous, caused shortening and widening of appendages, including the head and thoracic horns. However, scaling of horns to body size was not affected. Our results show that Fat signaling regulates horn growth in T. dichotomus as it does in appendage growth in other insects. However, we provide evidence that Fat signaling does not mediate the disproportionate, positive allometric growth of horns in T. dichotomus.

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.

Males of the Asian rhinoceros beetle, Trypoxylus dichotomus, possess exaggerated head and thoracic horns that scale dramatically out of proportion to body size. While RNAi-mediated knockdowns of the insulin receptor suggest that the insulin signaling pathway regulates nutrition-dependent growth including exaggerated horns, the genes that regulate disproportionate growth have yet to be identified. We used RNAi-mediated knockdown of several genes to investigate their potential role in growth and scaling of the sexually dimorphic, exaggerated head horns of T. dichotomus. Knockdown of the insulin signaling substrate chico and the ecdysone response element broad caused significant decreases in head horn length, while having no or minimal effects on other structures such as elytra and tibiae. However, scaling of horns to body size was not affected by either knockdown. In addition, knockdown of phosphatase and tensin homolog, a negative regulator of the insulin signaling pathway, had no significant effects on any trait. Our results do not identify any candidate genes that may specifically mediate the allometric aspect of horn growth, but they do confirm the insulin signaling pathway as a mediator of conditional trait expression, and importantly implicate the ecdysone signaling pathway, possibly in conjunction with insulin signaling, as an additional mediator of horn growth.

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 β (TGF-β) 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-β 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.

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., Author summary Goliath and Hercules beetles include some of the largest insects known, and the horns they wield are spectacular. These ‘rhinoceros’ beetles form a subfamily within the Scarabaeidae, a clade containing ~35,000 primarily hornless species. The other subfamily of horned scarabs, dung beetles, is distantly related and their horns are considered a separate origin and parallel radiation. We characterize horn development in a rhinoceros beetle and show that the details are surprisingly similar to the horns of dung beetles. Our results reveal exciting parallels at the level of underlying developmental mechanism. The superficial similarity of these two types of beetle horns mirrors an even deeper similarity in the pathways and genes responsible for their construction.

Juvenile hormone (JH) is a key insect growth regulator frequently involved in modulating phenotypically plastic traits such as caste determination in eusocial species, wing polymorphisms in aphids, and mandible size in stag beetles. The jaw morphology of stag beetles is sexually-dimorphic and condition-dependent; males have larger jaws than females and those developing under optimum conditions are larger in overall body size and have disproportionately larger jaws than males raised under poor conditions. We have previously shown that large males have higher JH titers than small males during development, and ectopic application of fenoxycarb (JH analog) to small males can induce mandibular growth similar to that of larger males. What remains unknown is whether JH regulates condition-dependent trait growth in other insects with extreme sexually selected structures. In this study, we tested the hypothesis that JH mediates the condition-dependent expression of the elaborate horns of the Asian rhinoceros beetle, Trypoxylus dichotomus. The sexually dimorphic head horn of this beetle is sensitive to nutritional state during larval development. Like stag beetles, male rhinoceros beetles receiving copious food produce disproportionately large horns for their body size compared with males under restricted diets. We show that JH titers are correlated with body size during the late feeding and early prepupal periods, but this correlation disappears by the late prepupal period, the period of maximum horn growth. While ectopic application of fenoxycarb during the third larval instar significantly delayed pupation, it had no effect on adult horn size relative to body size. Fenoxycarb application to late prepupae also had at most a marginal effect on relative horn size. We discuss our results in context of other endocrine signals of condition-dependent trait exaggeration and suggest that different beetle lineages may have co-opted different physiological signaling mechanisms to achieve heightened nutrient-sensitive weapon growth.

