Cuticle Degradation Research Articles

Fine structural survey of old cuticle degradation during pre-ecdysis in two European Atlantic crabs

Light and transmission electron microscopy were used to monitor changes due to the degradation of the old exoskeleton and related events in the sclerites, articular membranes, and gills of two decapod crustaceans ( Carcinus maenas and Macropipus puber) during pre-ecdysis. In both sclerites and articular membranes, degradation follows a similar general pattern in both crab species, while the gill cuticle appears unaltered. In early pre-ecdysis (D 0), the degradation of the old cuticle starts with the secretion of ecdysial droplets by the epidermis. Apolysis, occurring at stage D 1′, is re-defined as an event, not necessarily morphologically observable, consisting in the loss of adherence between the epidermis and the old cuticle during early pre-ecdysis of arthropods. At the stage D 1‴, the moulding of the epidermal cell surface occurs in preparation to the deposition of the new cuticle and causes the opening of the ecdysial cleft. In the principal layer of sclerites, degradation of the chitin-protein microfibres should precede mineral dissolution. In contrast to the other degraded cuticle layers, the membranous layer of sclerites and the innermost endocuticular lamellae of articular membranes are transformed into a digestion-resistant fibrous network resembling the ecdysial membrane of insects.

“Exolysosomes,” Enzyme-Containing Vesicles in the Ecdysial Space of Molting Crabs

Free vesicle-like bodies (VLBs) present in the ecdysial space of cuticle regions undergoing degradation during preecdysis of the Atlantic shore crabCarcinus maenashave been interpreted either as infectious organisms or as secretion structures associated with degradation of the old cuticle. Ultrastructural, cytochemical, and immunocytological investigations were performed to test these hypotheses and to see whether VLBs are peculiar to this crab species. Similar VLBs were systematically found in two other preecdysial crabs,Cancer pagurusandMacropipus puber.InCar. maenas,they originate during early premolt inside Golgi buddings and are often gathered into large vacuoles in epidermal cells. The histochemical azo-dye technique and a cerium-based cytochemical method revealed acid phosphatase activity in both the ecdysial space and the VLBs, while Feulgen's method and immunocytological labeling always failed to reveal any DNA or RNA in either the ecdysial space or the VLBs. We conclude that VLBs are not infectious organisms but “extracellular” cuticle-degrading organelles of lysosomal origin and propose to coin them “exolysosomes.”

Ultrastructural Effects of a Non-Steroidal Ecdysone Agonist, RH-5992, on the Sixth Instar Larva of the Spruce Budworm, Choristoneura fumiferana

Force feeding of RH-5992 (Tebufenozide), a non-steroidal ecdysone agonist to newly moulted sixth instar larvae of the spruce budworm, Choristoneura fumiferana, (Lepidoptera: Tortricidae) initiates a precocious, incomplete moult. Within 6 h post treatment (pt) the larva stops feeding and remains quiescent. Around 12 h pt, the head capsule slips partially revealing an untanned new head capsule that appears wrinkled and poorly formed. By 24 hr pt, the head capsule slippage is pronounced and there is a mid-dorsal split of the old cuticle in the thoracic region but there is no ecdysis. The larva remains moribund in this state and ultimately dies of starvation and desiccation. The temporal sequence of the external and internal changes of the integument were studied using both scanning and transmission electron microscopy. Within 3 h pt, there is hypertrophy of the Golgi complex indicating synthetic activity and soon after, large, putative ecdysial droplets are seen. Within 24 h, a new cuticle that lacks the endocuticular lamellae is formed. The formation of the various cuticular components, the degradation of the old cuticle and changes in the organelles of the epidermal cells of the mesothoracic tergite are described. The difference between the natural moult and the one induced by RH-5992 are explained on the basis of molecular events that take place during the moulting cycle. The persistence of this ecdysone agonist in the tissues permits the expression of all the genes that are up-regulated by the presence of the natural hormone but those that are turned on in the absence of the hormone are not expressed.

