Distribution of cuticular protein mRNAs in silk moth integument and imaginal discs

The distributions of mRNAs for two cuticular proteins of Hyalophora cecropia were examined with RT-PCR and in situ hybridization. For major regions of larval and pupal cuticle, there was a strong correspondence between the type of cuticle and the predominant cuticular protein message found. Epidermal cells underlying soft cuticle had mRNA for HCCP12, with a RR-1 consensus attributed to soft cuticle, while the epidermal cells associated with hard cuticle had predominantly mRNA for HCCP66, a protein with the RR-2 consensus attributed to hard cuticle. Both messages were found in all areas of the pupal fore- and hind-wings, with modest area-specific difference in concentration being much less than differences in the relative abundance of these cuticular proteins.

mRNA for HCCP12 was present in imaginal discs of feeding larvae of H cecropia. Data from Bombyx mori available at SilkBase (http://www.ab.a.u-tokyo.ac.jp/silkbase/) revealed that imaginal discs from feeding larvae had abundant mRNA for RR-1 cuticular proteins, representing six distinct gene products. Only discs from spinning larvae had mRNAs that coded for RR-2 proteins arising from 10 distinct genes. Thus, lepidopteran wing imaginal discs can no longer be regarded as inactive in larval cuticle production.     

Unique features of the structural model of 'hard' cuticle proteins: implications for chitin-protein interactions and cross-linking in cuticle

Cuticular proteins are one of the determinants of the physical properties of cuticle. A common consensus region (extended R&R Consensus) in these proteins binds to chitin, the other major component of cuticle. We previously predicted the preponderance of beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). Subsequently, we verified experimentally the abundance of antiparallel beta-pleated sheet in the structure of cuticle proteins (Iconomidou et al., Insect Biochem. Mol. Biol. 31 (2001) 877). Homology modelling of soft (RR-1) cuticular proteins using bovine plasma retinol binding protein (RBP) as a template revealed an antiparallel beta-sheet half-barrel structure as the basic folding motif (Hamodrakas et al., Insect Biochem. Molec. Biol. 32 (2002) 1577). The RR-2 proteins characteristic of hard cuticle, have a far more conserved consensus and frequently more histidine residues. Extension of modelling to this class of consensus, in this work, reveals in detail several unique features of the proposed structural model to serve as a chitin binding structural motif, thus providing the basis for elucidating cuticle's overall architecture and chitin-protein interactions in cuticle.     

Structure of the cuticle of the common house cricket with reference to the location of lipids

Cuticle segments from the thorax, abdomen, and jumping legs of the house cricket. Acheta domesticus, were examined using histological techniques for light microscopy, scanning and transmission electron microscopy, and direct examination of frozen-fractured cuticle. The surface of untreated cuticle is covered by a lipid film which obscures fine surface detail. Standard EM preparative procedures, as well as washing the cuticle with ethanol before examination, remove this film exposing previously covered openings to dermal gland ducts and wax canals. An epicuticle, exocuticle, mesocuticle, endocuticle, and a deposition layer were present in all transverse sections of cuticle. Light microscopy showed that the exocuticle and mesocuticle are heavily impregnated with lipids, whereas there is little lipid associated with the endocuticle. Frozen-fractured cuticle clearly shows the ‘plywood’ structure of the meso- and endocuticle, while the exocuticle fractures as if it were a solid sheet. The epicuticle is composed of a dense homogeneous layer, cuticulin, outer epicuticle, and the outer membrane. Superficial wax was detected only in cuticle samples prepared using vinylcyclohexane dioxide as a polar dehydrant. The results were used to construct a comprehensive model of the cuticle of A. domesticus.     