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

Male stag beetles possess colossal mandibles, which they wield in combat to obtain access to females. As with many other sexually selected weapons, males with longer mandibles win more fights. However, variation in the functional morphology of these structures, used in male-male combat, is less well understood. In this study, mandible bite force, gape, structural strength, and potential tradeoffs are examined across a wide size range for one species of stag beetle, Cyclommatus metallifer. We found that not only does male mandible size demonstrate steep positive allometry, but the shape, relative bite force, relative gape, and safety factor of the mandibles also change with male size. Allometry in these functionally important mandibular traits suggests that larger males with larger mandibles should be better fighters, and that the mandibles can be considered an honest signal of male fighting ability. However, negative allometry in mandible structural safety factor, wing size, and flight muscle mass suggest significant costs and a possible limit on the size of the mandibles. J. Exp. Zool. 325A:3-12, 2016. © 2015 Wiley Periodicals, Inc.

Many traits that are sexually dimorphic, appearing either differently or uniquely in one sex, are also sensitive to an organism's condition. This phenomenon seems to have evolved to limit genetic conflict between traits that are under different selective pressures in each sex. Recent work has shed light on the molecular and developmental mechanisms that govern this condition sensitive growth, and this work has now expanded to encompass both sexual dimorphism as well as conditionally plastic growth, as it seems the two phenomena are linked on a molecular level. In all cases studied the gene doublesex, a conserved regulator of sex differentiation, controls both sexual dimorphism as well as the condition-dependent plastic responses common to these traits. However, the advent of next-generation -omics technologies has allowed researchers to decipher the common and diverged mechanisms of sexually dimorphic plasticity and expand investigations beyond the foundation laid by studies utilizing beetle weapons.

The external organs of holometabolous insects are generated through two consecutive processes: the development of imaginal primordia and their subsequent transformation into the adult structures. During the latter process, many different phenomena at the cellular level (e.g. cell shape changes, cell migration, folding and unfolding of epithelial sheets) contribute to the drastic changes observed in size and shape. Because of this complexity, the logic behind the formation of the 3D structure of adult external organs remains largely unknown. In this report, we investigated the metamorphosis of the horn in the Japanese rhinoceros beetle Trypoxylus dichotomus. The horn primordia is essentially a 2D epithelial cell sheet with dense furrows. We experimentally unfolded these furrows using three different methods and found that the furrow pattern solely determines the 3D horn structure, indicating that horn formation in beetles occurs by two distinct processes: formation of the furrows and subsequently unfolding them. We postulate that this developmental simplicity offers an inherent advantage to understanding the principles that guide 3D morphogenesis in insects.

We have developed an imaging method designated as correlative light microscopy and block-face imaging (CoMBI), which contributes to improve the reliability of morphological analyses. This method can collect both the frozen sections and serial block-face images in a single specimen. The frozen section can be used for conventional light microscopic analysis to obtain 2-dimensional (2D) anatomical and molecular information, while serial block-face images can be used as 3-dimensional (3D) volume data for anatomical analysis. Thus, the sections maintain positional information in the specimen, and allows the correlation of 2D microscopic data and 3D volume data in a single specimen. The subjects can vary in size and type, and can cover most specimens encountered in biology. In addition, the required system for our method is characterized by cost-effectiveness. Here, we demonstrated the utility of CoMBI using specimens ranging in size from several millimeters to several centimeters, i.e., mouse embryos, human brainstem samples, and stag beetle larvae, and present successful correlation between the 2D light microscopic images and 3D volume data in a single specimen.

Various types of weapon traits found in insect order Coleoptera are known as outstanding examples of sexually selected exaggerated characters. It is known that the sex determination gene doublesex (dsx) plays a significant role in sex-specific expression of weapon traits in various beetles belonging to the superfamily Scarabaeoidea. Although sex-specific weapon traits have evolved independently in various Coleopteran groups, developmental mechanisms of sex-specific expression have not been studied outside of the Scarabaeoidea. In order to test the hypothesis that dsx-dependent sex-specific expression of weapon traits is a general mechanism among the Coleoptera, we have characterized the dsx in the sexually dimorphic broad-horned beetle Gnatocerus cornutus (Tenebrionidea, Tenebirionidae). By using molecular cloning, we identified five splicing variants of Gnatocerus cornutus dsx (Gcdsx), which are predicted to code four different isoforms. We found one male-specific variant (GcDsx-M), two female-specific variants (GcDsx-FL and GcDsx-FS) and two non-sex-specific variants (correspond to a single isoform, GcDsx-C). Knockdown of all Dsx isoforms resulted in intersex phenotype both in male and female. Also, knockdown of all female-specific isoforms transformed females to intersex phenotype, while did not affect male phenotype. Our results clearly illustrate the important function of Gcdsx in determining sex-specific trait expression in both sexes.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2522-8) contains supplementary material, which is available to authorized users.

