Postecdysial Cuticle Research Articles

Heterochrony of cuticular differentiation in eusocial corbiculate bees

AbstractThe exoskeleton (cuticle) of insects varies widely in shape, biomechanical properties, and functions, which are inherent to the biological species and developmental stage, besides showing a wealth of architectural specializations and nuances in the different body regions. The morphological pattern of exoskeleton maturation was studied in eusocial and solitary bees, including species of all three eusocial tribes of corbiculate bees as follows: Apini, Meliponini, and Bombini. The results showed striking differences in the rate of cuticle maturation between the solitary bees that leave the nest soon after the adult ecdysis, and the eusocial bees that take longer to leave the colony for foraging activities. There was a clear delay in the post-ecdysial cuticle tanning (melanization and sclerotization) in the eusocial bee species in comparison to the solitary species, suggesting adaptation to sociality and to the protective environment of the colony. Such heterochrony of cuticle maturation seems a conserved ontogenetic trait related to the way of life in corbiculate bees. The data generated a basic framework of exoskeleton maturation in corbiculate bees, under ontogenetic and evolutionary approaches, and give experimental support for further research on adaptation to the colony environment.

Bursicon, the insect cuticle-hardening hormone, is a heterodimeric cystine knot protein that activates G protein-coupled receptor LGR2

All arthropods periodically molt to replace their exoskeleton (cuticle). Immediately after shedding the old cuticle, the neurohormone bursicon causes the hardening and darkening of the new cuticle. Here we show that bursicon, to our knowledge the first heterodimeric cystine knot hormone found in insects, consists of two proteins encoded by the genes burs and pburs (partner of burs). The pburs/burs heterodimer from Drosophila melanogaster binds with high affinity and specificity to activate the G protein-coupled receptor DLGR2, leading to the stimulation of cAMP signaling in vitro and tanning in neck-ligated blowflies. Native bursicon from Periplaneta americana is also a heterodimer. In D. melanogaster the levels of pburs, burs, and DLGR2 transcripts are increased before ecdysis, consistent with their role in postecdysial cuticle changes. Immunohistochemical analyses in diverse insect species revealed the colocalization of pburs- and burs-immunoreactivity in some of the neurosecretory neurons that also express crustacean cardioactive peptide. Forty-three years after its initial description, the elucidation of the molecular identity of bursicon and the verification of its receptor allow for studies of bursicon actions in regulating cuticle tanning, wing expansion, and as yet unknown functions. Because bursicon subunit genes are homologous to the vertebrate bone morphogenetic protein antagonists, our findings also facilitate investigation on the function of these proteins during vertebrate development.

Effects of exogenous N-acetylhexosaminidase on the structure and mineralization of the post-ecdysial exoskeleton of the blue crab, Callinectes sapidus

A cuticular glycosidase with characteristics of N-acetyl-β- d-hexosaminidase (HexNAcase) was identified in post-ecdysial crab cuticle. Its appearance coincided with changes in cuticular glycoproteins and the onset of mineralization. To test if HexNAcase might be the causative agent in the alteration of the glycans and initiation of calcification, newly molted crab cuticle was treated with exogenous HexNAcase. Treating cuticular extracts from crabs at 0 h post-ecdysis with exogenous HexNAcase mimicked those changes observed in vivo. Specifically, the enzyme decreased the concanavalin A affinity of an 83-kDa glycoprotein that binds to calcite crystals in vitro. Treating pieces of 0 h post-ecdysial cuticle with HexNAcase rendered them capable of nucleating calcite in vitro (similar to 5 h post-ecdysial cuticle), while untreated, 0 h controls remained uncalcified. The data imply a role of the cuticular HexNAcase-like enzyme in the initiation of calcite nucleation in the newly formed exoskeleton.