Induction of resistance in barley against Erysiphe graminisf.sp. hordeiby free cutin monomers

Enzymatic degradation of plant cuticles at sites of fungal attack may release free cutin monomers. The hypothesis that free cutin monomers are signals for the activation of plant defence reactions was tested in the interaction of barley with the fungal pathogen Erysiphe graminisf.sp. hordei. Topical spray application of cutin monomers partially protected leaves of a highly susceptible barley cultivar against E. graminis, the plants being challenge-inoculated 1–3 days after the application. The degree of protection obtained by the C18 family of cutin monomers equaled protection obtained by the biological inducer E. graminisf.sp. tritici.Microscopic inspection of infection sites indicated enhanced resistance in barley epidermal cells to fungal penetration. Although the cutin monomers tested did not induce a number of defence-related barley genes, protection was eliminated by inhibiting host transcription with cordycepin, suggesting the involvement of inducible host reactions in the observed protection. Two-dimensional display of in vitrotranslation products from barley mRNA revealed the accumulation of one cutin monomer-induced mRNA, the same mRNA also being induced by pathogen attack. The data are compatible with a model that includes cutin monomers as signal molecules for the induction of disease resistance in barley.

Perception of free cutin monomers by plant cells

Enzymatic degradation of plant cuticles by fungal pathogens results in the release of free cutin monomers. The hypothesis that free cutin monomers are recognized by plant cells as endogenous stress‐related signals was tested in a model system consisting of cultured potato cells. Addition of cutin monomers in the micromolar range induced a transient alkalinization of the culture medium, similar to that observed with chitin or chitotetraose that served as positive control. The cutin monomers tested varied considerably in their potential to induce alkalinization, the most and least active compounds being cis‐9,10‐epoxy‐18‐hydroxystearic acid and palmitic acid, respectively. n,16‐dihydroxypalmitic acid (n = 8, 9 or 10) was found to be the major component of potato leaf cuticle and was among the most active cutin monomers. 9,10‐Dihydroxystearic acid, an analogue of the cutin monomer threo‐9,10,18‐trihydroxystearic acid, exhibited biological activity in a stereoselective manner, only the naturally occurring threo‐stereoisomer inducing a rapid and strong alkalinization response. Alkalinization of the culture medium was inhibited by addition of the protein‐kinase inhibitor K‐252a, and the onset of alkalinization was paralleled by changes in phosphorylation of specific proteins. The active cutin monomers also stimulated the production of the plant stress hormone ethylene and activated defence‐related genes at the mRNA level. The data provide evidence for a role of enzymatic breakdown products of plant cuticles as a new class of endogenous signal molecules.

The role of cuticle-degrading proteases in fungal pathogenesis of insects

The proteinaceous outer integument of insects forms an effective barrier against most microbes. Only the 700 known species of entomopathogenic fungi effect entry into their hosts by breaching the cuticle. There is accumulating evidence that the ability of fungi to degrade protein may aid their invasion of and growth in this orderly complex structure. Evidence for the particular importance of proteinases derives largely from studies of their production in infected cuticles associated with cuticle degradation, the effects of proteinase inhibitors on pathogen behavior, and by the analysis of protease-deficient mutants. More recently, studies have included the cloning, identification, and manipulation of specific protease genes of Metarhizium anisopliae, particularly those of the subtilisin (chymoelastase) type (designated Pr1) also produced by many other entomopathogenic fungi. Following solubilization of cuticle proteins by Pr1-type endoproteases, complete degradation of the cuticle involves a number of interacting enzyme species including a family of trypsin-like proteinases, metalloproteinases, several aminopeptidases, and a carboxypeptidase. Testing genetically engineered M. anisopliae null mutants of Pr1 indicated that the other endopeptidases can partially substitute for Pr1. The exopeptidases further degrade peptides released by the endopeptidases producing free amino acids for uptake and metabolism. Utilization of these enzymes has assisted investigators in understanding cuticle structure and how the cuticle is degraded naturally, and could lead to improved strain selection of entomopathogenic fungi or the introduction of their genes into other microbes and plants for the purpose of insect control. Key words: proteinaceous insect cuticle, pathogen endopeptidases, exopeptidases, multiple isozymes, enzyme regulation.

Degradation of cuticle during larval‐pupal and pupal‐adult development of the housefly, Musca domestica

Abstract. The patterns of changes in cuticle weight, its chitin content and chitinase activity have been studied during postembryonic development of the housefly, Musca domestica L. During pupariation the larval cuticle loses weight. During the early part of this weight‐loss the decline in chitin content parallels the overall change in cuticle weight. A simultaneous elevation in chitinase activity suggests that at this time the larval cuticle is being enzymatically degraded. Later weight loss may be due to sclerotization. No significant changes in cuticle weight or its chitin content occur in pharate cuticle until one day before eclosion. However, a peak of chitinase activity found at mid‐late pupal stage suggests the timing of pupal cuticle breakdown.

