Hard Cuticle Research Articles

Exploring the structure and dynamics of soft and hard cuticle of Bombyx mori using solid-state NMR techniques

This study conducts a comprehensive analysis and comparison of Bombyx mori cuticles across different developmental stages, ranging from larval to adult, utilizing advanced solid-state NMR techniques. The primary objective is to elucidate the underlying reasons for the contrasting hardness of adult cuticles and softness of larval cuticles. Notably, PXRD analysis reveals a prominent broad peak at 19.34°, indicating the predominantly amorphous nature of both larval and adult cuticles. Analysis of 13C CP-MAS SSNMR spectra highlights an elevated proportion of phenoxy carbon in adult cuticles (6.77%) compared to larval cuticles (1.24%). Furthermore, a distinctive resonance line at 144 ppm is exclusively observed in adult cuticles, due to catechols, suggesting potential biochemical pathway variations during development. Significant variations in the primary components of 13C chemical shift anisotropy (CSA) tensors for aliphatic carbons of amino acids, catechols, and lipids between adult and larval cuticles indicate alterations in electronic environments. Additionally, the shorter spin–lattice relaxation time of carbon nuclei in larval cuticles compared to adult cuticles implies slower motional dynamics with enhanced degree of sclerotization in adults. By investigating the internal structure and dynamics of cuticles, this research not only contributes to biomimetic material development but also enhances our understanding of structural changes across different developmental stages of B. mori.

Steroid hormone-dependent changes in trehalose physiology in the silkworm, Bombyx mori.

Holometabolous insects undergo metamorphosis to reconstruct their body to the adult form during pupal period. Since pupae cannot take any diets from the outside because of a hard pupal cuticle, those insects stock up on nutrients sufficient for successful metamorphosis during larval feeding period. Among those nutrients, carbohydrates are stored as glycogen or trehalose, which is the major blood sugar in insects. The hemolymph trehalose is constantly high during the feeding period but suddenly decreases at the beginning of the prepupal period. It is believed that trehalase, which is a trehalose-hydrolyzing enzyme, becomes highly active to reduce hemolymph trehalose level during prepupal period. This change in the hemolymph trehalose level has been interpreted as the physiological shift from storage to utilization of trehalose at that stage. Although this shift in trehalose physiology is indispensable for energy production required for successful metamorphosis, little is known on the regulatory mechanisms of trehalose metabolism in accordance with developmental progress. Here, we show that ecdysone, an insect steroid hormone, plays essential roles in the regulation of soluble trehalase activity and its distribution in the midgut of silkworm, Bombyx mori. In the end of larval period, soluble trehalase was highly activated in the midgut lumen. This activation was disappeared in the absence of ecdysone and also restored by ecdysone administration. Our present results suggest that ecdysone is essentially required for the changes in the function of the midgut on trehalose physiology as development progresses.

Cephalic biomechanics underpins the evolutionary success of trilobites

AbstractArthropods (i.e. insects, spiders, crustaceans, myriapods and others), are the most successful Phanerozoic animals. The group is characterized by the possession of a segmented body, jointed limbs and a hard cuticle that is episodically moulted. One highly successful but now extinct group of arthropods is the trilobites. Trilobites underwent episodic moulting (ecdysis), and most trilobites possess facial sutures, lines of weakness in the cephalon, via which the exuviae is shed and the animal emerges. However, zones of weakness appear to represent a structural trade‐off or constraint, particularly during burrowing; sacrificing a consolidated head region useful in burrowing for the ability to moult. Here we reconcile this trade‐off by using biomechanical modelling to demonstrate that facial sutures exist in regions of low stress during the application of burrowing loads. Furthermore, facial sutures and the structure of the cephalon enable sutured trilobites to withstand greater stresses than their non‐suture counterparts. We suggest that this ability to withstand greater burrowing loads enabled trilobites to successfully invade bioturbated and more consolidated sediments of the Cambrian Sediment Revolution, thus facilitating their diversification in the Cambrian and Ordovician and contributing to the evolutionary success of this iconic arthropod group.

Post-eclosion temperature effects on insect cuticular hydrocarbon profiles.

