Comparison between the sclerotization of adult and larval cuticle in Schistocerca gregaria

Femur cuticle from fifth instar larvae of the desert locust, Schistocerca gregaria, has been characterized with respect to composition, rate of deposition, and rate of sclerotization. The results are compared with those from adult cuticle of the same species. The protein compositions of the two types of cuticle are very similar, but the rates of deposition of both protein and chitin are different. The main difference is, however, that sclerotization is restricted to the first day after ecdysis in larval cuticle, whereas in adult cuticle sclerotization continues for at least a couple of weeks. The result is that the endocuticle remains untanned in the larvae, whereas in the adults the whole cuticle becomes tanned.     

Synthesis of the major adult cuticle proteins of Drosophila melanogaster during hypoderm differentiation

Differentiating imaginal hypodermal cells of Drosophila melanogaster form adult cuticle during the second half of the pupal stage (about 40 to 93 hr postpupariation). A group of proteins with molecular weights of 23,000, 20,000, and 14,000 is identified as putative major wing cuticle proteins with the following biological properties: These proteins are abundant components of cuticle and are major synthetic products of cuticle-secreting hypodermal cells. They are leucine-rich and methionine-free and are the most prominent proteins of this type synthesized by wing hypoderm at 65 hr, during the period of procuticle formation. Electron microscopic autoradiography shows that leucine-rich, methionine-free proteins specifically localize to the apical cell surface and newly secreted cuticle of 65-hr wing cells. This strongly suggests the export of these proteins to the cuticle. Lastly, these proteins undergo a reduction in extractability just after eclosion, during the period of cuticle protein crosslinking (sclerotization). The synthesis of these major hypoderm proteins is temporally regulated in development. In wing cells, the 14-kDa proteins are synthesized first, from 53 to 78 hr, and the 20- and 23-kDa proteins are synthesized from 63 to 93 hr. The pattern of synthesis for these proteins is similar in abdominal cells but delayed by 6 to 10 hr. Two-dimensional gel electrophoresis shows that each of the 23-, 20-, and 14-kDa size classes contains at least two component polypeptides. Patterns of protein synthesis in cells of the imaginal hypodermis are regulated in a precise temporal sequence during the production of adult cuticle. Their study yields a useful system for the analysis of molecular events in gene control and cell differentiation.     

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.     

Identification of a cuticle protein with unique repeated motifs in the silkworm,Bombyx mori

The insect cuticle is non-cellular matrix secreted from a monolayer of epidermal cells. After abrasion of the larval cuticle of the silkworm, Bombyx mori, a protein with molecular mass of 135 kDa is newly detected in the cuticle. Mass spectrometric analysis of the tryptic fragments from this protein revealed that the 135-kDa protein is encoded by the Cb10 gene. In the predicted amino acid sequence of Cb10, three repeated motifs with [YxGGFGGppG(L/V)L] sequence are found in the C-terminal region. In addition to the repeated motifs, Cb10 has seventeen CxxxxC motifs randomly distributed throughout the polypeptide chain and serine rich region at the N-terminal region. The Cb10 gene is strongly expressed in epidermal cells after pupal ecdysis, and its expression in the larval epidermal cells is induced not only by cuticular abrasion, but also by bacterial infection. These expression patterns suggest some specific roles of this protein in pupal cuticle formation and defense reactions.     

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.     

Cuticle proteins of the boll weevil,Anthonomus grandis,abdomen: Structural similarities and glycosylation

Cuticle proteins are thought to be important in defining the structural and functional differences occurring in insect cuticle. In order to explain and better understand the structural similarities among the cuticle proteins of the cotton boll weevil, Anthonomus grandis Boheman, described in a previous study (Stiles and Leopold, 1990, Insect Biochem.20, 113–125) three series of monoclonal antibody producing hybridoma cell lines were produced. Larval, pupal or adult cuticle proteins were used as antigens. While some of the monoclonal antibodies were specific for one or two cuticle proteins from a single developmental stage, the majority showed multiple cuticle protein binding patterns on Western blots. To determine whether this cross-reaction was due to common oligosaccharide chains bound to the proteins, lectins were used to probe Western blots. Many of the cuticle proteins were found to be glycosylated. The majority of the Con A reactive carbohydrate could be removed from the protein by N-glycosidase F digestion (specific for N-asparagine linked carbohydrate). N-glycosidase F digestion did not reduce the multiple cross-reactions of the monoclonal antibodies, nor did periodate oxidation of the CP. The carbohydrate remaining after enzyme digestion is presumably O-linked to serine/threonine.     

