Role of beta-alanine in cuticular tanning,sclerotization, and temperature regulation in Drosophila melanogaster

Injection of 20 nl of 1.0 M beta-alanine, about the minimal amount needed to produce wild-type tanned phenomenocopies from newly eclosed mutant black Drosophila melanogaster, increases stiffness and puncture-resistance of the wing cuticle. Increasing the concentration of beta-alanine to 2.0 M increases puncture-resistance further. Injection of 1.0 M of the beta-alanine analogue beta-aminoisobutyric acid, does not induce tanning or puncture-resistance, nor does injection of 1.0 M dopamine. However, injection of 1.0 M beta-alanine and 1.0 M dopamine increases puncture-resistance more than an injection of 1.0 M beta-alanine, though not more than an injection of 2.0 M beta-alanine alone. Within 10 min after injection of [3-³H]beta-alanine into newly eclosed normal flies, ³H becomes 8.7 times more concentrated in the cuticle than in an equal area of underlying epidermis. ³H is excluded from the epidermis or cuticle of ebony strains. Ebony strains show a deficiency of cuticular electron-absorbing material, and the cuticular lamellae show a tendency to separate from each other. Compaction of the chitinous lamellae is induced in alkali-detanned pupal sheaths by exposure to nascent quinones of N-acetyldopamine or N-beta-alanyldopamine. Glucosamine, but not N-acetylglucosamine, reacts with such quinones in tanning reactions. Under an infrared beam, black cuticular pigmentation induces more rapid heating of haemocoel fluids than does tan pigmentation. A theory of pigmentation and sclerotization relative to environmental adaptation is presented.     

The expanding alloscutal cuticle in adults of the argasid tick Argas (Persicargas) robertsi (Acari: Ixodoidea)

The extensible cuticle of Argas (P.) robertsi is tuberculate and deeply folded when the tick is unfed but expands rapidly during feeding. During this expansion the epicuticle becomes less convoluted and the underlying endocuticle stretches but there is no significant alteration in thickness. However, the stretched cuticle has taken on a more open structure. Increase in surface area is restricted to a blister-like expansion because of an inextensible lateral suture which separates the dorsal and ventral surfaces. The cuticle is very hydrophobic, contains 9.9% chitin in the female and 8.9% in the male and the cuticular proteins are largely basic. The cuticle has similar properties to that of the ixodid tick Boophilus microplus but differs from it in fine structure. These differences appear to be related to the time sequence of cuticle synthesis and deposition and to the cycle of expansion and contraction which takes place each time A. (P.) robertsi feeds.     

Functional characterization of bursicon receptor and genome-wide analysis for identification of genes affected by bursicon receptor RNAi

Bursicon is an insect neuropeptide hormone that is secreted from the central nervous system into the hemolymph and initiates cuticle tanning. The receptor for bursicon is encoded by the rickets (rk) gene and belongs to the G protein-coupled receptor (GPCR) superfamily. The bursicon and its receptor regulate cuticle tanning as well as wing expansion after adult eclosion. However, the molecular action of bursicon signaling remains unclear. We utilized RNA interference (RNAi) and microarray to study the function of the bursicon receptor (Tcrk) in the model insect, Tribolium castaneum. The data included here showed that in addition to cuticle tanning and wing expansion reported previously, Tcrk is also required for development and expansion of integumentary structures and adult eclosion. Using custom microarrays, we identified 24 genes that are differentially expressed between Tcrk RNAi and control insects. Knockdown in the expression of one of these genes, TC004091, resulted in the arrest of adult eclosion. Identification of genes that are involved in bursicon receptor mediated biological processes will provide tools for future studies on mechanisms of bursicon action.     

