The cuticular proteins of Tenebrio molitor: I. Electrophoretic banding patterns during postembryonic development

Buffer-soluble cuticular proteins of the abdomen of the yellow mealworm, Tenebrio molitor, were analyzed by SDS-polyacrylamide gel electrophoresis. Since the abdominal epidermis of Tenebrio persists throughout the insect's life, these cuticular proteins reflect the secretory history of a continuous line of cells during its entire metamorphic developmental program. Twenty-two to thirty-eight bands were detected in extracts of larval cuticle, 11 to 35 in pupal cuticular extracts, and 30 to 41 in extracts from adults. No population polymorphism was apparent, nor was there any sexual dimorphism, in these cuticular proteins. At each metamorphic stage, the cuticular proteins formed a unique banding pattern. Bands unique to the larval and to the adult exocuticular extracts were observed. Extracts from cuticles of freshly ecdysed animals (exocuticle) differed from extracts from animals in which sclerotization and postecdysial (endocuticle) deposition had occurred, both in number of hands and in their molecular weight distributions. Some proteins became less soluble during sclerotization. The majority of the exocuticular bands from all three stages had molecular weights below 25,000; higher-molecular-weight proteins were extracted from postecdysial animals of each stage.     

Partial amino acid sequences of cuticular proteins from Hyalophora cecropia

The amino-terminal amino acid sequences for seven cuticular proteins from Hyalophora cecropia are reported. Proteins were purified by blotting two dimensional acrylamide gels onto acid-etched glass fiber filters, and the proteins were sequenced without further elution. The sequences of the serine-rich proteins from rigid cuticles revealed a new family of cuticular proteins, with features reminiscent of the amino-termini of certain vertebrate neurofilament proteins, members of the intermediate filament protein family which includes keratins. The proteins from flexible cuticles showed sequence similarity to proteins previously sequenced for Drosophila, Manduca and Sarcophaga. Proteins with identical electrophoretic mobility from two different metamorphic stages or from two anatomical regions within a single stage had identical amino-terminal sequences.     

Analysis of the cuticular proteins of Hyalophora cecropia with two dimensional electrophoresis

The soluble cuticular proteins of defined anatomical regions from different metamorphic stages of the giant silkmoth, Hyalophora cecropia, were characterized by two dimensional electrophoresis. As urea concentrations in 2D gels were increased, some of the cuticular proteins from the larval dorsal abdomen decreased in mobility relative to the molecular weight standards. This decrease was also influenced by the pH and ionic strength of the resolving gel. Clustering of proteins into groups, whose members showed similar behavior under different electrophoretic conditions, was indicative of membership in multigene families. By such criteria, common families were found in cuticles with similar mechanical properties from different metamorphic stages, yet there was evidence that different members of a single family were independently regulated.     

The cuticular proteins of Tenebrio molitor: II. Patterns of synthesis during postembryonic development

The program of synthesis for the soluble cuticular proteins of Tenebrio molitor was determined by following the incorporation of labeled leucine after a 4-hr pulse in vivo. Soluble proteins were extracted from labeled cuticles and separated on SDS-polyacrylamide slab gels; individual gel slices were counted. The synthetic patterns of larvae and pupae were similar to one another but distinct from the adult pattern. At each stage, the preecdysial pattern was unlike that of postecdysial animals. Distinct periods of synthesis were detected for different proteins. One protein was synthesized and deposited throughout cuticle formation in all three metamorphic stages. One group was synthesized only after ecdysis, while synthesis and secretion of other proteins were restricted to the preecdysial period. Some cuticular proteins never acquired detectable label.     

Larval cuticle proteins of Lucilia cuprina: Electrophoretic separation,quantification and developmental changes

Proteins from isolated cuticles of third instar larvae of the sheep blowfly, Lucilia cuprina, have been solubilized with water or 7 M urea or 2% SDS. While 7 M urea or 2% SDS extract significantly more protein than water, the same major proteins, in the same relative proportions, are extracted by all three solutions. More than 80% of the cuticular protein is extracted by 7 M urea or 2% SDS. Extracted proteins resolve into nine major bands when analysed by gradient polyacrylamide gel electrophoresis. These proteins are anionic, relatively low in molecular weight (13–28 kd) and are essentially free of carbohydrate. Only minor differences exist between the proteins of two morphologically distinct cuticular regions. Cuticle proteins, extracted from larvae at different developmental stages (first, second and third instars) display quantitatively and qualitatively unique electrophoretic profiles. A number of proteins are common to all stages however. The electrophoretic profiles of proteins extracted from larval cuticles at various times within an instar also differ although the differences are largely quantitative. This is particularly evident during the transition from the feeding to the wandering stages of the third instar; the weight of the cuticle relative to that of the larva increases and this is accompanied by marked changes in the electrophoretic profile of the cuticle proteins.     