The insect cuticle is the interface between internal homeostasis and the often harsh external environment. Cuticular hydrocarbons (CHCs) are key constituents of this hard cuticle and are associated with a variety of functions including stress response and communication. CHC production and deposition on the insect cuticle vary among natural populations and are affected by developmental temperature; however, little is known about CHC plasticity in response to the environment experienced following eclosion, during which time the insect cuticle undergoes several crucial changes. We targeted this crucial to important phase and studied post‐eclosion temperature effects on CHC profiles in two natural populations of Drosophila melanogaster. A forty‐eight hour post‐eclosion exposure to three different temperatures (18, 25, and 30°C) significantly affected CHCs in both ancestral African and more recently derived North American populations of D. melanogaster. A clear shift from shorter to longer CHCs chain length was observed with increasing temperature, and the effects of post‐eclosion temperature varied across populations and between sexes. The quantitative differences in CHCs were associated with variation in desiccation tolerance among populations. Surprisingly, we did not detect any significant differences in water loss rate between African and North American populations. Overall, our results demonstrate strong genetic and plasticity effects in CHC profiles in response to environmental temperatures experienced at the adult stage as well as associations with desiccation tolerance, which is crucial in understanding holometabolan responses to stress.

Elytra-Mimetic Aligned Composites with Air-Water-Responsive Self-Healing and Self-Growing Capability.

Room-temperature self-healing and self-growing of the exoskeleton with aligned structures in insects has few analogs in synthetic materials. Insect cuticle, such as elytra in beetles, with a typical lightweight lamellar structure, has shown this capability, which is attributed to the accumulation of phenol oxidase with polyphenol and amine-rich compounds in the hard cuticle. In this study, laminar-structure-based intelligence is imitated by incorporating adaptable and growable pyrogallol (PG)-borax dynamic-covalent bonds into a poly(acrylamide)-clay network. The events that lead to crack formation and water accumulation quickly trigger the deprotection of PG. Subsequently, atmospheric O2, as a regeneration source, activates PG oxidative self-polymerization. Multiple permanent and dynamic cross-links, with the involvement of the sacrificed borax, and initiation of a series of intelligent responses occur. The fabricated composites with an aligned lamellar structure exhibit outstanding characteristics, such as air/water-triggered superstrong adhesion, self-repairing, self-sealing and resealing, and reprocessing. Moreover, the strategy endows the composites with a self-growing capability, which leads to a 4- to 10-fold increase in its strength in an outdoor climate (up to 51 MPa). This study could lead to advances in the development of air/water-responsive composite materials for applications such as adaptive barriers.

Stiffness distribution in natural insect cuticle reveals an impact resistance strategy

In nature, many insects have evolved hard cuticles to shelter their soft body, which is thought to be the “body-armour” for insects to protect against predators' sharp teeth or dynamic load damage caused by harsh environments. In recent years, researchers have found that the “body-armour” is composed of multi-layer materials with different elastic modulus from inside to outside. The gradient change rule of the formed material modulus reflects the evolutionary history of insect cuticle's adaptation to external changes. In this article, the mechanical properties of spatial hierarchical architecture of insect cuticle, especially for the shield-like beetle elytra under impact loading, was investigated to reveal the impact resistance strategy and in-depth mechanisms of crashworthy protection. The results show that both discontinuity at the cuticle layers interface and the distributions of stiffness gradients through layers’ thickness have a great influence on preventing stress wave propagation and improving impact-tolerance. Insect cuticle such as beetle elytra with discontinuous exponential stiffness gradient (DC-EXP) along the thickness has been identified to result in the minimum values of stress and interaction force under impact loading, which leads to the best impact resistance property and defensive effect. Furthermore, we compared and discussed the protective properties of insect elytra with different sclerotized endocuticle under quasi-static compression and impact loading, respectively. The knowledge gained from this work reveals the advantages of nature’s choice of the stiffness distribution and may serve to inspire further research of developing advanced multifunctional structures with improved impact resistance capability by programming reasonable stiffness distribution.