Caenorhabditis elegans BAH-1 Is a DUF23 Protein Expressed in Seam Cells and Required for Microbial Biofilm Binding to the Cuticle

The cuticle of Caenorhabditis elegans, a complex, multi-layered extracellular matrix, is a major interface between the animal and its environment. Biofilms produced by the bacterial genus Yersinia attach to the cuticle of the worm, providing an assay for surface characteristics. A C. elegans gene required for biofilm attachment, bah-1, encodes a protein containing the domain of unknown function DUF23. The DUF23 domain is found in 61 predicted proteins in C. elegans, which can be divided into three distinct phylogenetic clades. bah-1 is expressed in seam cells, which are among the hypodermal cells that synthesize the cuticle, and is regulated by a TGF-β signaling pathway.     

A study on the cuticle of Stegoalpheon kempi Chopra (Isopoda: Crustacea) in relation to its parasitic mode of life

Histological, histochemical, and possible functional properties of the cuticle in the bopyrid isopod parasite Stegoalpheon kempi Chopra are described. In the male, the epi- and the procuticle show similar histochemical characters. The protein constituent of the cuticle suggests a similarity to collagen and its significance is discussed in relation to the parasitic mode of life.In the female, the epicuticle is fuchsin-positive in Mallory's Triple and Masson's Trichrome stains, positive to tests for aromatic amino acids, but negative to tests for phenol, phenol oxidase, and quinone. In addition, the epicuticle shows auto-fluorescence and investigations on the fluorescent compounds present in the epicuticle protein show the presence of di- and tri-tyrosine. The function of these two unusual amino acids as possible cross links is discussed.The cuticle covering the pleopods lacks epicuticle, but it has a simple protein positive to the biuret test, and also a protein containing sulphydryl groups, perhaps as an adaptation for providing respiratory exchanges of gases through cuticle.     

Structure and expression of mRNA for a pupal cuticle protein of the silkworm,Bombyx mori

Electrophoretic and immunoblot analyses of proteins extracted from the salt-washed integuments of the silkworm Bombyx mori demonstrated that the pupal cuticle contains structural proteins distinct from those present in the larval cuticle. The cDNA clone encoding a pupal cuticle protein was isolated from the cDNA library constructed from epidermal mRNA of pharate pupae. Northern blot hybridization by use of a cDNA probe provided evidence that mRNA for the pupal cuticle protein accumulate in integument during larval-pupal transformation, though temporal rise of the mRNA level was also noticed at the stages of larval molting. Primary structure of the pupal cuticle protein was deduced from the nucleotide sequence of cDNA. The cloned mRNA sequence encodes a 27 kDa protein rich in alanine and proline, containing characteristic repeats of Ala-Pro-Ala-His-Gln-(Asp/Ser)-Trp-Asn sequence in the carboxyl-proximal domain. The sequence (Ile/Val)-(Leu/Ala)-(Asp/Glu)-Thr-Pro-Glu-Val-Ala-(Gln/Ala)-Ala-Arg-Ala-Ala-His-(Leu/Ile)-(Ala/Ser)-Ala-(Leu/His) occurs in three hydrophobic domains of the molecule.     

Retroactive Maintains Cuticle Integrity by Promoting the Trafficking of Knickkopf into the Procuticle of Tribolium castaneum

Molting, or the replacement of the old exoskeleton with a new cuticle, is a complex developmental process that all insects must undergo to allow unhindered growth and development. Prior to each molt, the developing new cuticle must resist the actions of potent chitinolytic enzymes that degrade the overlying old cuticle. We recently disproved the classical dogma that a physical barrier prevents chitinases from accessing the new cuticle and showed that the chitin-binding protein Knickkopf (Knk) protects the new cuticle from degradation. Here we demonstrate that, in Tribolium castaneum, the protein Retroactive (TcRtv) is an essential mediator of this protective effect of Knk. TcRtv localizes within epidermal cells and specifically confers protection to the new cuticle against chitinases by facilitating the trafficking of TcKnk into the procuticle. Down-regulation of TcRtv resulted in entrapment of TcKnk within the epidermal cells and caused molting defects and lethality in all stages of insect growth, consistent with the loss of TcKnk function. Given the ubiquity of Rtv and Knk orthologs in arthropods, we propose that this mechanism of new cuticle protection is conserved throughout the phylum.     