In vitro translation of epidermal RNA from different anatomical regions and metamorphic stages of Hyalophora cecropia

Epidermal RNA isolated from different anatomical regions and metamorphic stages of Hyalophora cecropia was translated in vitro with commercial wheat germ and rabbit reticulocyte systems. The translation products were analyzed by 2D gel electrophoresis. The two systems yielded identical products if canine microsomal membranes were added to remove signal peptides from the reticulocyte products. The endogenous processing by the wheat germ extract occurred even in the presence of protease inhibitors. Some of the processed translation products co-migrated with unlabeled cuticular protein standards. All of the major cuticular proteins could be identified, but only when translations were carried out with RNA from epidermis underlying cuticle containing these proteins. Hence, cuticular protein distribution is due to differential synthesis and not to differential extractability. For larval abdominal RNA, most of the major translation products did not co-migrate with known larval cuticular proteins or with proteins synthesized and retained by the epidermis. These unknown products were lower in apparent molecular weight than the cuticular proteins. Their identity remains unknown; they may be premature translation products, but altering translation conditions did not reduce their abundance.     

Post-translational modifications of the cuticular proteins of Hyalophora cecropia from different anatomical regions and metamorphic stages

Post-translational modifications are a conspicuous feature of the proteins of vertebrate extracellular matrices such as cartilage. Yet this feature remains virtually unexplored with insect cuticle, a situation this paper begins to remedy. Cuticular proteins were extracted from cuticles of Hyalophora cecropia and separated on isoelectrofocusing and 2D gels. Periodic acid-Schiff reagent stained several proteins from flexible cuticles and a few proteins from rigid cuticles, indicating that some proteins were glycosylated. Elucidation of the specific nature of this glycosylation came from probing electrophoretically separated cuticular proteins blotted onto nitrocellulose with biotinylated lectins. Most major cuticular proteins did not react; minor cuticular proteins and molecules which do not stain with Coomassie blue were found to bind lectins specific for mannose and N-acetylgalactosamine. Limited binding was also detected with lectins specific for N-acetylglucosamine, galactose and fucose. No sialic acid was detected using either lectins or neuraminidase digestion. The amount of glycosylation was greatest in proteins extracted from flexible cuticles. Although several proteins stained with Alcian blue indicating presence of sulfation, ³⁵S which had been incorporated at low levels in cuticular proteins corresponded to [³⁵S]methionine. No indication of the presence of mammalian-type glycosaminoglycans in insect cuticles was obtained after treatment with chondroitinase or nitrous acid. The functional significance of the modifications detected remains unknown. No evidence for phosphorylated proteins or lipoproteins was found.     

Developmental changes in cuticular proteins of Ascaris suum

1. Cuticles were isolated from developmental stages of the swine nematode Ascaris suum by a combination of mechanical disruption and detergent treatment of larvae or by surgical removal of cuticle from adults. Proteins from the isolated cuticles were solubilized with 2-mercaptoethanol (2ME) and analyzed by SDS-PAGE. 2. 2ME soluble, cuticular proteins from adults consisted of 5 to 6 bands with 80% of proteins in 2 bands with mol. wts of 106,000 and 93,000. Cuticular proteins from the third and fourth larval stages (L3 and L4) were comparable to adult, but differences in the number of bands were observed. The soluble proteins from the adult, L3 and L4 were readily degraded by a bacterial collagenase suggesting that these proteins are collagen-like structural elements of the cuticle. 3. The soluble proteins from the second stage (L2) differed from the adult and other larval stages in both the number and mol. wt of protein bands and their lack of degradation by bacterial collagenase. Amino acid composition of soluble cuticular proteins were similar for adult and L4, but glycine and proline were present in lower amounts in the L2. 4. These results support a hypothesis that there are stage specific differences in cuticular proteins from A. suum and that the greatest differences appear to exist between L2 and other stages.     