Retrieving Leaf Chlorophyll Content by Incorporating Variable Leaf Surface Reflectance in the PROSPECT Model

Leaf chlorophyll content plays a vital role in plant photosynthesis. The PROSPECT model has been widely used for retrieving leaf chlorophyll content from remote sensing data over various plant species. However, despite wide variations in leaf surface reflectance across different plant species and environmental conditions, leaf surface reflectance is assumed to be the same for different leaves in the PROSPECT model. This work extends the PROSPECT model by taking into account the variation of leaf surface reflection. In the modified model named PROSPECT-Rsurf, an additional surface layer with a variable refractive index is bounded on the N elementary layers. Leaf surface reflectance (Rs) is characterized by the difference between the refractive indices of leaf surface and interior layers. The specific absorption coefficients of the leaf total chlorophyll and carotenoids were recalibrated using a cross-calibration method and the refractive indices of leaf surface and interior layers were obtained during model inversion. Chlorophyll content (Cab) retrieval and spectral reconstruction in the visible spectral region (VIS, 400–750 nm) were greatly improved using PROSPECT-Rsurf, especially for leaves covered by heavy wax or hard cuticles that lead to high surface reflectance. The root mean square error (RMSE) of chlorophyll estimates decreased from 11.1 µg/cm2 to 8.9 µg/cm2 and the Pearson’s correlation coefficient (r) increased from 0.81 to 0.88 (p < 0.01) for broadleaf samples in validation, compared to PROSPECT-5. For needle leaves, r increased from 0.71 to 0.89 (p < 0.01), but systematic overestimation of Cab was found due to the edge effects of needles. After incorporating the edge effects in PROSPECT-Rsurf, the overestimation of Cab was alleviated and its estimation was improved for needle leaves. This study explores the influence of leaf surface reflectance on Cab estimation at the leaf level. By coupling PROSPECT-Rsurf with canopy models, the influence of leaf surface reflectance on canopy reflectance and therefore canopy chlorophyll content retrieval can be investigated across different spatial and temporal scales.

Not As Clear As It May Appear: Challenges Associated with Transparent Camouflage in the Ocean.

The "superpower" of invisibility is a reality and a necessity for many animals that live in featureless environments like the open ocean, where there is nowhere to hide. How do animals achieve invisibility? Many animals match their color patterns to their background, but this strategy is limited when the background scene is dynamic. Transparency allows organisms to match any background all the time. However, it is challenging for an organism to maintain transparency across its entire body volume. To be transparent, tissues must minimize light scattering, both at the surface and within. Until recently, it has been unclear how clear animals with complex bodies (such as many crustaceans with hard cuticles, thick muscles, and other internal organs) minimize such light scattering. This is especially challenging in an environment where light can come from many directions: reflections from downwelling sunlight and bioluminescent searchlights from predators. This review summarizes several recent discoveries of multiple unique adaptations for minimizing light scattering both on the exterior cuticle surface and throughout the body volume of transparent crustaceans, as well as the potential tradeoffs and challenges associated with transparent camouflage.

Group I chitin deacetylases are essential for higher order organization of chitin fibers in beetle cuticle

Roles in the organization of the cuticle (exoskeleton) of two chitin deacetylases (CDAs) belonging to group I, TcCDA1 and TcCDA2, as well as two alternatively spliced forms of the latter, TcCDA2a and TcCDA2b, from the red flour beetle, Tribolium castaneum, were examined in different body parts using transmission EM and RNAi. Even though all TcCDAs are co-expressed in cuticle-forming cells from the hardened forewing (elytron) and ventral abdomen, as well as in the softer hindwing and dorsal abdomen, there are significant differences in the tissue specificity of expression of the alternatively spliced transcripts. Loss of either TcCDA1 or TcCDA2 protein by RNAi causes abnormalities in organization of chitinous horizontal laminae and vertical pore canals in all regions of the procuticle of both the hard and soft cuticles. Simultaneous RNAi for TcCDA1 and TcCDA2 produces the most serious abnormalities. RNAi of either TcCDA2a or TcCDA2b affects cuticle integrity to some extent. Following RNAi, there is accumulation of smaller disorganized fibers in both the horizontal laminae and pore canals, indicating that TcCDAs play a critical role in elongation/organization of smaller nanofibers into longer fibers, which is essential for structural integrity of both hard/thick and soft/thin cuticles. Immunolocalization of TcCDA1 and TcCDA2 proteins and effects of RNAi on their accumulation indicate that these two proteins function in concert exclusively in the assembly zone in a step involving the higher order organization of the procuticle.

