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Concept: Pupa


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.

Concepts: Insect, Developmental biology, Epithelium, Complexity, Scarabaeidae, Pupa, Japanese rhinoceros beetle, Rhinoceros beetle


Studies of model insects have greatly increased our understanding of animal development. Yet, they are limited in scope to this small pool of model species: a small number of representatives for a hyperdiverse group with highly varied developmental processes. One factor behind this narrow scope is the challenging nature of traditional methods of study, such as histology and dissection, which can preclude quantitative analysis and do not allow the development of a single individual to be followed. Here, we use high-resolution X-ray computed tomography (CT) to overcome these issues, and three-dimensionally image numerous lepidopteran pupae throughout their development. The resulting models are presented in the electronic supplementary material, as are figures and videos, documenting a single individual throughout development. They provide new insight and details of lepidopteran metamorphosis, and allow the measurement of tracheal and gut volume. Furthermore, this study demonstrates early and rapid development of the tracheae, which become visible in scans just 12 h after pupation. This suggests that there is less remodelling of the tracheal system than previously expected, and is methodologically important because the tracheal system is an often-understudied character system in development. In the future, this form of time-lapse CT-scanning could allow faster and more detailed developmental studies on a wider range of taxa than is presently possible.

Concepts: Insect, Developmental biology, Larva, Medical imaging, Model organism, Evolutionary developmental biology, Metamorphosis, Pupa


Post-mortem interval (PMI) is frequently calculated using immature stages of carrion frequenting Calliphoridae (Diptera). This is based on identification to species level, followed by age estimation of the samples. These two processes depend on suitable preservation of insects for subsequent analyses, yet preservation methods for the pupal stage are poorly defined and inappropriate methods may result in discolouration or nucleic acid degradation. This study examined the effects of 21 common preservation methods on Calliphora vicina pupae of 4 and 7d old, assessing consequences of the various methods for DNA-based species identification, age estimation using morphological analyses, and differential gene expression (DGE) studies. Pupae were examined within two weeks of preservation and again after 6-8 months. Of the methods tested, hot-water-killing (HWK) followed by storage in 80% ethanol at -20°C or 4°C was the best treatment for external morphology and histological analyses respectively. DNA based species identification was possible following all methods. RNA integrity and amplification were best when pupae were stored at -80°C or in RNAlater (-20°C), however HWK and storage in 80% ethanol at -20°C was also acceptable, and thus the latter is proposed as a universal preservative method for pupae. This study proposes a preservation method for pupae that enables DNA-based species identification, internal and external morphological analysis for age estimation, and DGE study to be carried out on a single specimen, enabling a multidisciplinary approach to age estimation from a single pupa.

Concepts: DNA, Gene, Insect, Food preservation, Calliphoridae, Calliphora vicina, Pupa, Calliphora


The brood of ants and other social insects is highly susceptible to pathogens, particularly those that penetrate the soft larval and pupal cuticle. We here test whether the presence of a pupal cocoon, which occurs in some ant species but not in others, affects the sanitary brood care and fungal infection patterns after exposure to the entomopathogenic fungus Metarhizium brunneum. We use a) a comparative approach analysing four species with either naked or cocooned pupae and b) a within-species analysis of a single ant species, in which both pupal types co-exist in the same colony.

Concepts: Bacteria, Evolution, Insect, Plant, Caterpillar, Ant, Metarhizium anisopliae, Pupa


Explaining the taxonomic richness of the insects, comprising over half of all described species, is a major challenge in evolutionary biology. Previously, several evolutionary novelties (key innovations) have been posited to contribute to that richness, including the insect bauplan, wings, wing folding and complete metamorphosis, but evidence over their relative importance and modes of action is sparse and equivocal. Here, a new dataset on the first and last occurrences of fossil hexapod (insects and close relatives) families is used to show that basal families of winged insects (Palaeoptera, e.g. dragonflies) show higher origination and extinction rates in the fossil record than basal wingless groups (Apterygota, e.g. silverfish). Origination and extinction rates were maintained at levels similar to Palaeoptera in the more derived Polyneoptera (e.g. cockroaches) and Paraneoptera (e.g. true bugs), but extinction rates subsequently reduced in the very rich group of insects with complete metamorphosis (Holometabola, e.g. beetles). Holometabola show evidence of a recent slow-down in their high net diversification rate, whereas other winged taxa continue to diversify at constant but low rates. These data suggest that wings and complete metamorphosis have had the most effect on family-level insect macroevolution, and point to specific mechanisms by which they have influenced insect diversity through time.