Ultrastructure and cuticle formation of the carapace in the myodocopan ostracod exemplified by Euphilomedes japonica (Crustacea: Ostracoda)

The ultrastructure and formation of the cuticle of a myodocopan ostracod, Euphilomedes japonica, are investigated utilizing scanning and transmission electron microscopy. The outer lamella cuticle consists of four layers; epicuticle, exocuticle, endocuticle, and membranous layer like in the cuticle of other arthropods. The exocuticle and endocuticle are well-calcified and the organic matrix develops within the both cuticles. The outermost layer of new cuticle (epicuticle) is secreted first and the inner layers (exocuticle, endocuticle and membranous layer) are added proximally in the pre-, and postmoult stages. The calcification takes place in the whole area of carapace at the same time together with the synthesis of organic matrix within the endocuticle. This study demonstrates that the ultrastructure and formation of the cuticle in myodocopans are different from those in podocopans, and that the myodocopan carapaces have achieved a structural diversity for adaptation to different lifestyles.     

Electron microscopical localization of chitin in the cuticle of Halicryptus spinulosus and Priapulus caudatus (Priapulida) using gold-labelled wheat germ agglutinin: phylogenetic implications for the evolution of the cuticle within the Nemathelminthes

 The ultrastructure of the cuticle of adult and larval Priapulus caudatus and Halicryptus spinulosus is investigated and new features of cuticle formation during moulting are described. For the localization of chitin by TEM wheat germ agglutinin coupled to colloidal gold was used as a marker. Proteinaceous layers of the cuticle are revealed by digestion with pronase. The cuticle of larval and adult specimens of both species consists of three main layers: the outer, very thin, electron-dense epicuticle, the electron-dense exocuticle and the fibrillar, electron-lucent endocuticle. Depending on the body region, the exocuticle comprises two or three sublayers. The endocuticle can be subdivided into two sublayers as well. In strengthened parts such as the teeth, the endocuticle becomes sclerotized and appears electron-dense. Only all endocuticular layers show an intense labelling with wheat germ agglutinin-gold conjugates in all investigated specimens. Additional weak labelling is observed in the exocuticle III layer of the larval lorica of P. caudatus. All other cuticular layers remain unlabelled. Chitinase dissolves the unsclerotized endocuticular layers almost completely, but also exocuticle II and partly the loricate exocuticle III. The epicuticle, the homogeneous exocuticle I and the sclerotized endocuticle are not affected by chitinase. The labelling is completely prevented in all layers after incubation with chitinase. Pronase dissolves all exocuticular layers, but not evenly. The presumably sclerotized regions of exocuticle I are not affected as well as the complete epicuticle and the endocuticle. All cuticular features of the Priapulida are compared with the cuticle of each high-ranked taxon within the Nemathelminthes with special regard to the occurrence of chitin. Based on this out-group comparison it can be concluded that: (1) a two-layered cuticle with a trilaminate epicuticle and a proteinaceous basal layer represents an autapomorphic feature of the Nemathelminthes, (2) the stem species of the Cycloneuralia have already evolved an additional basal chitinous layer, (3) such a three-layered cuticle is maintained as a plesiomophy in the ground pattern of the Scalidophora and (4) in the Nematoida, the chitinous basal layer is replaced by a collagenous one at least in the adults; the synthesis of chitin is restricted to early developmental phases or the pharyngeal cuticle. Accepted: 12 March 1998     

The transfer of lipid in insects from the epidermal cells to the cuticle

The structure of the pore canals and the tubular filaments they contain are described in a series of insects and types of cuticle. In all these cuticles the tubular filaments arise from the plasma membrane of the epidermal cells and they contain argentaffin material, regarded as sclerotin precursors, and lipid-staining material, regarded as wax precursors. These materials are transferred to the inner epicuticle and are exuded over the surface of the outer epicuticle to form the waterproofing layer as described in the preceding paper. They are also transported to those parts of the endocuticle destined to form hard exocuticle. There are no terminations of tubular filaments in the soft cuticle of Manduca larva, in the soft expanding cuticle of Rhodnius, and in the non-sclerotized post-ecdysial endocuticle of Tenebrio. Apis. etc. In the puparium of Calliphora lipid appears to be added by the epidermal cells directly and not by way of tubular filaments. It is confirmed that lipid is a component of sclerotized cuticle.     