Study on the Influence of Nitrogen Plasma on Dyeing Properties of Rex Rabbit Fibers

The surface of rex rabbit fibers is hydrophobic in nature because of the presence of the hard cuticle on its surface, and this hydrophobicity may give rise to many problems in the dyeing and finishing processes. In order to improve its dyeability and decrease dye pollution in sewage discharge, nitrogen plasma was used to modify rabbit fibers and after that the modified fibers were dyed with the anionic dyestuffs (C.I. number 16185). The effects of nitrogen plasma on the dyeing properties and the dyeing behavior for the rex rabbit fibers were studied, the related parameters including the treatment time and discharge power were optimized. Surface morphology and roughness of rex rabbit fibers were characterized by scanning electron microscope and atomic force microscopy. XPS and FTIR-ATR were further performed to determine the surface chemical compositions of rex rabbit fibers. The physical properties of rex rabbit fibers were characterized by tensile strength tests. The results show that nitrogen plasma treatment can remove surface scales on the rex rabbit fibers and introduce more active groups such as hydroxyl (–OH), carbonyl (–C=O), and amino (–NH2) on the surface of the fibers, which makes rex rabbit fibers have better dyeability, and effectively improves dyeing rate and dye fixation rate.

A discrete transition zone between cuticle and cortex layers of a human hair fibre: changes observed in the presence of breast cancer.

Attenuated total reflection Fourier transform infrared (ATR–FT-IR) spectroscopy of hair fibres shows a discrete transition zone (DTZ) between the hard protective cuticle layer and the softer elongated cortical cells of the cortex. The DTZ is composed of flattened orthocortical cells located on the outer perimeter of the cortex and appears to range in thickness between 2 and 3.6 μm. The inner surface of the DTZ, adjacent to the elongated cortical cells that make up the core of the hair fibre, is irregular. ATR–FT-IR analyses of these flattened orthocortical cells indicate major changes in the molecular structure of keratins found in this transition zone. Other studies have identified cells that produce keratins that are distinct from alpha keratins found in the elongated heterocortical cells in the hair fibre core. These distinct keratins appear to be produced in the lower region of the hair follicle at the interface between the cuticle and cortex.The DTZ is also the region where ATR–FT-IR spectroscopy studies identified changes in C−H bending of lipid esters indicative of breast cancer. Lipid ester absorption bands at 1738 and 1732 cm−1, present in non-cancer hair, are absent in the cancer hair and a new ester band absorbing at 1736 cm−1 is observed. When the breast cancer is clinically removed, the 1736 cm−1 ester band absorption and the increase in the 1446–1456 C−H-bending absorption ratio are no longer observed. This suggests that biomarkers produced by the breast cancer interact with stem or other cells near the lower region of the follicle, controlling the amount and type of lipid esters in the DTZ.

Small genome symbiont underlies cuticle hardness in beetles

Beetles, representing the majority of the insect species diversity, are characterized by thick and hard cuticle, which plays important roles for their environmental adaptation and underpins their inordinate diversity and prosperity. Here, we report a bacterial endosymbiont extremely specialized for sustaining beetle's cuticle formation. Many weevils are associated with a γ-proteobacterial endosymbiont lineage Nardonella, whose evolutionary origin is estimated as older than 100 million years, but its functional aspect has been elusive. Sequencing of Nardonella genomes from diverse weevils unveiled drastic size reduction to 0.2 Mb, in which minimal complete gene sets for bacterial replication, transcription, and translation were present but almost all of the other metabolic pathway genes were missing. Notably, the only metabolic pathway retained in the Nardonella genomes was the tyrosine synthesis pathway, identifying tyrosine provisioning as Nardonella's sole biological role. Weevils are armored with hard cuticle, tyrosine is the principal precursor for cuticle formation, and experimental suppression of Nardonella resulted in emergence of reddish and soft weevils with low tyrosine titer, confirming the importance of Nardonella-mediated tyrosine production for host's cuticle formation and hardening. Notably, Nardonella's tyrosine synthesis pathway was incomplete, lacking the final step transaminase gene. RNA sequencing identified host's aminotransferase genes up-regulated in the bacteriome. RNA interference targeting the aminotransferase genes induced reddish and soft weevils with low tyrosine titer, verifying host's final step regulation of the tyrosine synthesis pathway. Our finding highlights an impressively intimate and focused aspect of the host-symbiont metabolic integrity via streamlined evolution for a single biological function of ecological relevance.

