The isolation and immunogenicity of the cuticle of Dipetalonema viteae (Filarioidea)

The cuticle of the filaria Dipetalonema viteae was isolated by sonication in 1% sodiumdodecylsulphate (SDS) and in a mixture of 1% SDS and 5% B-mercaptoethanol (BME). Sonication in SDS removed all internal parts and left the cuticle intact; this was verified by light- and electronmicroscopy. Sonication and incubation of the cuticle in the mixture of SDS-BME at pH 6.8 dissolved the basal and part of the median zone of the cuticle. The epicuticle and the cortical zone remained intact. The extracts were investigated using SDS-polyacrylamide gel electrophoresis; the early extracts contained a wide variety of proteins, whereas the later steps showed a consistent pattern with a smaller number of bands. Cuticles after SDS-purification, the extract of cuticular material in SDS-BME, and the cuticles insoluble in SDS-BME were used to immunize mice; the antibodies produced were visualized by an indirect fluorescent antibody test on cryostat sections of female worms. When SDS-purified cuticles were used for immunization, antibodies directed against all organs in the filariae were found. The SDS-BME extract and the insoluble cuticular pellet stimulated the production of antibodies restricted to the cuticle of adult worms and microfilariae. The purification method opens up the possibility of further isolation and characterization of antigens from the cuticle.     

CUTICLES FROM CHONDRUS CRISPUS (RHODOPHYTA)1

A simple, nondestructive physical process was developed for routinely isolating the outermost layers from female, male, and sporophyte fronds of Chondrus crispus Stack-house. Yields of pure cuticles from apical segments ranged from 0.74 to 2.35% on a dry weight basis after 5–7 d of culture. These undegraded cuticles were examined by electron microscopy (scanning and transmission electron microscopy), spectroscopy (infrared and X-ray), and chemical means. Cuticles isolated from female or male fronds were characterized by parallel arrays of electron-dense lamellae (typically 6–14) alternating with more electron-transparent regions. The thickness and uniformity of these lamellae provide the physical basis for the iridescence characteristic of C. crispus fronds. Sporophyte fronds are not iridescent. This phenomenon may be explained by the fewer electron-dense cuticular lamellae (usually three to seven) and the fact that these lamellae anastomose freely to form a thin cuticle with a highly irregular substructure. Elements detected by X-ray analysis, in addition to carbon and oxygen, included Mg, Br, S, and Ca in both gametophyte and sporophyte cuticles. Major features of FTIR spectra of all cuticles were absorbances due to proteins. A strong band, indicative of sulfate ester, occurred near 1250 cm^?1 in all cuticle preparations. Gametophyte, but not sporophyte, cuticles absorbed at 935, 846, and 800 cm^?1 consistent with the presence of kappa and/or iota carrageenan. Amino acid analyses showed that sporophyte and gametophyte cuticles were generally similar in gross composition. All contained proline as the principal residue together with significant amounts of cysteine, methionine, and lysine. Protein contents calculated from these analyses ranged from 37.6 to 44.4% on a dry weight basis as compared to 51.5–56.7% calculated from total nitrogen values. Up to 75% of the cuticle mass was solubilized by sodium dodecyl sulfate-β-mercaptoethanol. Three similar migrating bands were seen in female and male cuticle extracts on sodium dodecyl sulfate–polyacrylamide gel electrophoresis; however, none of the three weaker bands from sporophyte cuticles comigrated with those from gametophytes. Chloroform-methanol extraction removed < 3.3% of the cuticle mass, suggesting that lipids were minor components.     