Localization of RR-1 and RR-2 cuticular proteins within the cuticle of Anopheles gambiae

The largest arthropod cuticular protein family, CPR, has the Rebers and Riddiford (R&R) Consensus that in an extended form confers chitin-binding properties. Two forms of the Consensus, RR-1 and RR-2, have been recognized and initial data suggested that the RR-1 and RR-2 proteins were present in different regions within the cuticle itself. Thus, RR-2 proteins would contribute to exocuticle that becomes sclerotized, while RR-1s would be found in endocuticle that remains soft. An alternative, and more common, suggestion is that RR-1 proteins are used for soft, flexible cuticles such as intersegmental membranes, while RR-2s are associated with hard cuticle such as sclerites and head capsules. We used TEM immunogold detection to localize the position of several RR-1 and RR-2 proteins in the cuticle of Anopheles gambiae. RR-1s were localized in the procuticle of the soft intersegmental membrane except for one protein found in the endocuticle of hard cuticle. RR-2s were consistently found in hard cuticle and not in flexible cuticle. All RR-2 antibodies localized to the exocuticle and four out of six were also found in the endocuticle. Hence the location of RR-1s and RR-2s depends more on properties of individual proteins than on either hypothesis.     

Identification and molecular analysis of the cuticle protein genes of Dacus oleae

A 11.9 Kb DNA segment of the Dacus oleae genome that contains three cuticle protein genes has been cloned and characterized. These three genes are clustered within 8.04 Kb of DNA; the restriction sites for eight enzymes and the organization of the cuticle genes in this 11.9 Kb cloned fragment were determined. Using this clone as a radioactive probe, it was shown that the three cuticle genes are expressed as poly(A) RNA in the epidermis of late third instar larvae but are not abundantly expressed in other tissues.     

Changes in the mechanical properties of the extensible cuticle of Rhodnius through the fifth larval instar

The ultimate tensile strength (σ_UT) and the modulus of elasticity (E) of Rhodnius extensible cuticle are σ_UT = 2.20 × 10⁷ Nm^?2, E = 2.43 × 10⁸ Nm^?2 (unplasticised); σ_UT = 1.43 × 10⁷ Nm^?2, E = 9.45 × 10⁶ Nm^?2 (plasticised with 5HT) and σ_UT = 9.05 × 10⁶ Nm, E = 2.46 × 10⁶ Nm^?2 (plasticised in pH 5 buffer).The mechanical properties of cuticle from insects which have deposited additional layers of cuticle after they have been fed differ from those of cuticle from unfed insects. This is possibly due to the different composition of the additional cuticle: it is suggested that the post-feeding cuticle is providing protection and a template for the next instars cuticle.The maximum strain of extensible cuticle from starved insects is related to the amount of matrix protein present.     

Heat-induced aggregation of XRCC5 (Ku80) in nontolerant and thermotolerant cells.

XRCC5 (also known as Ku80) is a component of the DNA-dependent protein kinase (DNA-PK), existing as a heterodimer with G22P1 (also known as Ku70). DNA-PK is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair, and kinase activity is dependent upon interaction of the Ku subunits with the resultant DNA ends. Nuclear XRCC5 is normally extractable with non-ionic detergent; it is found in the soluble cytoplasmic fraction after nuclear isolation with Triton X-100. In this study, we found that heating at 45.5 degrees C causes a decreased extractability of XRCC5 from the nuclei of human U-1 melanoma or HeLa cells. Such decreases in extractability are indicative of protein aggregation within nuclei. Recovery of extractability of XRCC5 to that of unheated control cells was observed after incubation at 37 degrees C after heat shock. The decrease in extractability and the kinetics of recovery were dependent on dose, although the decrease in extractability reached a plateau after heating for 15 min or more. Thermotolerant U-1 cells also showed decreased extractability of XRCC5, but to a lesser degree compared to nontolerant cells. When a comparable initial reduction of extractability of XRCC5 was induced in both thermotolerant and nontolerant cells, the kinetics of recovery was nearly identical. The kinetics of recovery of the extractability of XRCC5 was different from that of total nuclear protein in nontolerant cells; recovery of extractability of XRCC5 occurred faster initially and returned to the level in unheated cells faster than total nuclear protein. Similar results were obtained for thermotolerant cells, with differences between the initial recovery of the extractability of XRCC5 and total protein being particularly evident after longer heating times. Heat has been shown to inactivate XRCC5. We speculate that inactivation of XRCC5 after heat shock results from protein aggregation, and that changes in XRCC5 may, in part, lead to inhibition of DSB repair through inactivation of the NHEJ pathway.     