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.

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.

Synopsis The exaggerated weapons and ornaments of sexual selection are condition-dependent traits that often grow to exaggerated proportions. The horns of male rhinoceros beetles are extremely sensitive to the larval nutritional environment and are used by rival males in combat over access to females. In contrast to horns, other parts of the body, such as wings, eyes, and legs, scale proportionally with body size, whereas others, such as males’ external genitalia, are invariant with body size, regardless of nutrition. We document how body parts of the Asian rhinoceros beetle, Trypoxylus dichotomus, exhibit plasticity and constraint in response to nutritional condition. We discuss the implications of these results for the evolution of condition-dependent and condition-independent traits in animals.

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.

Most aphids exhibit wing polyphenism, in which winged and wingless females are produced depending on aphid densities. Although juvenile hormone (JH) has been implicated in the regulation of aphid wing polyphenism, relatively few studies examining the direct relationship between JH titer and resultant wing morphs have been undertaken. We therefore investigated the relationship between JH III titer and the development of wing morphs in the vetch aphid Megoura crassicauda during postembryonic development. JH III measurements by liquid chromatography-mass spectrometry (LC-MS) revealed that, at the third instar, presumptive wingless nymphs had significantly higher JH III titers than winged nymphs. In winged nymphs at the third instar, JH III application inhibited wing development resulting in the appearance of winged/wingless intermediates as well as juvenilized individuals with supernumerary molting. These results suggest that JH III plays an important role in wing-morph differentiation during postembryonic development.

In Daphnia pulex, juveniles form "neckteeth" a defensive structure on their heads, in response to predatory kairomones released by Chaoborus larvae. This phenomenon provides a model experimental system for the study of developmental mechanisms and evolutionary processes in predator-induced polyphenisms. Although it is thought that kairomone signals are sensed and converted into physiological signals resulting in morphological changes, little is known about the endocrine and physiological mechanisms of this process. Juvenile hormones and related chemicals, that is, juvenoids, are key hormones responsible for various physiological events in insects, including polyphenisms. In some crustaceans, methyl farnesoate (MF) is known to act as a juvenoid. In order to investigate the functions of juvenoids in defense morph formation, we treated daphnids with MF as well as JHIII (Juvenile Hormone III, an insect juvenoid) and fenoxycarb (a synthetic juvenile hormone analog) during their developmental stages. Strikingly, in the first-instar juveniles, all examined juvenoids stimulated the formation of neckteeth only in the presence of kairomones, not by themselves. This juvenoid effect on the neckteeth formation might be due to disturbance of the JH pathway. Juvenoid treatments reduced tail-spine length, whereas predatory kairomones are known to elongate tail spine. These results suggest that other physiological factors are responsible for the tail-spine elongation.

The regulation of caste differentiation is essential to insect eusociality. Termite soldiers are sterile and cannot eat by themselves because they have specialized mouth morphology. Almost all termite species have a soldier caste, and the soldier ratio per colony is maintained at a low level, probably by elaborate regulatory mechanisms. Although the soldier presence is considered to negatively affect soldier differentiation in all examined species, the detailed mechanism remains unclear. Presoldier differentiation can be induced artificially by juvenile hormone (JH) application to workers, showing that JH is a key factor underlying the regulation of soldier differentiation. In this study, to elucidate physiological changes in workers because of the soldier presence during the molt into presoldiers, JH III applications and JH titer quantifications were carried out in the rhinotermitid termite Reticulitermes speratus. Firstly, the effects of soldier presence before the molt into presoldiers induced by JH III application to workers were investigated. The rates of presoldier molt induced by the treatments with soldiers were significantly lower than those without soldiers. Secondly, worker JH titers in the presence or absence of soldiers were quantified by LC-MS on day 0, 5, 10, and 15 after JH application. Results indicated that the worker JH titers (endogenous+applied JH III) in the presence of soldiers were significantly lower than those without soldiers on day 5 after the JH treatment. On days 10 and 15, such soldier effects were not observed. Finally, the effective duration of soldier presence after the JH application was elucidated. A 4 day period of co-existence with soldiers suppressed presoldier differentiation, suggesting that the soldier presence rapidly decreased the JH titer in other colony members (i.e., workers), resulting in the inhibition of presoldier production.