Development of plant cuticles: fine structure and cutin composition of Clivia miniata Reg. leaves

The fine structure and monomeric composition of the ester-cutin fraction (susceptible to BF₃/CH₃OH transesterification) of the adaxial leaf cuticle of Clivia miniata Reg. were studied in relation to leaf and cuticle development. Clivia leaves grow at their base such that cuticle and tissues increase in age from the base to the tip. The zone of maximum growth (cell expansion) was located between 1 and 4 cm from the base. During cell expansion, the projected surface area of the upper epidermal cells increased by a factor of nine. In the growth region the cuticle consists mainly of a polylamellate cuticle proper of 100–250 nm thickness. After cell expansion has ceased both the outer epidermal wall and the cuticle increase in thickness. Thickening of the cuticle is accomplished by interposition of a cuticular layer between the cuticle proper and the cell wall. The cuticular layer exhibits a reticulate fine structure and contributes most of the total mass of the cuticle at positions above 6 cm from the leaf base. The composition of ester cutin changed with the age of cuticles. In depolymerisates from young cuticles, 26 different monomers could be detected whereas in older ones their number decreased to 13. At all developmental stages, 9,16-/10,16-dihydroxyhexadecanoic acid (positional isomers not separated), 18-hydroxy-9-octadecenoic acid, 9,10,18-trihydroxyoctadecanoic acid and 9,10-epoxy-18-hydroxyoctadecanoic acid were most frequent with the epoxy alkanoic acid clearly predominating (47% at 16 cm). The results are discussed as to (i) the age dependence of cutin composition, (ii) the relationship between fine structure and composition, (iii) the composition of the cuticle proper, the cuticular layer and the non-depolymerizable cutin fraction, and (iv) the polymeric structure of cutin.Abbreviations CL cuticular layer - CP cuticle proper - MX cutin polymer matrix     

Development of plant cuticles: occurrence and role of non-ester bonds in cutin of Clivia miniata Reg. leaves

The effect of BF₃-methanol treatment on the mass and fine structure of isolated Clivia leaf cuticles at different stages of development has been investigated. BF₃-methanol cleaves ester linkages in cutin; however, the cuticles are not completely depolymerized. With increasing age, the residue left after BF₃-methanol treatment increases in mass. In very young cuticles, 10% of the total cutin resisted BF₃-methanol and the fraction of nonester cutin increased up to 62% in mature leaves. Transmission electron microscopy shows that fine structure of the cuticle proper is severely distorted but not destroyed. The internal cuticular layer, which exhibits a heavy contrast when fixed with KMnO₄, is completely depolymerized, while the external cuticular layer is hardly affected. The results are discussed in relation to cuticle development and to the function of cuticles as transpiration resistances.Abbreviation CP cuticle proper - ECL external cuticular layer - E cutin ester bonded cutin - ICL internal cuticular layer - MX-membrane polymer matrix membrane - NE-cutin non-ester bonded cutin - TEM transmission electron microscopy     

Role of cuticular lipids and water-soluble compounds in tick susceptibility to Metarhizium infection

The arthropod cuticle acts as a physiochemical barrier protecting the organism from pathogens' entry. Entomopathogenic fungi actively penetrate the cuticles of arthropod hosts and are therefore directly affected by cuticle composition. Previously we have observed that Metarhizium spp. developing on resistant ticks ultimately die without penetrating tick's cuticle, suggesting that the cuticles of resistant ticks have antifungal compounds. In the present study, lipids and water-soluble cuticular components were extracted from engorged female tick cuticles, of one susceptible and one resistant tick species to Metarhizium spp. While conidia exposed to lipids from the susceptible tick, Rhipicephalus annulatus, germinated and differentiated into appressorium, conidia exposed to lipids from the resistant tick, Hyalomma excavatum, were inhibited. Soluble cuticular component extracts from both susceptible and resistant ticks stimulated conidial germination but not appressorium differentiation. A comparative analysis of the fatty acid profile in lipid extract of each tick exhibited similar compositions, but the relative abundance of C16:0, C18:0, C18:1ω9C and C20:0 was 2–5 times higher in the extracts from resistant ticks. All of these fatty acids inhibited conidial germination in vitro at 1% and 0.1% w/v concentration, but C20:0 stimulated appressorium differentiation at low concentration. This is the first report demonstrating a possible link between the presence of antifungal compounds in a specific concentration in tick cuticle and tick resistance to infection.     