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.     

The cuticle of Caenorhabditis elegans. II. Stage-specific changes in ultrastructure and protein composition during postembryonic development

The cuticle of the free-living nematode Caenorhabditis elegans is a proteinaceous extracellular structure that is replaced at each of four postembryonic molts by the underlying hypodermis. The cuticles of the adult and three juvenile stages (L1, Dauer larva, L4) have been compared ultrastructurally and biochemically. Each cuticle has an annulated surface and comprises two main layers, an inner basal layer and an outer cortical layer. The adult cuticle has an additional clear layer which separates the basal and cortical layers and is traversed by regularly arranged columns of electron-dense material. The fine structure of the cortical layer is similar in cuticles from different stages while that of the basal layer is stage specific. Purified cuticles were obtained by sonication and treatment with sodium dodecyl sulfate (SDS) and their component proteins solubilized with a sulfhydryl reducing agent. The degree of cuticle solubility is stage specific and the insoluble structures for each cuticle were localized by electron microscopy. Analysis of ³⁵S-labeled soluble cuticle proteins by SDS-polyacrylamide gel electrophoresis yields unique banding patterns for each stage. Most proteins are of high molecular weight (100–200 K) and are restricted to particular stages. Sixteen of the nineteen major proteins characterized are specifically degraded by bacterial collagenase. The results indicate that the different molts are not reiterative, but require the integration of both unique and shared gene functions. The potential use of stage-specific cuticle differences to identify and characterize regulatory genes controlling cuticle-type switching during development is discussed.     

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.     

Changes in the activity of dopa quinone imine conversion factor during the development of Bombyx mori

The activity of DOPA quinone imine conversion factor (QICF) in tissues at different developmental stages of the silkworm, Bombyx mori, was determined. QICF activity was detected in all developmental stages from egg to pupa although the activities, other than in fifth instar larvae, were quite low. Activity in whole larvae peaked one day before the onset of larval-pupal development and declined to a low level shortly before ecdysis. In whole pupae, maximal QICF activity was obtained 1 h after pupation. The activity in larval cuticles was elevated on the last day of the fourth instar and again between days 4 and 8 of the fifth instar, decreasing to very low levels before pupal ecdysis. QICF was detectable in pupal cuticles with most of the pupal activity found in homogenates of mid and hind guts. A major part of the total larval QICF activity was found in hemolymph. Activity in hemolymph varied in a different manner from that in cuticles, with markedly raised levels immediately before pupal ecdysis when the cuticular activity had declined. It is postulated that QICF in cuticles plays some role in wound healing and/or sclerotizatio,, while QICF in hemolymph participates in melanization in the humoral immune system.     

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.     

Dauer Larvae of Caenorhabditis briggsae in Axenic Culture

The free-living hermaphroditic nematode, Caenorhabditis briggsae, enters a dauer stage under certain conditions in axenic culture. Dauer larvae differ from directly-developing third-stage larvae in internal structure, size at time of second molt, morphology of second and third cuticles, separation zone of cuticular caps, and survival at 4 C and 37 C, temperatures fatal to other stages. Males, which occur rarely in liquid medium, may mature under conditions which cause most of the hermaphrodites to go into the dauer stage, resulting in a culture with increased male-to-hermaphrodite ratio.     

Haemonchus contortus: evidence that the 3A3 collagen gene is a member of an evolutionarily conserved family of nematode cuticle collagens

Rabbit antisera were raised against an 18 amino acid-long peptide that corresponds to the predicted sequence of the carboxy-terminal, nontriple helical region of the Haemonchus contortus 3A3 collagen gene. This sequence is highly conserved and diagnostic for members of the col-l collagen family, which includes the 3A3 gene. We find that these antisera react predominantly with multiple, high molecular weight (greater than 68 kDa) proteins on Western blots of whole worm extracts. The number and molecular weights of the reacting proteins vary depending upon the developmental stage of the worms analyzed. All of the reacting proteins are collagenase sensitive. The reacting collagens copurify with cuticles and are released from cuticles by reducing agents. In indirect immunofluorescence assays the antisera react only with the broken edges of isolated cuticles, suggesting that the antisera are reacting with an internal cuticle layer. This layer appears to be circular and to extend throughout the length of the worm. The antisera react on Western blots with multiple, high molecular weight collagens of eight other nematodes examined, representing two classes and several orders. These data provide additional support for the notion that the 3A3 collagen gene, and other members of the col-l collagen family, encode cuticle collagens. Collagens with this peptide sequence, presumably other members of the col-l collagen family, appear to be widely distributed in the phylum Nematoda.     