Concepts: Evolution, Insect, Charles Darwin, Insects, Pterygota, Insect wing, Endopterygota, Pupa


Diapause is an actively induced dormancy that has evolved in Metazoa to resist environmental stresses. In temperate regions, many diapausing insects overwinter at low temperatures by blocking embryonic, larval or adult development. Despite its Afro-tropical origin, Drosophila melanogaster migrated to temperate regions of Asia and Europe where females overwinter as adults by arresting gonadal development (reproductive diapause) at temperatures <13°C. Recent work in D. melanogaster has implicated the developmental hormones dILPs-2 and/or dILP3, and dILP5, homologues of vertebrate insulin/insulin-like growth factors (IGFs), in reproductive arrest. However, polymorphisms in timeless (tim) and couch potato (cpo) dramatically affect diapause inducibility and these dILP experiments could not exclude this common genetic variation contributing to the diapause phenotype. Here, we apply an extensive genetic dissection of the insulin signaling pathway which allows us to see both enhancements and reductions in egg development that are independent of tim and cpo variations. We show that a number of manipulations dramatically enhance diapause to ~100%. These include ablating, or reducing the excitability of the insulin-producing cells (IPCs) that express dILPs-2,3,5 employing the dilp2,3,5-/- triple mutant, desensitizing insulin signaling using a chico mutation, or inhibiting dILP2 and 5 in the hemolymph by over-expressing Imaginal Morphogenesis Protein-Late 2 (Imp-L2). In addition, triple mutant dilp2,3,5-/- females maintain high levels of diapause even when temperatures are raised in adulthood to 19°C. However at 22°C, these females all show egg development revealing that the effects are conditional on temperature and not a general female sterility. In contrast, over-expression of dilps-2/5 or enhancing IPC excitability, led to levels of ovarian arrest that approached zero, underscoring dILPs-2 and 5 as key antagonists of diapause.

Concepts: Genetics, Signal transduction, Biology, Insect, Developmental biology, Drosophila melanogaster, Drosophila, Pupa


Transmembrane glycerol transport is typically facilitated by aquaglyceroporins in Prokaryota and Eukaryota. In holometabolan insects however, aquaglyceroporins are absent, yet several species possess polyol permeable aquaporins. It thus remains unknown how glycerol transport evolved in the Holometabola. By combining phylogenetic and functional studies, here we show that a more efficient form of glycerol transporter related to the water-selective channel AQP4 specifically evolved and multiplied in the insect lineage, resulting in the replacement of the ancestral branch of aquaglyceroporins in holometabolan insects. To recapitulate this evolutionary process, we generate specific mutants in distantly related insect aquaporins and human AQP4 and show that a single mutation in the selectivity filter converted a water-selective channel into a glycerol transporter at the root of the crown clade of hexapod insects. Integration of phanerozoic climate models suggests that these events were associated with the emergence of complete metamorphosis and the unparalleled radiation of insects.

Concepts: DNA, Evolution, Species, Insect, Phylogenetics, Transport, Endopterygota, Pupa