Characterization of cuticular proteins in the red flour beetle, Tribolium castaneum

We are characterizing the cuticular proteins of Tribolium castaneum (Herbst) (Coleoptera:Tenebrionidae) to determine their role in the function of the exoskeleton. Based on qualitative analyses of cuticles, we focused on the sodium dodecyl sulfate (SDS)-extractable proteins. A small-scale cuticle "mini-prep" procedure was devised that yields preparations virtually free of contaminating cellular material compared to hand-dissected preparations, as assessed by fluorescent microscopy using DAPI to stain nuclei. Proteins extracted in 1% SDS from various developmental stages (last larval instar, pupal, adult) were analyzed by one-dimensional denaturing polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. The cuticular protein profiles show both similarities and differences among the stages examined. The amino acid composition, glycosylation, and partial amino acid sequence of several abundant cuticular proteins indicate similarity to cuticular proteins of other insects.     

Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry

Identification of authenticated cuticular proteins has been based on isolation and sequencing of individual proteins extracted from cleaned cuticles. These data facilitated classification of sequences from conceptual translation of cDNA or genomic sequences. The question arises whether such putative cuticular proteins actually are incorporated into the cuticle. This paper describes the profiling of cuticular proteins from Anopheles gambiae starting with cuticle cleaned by the insect itself in the course of molting. Proteins extracted from cast larval head capsules and cast pupal cuticles were fractionated by 1D SDS gel electrophoresis. Large gel slices were reduced, carbamidomethylated and digested with trypsin. The pellet remaining after SDS extraction was also treated with trypsin. The resulting peptides were separated on a C18 column and then analyzed by tandem mass spectrometry. Two-hundred-ninety-five peptides from putative cuticular proteins were identified; these corresponded to a minimum of 69 and a maximum of 119 different proteins. Each is reported as an authentic Anopheles cuticular protein for the first time. In addition to members of two known cuticular protein families, members of additional families likely to be structural components of the cuticle were identified. Furthermore, other peptides were identified that can be attributed to molting fluid, muscle and sclerotizing agents.     

Ascaris lumbricoides: characterization of the collagenous components of the adult cuticle

The proteins of the cuticle of adult Ascaris lumbricoides suum were characterized with respect to heterogeneity, glycosylation, and susceptibility to collagenase. Pepsin digestion of intact cuticles was used to determine the extent of stable triple-helical structures of the cuticular components. With sodium dodecyl sulfate-poly-acrylamide gel electrophoresis, it was shown that treatment of purified cuticles with beta-mercaptoethanol released three components (99, 90, and 68 kDa) which comprise 95% of the total solubilized material. The remaining fraction consists of at least four components (16, 28, 154, and 173 kDa). Periodic acid-Schiff staining showed that the only glycoprotein was the 173-kDa component. All cuticular components, except the 173-kDa protein, were degraded by bacterial collagenase. Pepsin digestion of intact cuticles for 24 hr at 4 C produced, after reduction, a 95-kDa fragment; by 96 hr, four fragments (95, 90, 83, and 77 kDa) were evident. When the 96-hr pepsin digest was treated with fresh pepsin, the 77-kDa fragment became the major constituent. With agarose gel electrophoresis, analysis of non-reduced, pepsin-released material revealed intact aggregates that were greater than 2 X 10(3) kDa. The enzyme digestion studies indicate that, with the exception of the 173-kDa component, each cuticular protein contains collagenous domains and that, within the cuticle, the longest contiguous collagen chain in a triple-helical conformation has a uniform molecular size of 77 kDa.     

