Publications by authors named "Hiroki Gotoh"

33 Publications

Epithelial folding determines the final shape of beetle horns.

Curr Opin Genet Dev 2021 Apr 10;69:122-128. Epub 2021 Apr 10.

Department of Entomology, Washington State University, Pullman, WA, 99163 USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.gde.2021.03.003DOI Listing
April 2021

Computational analyses decipher the primordial folding coding the 3D structure of the beetle horn.

Sci Rep 2021 Jan 13;11(1):1017. Epub 2021 Jan 13.

Pattern Formation Laboratory, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, 565-0871, Japan.

The beetle horn primordium is a complex and compactly folded epithelial sheet located beneath the larval cuticle. Only by unfolding the primordium can the complete 3D shape of the horn appear, suggesting that the morphology of beetle horns is encoded in the primordial folding pattern. To decipher the folding pattern, we developed a method to manipulate the primordial local folding 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 also differs according to the region. The computational methods we used are applicable to the morphological study of other exoskeletal animals.
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http://dx.doi.org/10.1038/s41598-020-79757-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806817PMC
January 2021

Genetical control of 2D pattern and depth of the primordial furrow that prefigures 3D shape of the rhinoceros beetle horn.

Sci Rep 2020 10 29;10(1):18687. Epub 2020 Oct 29.

Ecological Genetics Laboratory, Department of Genomics and Evolutionary Biology, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.

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.
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http://dx.doi.org/10.1038/s41598-020-75709-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596553PMC
October 2020

Structure and development of the complex helmet of treehoppers (Insecta: Hemiptera: Membracidae).

Zoological Lett 2020 24;6. Epub 2020 Feb 24.

4Faculty of Environmental Earth Sciences, Hokkaido University, Sapporo, Hokkaido Japan.

Some insects possess complex three-dimensional (3D) structures that develop under the old cuticle prior to the last imaginal molt. Adult treehoppers (Insecta: Hemiptera: Auchenorrhyncha: Membracidae) have one such complex 3D structure, known as a helmet, on their dorsal side. The adult helmet likely forms inside the nymphal pronotum during the final instar nymphal stage. Previous morphological studies have reported that the adult helmet is a large, bi-layered, plywood-like structure, whereas the nymphal pronotum is a monolayer, sheath-like structure. The adult helmet is much larger than nymphal helmet. Thus, the emergence of the adult helmet involves two structural transitions: a transition from a monolayer, sheath-like pronotum to a bi-layer, plywood-like helmet, and a transition in size from small to large. However, when, how, and in what order these transitions occur within the nymphal cuticle is largely unknown. To determine how adult helmet development occurs under the nymphal cuticle, in the present study we describe the morphology of the final adult helmet and investigate developmental trajectories of the helmet during the final instar nymphal stage. We used micro-CT, scanning electron microscope and paraffin sections for morphological observations, and used as a model species. We found that the structural transition (from monolayer, sheath-like structure to bi-layer, roof-like structure) occurs through the formation of a "miniature" of the adult helmet during the middle stage of development and that subsequently, extensive folding and furrows form, which account for the increase in size. We suggest that the making of a "miniature" is the key developmental step for the formation of various 3D structures of treehopper helmets.
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http://dx.doi.org/10.1186/s40851-020-00155-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041272PMC
February 2020

The Insulin Signaling Substrate Chico and the Ecdysone Response Element Broad Both Regulate Growth of the Head Horns in the Asian Rhinoceros Beetle, Trypoxylus dichotomus.

Integr Comp Biol 2019 11;59(5):1338-1345

Department of Entomology, Washington State University, Pullman, WA 99164, USA.

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.
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http://dx.doi.org/10.1093/icb/icz093DOI Listing
November 2019

Dimorphic sperm formation by .

Proc Natl Acad Sci U S A 2019 05 29;116(21):10412-10417. Epub 2019 Apr 29.

Division of Evolutionary Developmental Biology, National Institute for Basic Biology, Myodaiji, 444-8585 Okazaki, Japan;

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 (), which is the master sex-determination gene in and is necessary for female germline development. It has been widely shown that the sex-determination function of in Drosophilidae species is not conserved in other insects of different orders. We investigated the function of in the lepidopteran insect 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 mutants revealed that the gene is indispensable for proper morphogenesis of apyrene sperm. Similarly, our analyses using 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 Although is essential for oogenesis in , it also plays important roles in spermatogenesis in Therefore, the ancestral function of might be related to germline development.
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http://dx.doi.org/10.1073/pnas.1820101116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535010PMC
May 2019

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

Development 2019 03 4;146(5). Epub 2019 Mar 4.

Graduate School of Environmental Science, Hokkaido University, Sapporo, Hokkaido, 060-0810, Japan

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 Among those, (), 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 by RNAi reduced the mandibular length and distorted its morphology. Furthermore, the epistatic relationships among , , () and were revealed by combined RNAi and qRT-PCR analyses, suggesting that is regulated by , 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.
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http://dx.doi.org/10.1242/dev.171942DOI Listing
March 2019

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

Evol Dev 2019 01 26;21(1):44-55. Epub 2018 Dec 26.