Glycosidase activity in the post-ecdysial cuticle of the blue crab, Callinectes sapidus

We have previously demonstrated a marked change in sugar moieties of glycoproteins of the cuticle of the blue crab, Callinectes sapidus, between 0.5 and 3 h post-ecdysis. The present study has identified a glycosidase that appears in the cuticle during the early post-ecdysial hours. The enzyme has affinities for p-nitrophenyl derivatives of both N-acetylglucosamine and N-acetylgalactosamine. Both activities are competitively inhibited by chitobiose, suggesting that the enzyme could be a N-acetylhexosaminidase (EC 3.2.1.52). Atypical of N-acetylhexosaminidases described to date, this enzyme has a pH optimum of 7.0. The enzyme activity is high during the post-ecdysial period coincident with the changes in glycoprotein profiles observed in vivo. Partial purification of the enzyme has been accomplished by Sephacryl size-exclusion chromatography followed by concanavalin A affinity chromatography.

Formation of the inner branchiostegal cuticle of the blue crab, Callinectes sapidus.

The noncalcified inner branchiostegal cuticle, which lines the branchial chamber, was examined histologically and ultrastructurally over the molt cycle in the blue crab, Callinectes sapidus. In intermolt crabs (stage C4 ) the epithelium underlying the inner cuticle is cuboidal and has abundant intercellular spaces and a prominent basement membrane. Apolysis occurs at stage D0 and dissolution of the cuticle is accompanied by the formation of numerous lysosomes in the epithelium. During stage D1 , cells increase in height, apical mitochondria become more abundant, and the cuticle continues to be resorbed. An epicuticle is formed in early D2 , arising from a fusion of small subunits apparently attached to short apical microvilli. Cuticle deposition continues through D2 and is complete by stage D3 . By the time cuticle deposition is complete, the epithelium has become extremely columnar and cells are filled with bundles of microtubules. In stage D4 , an amorphous electron-dense core appears in the microtubule-filled cells, which are attached to the cuticle at their apical end and anchored to their basement membrane at the basal surface. These microtubule-filled cells persist through ecdysis, stage E, but during stage A1 the cores disappear and some organelles begin to reappear in the cytoplasm. By stage A2 , the cells return to the cuboidal morphology seen in intermolt and remain so throughout the remainder of the molt cycle. This new pattern of cuticle deposition resembles that observed in the gills of crustaceans in that the cuticle is uncalcified and there is no postecdysial cuticle formation. However, instead of apolysis being delayed until just before ecdysis, the inner cuticle is formed during the first half of premolt, allowing the epithelial cells time to differentiate into a morphology that provides tensile strength for the stress of ecdysis. These new observations demonstrate that cuticle formation can follow very diverse structural and temporal patterns. In order to integrate and coordinate these diverse patterns, it is suggested that a suite of feedback mechanisms must be present. J. Morphol. 240:267-281, 1999. © 1999 Wiley-Liss, Inc.

Characterization of two new cuticular genes specifically expressed during the post-ecdysial molting period in Tenebrio molitor.

In a previous study, we have isolated a cDNA, TM-ACP17, coding for a post-ecdysial adult protein of Tenebrio molitor. After screening of a genomic library with TM-ACP17, we report isolation and sequencing of TM-ACP17 gene and a new gene, TM-LPCP29, coding for a larval-pupal protein. These two genes exhibit a common sequence of 15 nucleotides and a characteristic of most cuticular protein genes so far described: an intron interrupting the signal peptide. The deduced aa sequence of TM-LPCP29 exhibits a high percentage of Ala (26.5%) and Val (17.5%) and is highly hydrophobic. In the N-terminal part, the motif VAAPV is repeated ten times. Numerous histidine residues are present in the C- and N-terminal regions. A comparison is made with other cuticle protein sequences. Northern hybridization analysis showed that TM-LPCP29 is present during larval and mainly pupal post-ecdysial cuticle secretion. In-situ hybridization revealed that TM-LPCP29 mRNA is expressed in epidermis and not in muscles or fat body.