GROWTH, INTERMOULT PERIOD AND WEIGHT OF EDIBLE PORTIONS OF GIANT PRAWN AND TIGER SHRIMP

Since a prawn's body is covered by a hard cuticle, its growing mechanism develops through a moulting procese. The development of the adult giant prawn and tiger shri-p during the moulting cycles has been studied

High-resolution AP-SMALDI mass spectrometry imaging of Drosophila melanogaster

The pomace fly or fruit fly Drosophila melanogaster is known as a cost-effective model organism widely used to study neurological diseases and metabolite-related diseases. Among these metabolites, lipids play important roles in energy homeostasis, metabolism, membrane structure, and signaling. Although the Drosophila lipidome has been described in previous studies, there is only a little information on the localization and distribution of the various lipid classes and species in the fly. In this work, high-resolution atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging was performed in order to determine the spatially resolved distribution of metabolites of D. melanogaster, with a focus on the tissue-specific characteristic regional distributions of identified compounds from 20μm thick cryosections of the fly. We identified and characterized the anatomical distribution of a total of 97 lipids, 62 of them identified as glycerophospholipids and sphingolipids and 35 of them as glycerolipids, in three biological replicates of the fly with a consistent anatomical distribution using 2,5-dihydroxybenzoic acid (DHB) as a matrix for soft ionization in positive-ion mode with a pixel size of 5–10μm. Furthermore, we used para-nitroaniline as a matrix for both the positive- and negative-ion mode, enabling the identification of 89 deprotonated lipids in negative-ion mode. Among them, 48 have been identified in both positive- (protonated) and negative-ion (deprotonated) mode within the mass accuracy of ±3ppm. All ion images were separated according to their localization principally into head, posterior region and whole body of the fly. The spatial identity especially of Drosophila brain metabolites, including lipids, small metabolites and neuropeptides, will provide the possibility of studying neurodegenerative diseases. Therefore, we additionally mapped the distribution of neuropeptides in coronal Drosophila brain sections. Furthermore, several lipophilic male- and female-specific sex pheromones were identified, differentiated, and characterized according to their typical distribution pattern in mated and virgin flies. We report on an efficient method for the preparation of improved tissue sections after ethanol dehydration and 5% carboxymethylcellulose and gelatin embedding, capable of maintaining the tissue integrity of the whole fly, which was a challenge due to the hard cuticle and heterogeneous tissue types of this insect. Moreover, our instrumentation with a high spatial resolution, mass resolution and mass accuracy combined with on-tissue MS/MS imaging overcomes common limitations typically observed with low resolution mass spectrometric imaging, such as insufficient spatial resolution which cannot deliver precise and detailed information from the internal organ-specific tissues, and uncertainties of generated signals based on low mass accuracy and resolution. By using DHB, our approach allowed the identification of protonated, sodiated and potassiated lipid species within a mass accuracy of ±1ppm and lipophilic pheromones within ±3ppm with a spatial resolution set up within 5–15μm at a high detection sensitivity of the instrumentation.

Localization of RR-1 and RR-2 cuticular proteins within the cuticle of Anopheles gambiae

The largest arthropod cuticular protein family, CPR, has the Rebers and Riddiford (R&R) Consensus that in an extended form confers chitin-binding properties. Two forms of the Consensus, RR-1 and RR-2, have been recognized and initial data suggested that the RR-1 and RR-2 proteins were present in different regions within the cuticle itself. Thus, RR-2 proteins would contribute to exocuticle that becomes sclerotized, while RR-1s would be found in endocuticle that remains soft. An alternative, and more common, suggestion is that RR-1 proteins are used for soft, flexible cuticles such as intersegmental membranes, while RR-2s are associated with hard cuticle such as sclerites and head capsules. We used TEM immunogold detection to localize the position of several RR-1 and RR-2 proteins in the cuticle of Anopheles gambiae. RR-1s were localized in the procuticle of the soft intersegmental membrane except for one protein found in the endocuticle of hard cuticle. RR-2s were consistently found in hard cuticle and not in flexible cuticle. All RR-2 antibodies localized to the exocuticle and four out of six were also found in the endocuticle. Hence the location of RR-1s and RR-2s depends more on properties of individual proteins than on either hypothesis.