Conidial morphology, host colonization, and development of shot hole of almond caused by Wilsonomyces carpophilus

Morphology and ultrastructure of shot hole disease of almond infected by conidia of Wilsonomyces carpophilus were examined using light, scanning, and transmission electron microscopy. The multicelled conidia of the fungus were thick-walled and darkly pigmented. The conidial wall was multilayered and mainly consisted of an electron-dense, outer-wall layer and an electron-translucent, inner-wall layer. Septa of conidia were also multilayered. An electron-translucent zone separated the electron-dense, septa-wall layers of adjacent cells, and this zone extended to the inside of the outer-wall layer of a conidium. Conidia lacked true septa (distoseptate) and germinated by rupturing the outer-wall layers. Germination hyphae penetrated indirectly through stomata or directly through the cuticle into leaf tissue from appressoria that were produced terminally or on lateral branches of germ tubes. An extracellular mucilaginous matrix was commonly observed around hyphae and germ tubes in contact with leaf surfaces. Within leaf tissue, hyphae ramified throughout intercellular spaces and degraded cell walls of epidermal cells apparently without cuticle degradation. Diseased host tissue was tan brown and collapsed; ultrastracturally, diseased leaf cells were reduced in size, nonvacuolated, and had disrupted chloroplasts. A healthy host tissue response, adjacent to an infection site on leaves of potted plants, was the formation of a wound periderm. Within 10–14 days after an infection period (16 h of wetness), the periderm became a lignified–suberized barrier at 15 °C or a suberized abscission layer at 22 °C based on the histological stains Safranin O, Sudan III, and Sudan Black. At 15 °C, no abscission occurred and meristematic cells remained isodiametric but their walls became suberized and lignified, whereas cells adjacent to diseased tissue became lignified. At 22 °C, abscission occurred as cells adjacent to diseased tissue became vacuolated, enlarged, and suberized. Subsequently, the epidermis ruptured and the enlarged cells separated along the middle lamella to form an abscission layer. Hyphal growth was limited to the boundaries of the walled-off diseased tissue. Under favorable environmental conditions, hyphae replaced host epidermal cells and aggregated above the palisade layer to form pulvinate sporodochia. Sporodochia consisted of hyphae, numerous sympodially developing conidiogenous cells, and conidia that ruptured the host cuticle. Key words: fungal morphology, fungal foliar diseases, wound periderm, host–parasite interactions.

Analysis of aminopeptidase and dipeptidylpeptidase IV from the entomopathogenic fungus Metarhizium anisopliae

Analytical and preparative isoelectric focusing were used to separate extracellular isoenzymes of aminopeptidase (pI 4.51, M(r) 45,000, pH optimum 7.0) and prolyl-dipeptidylpeptidase (pI 4.01, M(r) 74,000, pH optimum 8.0) produced by the entomopathogenic fungus Metarhizium anisopliae during growth on locust cuticle. Production of both activities is repressed by readily utilized nitrogen sources, but unlike the aminopeptidase, the dipeptidylpeptidase was also excreted at high levels during growth on casein. Casein-grown cultures contained additional isoenzymes with activity against lysyl-alanyl-4-methoxy-2-naphthylamine indicating M. anisopliae possesses multiple peptidases as an adaptation to different nutrient conditions. The aminopeptidase hydrolysed alanyl-leucyl-alanine and showed a broad specificity versus monoaminoacyl beta-naphthylamine (beta NA) substrates with alanine beta NA being the most rapidly hydrolysed. Inhibition by both bestatin and amastatin indicated similarities to the class of alanyl aminopeptidases (aminopeptidase M). Metal complexing agents also inhibited the aminopeptidase indicating a metal ion requirement. A specific inhibitor for serine proteases [diisopropyl fluorophosphate (DFP)] was without effect. The dipeptidylpeptidase showed a strong preference for substrates having a penultimate proline residue including alanyl-prolyl-glycine and aa-prolyl-beta NA substrates. The enzyme showed a broad specificity at the N-terminal amino acid. Inhibition by diprotin A indicates similarities with mammalian prolyl-dipeptidylpeptidases. The enzyme was also inhibited by DFP, implying involvement of a serine residue in catalysis. The results are discussed in the context of cuticle degradation and the participation of exopeptidases as mediators in releasing amino acids necessary for pathogen growth.