POSTECDYSIAL CHANGES IN THE PROTEIN AND GLYCOPROTEIN COMPOSITION OF THE CUTICLE OF THE BLUE CRAB CALLINECTES SAPID US

ABSTRACT Postecdysial modifications of the cuticle of Callinectes sapidus were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of soluble protein extracts. Gels stained with Coomassie blue and with silver demonstrated that major changes in protein composition or mobility occurred immediately after ecdysis. Electroblots of SDS-PAGE gels were probed with various biotinylated lectins. The patterns of lectin binding were also markedly different between blots of pre- and postecdysial cuticle extracts, demonstrating a change in glycosylation or glycoprotein mobility in the early postecdysial period. The dramatic alteration in cuticular composition is concurrent with the onset of sclerotization and mineralization of the preexuvially deposited layers of the cuticle.

Monoclonal antibodies recognizing larval- and pupal-specific cuticular proteins of Tenebrio molitor (Insecta, Coleoptera)

To study the sequential expression of insect epidermal cells during metamorphosis, a library of monoclonal antibodies (MABs) was prepared against the water-soluble proteins from preecdysial pupal cuticle of Tenebrio molitor. Six selected MABs recognizing only larval and pupal cuticular proteins (CPs) in immunoblot analysis were classified into three types. Type 1 recognized a 21.5 and a 22 kDa polypeptide, type 2, a 26 kDa polypeptide, and type 3, three polypeptides of 18.5, 19.5 and 21.5 kDa. They did not immunoreact with any protein of fat bodies or haemolymph from pharate pupae, suggesting that the antigens originate from the epidermis. The stage-specificity was confirmed by electron microscopic immunogold labelling. Type 1 and 3 MABs recognized antigens characterizing larval and pupal preecdysial sclerotized cuticles, while the antigens recognized by type 2 were localized in the first few lamellae of unsclerotized postecdysial cuticle. When the expression of the adult programme was inhibited by application of a juvenile hormone analogue, the larval-/pupal-specific CPs were detected in the supernumerary pupal cuticle. These results suggest that the genes encoding these proteins are juvenile hormone dependent. These MABs should be useful tools to isolate pupal-specific genes whose regulation sems to be different from that of the adult-specific ones.

Ultrastructural study of the penetration by Metarhizium anisopliae through Dimilin-affected cuticle of Manduca sexta

Fourth instar larvae of the tobacco hornworm, Manduca sexta, were inoculated with conidia of Metarhizium anisopliae then fed a diet containing the insect chitin synthesis inhibitor Dimilin. Cuticle of Dimilin-treated insects provided reduced resistance to penetration by hyphae of M. anisopliae. Widespread histolysis of postecdysial (Dimilin-affected) cuticle occurred. In addition, although lamellate preecdysial cuticle was not affected by Dimilin, the majority of the cuticle in the vertical cuticular columns was laid down at the same time as the postecdysial cuticle. Therefore, the vertical cuticular columns were areas of weakness in the preecdysial cuticle which as a consequence failed to provide a mechanical barrier to the penetrating fungus.

The effect of Dimilin on the ultrastructure of the integument of Manduca sexta

The insecticide Dimilin was fed to larvae of the tobacco hornworm, Manduca sexta, throughout the 4th stadium. The effects were observed on the ultrastructure of the 4th-instar integument. Although post-ecdysial abdominal cuticle was laid down to the same extent in both experimental and control insects, the fine structure of the integument was very different after the two treatments. In particular the post-ecdysial cuticle of Dimilin-treated insects was granular in appearance and devoid of lamellae and the epidermal cells extruded cytoplasm apically. New 5th-instar cuticle was produced on time in experimental insects but the procuticle was again granular and non-lamellate.