The CPCFC cuticular protein family: Anatomical and cuticular locations in Anopheles gambiae and distribution throughout Pancrustacea

Arthropod cuticles have, in addition to chitin, many structural proteins belonging to diverse families. Information is sparse about how these different cuticular proteins contribute to the cuticle. Most cuticular proteins lack cysteine with the exception of two families (CPAP1 and CPAP3), recently described, and the one other that we now report on that has a motif of 16 amino acids first identified in a protein, Bc-NCP1, from the cuticle of nymphs of the cockroach, Blaberus craniifer (Jensen et al., 1997). This motif turns out to be present as two or three copies in one or two proteins in species from many orders of Hexapoda. We have named the family of cuticular proteins with this motif CPCFC, based on its unique feature of having two cysteines interrupted by five amino acids (C-X(5)-C). Analysis of the single member of the family in Anopheles gambiae (AgamCPCFC1) revealed that its mRNA is most abundant immediately following ecdysis in larvae, pupae and adults. The mRNA is localized primarily in epidermis that secretes hard cuticle, sclerites, setae, head capsules, appendages and spermatheca. EM immunolocalization revealed the presence of the protein, generally in endocuticle of legs and antennae. A phylogenetic analysis found proteins bearing this motif in 14 orders of Hexapoda, but not in some species for which there are complete genomic data. Proteins were much longer in Coleoptera and Diptera than in other orders. In contrast to the 1 and occasionally 2 copies in other species, a dragonfly, Ladona fulva, has at least 14 genes coding for family members. CPCFC proteins were present in four classes of Crustacea with 5 repeats in one species, and motifs that ended C-X(7)-C in Malacostraca. They were not detected, except as obvious contaminants, in any other arthropod subphyla or in any other phylum.The conservation of CPCFC proteins throughout the Pancrustacea and the small number of copies in individual species indicate that, when present, these proteins are serving important functions worthy of further study.

Studies of Laboulbeniales (Fungi, Ascomycota) on Myrmica ants (II): variation of infection by Rickia wasmannii over habitats and time

One group of important insect parasites are the Laboulbeniales (Ascomycota), microscopic fungi that live attached to the exterior of their hosts, mainly beetles, but also mites, millipedes, earwigs, and ants. Rickia wasmannii is a common fungus in Europe and is limited to the ant genus Myrmica (Hymenoptera, Formicidae). This paper presents patterns of R. wasmannii infection in the Netherlands from three host species collected in three series of pitfall traps: Myrmica ruginodis, M. sabuleti, and M. scabrinodis. The infection rate of especially M. sabuleti was so high, that it allowed analyses of infection patterns over time and habitats. We found that only workers were infected, mostly the older ones with a hard cuticle. Gynes are probably never infected. This is supported with data from a nature restoration site: in this young area R. wassmannii is not abundant in contrast to close-by sites, so there probably is a build-up of infection by Rickia over time through worker contact. Taken over three periods throughout the year (spring, summer, autumn), parasite prevalence declined significantly in M. sabuleti, with a non-significant declining trend in M. scabrinodis. Increased allogrooming behavior in the nest in the winter may be the main contributing factor for this. New, largely uninfected cohorts of workers lead to decreased infection rate during the reproduction season. Finally, Rickia wasmannii occurs throughout a wide variety of habitats, from moist and cool to dry and warm.