Organisation of wing cuticle in Locusta migratoria linnaeus,Tropidacris cristata linnaeus and Romalea microptera beauvais (Orthoptera : Acrididae)

The composite fibrous architectures of the wing cuticles of Locusta migratoria, Tropidacris (= Eutropidacris) cristata and Romalea microptera (Orthoptera : Acrididae) have been established. The wing cuticle in all the 3 species consists of: (i) an exocuticle, which is either pigmented or birefringent, and which under an electron microscope shows constantly helicoidal architecture of chitin microfibrils; (ii) endocuticle, which shows alternately birefringent and isotropic layers when sectioned transversely across the wing veins; these layers show helicoidal and unidirectional architecture, respectively of chitin microfibrils under the electron microscope. In transverse section, the chitin microfibrils appear as clear rods (2.8 nm in diameter) in a darkly stained matrix. However, in the hinge called the “claval furrow”, these microfibrils are considerably larger, being 25 nm in diameter. This presumably gives sufficient hardness to the claval hinge, which is the most vulnerable area for wear and tear during flight. The pore canals follow the parabolic pattern of microfibrils in the helicoidal layer, but remain straight in the unidirectional layers. The thickness of wing cuticle increases up to about 10–12 days, the time at which the acridids most probably attain the optimum flight ability. It is suggested that changes in the wing cuticle are related to increased wing beat frequency and speed of flight with age, and may help in resisting the simultaneous increase in the bending and twisting forces on the wing.     

Bursicon signaling mutations separate the epithelial-mesenchymal transition from programmed cell death during Drosophila melanogaster wing maturation

Following eclosion from the pupal case, wings of the immature adult fly unfold and expand to present a flat wing blade. During expansion the epithelia, which earlier produced the wing cuticle, delaminate from the cuticle, and the epithelial cells undergo an epithelial–mesenchymal transition (EMT). The resulting fibroblast-like cells then initiate a programmed cell death, produce an extracellular matrix that bonds dorsal and ventral wing cuticles, and exit the wing. Mutants that block wing expansion cause persistence of intact epithelia within the unexpanded wing. However, the normal progression of chromatin condensation and fragmentation accompanying programmed cell death in these cells proceeds with an approximately normal time course. These observations establish that the Bursicon/Rickets signaling pathway is necessary for both wing expansion and initiation of the EMT that leads to removal of the epithelial cells from the wing. They demonstrate that a different signal can be used to activate programmed cell death and show that two distinct genetic programs are in progress in these cells during wing maturation.     

Ultrastructural changes in intersegmental cuticle during rotation of the terminal abdominal segments in a mosquito

The terminal abdominal segments of male Aedes aegypti rotate 180° within 24 hr after adult emergence, rotation occurring in the intersegmental membrane between abdominal segments VII and VIII. The ultrastructure of this rotating membrane is compared with non-rotating intersegmental membranes at different developmental stages. The deposition of cuticle in both the rotating and non-rotating intersegments appears ultrastructurally similar, and follows the sequential pattern described for other insects. Shortly after adult emergence, however, disruptive changes occur in the membrane cuticle that are more pronounced in non-rotating intersegments. This disruption occurs initially 1 hr after adult emergence and becomes maximal within 3 hr. Disruption appears to occur by the addition of fluid to the cuticle and results in a ten-fold increase in cuticle thickness in non-rotating intersegments but only a two-fold increase in thickness in the rotating intersegment. While in the disrupted condition, the non-rotating intersegmental membranes become extensively folded whereas the cuticle in the rotating intersegment becomes stretched. During rotation, strain forces in the rotating intersegment result in a reorientation of microfibers in the cuticle from parabolic to parallel. This reorientation is presumably brought about by plastic flow.     

Tissue remodeling during maturation of the Drosophila wing

The final step in morphogenesis of the adult fly is wing maturation, a process not well understood at the cellular level due to the impermeable and refractive nature of cuticle synthesized some 30 h prior to eclosion from the pupal case. Advances in GFP technology now make it possible to visualize cells using fluorescence after cuticle synthesis is complete. We find that, between eclosion and wing expansion, the epithelia within the folded wing begin to delaminate from the cuticle and that delamination is complete when the wing has fully expanded. After expansion, epithelial cells lose contact with each other, adherens junctions are disrupted, and nuclei become pycnotic. The cells then change shape, elongate, and migrate from the wing into the thorax. During wing maturation, the Timp gene product, tissue inhibitor of metalloproteinases, and probably other components of an extracellular matrix are expressed that bond the dorsal and ventral cuticular surfaces of the wing following migration of the cells. These steps are dissected using the batone and Timp genes and ectopic expression of alphaPS integrin, inhibitors of Armadillo/beta-catenin nuclear activity and baculovirus caspase inhibitor p35. We conclude that an epithelial-mesenchymal transition is responsible for epithelial delamination and dissolution.     