Fine structure of the cuticle of the desert scorpion, Hadrurus arizonensis.

The structure of the sclerite and intersegmental cuticle of the opithosoma of the desert scorpion, Hadrurus arizonensis, has been examined by transmission electron microscopy. The sclerite cuticle contains a four-layered epicuticle, a hyaline exocuticle, an inner exocuticle and an endocuticle. The outer part of the hyaline exocuticle and the whole of the inner exocuticle are constructed of helicoidally arranged planes of microfibrils. Within the endocuticle, the overall architecture is not helicoidal as previously assumed, but consists of bundles of microfibrils oriented horizontally and vertically. Microbibrils of the inner exocuticle and the endocutile are seen as simple unstained rods, but those of the hyaline exocuticle are electron dense rods with an unstained central core. The intersegmental cuticle contains a four-layered epicuticle and a procuticle. In detail, its fine structure differs in most respects from that of the sclerite cuticle. Electron microscopy reveals that hyaline exocuticle, previously assumed to be continuous from sclerite to intersegmental membrane, is absent in the latter.     

Extractable proteins from abdominal cuticle of sexually mature locusts,Locusta migratoria

The urea-extractable proteins from the abdominal cuticle of mature locusts Locusta migratoria, have been characterized by two-dimensional electrophoresis and by amino acid analysis of purified components. The existence of sex-related differences in protein composition was confirmed in mature cuticle, whereas such differences are absent in pharate cuticle. The proteins from mature cuticle are mainly acidic, while the pharate proteins are mainly basic. The deposition of the proteins characteristic for mature cuticle starts within a few days after ecdysis; the different proteins do not appear simultaneously in the extracts. It is suggested that the pharate proteins represent exocuticle, that the proteins extracted from mature cuticle are derived from endocuticle, and that the differences between the two groups of proteins will give the two layers different physical properties.     

Characterization of RR-1 and RR-2 cuticular proteins from Myzus persicae

A cDNA library for Myzus persicae has served to identify sequences coding for cuticular proteins (CPs) with RR-1 and RR-2 consensus. Two putative CPs showed a common RR-2 chitin binding domain (CBD) but differed in their C and N terminals. Two other predicted CPs showed a typical RR-1 CBD but differed in size and sequence of the C and N terminals. An additional sequence encoding for a protein that showed terminal amino acid repeats similar to those of putative CPs from M. persicae, but lacked the R & R consensus, was also described. A comparison of the sequences obtained from the cDNA library with those attained from the genomic DNA, confirmed their identity as cuticular proteins genes. Presence of introns was revealed in the Mpcp4 and Mpcp5 genes coding for CPs with an RR-1 consensus. The Mpcp4 has a single large intron, while the Mpcp5 has two shorter ones. Introns were not found in the Mpcp2 and Mpcp3 genes encoding for CPs with RR-2 consensus. Differences were also noticed for 3' UTR and 5' UTR of both the RR-1 and RR-2 CPs. CPs genes were expressed in bacteria, and the resulting protein was identified as a CP by amino acid sequencing.     

Observations on the integument of the water mite Arrenurus major (Acari: Parasitengona)