CDNA cloning and analysis of alanine-rich cuticle protein gene of common fouling barnacle, Balanus amphitrite

Article history: Received on: 16/03/2015 Revised on: 31/03/2015 Accepted on: 22/04/2015 Available online: 20/05/2015 Hard cuticle of insects is a complex structure composed mainly of several proteins and chitin fibers. Cuticular proteins are important as presence of different cuticular proteins confer different mechanical properties to the exoskeletons of insects. Hence, there is growing interest in studying cuticular proteins as bioengineering of these proteins may help in synthesis of biomaterials for various applications. Till date many cuticle proteins have been identified from insects. These proteins show presence of characteristic motifs in them such as R&R motif, extended R&R motif, AAP (V/L) motif etc. We have identified one of such putative cuticle protein encoding gene from common fouling barnacle, Balanus amphitrite by cDNA cloning and sequencing. The gene was found to encode an open reading frame (ORF) of 102 amino acids. The deduced protein sequence was found rich in alanine forming almost 22% of the total amino acids. Other two predominant amino acids, valine and proline together contributed ~23% of total amino acids. The hydropathy plot of the amino acid sequence showed presence of two hydrophobic regions surrounding central hydrophilic region. The protein sequence showed presence of three characteristic YHAAP (V/L) motifs at the C-terminus.

Embryonic development ofCarabus insulicola(Insecta, Coleoptera, Carabidae) with special reference to external morphology and tangible evidence for the subcoxal theory

The egg morphology and successive changes in the developing embryos of the carabid ground beetle Carabus insulicola (Carabidae) are described based on light and scanning electron microscopy observations. Newly laid eggs of this species are ellipsoid and measure approximately 6.1 × 2.9 mm, before increasing to 6.6 × 3.4 mm at hatching. The egg period is about 11 days at 23°C. The egg shell is characterized by a thin fragile chorion covering a hard serosal cuticle. The embryo forms on the ventral egg surface, where it develops for the duration of the egg period. During the process of thoracic leg formation, two subcoxal rings, subcoxae-1 and 2, are clearly discernible at the basalmost region of the leg rudiments, and these subcoxae participate in the formation of the larval pleura and sterna. The result thus provides tangible evidence for the subcoxal theory, that is, that thoracic pleura and sterna are derived from subcoxal regions. Despite the complete absence of abdominal appendages in the larvae of this species, two pairs of appendage-like swellings, the medial and lateral ones, temporarily arise in the first eight abdominal segments during the middle of embryonic development. The medial swellings are assumed to be serially homologous with the coxal part of the thoracic leg, and they later flatten out and participate in the formation of the larval pleura (hypopleurites). In the light of the serially homologous relationships among gnathal appendages, thoracic legs, and abdominal appendage-like swellings, we identified the subcoxal regions in both the gnathal and abdominal segments. Although, the lateral swellings soon degenerate and disappear, it is considered that the swellings originate in the abdominal subcoxae-2 and may be homologous to the tracheal gills of larvae of Gyrinidae. Based on the embryological results, new interpretations for the constituent of gnathal appendages are proposed.

Locusts use a composite of resilin and hard cuticle as an energy store for jumping and kicking

Locusts jump and kick by using a catapult mechanism in which energy is first stored and then rapidly released to extend the large hind legs. The power is produced by a slow contraction of large muscles in the hind femora that bend paired semi-lunar processes in the distal part of each femur and store half the energy needed for a kick. We now show that these energy storage devices are composites of hard cuticle and the rubber-like protein resilin. The inside surface of a semi-lunar process consists of a layer of resilin, particularly thick along an inwardly pointing ridge and tightly bonded to the external, black cuticle. From the outside, resilin is visible only as a distal and ventral triangular area that tapers proximally. High-speed imaging showed that the semi-lunar processes were bent in all three dimensions during the prolonged muscular contractions that precede a kick. To reproduce these bending movements, the extensor tibiae muscle was stimulated electrically in a pattern that mimicked the normal sequence of its fast motor spikes recorded in natural kicking. Externally visible resilin was compressed and wrinkled as a semi-lunar process was bent. It then sprung back to restore the semi-lunar process rapidly to its original natural shape. Each of the five nymphal stages jumped and kicked and had a similar distribution of resilin in their semi-lunar processes as adults; the resilin was shed with the cuticle at each moult. It is suggested that composite storage devices that combine the elastic properties of resilin with the stiffness of hard cuticle allow energy to be stored by bending hard cuticle over only a small distance and without fracturing. In this way all the stored energy is returned and the natural shape of the femur is restored rapidly so that a jump or kick can be repeated.