The post-ecdysial development of the cuticle and the eye of the Devonian trilobitePhacops rana milleriStewart 1927

Specimens of the Devonian trilobitePhacops rana milleriStewart 1927 from the Silica Shale of Ohio are sometimes preserved in the early stages of the post-ecdysial cycle. Individuals that died in this early stage have pale, thin and wrinkled cuticles; in later stages the cuticle became rigid as it thickened and darkened. The post-ecdysial development of the cuticle and the schizochroal compound eye has been studied in a suite of specimens representing various stages, culminating in the intermoult condition. Scanning electron microscope studies on etched specimens, supplementing cathodoluminescence and light microscopy, have enabled primary structures to be distinguished from secondary diagenetic effects, and have been used to elucidate diagenetic changes. In our earliest post-ecdysial cuticle the 10 pm thick outer prismatic layer is already formed and the principal layer below this is only about 25 pm thick. The later thickening of the cuticle affects the principal layer alone. In subsequent development there is a division into three zones with a variety of vertical canals. The final intermoult cuticle can be up to 500 pm thick. In the developing eye, each of the post-ecdysial lenses has initially the form of a small, simple calcite cone hanging from the lower surface of the cornea. In later stages this lens spreads to the full width of the lens capsule, losing its conical form, taking on a Huygensian shape, and eventually acquiring its mature form in which there is a central core of massive texture and, proximally, a thin intralensar bowl, of much the same appearance as the core. The upper unit of the lens consists of thin calcite lamellae, radially arranged around the c-axis, each lamella consisting of palisade-like fibres, parallel in the lower part of the lens, but radiating outwards where they meet the convex outer lens surface. When the lens is mature, a ring of scleral material forms on the inside of the cylindrical wall of the lens capsule, together with an annular girdle of fine-grained material at the junction of lens and sclera. Some suggestions are made regarding function in the developing eye, and the need for complete replacement of calcium carbonate at each ecdysis is discussed.

An ultrastructural study of the integument during the moult cycle of the woodlouse,Oniscus asellus (Crustacea, Isopoda)

Woodlice are unique among the arthropods in moulting in two halves. The intramoult is 1.8 days inOniscus asellus and results from the two halves being out of synchrony throughout the cycle. In the integument, the initiation of a new moult cycle is heralded by epidermal cell vacuolation; a little later, the subepidermal tissue proliferates and, for a brief time, macrophages appear. The cuticle layers are produced in order, starting with epicuticular structures such as tricorns and plaques, followed by the biphasic epicuticle, the lamellate pre-ecdysial cuticle and, after ecdysis, the lamellate postecdysial cuticle. Epicuticle is formed in a continuous sheet along the distal epidermal membrane and postecdysial cuticle from fibres formed within the cell body — both features not reported for other arthropods. Immediately prior to ecdysis the epicuticle and pre-ecdysial cuticle are highly corrugated and the epidermis very constricted as the width of the ecdysing gap is increased by the new cuticle components withdrawing slightly beneath the old cuticle. At this time the space between the two cuticles appears devoid of the fluid that once occupied it, although the ecdysial membrane is still apparent. After ecdysis the appearance of the epicuticle changes due to chemical events which make it relatively impermeable. The pre-ecdysial cuticle also changes in appearance at this time due to the physical stress of expansion. Large cuticle precursor vesicles, similar to those of other Crustacea, are particularly associated with postecdysial cuticle production. In addition there are numerous small vesicles associated with pre-ecdysial cuticle formation. The epidermal vacuoles disappear as postecdysial cuticle is produced so that, when it is complete, the epidermis is again a narrow cell layer. Apolysis occurs straight after this and shortly afterwards the cycle recommences.

The formation of the epicuticle and associated structures inOniscus asellus (Crustacea, Isopoda)

The integument of the woodlouse,Oniscus asellus, consists of a two-layered epicuticle, a largely lamellate procuticle — itself divided into two regions (pre-and postecdysial cuticles), and the epidermis. At the initiation of new cuticle production the epidermal cells become vacuolated and retract away from the cuticle. Apolysis occurs immediately after the cessation of postecdysial cuticle production. The formation of the epicuticle is unique among the arthropods since material aggregates along the distal epidermal membrane. By indenting, doubling back on itself, and incorporating septa, the epicuticle forms surface structures such as plaques and tricorns.

Preparation of some amidic 6-ketosteroids as potential inhibitors of postecdysial cuticle sclerotization in Pyrrhocoris apterus L.

Preparation of some amidic 6-ketosteroids as potential inhibitors of postecdysial cuticle sclerotization in Pyrrhocoris apterus L.