Spatial and temporal variations in cuticle proteins as revealed by monoclonal antibodies. Immunoblotting analysis and ultrastructural immunolocalization in a beetle, Tenebrio molitor

A library of monoclonal antibodies (Mabs) against adult cuticle of Tenebrio was used to visualize the secretion of cuticular antigens during metamorphosis. Immunoblots of water- and urea-soluble proteins, and high resolution immunogold labelling has shown that, except in one clone, the Mabs recognize antigens in the three developmental stages. However, the MW of larval and pupal antigens are different from the adult ones, though sharing common epitopes. Blots of cuticle proteins (CPs) bound to different lectins shown few water-soluble glycosylated proteins weakly or not recognized by the Mabs, suggesting that the majority of the Mabs do not recognize glycosylated epitopes. The immunolocalization of the different antigens suggests a molecular basis for both developmental and regional variations in cuticular architecture and to the modifications due to sclerotization, which differ between pre- and postecdysial cuticles of the three developmental stages.     

High Laccase2 expression is likely involved in the formation of specific cuticular structures during soldier differentiation of the termite Reticulitermes speratus

Termite soldiers are morphologically specialized for colony defense. Analysis of the mechanisms of soldier differentiation is important for understanding the establishment of termite societies. Soldiers differentiate from workers through a presoldier stage and have well-sclerotized and pigmented cuticles. These characteristics are important for nest defense and are likely to be caused by soldier-specific mechanisms of cuticular tanning. The molecular mechanisms leading to cuticular tanning have not been elucidated. Laccase2 (Lac2) plays important roles in this process in insects, and we hypothesized that Lac2 expression may be involved in soldier-specific cuticular tanning. We observed inner and outer head cuticle changes and compared the Lac2 expression patterns among three molts (worker–worker, worker–presoldier and presoldier–soldier) in the termite Reticulitermes speratus. Quantitative analyses of head cuticle colors showed that the color properties changed more conspicuously in presoldier–soldier molts than in the other two molts. Histological observations showed that the exocuticles of soldier heads were substantially thicker than those of worker and presoldier heads, underwent tanning before or just after ecdysis, and were pigmented at earlier time points than other molts. Finally, markedly higher Lac2 expression levels were observed just before and after ecdysis only in the presoldier–soldier molt. These results suggest that specific cuticular formation occurs in the exocuticles during soldier differentiation, and that the high level of Lac2 expression during the presoldier–soldier molt is related to soldier-specific cuticular tanning. We speculate that evolution of the regulatory mechanisms of Lac2 expression were important for the acquisition of soldier-specific cuticles.     

Wheat Germ Agglutinin Bound to the Outer Cuticle of the Seed Gall Nematodes Anguina agrostis and A. tritici

The presence of wheat germ agglutinin (WGA) on the cuticular surface of the seed gall nematodes Anguina agrostis and Anguina tritici was demonstrated, and the nature of its binding was examined. Crude extracts from the cuticles of A. tritici agglutinated human red blood cells, and only N-acetylglucosamine (GlucNAc) inhibited the agglutination. Distribution of the lectin was visualized by treating live infective juveniles (J2) with rabbit anti-WGA antibody and staining with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. The lectin bound to the outer cuticular surface of the whole body wall. Pretreatment with GlucNAc oligomers did not reduce the fluorescence created by the anti-WGA-WGA binding, indicating at least a partial nonspeciflc adhesion of the WGA to the nematode surface. Proteolytic enzyme pretreatments diminished the fluorescence, whereas lipase and periodate pretreatments increased the fluorescence. Adult females and males were labeled only on the head and tail, whereas eggs were not labeled at all. It was concluded that the WGA on the J2 cuticle originates from the host.     

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.