Worms under Pressure: Bulk Mechanical Properties of C. elegans Are Independent of the Cuticle

The mechanical properties of cells and tissues play a well-known role in physiology and disease. The model organism Caenorhabditis elegans exhibits mechanical properties that are still poorly understood, but are thought to be dominated by its collagen-rich outer cuticle. To our knowledge, we use a novel microfluidic technique to reveal that the worm responds linearly to low applied hydrostatic stress, exhibiting a volumetric compression with a bulk modulus, κ = 140 ± 20 kPa; applying negative pressures leads to volumetric expansion of the worm, with a similar bulk modulus. Surprisingly, however, we find that a variety of collagen mutants and pharmacological perturbations targeting the cuticle do not impact the bulk modulus. Moreover, the worm exhibits dramatic stiffening at higher stresses—behavior that is also independent of the cuticle. The stress-strain curves for all conditions can be scaled onto a master equation, suggesting that C. elegans exhibits a universal elastic response dominated by the mechanics of pressurized internal organs.     

Avoidance of maladaptive,precocious copulation in the cabbage white butterfly,Pieris rapae crucivora

In the cabbage white butterfly, Pieris rapae crucivora, copulation hinders normal expansion and hardening of the wings of newly emerged females. The resulting permanent wing deformation makes it impossible for females to fly and therefore, to find an oviposition site and nectar sources. An attempt was made to clarify whether the newly emerged female butterfly avoids copulation. Observation of wing expansion and hardening reveals that the wings are fully expanded and hard by 20–30 min after emergence. In the field, presentation of females with soft wings to males shows that males will attempt to copulate with these females. However, newly emerged females prevent successful completion of copulation by assuming the mate refusal posture, and thereby avoid a potentially maladaptive copulation. The discussion focusses on the question as to why females and not males avoid early copulation.     

Design and mechanical properties of insect cuticle

Since nearly all adult insects fly, the cuticle has to provide a very efficient and lightweight skeleton. Information is available about the mechanical properties of cuticle-Young's modulus of resilin is about 1 MPa, of soft cuticles about 1 kPa to 50 MPa, of sclerotised cuticles 1-20 GPa; Vicker's Hardness of sclerotised cuticle ranges between 25 and 80 kgf mm(-2); density is 1-1.3 kg m(-3)-and one of its components, chitin nanofibres, the Young's modulus of which is more than 150 GPa. Experiments based on fracture mechanics have not been performed although the layered structure probably provides some toughening. The structural performance of wings and legs has been measured, but our understanding of the importance of buckling is lacking: it can stiffen the structure (by elastic postbuckling in wings, for example) or be a failure mode. We know nothing of fatigue properties (yet, for instance, the insect wing must undergo millions of cycles, flexing or buckling on each cycle). The remarkable mechanical performance and efficiency of cuticle can be analysed and compared with those of other materials using material property charts and material indices. Presented in this paper are four: Young's modulus-density (stiffness per unit weight), specific Young's modulus-specific strength (elastic hinges, elastic energy storage per unit weight), toughness-Young's modulus (fracture resistance under various loading conditions), and hardness (wear resistance). In conjunction with a structural analysis of cuticle these charts help to understand the relevance of microstructure (fibre orientation effects in tendons, joints and sense organs, for example) and shape (including surface structure) of this fibrous composite for a given function. With modern techniques for analysis of structure and material, and emphasis on nanocomposites and self-assembly, insect cuticle should be the archetype for composites at all levels of scale.     