Cuticle laccase of the silkworm,Bombyx mori: Purification,gene identification and presence of its inactive precursor in the cuticle.

Laccase is a multi-copper enzyme found in variety of organisms including plants, fungi and bacteria. In insects, laccase is thought to play an important role in cuticle sclerotization with its ability to catalyze the oxidation of phenolic compounds to their corresponding quinones. From the newly ecdysed pupae of the silkworm, Bombyx mori, we purified a dimer form of cuticular laccase with 70-kDa polypeptides. Mass spectrometric analysis of the tryptic fragments and cDNA sequence analysis revealed that the gene for the purified laccase (BmLaccase2) is an ortholog of laccase2, one of the multiple laccase genes found in insect genomes. BmLaccase2 is highly expressed in the epidermis prior to ecdysis, suggesting that the BmLaccase2 protein accumulates before ecdysis. However, the cuticle of newly ecdysed pupa does not have laccase activity, and the activity only becomes detectable several hours after ecdysis. These data suggest that cuticle laccase is synthesized as an inactive precursor, which is later activated after ecdysis. We also found that urea-solubilized cuticle protein extract contains an inactive form of laccase that can be activated by trypsin treatment.     

A novel class of sticky peel and light green mutations causes cuticle deficiency in leaves and fruits of tomato (Solanum lycopersicum)

The plant cuticle consists of aliphatic wax and cutin, and covers all the aerial tissues, conferring resistance to both biotic and abiotic stresses. In this study, we performed phenotypic characterizations of tomato mutants having both sticky peel (pe) and light green (lg) mutations. Our genetic analysis showed that these two mutations are tightly linked and behave like a monogenic recessive mutation. The double mutant (pe lg) produced glossy soft fruits with light green leaves, most likely due to defects in cuticle formation. Cytological analysis revealed that the thickness of the fruit cuticle layer was dramatically reduced in the pe lg mutant. The epidermal cells of the leaves were also deformed in the pe lg mutant, suggesting that leaf cuticle formation was also disrupted in the mutant. Consistent with this, transmission electron microscopic analysis showed that the electron density of the cuticle layer of the adaxial surface of the leaf was reduced in the pe lg mutant compared to WT, suggesting that there are changes in cuticle structure and/or composition in the pe lg mutant. Both physiological analysis to measure the rate of transpiration, and staining of the fruits and leaves with toluidine blue, revealed that water permeability was enhanced in the pe lg mutant, consistent with the reduced thickness of its cuticle layer. Taken together the preliminary analyses of the cuticle components, the PE LG is most likely involved in proper cuticle formation.     

Extractability and biological activity of tannins from various tree leaves determined by chemical and biological assays as affected by drying procedure

Leaf samples of Juglans regia (walnut), Rhus typhina (sumach), Aesculus hippocastanum (chestnut) and Salix alba (willow) were either freeze dried or oven dried and the amount of tannins was determined by two chemical assays (Folin Ciocalteau and ferric chloride method). In addition, the biological activity of these tannins was determined by their protein precipitation capacity and by a bioassay based on the incubation to the leaves in vitro in buffered rumen fluid with and without polyethylene glycol. Generally, oven drying is recognised to decrease the extractability and/or the biological activity of tannins. Our results however do not indicate a general decrease in extractability of tannins due to oven drying. The effects observed were plant specific with negative effects of oven drying in walnut and willow leafs, positive effects in chestnut leaves and no effects were observed for the sumach. No negative effects of oven drying were detected for the biological activity of the tannins. The specific protein precipitation capacity was nearly identical for the freeze dried and the oven dried tannins. Oven drying however decreased the solubility of the cell contents and the plant cell wall, which led to changes in fermentation end products, but the biological activity of the tannins was not negatively affected by the oven drying. Although some differences in the extractability and activity of the individual plant species were observed, we conclude from this work that the drying procedure has no negative effect on the biological activity of the tannins examined.