A study of the integument of the aquatic mite Arrenurus major Marshall is presented. When the cuticle is examined with the unaided eye and the light microscope, it appears to possess numerous tiny pits. However, scanning electron micrographs of the cuticle reveal that it is a solid surface with topographical sculpturing of the epicuticle, indicating that the “pits” are an internal phenomenon. In cuticle which has been sectioned, areas devoid of cuticular material beneath the thin exocuticle are revealed. These areas are the pits which are goblet-shaped. The integument consists of five major strata. These are from the outside to the inside: (1) a superficial layer with a maximum observed thickness of 725 Å, (2) an epicuticle with a thickness of about 900 Å and composed of at least four sublayers, (3) an exocuticle with a thickness of about 1.5 Å. Fibers of the exocuticle are arranged in a Bouligand pattern and exhibit a regularly occurring discontinuity with a spacing of 200 Å. (4) An endocuticle ranging from 15 to 20 μ in thickness. The endocuticle is characterized by bandings which superficially resemble the lamellae of insects but are not homologous, microfibers which exhibit a preferred orientation, and the presence of the pits; and (5) an epidermis lying beneath the endocuticle and extending into the pits. Pore canals are present only in the exocuticle and have their origin at the apices of the pits. The pore canals contain a central filament, and a plug is present just beneath the epicuticle.     

Pupal cuticle proteins of Manduca sexta: characterization and profiles during sclerotization

Proteins in pupal abdominal cuticle of the tobacco hornworm, Manduca sexta, were characterized during the pre-ecdysial and post-ecdysial periods of sclerotization and endocuticle formation. Protein extractability decreased dramatically as the cuticle became sclerotized through 6 h post-ecdysis, but increased rapidly from 9 to 48 h as endocuticular layers were secreted. Nearly 100 proteins that were extracted from pre-ecdysial cuticle became largely insoluble during sclerotization. Three major proteins in this group destined to become exocuticle had apparent molecular masses (Mapp) of 20, 27 and 36 kDa, and were designated MS-PCP20, MS-PCP27, and MS-PCP36. Amino acid analysis revealed glycine to predominate in all three proteins, and alanine, aspartate, glutamate, proline and serine were also relatively abundant. Histidine residues, which provide sites for adduct and cross-link formation with quinone metabolites of N-beta-alanyldopamine during sclerotization of pupal cuticle, ranged from 2 to 3 mol %. N-Terminal amino acid analysis of MSPC-20 and MSPC-36 also revealed some sequence similarities indicating they may be related. An almost entirely new group of proteins appeared by 9 h as endocuticule secretion began, and these increased in abundance through 48 h post-ecdysis. Two of these were major proteins with Mapps of 33 and 34 kDa, and they also had close similarities in their N-terminal amino acid sequences. This study showed that the large number of proteins secreted into the presumptive exocuticle of the pupa before ecdysis are involved in sclerotization reactions and as a consequence become largely insoluble. The epidermis then switches to the secretion of an entirely new group of proteins that are involved in formation of the endocuticle.     

Electron microscopic and preparative methods for the analysis of isopod cuticle

The crustacean cuticle consists of a complex organic matrix and a mineral phase. The physical and chemical properties of the cuticle are corellated to the specific functions of cuticular elements, leading to a large variety in its structure and composition. Investigation of the structure-function relationship in crustacean cuticle requires sophisticated methodological tools for the analysis of different aspects of the cuticular architecture. In the present paper we report improved preparation methods that, in combination with various electron microscopic techniques, have led to new insights of cuticle structure and composition in the tergite cuticle of Porcellio scaber. We used thin sections of non-decalcified tergites and decalcified resin embedded material for transmission electron microscopy and scanning transmission electron microscopy. Etched sagittal planes of bulk tergite samples were analysed with field emission scanning electron microscopy. We have found a distinct distal region within the exocuticle that differs from the subjacent proximal exocuticle in the arrangement of fibres. Within this distal exocuticle chitin-protein fibrils assemble to fibres with diameters between 15 and 50 nm that are embedded in a mineral matrix. In the proximal exocuticle and the endocuticle fibrils do not assemble to fibres and are surrounded by mineral individually. Furthermore, we show that the pore canals are filled with mineral, and demonstrate that mild etching of polished sagittal cuticle surfaces reveals regions containing mineral of diverse solubility.     