Protecting against water loss: analysis of the barrier properties of plant cuticles

The cuticle is the major barrier against uncontrolled water loss from leaves, fruits and other primary parts of higher plants. More than 100 mean values for water permeabilities determined with isolated leaf and fruit cuticles from 61 plant species are compiled and discussed in relation to plant organ, natural habitat and morphology. The maximum barrier properties of plant cuticles exceed that of synthetic polymeric films of equal thickness. Cuticular water permeability is not correlated to the thickness of the cuticle or to wax coverage. Relationships between cuticular permeability, wax composition and physical properties of the cuticle are evaluated. Cuticular permeability to water increases on the average by a factor of 2 when leaf surface temperature is raised from 15 degrees C to 35 degrees C. Organic compounds of anthropogenic and biogenic origin may enhance cuticular permeability. The pathway taken by water across the cuticular transport barrier is reviewed. The conclusion from this discussion is that the bulk of water diffuses as single molecules across a lipophilic barrier while a minor fraction travels along polar pores. Open questions concerning the mechanistic understanding of the plant cuticular transport barrier and the role the plant cuticle plays in ensuring the survival and reproductive success of an individual plant are indicated.     

Novel cuticular proteins revealed by the honey bee genome

With the completion of the honey bee genome project, a transition is now occurring from the acquisition of gene sequence to understanding the role and context of gene products within the genome. Here we annotated and characterised a cluster of three genes in a GC-rich 11 kb genomic region on the linkage group 4 encoding highly hydrophobic polypeptides (named apidermins; APD 1-3) containing both sequence motifs characteristic of cuticular proteins and distinctly novel features. Five amino acids, Ala, Gly, Leu, Pro and Val, account for 74-86% of their respective sequences with Ala being the most abundant residue (at least 30% of each peptide). A conserved tetra-peptide AAPA/V is found in all three proteins, but none has the 'R and R' signature implicated in chitin binding. Two proteins, APD-1 and APD-2, contain an arginine-rich motif RERR in short non-hydrophobic stretches near the N-terminal of mature proteins and in both proteins tryptophan is the C-terminal residue. All three genes are spliced and highly expressed in a defined spatio-temporal pattern. apd-1 is expressed in the exoskeletal epidermis, but only during a restricted period of a few days of late pupal and early adult life when the cuticle becomes dark. APD2 appears to be a protein of "internal" cuticles and is expressed in the tracheas, oesophagus and stomach, and also in the embryo. The expression of apd-3 partly overlaps with both apd-1 and apd-3, but apd-3 also is uniquely associated with non-pigmented cuticles such as the eye cover and external cuticle of white pupae. This study expands the collection of genes encoding cuticular proteins by three novel and well characterised members.     

Quantitative determination of catecholic degradation products from insect sclerotized cuticles

Acid hydrolysates of cuticle from various insect species were quantitatively analyzed for five catecholic amino acid adducts. Four of the adducts are ketocatechols; in three of them the amino acid moiety, either lysine, glycine or beta-alanine, is connected via its amino group to the alpha-carbon atom of 3,4-dihydroxyacetophenone, in the fourth a tyrosine residue is connected to the same position via its phenolic group. The fifth adduct contains histidine linked via its imidazole-ring to the beta-position of the dopamine sidechain. The three ketocatecholic adducts containing alpha-amino acids were obtained in significant yields from adult cuticles of the locust Schistocerca gregaria, the cockroaches Blaberus craniifer and Periplaneta americana, and the beetles Pachynoda sinuata and Tenebrio molitor, but only in trace amounts from larval and pupal cuticles of T. molitor, pupal cuticles of the moths Manduca sexta and Hyalophora cecropia, and puparia of the blowfly Calliphora vicina. The beta-alanine-containing ketocatechol was not obtained from cuticle of locusts and T. molitor larvae and pupae, but it was present in the hydrolysates of the other cuticles. The beta-histidine-dopamine adduct was obtained from all the cuticles, the highest yield was obtained from adult P. sinuata and the lowest yield was from adult S. gregaria. The beta-histidine-dopamine adduct is derived from the product formed by reaction of p-quinone methides of N-acetyldopamine (NADA) or N-beta-alanyldopamine (NBAD) with histidine residues in the cuticular proteins. The ketocatecholic adducts are assumed to be degradation products of crosslinks formed when oxidized dehydro-NADA reacts with the cuticular proteins. The insect species investigated appear to use both pathways for sclerotization, but to widely differing extents; the dehydro-NADA pathway dominates in cuticles which are exposed to strong deforming forces, such as those of adult locusts and cockroaches, and the p-quinone methide pathway dominates in cuticle of lepidopteran pupae and blowfly puparia, which are not exposed to strong mechanical forces but have to be effectively protected against microbial and fungal attacks.     