Molting fluid enzymes of the tobacco hornworm, Manduca sexta: Timing of proteolytic and chitinolytic activity in relation to pre‐ecdysial development

AbstractDevelopmental profiles for a number of molting fluid (MF) enzyme activities were established and related to the progress of pupal cuticle degradation during the four days that precede the eclosion of adult tobacco hornworms. Cuticle degrading activity, molting fluid protease 1 (MFP‐1), and molting fluid protease 2 (MFP‐2) all increased in activity at the time that loss of material from the old cuticle occurred. In contrast, chitinase and β‐acetylglucosaminidase activities did not parallel weight loss from the old cuticle. These results are consistent with the hypothesis that proteolytic activity is a prerequisite for the action of chitinase on cuticle chitin. © 1993 Wiley‐Liss, Inc.

Prepenetration events during infection of host cuticle by Metarhizium anisopliae

The early involvement of cuticle degrading enzymes in infection was suggested by the presence of chymoelastase protease (Prl), esterase, and N-acetylglucosaminidase in ungerminated spores of Metarhizium anisopliae. Enzymes were released from spores by washing in water or dilute buffer solutions. This, together with the degradation of enzyme substrates by intact spores, indicates that enzyme activities are located at the surface of spores, and probably parallels the availability or release of enzymes under natural conditions. Levels of enzymes on conidia from infected Manduca sexta larvae were higher than those harvested from Sabauraud dextrose agar (SDA), indicating that environmental conditions in which spores develop can preadapt them for the pathogenic life style. Cell-bound enzymes were released from differentiating germlings in varying degrees by water, salt solutions, detergents, and mercaptoethanol. Release of Pr1 and esterase activities by salt solutions suggests that their binding to cell walls involves ionic bonds. By contrast, release of N-acetylglucosaminidase required the disruption of membranes. We suggest that binding of enzymes to fungal walls could, in part, explain localized action of cuticle degrading enzymes during host penetration. The esterase activity in conidia is represented by a single major activity (p I 7) and in infection structures by multiple isoenzymes, all characterizable as β-esterases. Esterase activites were greatest against short and intermediate length fatty acids with little activity above C 10. The effects of surface topography on appressorium formation was studied using M. sexta cuticles and plastic replicas of the cuticles. Appressoria were only produced after extensive growth over the microfolds of the cuticle surface of early (1 day) fifth instar larvae. By contrast germination on the comparatively flat surface of 5-day fifth instar larvae allows appressorium formation close to the conidium. The results are discussed in the context of cuticle degradation during prepenetration fungal growth, and the effects of host morphology on pathogen behavior.

Preparation of the infection court by Erysiphe graminis: Degradation of the host cuticle

Contact with a surface stimulates the release of a liquid from conidia of Erysiphe graminis and the liquid has been shown to contain esterase activity. The present investigation demonstrates that the liquid exudate partially erodes the cuticle of barley leaves. The release of esterase into the liquid occurs in two stages. The second stage of release occurs between 10 and 15 min after contact and was shown to be temperature dependent and to be inhibited by cycloheximide. Cuticular erosion may provide a stimulus for subsequent morphological differentiation, such as appressorium formation, or it may enhance appressorial adhesion.

Degenerative changes in the epidermal cell wall of Allium cepa caused by Botrytis allii: Loss of dry mass, pectin, and cellulose in halos

Halos detected using interference microscopy (even- and fringle-field modes with monoand poly-chromatic light) around penetration sites of Botrytis allii in cell walls of normal and protoplast-free outer epidermal tissue of white, yellow, and red onions were alike. Halos in protoplast-free cell walls contained 33% less dry mass than areas of these walls adjacent to halos (quantitative interference miscroscopy with 546 nm light in the even-field mode). Halos were significantly larger in the white onion than in the yellow and red varieties. The loss of cell wall dry mass during the production of halos involved the loss of pectin and cellulose. We infer that this is caused by enzymes released from the pathogen. Cuticle degradation at penetration sites was not observed.

Cuticle-degrading enzymes of entomopathogenic fungi: Mechanisms of interaction between pathogen enzymes and insect cuticle

Extracellular enzymes of Metarhizium anisopliae had considerable affinity for insect cuticle. Binding of proteases immobilized over 70% of soluble enzyme activity, which in vivo could have a significant influence on the extent and nature of cuticle degradation. Adsorbed protease, carboxypeptidase, and N-acetylglucosaminidase activities were recoverable with 0.2 m buffer suggesting nonspecific ionic binding. Chitinase bound irreversibly as a specific enzyme-substrate complex. Cuticle degradation by an alkaline (optimum pH 9) basic ( pI 9.5) protease was inhibited by increasing salt concentrations while anilide hydrolysis was unaffected. Inhibition arose from interference with essential electrostatic adsorption of the enzyme on to the cuticle. An anionic detergent enhanced enzymic solubilization of cuticle proteins (probably due to increased electronegativity of cuticle) at the expense of continued proteolysis of released peptides, clearly distinguishing between the two processes. A cationic detergent inhibited cuticle degradation, indicating that salt labile bonds form between the negative (probably carboxyl) groups of cuticle and the positively charged groups of the protease. The significance of these results in understanding the mechanism of cuticle degradation are discussed.