A comparison of the cuticular properties of the female ticks <Emphasis Type="Italic">Ixodes pacificus</Emphasis> and <Emphasis Type="Italic">Amblyomma hebraeum</Emphasis> (Acari: Ixodidae) throughout the feeding period

The mechanical properties of the cuticle of Ixodes pacificus (Ip) are compared to those of Amblyomma hebraeum (Ah) from our earlier work. The 10-fold size difference between the species is expected to lead to significant differences in mechanical properties, because cuticular stretch depends on high internal hydrostatic pressure during the rapid phase of engorgement. We demonstrate here: (1) The cuticle of partially fed Ip is less stiff and viscous than that of Ah. (2) A stretch-recoil cycle in both ticks consists of recoverable deformation (ESv) and permanent deformation (ESp); ESp is higher in Ip, and increases sharply during the slow phase of engorgement, but not in Ah. (3) Injected dopamine (DA) increases ESp and reduces all measures of stiffness and viscosity, suggesting that a catecholaminergic neurotransmitter plays a fundamental role in modulating mechanical properties of the cuticle. However, unlike Ah, DA’s effect was not different from that of the control (1.2% NaCl). Mere insertion of the needle may have punctured the gut, causing the release of perhaps a catecholamine that increases ESp, an hypothesis supported by the fact that inserting a needle without any injection also caused an increase in ESp. (4) Stretch reduces ESp, but subjecting loops to pH 6.5 in vitro restores it. (5) Despite the smaller size of Ip, later onset of the rapid phase of engorgement, a thinner cuticle and different mechanical properties all reduce the internal pressure needed for stretch.     

Morphology of hindwing veins in the shield bug Graphosoma italicum (Heteroptera: Pentatomidae)

Light, fluorescence, and electron microscopy were applied to cross sections and -breakage and whole-mount preparations of the anterior hindwing vein of the shield bug Graphosoma italicum. These analyses were complemented by investigations of the basal part of the forewing Corium and Clavus. The integration of structural, histological, and fluorescence data revealed a complex arrangement of both rigid and elastic structures in the wall of wing veins and provided insights into the constitution of transition zones between rigid and elastic regions. Beneath the exocuticular layers, which are continuous with the dorsal and ventral cuticle of the wing membrane, the lumen of the veins is encompassed by a mesocuticular layer, an internal circular exocuticular layer, and an internal longitudinal endocuticular layer. Separate parallel lumina within the anterior longitudinal vein of the hindwing, arranged side-by-side rostro-caudally, suggest that several veins have fused in the phylogenetic context of vein reduction in the pentatomid hindwing. Gradual structural transition zones and resilin enrichment between sclerotized layers of the vein wall and along the edges of the claval furrow are interpreted as mechanical adaptations to enhance the reliability and durability of the mechanically stressed wing veins.     

Cuticular morphogenesis during continuous growth of the final instar larva of a moth

The larvae of the tobacco hornworm, Manduca sexta, grow continuously. During the feeding period of the fifth larval instar their weight increases ten-fold (ca. 1·2–12 g) accompanied by a four-fold expansion of the surface area of the abdominal cuticle. We have found that this cuticle contains structures which facilitate its expansion. Folds in the epicuticle (papillae) flatten as the cuticle expands. The endocuticle, in contrast, does not unfold but rather is plastically deformed. This plastic deformation is assisted by vertical structures in the cuticle (cuticular columns) which are more easily deformed than the surrounding lamellate cuticle. The head capsule cuticle, which does not expand as the larva grows, lacks papillae and cuticular columns. Thus, these are specialized structures that are reserved for cuticle that must expand as the larva grows.     

A post-ecdysial plasticization of the abdominal cuticle in Rhodnius

The ecdysis and emergence of fifth instar larvae of Rhodnius prolixus have been closely observed. At the time of ecdysis the cuticle of the head, legs, and wingpads is soft and readily deformed. it does not become sufficiently rigid for normal use until about 90 min later. The cuticle of the abdomen is however hard and inextensible at the time of ecdysis. From about 60 min onward this cuticle undergoes a plasticization; it is maximally extensible at about 180 min, thereafter becoming inextensible again. Unlike the plasticization of the abdominal cuticle which occurs after feeding, this post-ecdysial plasticization is not under direct nervous control. Although it seems that there is some temporal link with the darkening of the cuticle, it is considered unlikely that plasticization is a direct consequence of the tanning process. The significance of this post-ecdysial plasticization is not obvious.     