Consideration of the importance of hydrophobic interactions in stabilizing insect cuticle

Protein fractions of insect cuticles with different mechanical properties have related values of polarity and hydrophobicity. Hydrophobicity is important for the self-assembly of cuticle which is produced prior to the moult and in plasticization of cuticle. The cuticles of soft-bodied fly larvae are quite distinct from those of exopterygotes (e.g. locusts) and this can be related to the chemistry and mode of tanning. The properties of cuticular proteins are compared: the proteins of the pliant cuticles most closely resemble globulins, and the proteins in stiff cuticles are more like fibrous and hydrophobic structural proteins. Changes in the environment of the proteins may alter their shape and hence the mechanical properties of the cuticle.     

Studies on proteins in post-ecdysial nymphal cuticle of locust, Locusta migratoria, and cockroach, Blaberus craniifer

Proteins were extracted from the cuticle of mid-instar nymphs of locusts, Locusta migratoria, and cockroaches, Blaberus craniifer. Seven proteins were purified from the locust extract and five from the cockroach extract, and their amino acid sequences were determined. Polyacrylamide gel electrophoresis indicates that the proteins are present only in the post-ecdysially deposited layer of the nymphal cuticles. One of the locust and one of the cockroach nymphal proteins contain a 68-residue motif, the RR-2 sequence, which has been reported for several proteins from the solid cuticles of other insect species. Two of the cockroach proteins contain a 75-residue motif, which is also present in a protein from the larval/pupal cuticle of a beetle, Tenebrio molitor, and in proteins from the exoskeletons of a lobster, Homarus americanus, and a spider, Araneus diadematus. The motif contains a variant of the Rebers-Riddiford consensus sequence, and is called the RR-3 motif. One of the locust and three of the cockroach post-ecdysial proteins contain one or more copies of an 18-residue motif, previously reported in a protein from Bombyx mori pupal cuticle. The nymphal post-ecdysial proteins from both species have features in common with pre-ecdysial proteins (pharate proteins) in cuticles destined to be sclerotised; they show little similarity to the post-ecdysial cuticular proteins from adult locusts or to proteins from soft, pliable cuticles. Possible roles for post-ecdysial cuticular proteins are discussed in relation to the reported structures.     

Supercoil of collagen fibrils in the integument of Alvinella, an abyssal annelid

The polychaete annelid Alvinella pompejana was discovered near the hydrothermal vents, recently explored in the Eastern Pacific Ocean. This worm is protected by a cuticle deeply transformed over certain areas of the body and some changes are due to the presence of a very special bacterial flora. The present work however deals mainly with the supercoiled collagen fibrils, which are well visualized in thin sections observed by transmission electron microscopy. This character strongly differentiates this species from other annelids and worms in general, the cuticle of which includes straight and apparently non-coiled collagen fibrils. This indicates that fibrils are extensible in Alvinella, possibly under physiological conditions, and that internal pressure and local volume variations are regulated according to principles which depart from what is recognized in other worms, where cuticular fibrils are considered as inextensible. Possible models of this cuticle are discussed and particularly aspects which show a relationship with certain liquid crystals. Very different factors may be involved in morphogenesis of such cuticles: microvilli distribution, self-assembly of collagen fibrils, mechanical constraints. An appendix recalls some classical data on worm cuticle geometry and presents an estimate of volume variations resulting from coiling of fibrils.     