La formation des canaux cuticulaires chez l'adulte de Tenebrio molitor L. étude ultrastructurale et remarques histochimiques

The sclerotized cuticle of adult Tenebrio shows (1) an exocuticle composed of rotating lamellate layers and of columns of cuticular material, the fibres of which run perpendicularly through the lamellae, (2) an endocuticle composed of layers with preferred orientation. In the exocuticle, the pore canals are numerous and run along the columns; they do not rotate with the lamellate layers. They show several filaments some of which leave the canals and form a dense intracuticular network. In the last layers of exocuticle, the pericolumnar canals fuse and form large endocuticular canals which rotate in phase with the cuticular fibres. The formation of columns and canals is in relation with cellular expansions which penetrate into the cuticle during cuticle deposition. Exocuticular columns seem characteristic of highly sclerotized cuticles and the intracuticular filaments may have a role in the transport of sclerotisation precursors.     

Internal anatomy of Hypochthonius rufulus (acari: Oribatida)

The internal anatomy of juveniles and adults of Hypochthonius rufulus selected as a model species representing the lower Oribatida was investigated histologically and compared with the published characteristics of higher oribatid internal anatomy. In this species, the cuticle is weak and flexible, consisting of epicuticle and endocuticle on the body, but including an exocuticle between the epicuticle and endocuticle of the legs. Walls of the mesenteron in the digestive tract are of uniform thickness and structure without any regional thickening, and there are no proventricular glands. The hindgut is apparently divided into five parts: colon 1 and 2, rectum 1 and 2, and anal atrium; food bolus exhibits a multilamellar structure in this section. The glandular system is less diversified than in some other oribatids. Tracheae are apparently lacking. Females possess only two relatively large eggs, filling one-half of opistosoma, and they lack ovipositors. Eggs are present in females during the whole year. Gonad buds appear first in the protonymph stage. Only one male was found among 146 adults studied. No male external organ (aedeagus or penis) is present.     

Higher sequence coverage and improved confidence in the identification of cysteine-rich proteins from the wool cuticle using combined chemical and enzymatic digestion

Keratin-associated proteins (KAPs) are important constituents of the wool cuticle, comprised of the endo-, exocuticle and a-layers, which contribute significantly to the fibre's molecular and mechanical characteristics. Relatively little is known about the distribution of specific KAPs across these layers, and correct protein identification of individual KAPs is difficult due to extensive homology and identity among individual KAPs. We here present evidence that, by specifically exploiting the high-cysteine content of KAPs in the wool cuticle, using 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage in combination with tryptic digestion, a larger number of KAPs can be identified than with standard trypsin-only digests. A total of 27 KAPs were identified, six of which could only be identified using NTCB. Furthermore, NTCB-mediated cleavage of cuticle proteins generated unique peptides critical for unambiguous identification of two KAPs, as well as significantly increasing the overall sequence coverage of most identified KAPs. Interestingly, some of the peptides found to be unique to particular KAPs could only be found in either the exo- or endocuticle. We conclude that for the analysis of high sulphur proteomes, specific targeting of cysteine residues using chemical agents such as NTCB can provide critical information for unambiguous protein identification.     

Organization of the cuticle of an aquatic fly larva

The aquatic, apneustic larva of the midge, Chironomus riparius, has a very thin (up to 5 micrometers), readily deformable, post-cephalic cuticle. The ultrastructure of this cuticle from newly moulted and older final instar animals, and exuvia shed at pupation, has been examined using routine methods and also after the extraction of proteins with formamide and acetic acid. From the results described, and using established criteria, it is inferred that an exocuticle is present and represents about 25% of the thickness of the mature procuticle, the remainder being endocuticle. Therefore, it would seem that this exceptionally delicate cuticle conforms to the conventional plan of tanned or sclerotized solid cuticles, unlike those reported in the larvae of cyclorrhaphous dipterans such as blowflies or other soft-bodied insects. This is the first account, using experimental techniques, of the fine structure of sectioned cuticle from nematocerous dipteran larvae. It also indicates the value of the exuvium as a source of information about cuticle structure.