Repeated plasticization and recovery of cuticular stiffness in the blood-sucking bug Triatoma infestans in the feeding context

Triatominae bugs experience changes in the mechanical properties of their cuticle prior to feeding. This process-plasticization-allows a rapid stretching of the unsclerotized abdominal cuticle of triatominae larvae and it is evoked by sensory inputs related to feeding. We tested: (a) whether the cuticle recovers its original mechanical properties after plasticization, (b) whether repeated stimulation would be able to evoke recurrent plasticization along the same larval instar, (c) the temporal course of recovering cuticular stiffness. We injected Ringer solution into the body cavity of the bugs at constant pressure, using the injection rate (ml/min) as a measure of the cuticle extensibility. To trigger plasticization, individuals were allowed to feed on blood from an artificial feeder at 32+/-2 degrees C. After plasticization occurred, the abdominal cuticle gradually recovered its original mechanical properties. Bugs were capable of plasticizing for a second time when repeatedly stimulated. The effects of plasticization vanished between 1 and 2 h after stimulation. Although one full meal could suffice to accomplish moult in other Triatomine species, Triatoma infestans is able to feed repeatedly during a single larval instar. Accordingly to this, their cuticle recovers stiffness in some hours and becomes able to respond repeatedly to sensory inputs associated with feeding.     

Transformations of epithelial monolayers during wing development of Manduca sexta

The two epithelial monolayers of the insect wing undergo striking morphogenetic changes during the course of adult development, but the exact interactions between these monolayers were not evident until the ultrastructure of the cells was carefully examined. The interaction of the dorsal monolayer with the ventral monolayer continually changes as the two initially separate monolayers first lose their pupal basal laminae and then come together along a sharp interface to form microtubule-associated junctions. As blood space between the two monolayers expands 2 days later, new adult basal laminae and cuticle form. Concomitantly the epithelial cells stretch along their apicobasal axes to create a thin cellular M layer halfway between the dorsal and ventral surfaces of the wing that represents the site where connections between the monolayers are maintained at specialized basal junctions. The elongated processes of each monolayer that make up this M layer first fasciculate and then span the space separating the two monolayers, but only at relatively widely-spaced intervals. During later stages of adult development, dense aggregates of microtubules appear in these epithelial processes and presumably contract as cells dramatically shorten along their apicobasal axes during expansion of the wing. Examination of the ultrastructure of the developing adult wing has revealed how certain cellular events can account for the mechanics of cuticle and wing expansion after adult emergence.     

Butterfly Wings Are Three-Dimensional: Pupal Cuticle Focal Spots and Their Associated Structures in Junonia Butterflies

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.     

The contribution of surface waxes to pre-penetration growth of an entomopathogenic fungus on host cuticle

A locust wing bioassay, that allowed an entomopathogenic fungus to be removed from host cuticle before penetration, was used to investigate the role of surface lipids and waxes in pre-penetration growth of the specific locust pathogen Metarhizium anisopliae var. acridum. SEM and atomic force electron microscopy showed the impact of the fungus on the architecture of the cuticle surface. Although the fungus can germinate on authentic alkanes as the sole carbon source, only low levels of germination occurred on crude, non-polar wing cuticle extracts, containing a mixture of long-chain n-alkanes and other waxes (identified in particular by gas chromatography and mass spectroscopy). The fungus removed a large proportion of non-polar and polar components during pre-penetration growth on the wing. Polar crude extracts from Schistocerca gregaria hindwings, which contained fatty acids, fatty acid esters, glucose, amino acids and peptides, were strong promoters of germination, and poor germination was observed on a locust hindwing from which the extract had been taken. Thus simple polar compounds, also present on the surface, may be required to stimulate germination before the fungus can make use of a complex mixture of non-polar lipids.