Cuticle-degrading enzymes of entomopathogenic fungi: Cuticle degradation in vitro by enzymes from entomopathogens

Extracellular fluids from Metarhizium anisopliae, Beauveria bassiana, and Verticillium lecanii grown on cuticle as the sole carbon source released amino acids and N-acetylglucosamine from protein and chitin, respectively, in comminuted locust cuticle. An endoprotease, chitinase, and N-acetyl-β-glucosaminidase were each purified from culture filtrates of M. anisopliae until free of other cuticle-degrading enzymes and tested singly, in combination, or in sequence against “whole cuticle” (containing tanned and untanned proteins) and exuviae (tanned cuticle). The protease hydrolyzed ca. 25–30% of cuticle proteins ( w w ), releasing peptides (mean chain length, 4.7) containing all 15 amino acids found in locust cuticle. Small amounts of amino sugars were also liberated following protein solubilization. Chitinase tested separately released monomeric N-acetylglucosamine (equivalent to 3–4% of cuticle chitin); however, when combined simultaneously with protease, N-acetylglucosamine release was increased × 1.5. Pretreatment with protease considerably enhanced chitinase activity (ca. × 3.5) compared to controls (preincubated with autoclayed protease). This implies that cuticular chitin is shielded by protein. N-acetyl-β-glucosaminidase showed no detectable activity against cuticle either alone or in combination with protease or chitinase. Exuvia was comparatively resistant to both proteolytic and chitinolytic attack; pretreatment with protease had no effect on subsequent chitinase activity. The results are discussed in relation to cuticle structure and the role of host and fungal enzymes in degrading cuticle during molting or infection.

Biphasic expression and function of glucose dehydrogenase in Drosophila melanogaster.

Glucose dehydrogenase (GO) was found to be expressed during the pupal stage in both sexes of Drosophila melanogaster, but is limited to the male ejaculatory duct at the adult stage. During copulation GO is transferred from males to females. Mutational analysis of the Go locus indicates that a single structural gene encodes the pupal and ejaculatory duct GO. Thus an example of an enzyme structural gene switching from non-sex-limited to sex-limited expression has been found. Go mutants are recessive lethals exhibiting a late pupal effective lethal phase. These mutants can be rescued by excising the anterior end of the pupal case 0-2 days prior to the normal adult emergence time. It appears that the function of GO in pupae is to aid in the degradation of the puparium cuticle in preparation for the eclosion of the adult.

The chemical environment of leaf surfaces with special reference to spore germination of pathogenic fungi

AbstractThe release of diffusible substances from plant leaves, fungal spores or from pollen into water droplets on the surface of leaves is discussed in relation to (1) direct and indirect effects on spores of pathogens and (2) the effect on the growth of saprophytic micro‐organisms. Evidence suggests that competition for nutrients in droplets on leaves may limit germination of spores of some pathogens. Modification of the chemical environment of leaf surfaces by saprophytic micro‐organisms is discussed in relation to lytic enzyme and antibiotic production, cuticle degradation, production of growth substances and fixation of nitrogen.Examples are given of the effect on pathogenic fungi of diffusible toxins from leaves and inhibitors associated with surface wax. Treatments of leaves that remove or reduce surface wax can result in a stimulation of spore germination of some pathogenic fungi. The possible occurrence of phytoalexins on leaf surfaces prior to entry of pathogens into leaves is discussed in relation to differences in host‐pathogen interaction.

Aspects of cuticle formation in Drosophila melanogaster

AbstractThe molting process and some aspects of puparium formation in Drosophila melanogaster have been examined by electron microscopy and autoradiography. Results indicate clearly alternate phases of production of enzymes involved in cuticle synthesis and enzymes involved in cuticle degradation, in epithelial cells. The synthesis phase begins about four hours before each molt and appears to continue about three hours after puparium formation. Protein which is involved in cuticle structure appears to remain where deposited. On the other hand, metabolic products of tyrosine that are involved in sclerotization and tanning diffuse outward through the cuticle from their origin in the epithelial cells. Channels or canals for transport of materials from cells toward epicuticle have not been observed in larval or prepupal material.