Termites are social insects, presenting morphologically distinct castes, performing specific tasks in the colony. The developmental processes underlying caste differentiation are mainly controlled by juvenile hormone (JH). Although many fragmentary data support this fact, there was no comparative work on JH titers during the caste differentiation processes. In this study, JH titer variation was investigated using a liquid chromatography-mass spectrometry (LC-MS) quantification method in all castes of the Japanese damp-wood termite Hodotermopsis sjostedti, especially focusing on the soldier caste differentiation pathway, which was induced by treatment with a JH analog. Hemolymph JH titers fluctuated between 20 and 720 pg/μl. A peak of JH was observed during molting events for the pseudergate stationary molt and presoldier differentiation, but this peak was absent prior to the imaginal molt. Soldier caste differentiation was generally associated with high JH titers and nymph to alate differentiation with low JH titers. However, JH titer rose in females during alate maturation, probably in relation to vitellogenesis. In comparison, JH titer was surprisingly low in neotenics. On the basis of these results in both natural and artificial conditions, the current model for JH action on termite caste differentiation is discussed and re-appraised.

In this post-genomic era, genome-wide functional analysis is indispensable. The recent development of RNA interference techniques has enabled researchers to easily analyze gene function even in non-model organisms. On the other hand, little progress has been made in the identification and functional analyses of cis-regulatory elements in non-model organisms. In order to develop experimental platform for identification and analyses of cis-regulatory elements in a non-model organism, in this case, the ladybird beetle, Harmonia axyridis, we established transgenic transposon-tagged lines using a novel composite vector. This vector enables the generation of two types of insertion products (jumpstarter and mutator). The jumpstarter portion carries a transposase gene, while the mutator segment carries a reporter gene for detecting enhancers. The full-composite element is flanked by functional termini (required for movement); however, the mutator region has an extra terminus making it possible for the mutator to remobilize on its own, thus leaving an immobile jumpstarter element behind. Each insertion type is stable on its own, but once crossed, jumpstarters can remobilize mutators. After crossing a jumpstarter and mutator line, all tested G2 females gave rise to at least one new insertion line in the next generation. This jumping rate is equivalent to the P-element-mediated jumpstarter method in Drosophila. These established transgenic lines will offer us the ideal experimental materials for the effective screening and identification of enhancers in this species. In addition, this jumpstarter method has the potential to be as effective in other non-model insect species and thus applicable to any organism.

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., Author Summary Sexual dimorphisms such as the exaggerated antlers of deer, the enormous clawed chelae of crabs, and the horns and mandibles of beetles, are widespread across animal taxa and have fascinated biologists for centuries. Much recent work has uncovered the importance of the role of the sex-determination pathway in the expression of sexually dimorphic traits. However, critical interactions between this pathway and other growth regulatory mechanisms – for example, the physiological mechanisms involved in nutrition-dependent expression of these traits – are less well understood. In this study, we provide evidence of a developmental link between nutrition-sensitivity and sexual differentiation in the giant mandibles of the sexually dimorphic stag beetle, Cyclommatus metallifer. We examined the regulation and function of a key sex determination gene in animals, doublesex (dsx), and its interaction with juvenile hormone (JH), an important insect hormone known to regulate insect polyphenisms including the regulation of the disproportionate growth of male stag beetle mandibles. We found that Cmdsx changes mandibular responsiveness to JH in a sex-specific pattern. Based on these results, we hypothesize that sex-specific regulation of JH responsiveness is a developmental link between nutrition and sexual differentiation in stag beetles.