Butterfly wing color patterns often contain eyespots, which are developmentally determined at the late larval and early pupal stages by organizing activities of focal cells that can later form eyespot foci. In the pupal stage, the focal position of a future eyespot is often marked by a focal spot, one of the pupal cuticle spots, on the pupal surface. Here, we examined the possible relationships of the pupal focal spots with the underneath pupal wing tissues and with the adult wing eyespots using Junonia butterflies. Large pupal focal spots were found in two species with large adult eyespots, J. orithya and J. almana, whereas only small pupal focal spots were found in a species with small adult eyespots, J. hedonia. The size of five pupal focal spots on a single wing was correlated with the size of the corresponding adult eyespots in J. orithya. A pupal focal spot was a three-dimensional bulge of cuticle surface, and the underside of the major pupal focal spot exhibited a hollowed cuticle in a pupal case. Cross sections of a pupal wing revealed that the cuticle layer shows a curvature at a focal spot, and a positional correlation was observed between the cuticle layer thickness and its corresponding cell layer thickness. Adult major eyespots of J. orithya and J. almana exhibited surface elevations and depressions that approximately correspond to the coloration within an eyespot. Our results suggest that a pupal focal spot is produced by the organizing activity of focal cells underneath the focal spot. Probably because the focal cell layer immediately underneath a focal spot is thicker than that of its surrounding areas, eyespots of adult butterfly wings are three-dimensionally constructed. The color-height relationship in adult eyespots might have an implication in the developmental signaling for determining the eyespot color patterns.

Concepts: Insect, Larva, Lepidoptera, Butterfly, Butterflies, Pupa


Butterfly wings are covered with regularly arranged single-colored scales that are formed at the pupal stage. Understanding pupal wing development is therefore crucial to understand wing color pattern formation. Here, we successfully employed real-time in vivo imaging techniques to observe pupal hindwing development over time in the blue pansy butterfly, Junonia orithya. A transparent sheet of epithelial cells that were not yet regularly arranged was observed immediately after pupation. Bright-field imaging and autofluorescent imaging revealed free-moving hemocytes and tracheal branches of a crinoid-like structure underneath the epithelium. The wing tissue gradually became gray-white, epithelial cells were arranged regularly, and hemocytes disappeared, except in the bordering lacuna, after which scales grew. The dynamics of the epithelial cells and scale growth were also confirmed by fluorescent imaging. Fluorescent in vivo staining further revealed that these cells harbored many mitochondria at the surface of the epithelium. Organizing centers for the border symmetry system were apparent immediately after pupation, exhibiting a relatively dark optical character following treatment with fluorescent dyes, as well as in autofluorescent images. The wing tissue exhibited slow and low-frequency contraction pulses with a cycle of approximately 10 to 20 minutes, mainly occurring at 2 to 3 days postpupation. The pulses gradually became slower and weaker and eventually stopped. The wing tissue area became larger after contraction, which also coincided with an increase in the autofluorescence intensity that might have been caused by scale growth. Examination of the pattern of color development revealed that the black pigment was first deposited in patches in the central areas of an eyespot black ring and a parafocal element. These results of live in vivo imaging that covered wide wing area for a long time can serve as a foundation for studying the cellular dynamics of living wing tissues in butterflies.

Concepts: Insect, Epithelium, Tissues, Skin, Dyes, Lepidoptera, Butterfly, Pupa


The phylogeny of insects, one of the most spectacular radiations of life on earth, has received considerable attention. However, the evolutionary roots of one intriguing group of insects, the twisted-wing parasites (Strepsiptera), remain unclear despite centuries of study and debate. Strepsiptera exhibit exceptional larval developmental features, consistent with a predicted step from direct (hemimetabolous) larval development to complete metamorphosis that could have set the stage for the spectacular radiation of metamorphic (holometabolous) insects. Here we report the sequencing of a Strepsiptera genome and show that the analysis of sequence-based genomic data (comprising more than 18 million nucleotides from nearly 4,500 genes obtained from a total of 13 insect genomes), along with genomic metacharacters, clarifies the phylogenetic origin of Strepsiptera and sheds light on the evolution of holometabolous insect development. Our results provide overwhelming support for Strepsiptera as the closest living relatives of beetles (Coleoptera). They demonstrate that the larval developmental features of Strepsiptera, reminiscent of those of hemimetabolous insects, are the result of convergence. Our analyses solve the long-standing enigma of the evolutionary roots of Strepsiptera and reveal that the holometabolous mode of insect development is more malleable than previously thought.

Concepts: Gene, Evolution, Genome, Insect, Developmental biology, Larva, Lepidoptera, Pupa