Department of Entomology, Washington State University, Pullman, Washington.

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.
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http://dx.doi.org/10.1111/ede.12277DOI Listing
January 2019

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

Mol Ecol 2018 12 17;27(24):5049-5072. Epub 2018 Dec 17.

Department of Biology, McMaster University, Hamilton, Ontario, Canada.

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.
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http://dx.doi.org/10.1111/mec.14907DOI Listing
December 2018

Rhinoceros beetle horn development reveals deep parallels with dung beetles.

PLoS Genet 2018 10 4;14(10):e1007651. Epub 2018 Oct 4.

Division of Evolutionary Developmental Biology, National Institute for Basic Biology, Okazaki, Japan.

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.
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http://dx.doi.org/10.1371/journal.pgen.1007651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171792PMC
October 2018

Anisotropy of cell division and epithelial sheet bending via apical constriction shape the complex folding pattern of beetle horn primordia.

Mech Dev 2018 08 18;152:32-37. Epub 2018 Jun 18.

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi 464-8601, Japan; Faculty of Environmental Earth Sciences, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.mod.2018.06.003DOI Listing
August 2018

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

Zoolog Sci 2018 Jun;35(3):260-267

1 Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.

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.
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http://dx.doi.org/10.2108/zs170166DOI Listing
June 2018

Recent advances in understanding the mechanisms of sexually dimorphic plasticity: insights from beetle weapons and future directions.

Curr Opin Insect Sci 2018 02 24;25:35-41. Epub 2017 Nov 24.

Division of Evolutionary Developmental Biology, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki, 444-8585 Aichi, Japan.

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.
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http://dx.doi.org/10.1016/j.cois.2017.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880310PMC
February 2018

The Fat-Dachsous signaling pathway regulates growth of horns in Trypoxylus dichotomus, but does not affect horn allometry.

J Insect Physiol 2018 Feb - Mar;105:85-94. Epub 2018 Jan 31.

Department of Entomology, Washington State University, Pullman, WA 99164, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jinsphys.2018.01.006DOI Listing
September 2019

Complex furrows in a 2D epithelial sheet code the 3D structure of a beetle horn.

Sci Rep 2017 10 24;7(1):13939. Epub 2017 Oct 24.

Graduate School of Frontier Bioscience, Osaka University, Suita, Osaka, 565-0871, Japan.

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.
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http://dx.doi.org/10.1038/s41598-017-14170-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655322PMC
October 2017

A novel imaging method for correlating 2D light microscopic data and 3D volume data based on block-face imaging.

Sci Rep 2017 06 16;7(1):3645. Epub 2017 Jun 16.

Department of Anatomy, Graduate School of Medicine, Gunma University. 39-22 Showa-machi 3-chome, Maebashi, Gunma, 371-8511, Japan.

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.
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http://dx.doi.org/10.1038/s41598-017-03900-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473845PMC
June 2017

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

Dev Biol 2017 02 16;422(1):24-32. Epub 2016 Dec 16.

Graduate School of Environmental Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan.

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.
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http://dx.doi.org/10.1016/j.ydbio.2016.12.011DOI Listing
February 2017

Molecular cloning and functional characterization of the sex-determination gene doublesex in the sexually dimorphic broad-horned beetle Gnatocerus cornutus (Coleoptera, Tenebrionidae).

Sci Rep 2016 07 11;6:29337. Epub 2016 Jul 11.

Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.

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.
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http://dx.doi.org/10.1038/srep29337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941388PMC
July 2016

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

BMC Genomics 2016 Mar 22;17:250. Epub 2016 Mar 22.

Department of Entomology, Washington State University, Pullman, WA, 99164, USA.

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.
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http://dx.doi.org/10.1186/s12864-016-2522-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802893PMC
March 2016

Functional mechanics of beetle mandibles: Honest signaling in a sexually selected system.

J Exp Zool A Ecol Genet Physiol 2016 Jan 9;325(1):3-12. Epub 2015 Sep 9.

Department of Biology, Gonzaga University, Spokane, Washington.

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.
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http://dx.doi.org/10.1002/jez.1961DOI Listing
January 2016

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

Dev Dyn 2015 Sep 14;244(9):1039-1045. Epub 2015 Jul 14.

Department of Entomology, Washington State University, Pullman, Washington.

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.
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http://dx.doi.org/10.1002/dvdy.24296DOI Listing
September 2015

Exaggerated trait growth in insects.

Annu Rev Entomol 2015 Jan 20;60:453-72. Epub 2014 Oct 20.

Department of Entomology, Washington State University, Pullman, Washington 99164; email: ,

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.
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http://dx.doi.org/10.1146/annurev-ento-010814-021045DOI Listing
January 2015

Establishment of transgenic lines for jumpstarter method using a composite transposon vector in the ladybird beetle, Harmonia axyridis.

PLoS One 2014 24;9(6):e100804. Epub 2014 Jun 24.

Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0100804PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069169PMC
November 2015

Social interactions affecting caste development through physiological actions in termites.

