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

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

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

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

Butterfly wing color made of pigmented liquid.

A previously undescribed mechanism underlying butterfly wing coloration patterns was discovered in two distantly related butterfly species, Siproeta stelenes and Philaethria diatonica. These butterflies have bright green wings, but the color pattern is not derived from solid pigments or nanostructures of the scales or from the color of the cuticular membrane but rather from a liquid retained in the wing membrane. Wing structure differs between the green and non-green areas. In the non-green region, the upper and lower cuticular membranes are attached to each other, whereas in the green region, we observed a space of 5-10 µm where green liquid is held and living cells are present. A pigment analysis and tracer experiment revealed that the color of the liquid is derived from hemolymph components, bilin and carotenoid pigments. This discovery broadens our understanding of the diverse ways in which butterfly wings obtain their coloration and patterns.

Pivot burrowing of scarab beetle (Trypoxylus dichotomus) larva.

Many organisms live in the soil but only a little is known about their ecology especially movement style. Scarab beetle larvae do not have appendages to shovel soil and their trunk is thick compared to their body length. Hence, their movement through the soil is perplexing. Here, we established the observation and analysis system of larval movement and found that the last larval instars of Trypoxylus dichotomus burrow in two different ways, depending on the hardness of the soil. If the soil is soft, the larvae keep their body in a straight line and use longitudinal expansion and contraction; if the soil is hard, they flex and rotate their body. It is thought that the larvae adapt to diverse soil conditions using two different excavation methods. These results are important for understanding the soil ecology and pose a challenge to engineer of newer excavation technology.

Epithelial folding determines the final shape of beetle horns.

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.

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

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.

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

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.

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

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 Antianthe expansa 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.

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

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.