Characterization of aqueous pores in plant cuticles and permeation of ionic solutes

Plant cuticles are lipid membranes with separate diffusion paths for lipophilic non-electrolytes and hydrated ionic compounds. Ions are lipid insoluble and require an aqueous pathway across cuticles. Based on experimental data, the aqueous pathway in cuticles has been characterized. Aqueous pores arise by hydration of permanent dipoles and ionic functional groups. They can be localized using ionic fluorescent dyes, silver nitrate, and mercuric chloride. Aqueous pores preferentially occur in cuticular ledges, at the base of trichomes, and in cuticles over anticlinal walls. Average pore radii ranged from 0.45 to 1.18 nm. Penetration of ions was a first order process as the fraction of the salt remaining on the cuticle surface decreased exponentially with time. Permeability of cuticles to ions depended on humidity and was highest at 100% humidity. Wetting agents increased rate constants by factors of up to 12, which indicates that the pore openings are surrounded by waxes. The pores in cuticular ledges of Helxine soleirolii allowed passage of berberine sulphate, which has a molecular weight of 769 g mol(-1). Increasing the molecular weight of solutes from 100 to 500 g mol(-1) decreased the rate constants of penetration by factors of 7 (Vicia faba) and 13 (Populus canescens), respectively. Half-times of penetration of inorganic salts and organic ions across Populus cuticles and Vicia leaf surfaces varied between 1 and 12 h. This shows that penetration of ionic compounds can be fairly rapid, and ions with molecular weights of up to 800 g mol(-1) can penetrate cuticles that possess aqueous pores.     

Cuticular Pores and Transcuticular Canals in Diverse Fruit Varieties

Dewaxed thin-sectioned and dewaxed isolated mature fruit cuticlesrevealed the unequivocal presence in situ of visibly discrete,ubiquitous, cuticular pores or orifices concomitant with anticlinally-orientedtranscuticular canals in 51 varieties of fruit among 20 plantfamilies. More than 66 per cent of the fruit cuticles have poresand/or canals. No correlation exists between either fruit sizeor pore size and cuticle thickness. Dewaxed cuticles rangedfrom 1.25–22.5 µm in thickness. Canal lengths aredirectly related to cuticle thickness. Cuticular occlusionsof the epidermal cells were found in 76 per cent of the fruitsexamined. Evidence is provided by light microscopy photomicrographs. Fruit cuticles, cuticle morphology, cuticular pores, transcuticular canals     

Expression and hormonal control of a new larval cuticular multigene family at the onset of metamorphosis of the tobacco hornworm

The pattern of cuticular protein synthesis by the epidermis of the tobacco hornworm larva changes during the final day of feeding, leading to an alteration in cuticular structure and a stiffening of the cuticle. We have isolated a small multigene family which codes for at least three of the new cuticular proteins made at this time. The five genes which were isolated from this family map to two different genomic regions. Sequencing shows that one of the genes is 1.9 kb and consists of three exons coding for a 12.2-kDa acidic (pI = 5.26) protein that is predominantly hydrophilic. The deduced amino acid sequence shows regions of similarity to proteins from flexible lepidopteran cuticles and from Drosophila larval and pupal cuticles, but not to proteins found in highly sclerotized cuticles. This gene family is first expressed late on the penultimate day (Day 2) of feeding in the final larval instar and ceases expression 2 days later when metamorphosis begins. In situ hybridization shows that this gene family is expressed in all the epidermal cells of Day 3 larvae except the bristle cells and those at the muscle attachment site. Expression can be induced in Day 1 epidermis by exposure to 50 ng/ml 20-hydroxyecdysone in vitro, but only if juvenile hormone is absent. Its developmental expression, tissue specificity, and hormonal regulation strongly suggest that this multigene family is involved in the structural changes that occur in the larval cuticle just prior to the onset of metamorphosis.