Front Physiol 2014 9;5:127. Epub 2014 Apr 9.

Department of Biology, Graduate School of Science and Engineering, University of Toyama Toyama, Japan.

A colony of social insects is not only an aggregation of individuals but also a functional unit. To achieve adaptive social behavior in fluctuating environmental conditions, in addition to coordination of physiological status in each individual, the whole colony is coordinated by interactions among colony members. The study on the regulation of social-insect colonies is termed "social physiology." Termites, a major group of social insects, exhibit many interesting phenomena related to social physiology, such as mechanisms of caste regulation in a colony. In their colonies, there are different types of individuals, i.e., castes, which show distinctive phenotypes specialized in specific colony tasks. Termite castes comprise reproductives, soldiers and workers, and the caste composition can be altered depending on circumstances. For the regulation of caste compositions, interactions among individuals, i.e., social interactions, are thought to be important. In this article, we review previous studies on the adaptive meanings and those on the proximate mechanisms of the caste regulation in termites, and try to understand those comprehensively in terms of social physiology. Firstly, we summarize classical studies on the social interactions. Secondly, previous studies on the pheromone substances that mediate the caste regulatory mechanisms are overviewed. Then, we discuss the roles of a physiological factor, juvenile hormone (JH) in the regulation of caste differentiation. Finally, we introduce the achievements of molecular studies on the animal sociality (i.e., sociogenomics) in terms of social physiology. By comparing the proximate mechanisms of social physiology in termites with those in hymenopterans, we try to get insights into the general principles of social physiology in social animals.
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http://dx.doi.org/10.3389/fphys.2014.00127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988372PMC
June 2014

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

PLoS Genet 2014 Jan 16;10(1):e1004098. Epub 2014 Jan 16.

Graduate School of Environmental Science, Hokkaido University, Sapporo, Hokkaido, Japan.

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.
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http://dx.doi.org/10.1371/journal.pgen.1004098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3894178PMC
January 2014

Identification of an alternative knockdown resistance (kdr)-like mutation, M918L, and a novel mutation, V1010A, in the Thrips tabaci voltage-gated sodium channel gene.

Pest Manag Sci 2014 Jun 30;70(6):977-81. Epub 2013 Sep 30.

College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, Fujian, P.R. China; Department of Entomology, Washington State University, Pullman, WA, USA.

Background: Knockdown resistance (kdr) has been identified as a main mechanism against pyrethroid insecticides in many arthropod pests including in the onion thrips, Thrips tabaci. To characterize and identify pyrethroid-resistance in onion thrips in Washington state, we conducted insecticide bioassays and sequenced a region of the voltage gated sodium channel gene from several different T. tabaci populations.

Results: Field collected Thrips tabaci were found to have large variations in resistance to the pyrethroid insecticide lambda-cyhalothrin. We identified two single nucleotide substitutions in our analysis of a partial sequence of the T. tabaci voltage-gated sodium channel gene. One mutation resulted in the non-synonymous substitution of methionine with leucine (M918L), which is well known to be responsible for super knockdown resistance in some pest species. Another non-synonymous substitution, a valine (GTT) to alanine (GCT) replacement at amino acid 1010 (V1010A) was identified in our study and was associated with lambda-cyhalothrin resistance.

Conclusion: We have characterized a known kdr mutation and identified a novel mutation in the voltage-gated sodium channel gene of Thrips tabaci associated with resistance to lambda-cyhalothrin. This gene region and these mutations are expected to be useful in the development of a diagnostic test to detect kdr resistance in many onion thrips populations.
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http://dx.doi.org/10.1002/ps.3638DOI Listing
June 2014

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

Bioessays 2013 Oct 12;35(10):889-99. Epub 2013 Jul 12.

School of Biological Sciences, University of Bristol, UK.

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.
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http://dx.doi.org/10.1002/bies.201300031DOI Listing
October 2013

Effect of juvenoids on predator-induced polyphenism in the water flea, Daphnia pulex.

J Exp Zool A Ecol Genet Physiol 2013 Oct 11;319(8):440-50. Epub 2013 Jun 11.

Laboratory of Ecological Genetics, Graduate School of Environmental Science, Hokkaido University, Sapporo, Hokkaido, Japan; Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Myodaiji, Okazaki, Aichi, Japan.

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.
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http://dx.doi.org/10.1002/jez.1807DOI Listing
October 2013

Juvenile hormone titer and wing-morph differentiation in the vetch aphid Megoura crassicauda.

J Insect Physiol 2013 Apr 19;59(4):444-9. Epub 2013 Feb 19.

Laboratory of Ecological Genetics, Graduate School of Environmental Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan.

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.
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http://dx.doi.org/10.1016/j.jinsphys.2013.02.004DOI Listing
April 2013

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

Evol Dev 2012 Jul;14(4):363-71

Laboratory of Evolutionary Ecology, Graduate School of Environmental Science, Okayama University, Japan.

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.
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http://dx.doi.org/10.1111/j.1525-142X.2012.00554.xDOI Listing
July 2012