Biochemistry of insect epicuticle degradation by entomopathogenic fungi

The biochemical interaction between fungal pathogens and their insect host epicuticle was studied by examining fungal hydrocarbon degrading ability. As a contact insecticide, entomopathogenic fungi invade their host through the cuticle, covered by an outermost lipid layer mainly composed of highly stable, very long chain structures. Strains of Beauveria bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes), pathogenic both to the blood-sucking bug Triatoma infestans (Hemiptera: Reduviidae) and the bean-weevil Acanthoscelides obtectus (Coleoptera, Bruchidae), were grown on different carbon sources. Alkane-grown cells showed a lipid pattern different from that of glucose-grown cells, evidenced by a major switch in the triacylglycerol and sterol components. Radiolabelled hydrocarbons were used to investigate the catabolic pathway and the by-product incorporation into fungal cellular components. The first oxidation round is presumably carried out by a cytochrome P450 enzyme system, the metabolites will traverse the peroxisomal membrane, and after successive transformations will eventually provide the appropriate fatty acyl CoA for complete degradation in the peroxisomes, the site of beta-oxidation in fungi. In this review, we will show the relationship between fungal ability to catabolize very long chain hydrocarbons and virulence parameters.     

Toxocara canis: a labile antigenic surface coat overlying the epicuticle of infective larvae.

An electron-dense coat covering the surface of Toxocara canis infective-stage larvae is described. This coat readily binds to cationized ferritin and ruthenium red, indicating a net negative charge and mucopolysaccharide content, and can be visualized by immuno-electron microscopy only if cryosectioning is employed. Monoclonal antibodies reactive to the surface of live larvae bind the surface coat but not the underlying cuticle in ultrathin cryosections. The surface coat is dissipated on exposure to ethanol, explaining the lack of surface reactivity of conventionally prepared immunoelectron microscopy sections of T. canis. Differential ethanol extraction of surface-iodinated larvae demonstrates that the major component associated with the coat is TES-120, a 120-kDa glycoprotein previously identified by surface iodination, which is also a dominant secreted product. The surface-labeled TES-70 glycoprotein is linked with a more hydrophobic stratum at the surface, while a prominent 32-kDa glycoprotein, TES-32, is more strongly represented within the cuticle itself. Antibody binding to the coat under physiological conditions results in the loss of the surface coat, but this process is arrested at 4 degrees C. This result gives a physical basis to earlier observations on the shedding of surface-bound antibodies by this parasite. An extracuticular surface coat has been demonstrated on Toxocara larvae prior to hatching from the egg and during all stages of in vitro culture, suggesting that it may play a role both in protecting the parasite on hatching in the gastrointestinal tract and on subsequent tissue invasion in evading host immune responses directed at surface antigens.     

Strongyloides ratti and Trichinella spiralis: net charge of epicuticle

The intact epicuticles of Strongyloides ratti stage-3 larvae and Trichinella spiralis stage-1 larvae were found to have a surface net negative charge. Ultrastructural studies on S. ratti using cationized ferritin and ruthenium red showed the negative charge to be dense and uniformly distributed over the epicuticular surface. Staining with acetic acid-ferric oxide hydrosol occurred at pH 1.65 and suggests that amino acid carboxyl groups were not responsible for the negative charge property. Alcian blue staining occurred at pH 0.5 and at a critical electrolyte concentration (CEC) of 0.9 M MgCl2, a property similar to that of highly sulfated mucopolysaccharides such as the proteoglycan keratan sulfate. In contrast, T. spiralis larvae failed to stain with alcian blue below pH 5.0 or at a CEC of 0.1 M, suggesting its negative charge is associated with dissociated amino acid carboxyl groups. Attempts to remove the negative charge-bearing components in the epicuticle of S. ratti by detergents, organic solvents, denaturing agents, proteases, uronidases, neuraminidases, and lipases were unsuccessful. The presence of elastin in the S. ratti larval outer cortical layer was indicated by its vulnerability to elastase and its reaction to aldehyde fuchsin-alcian blue stain. These results show that the epicuticle of S. ratti is not a typical cell membrane, although it appears to have ultrastructural similarities. It is suggested that the association of highly sulfated mucopolysaccharides with the epicuticular surface of free-living nematodes such as S. ratti L3 may reflect a greater need to protect against surface desiccation. It is also postulated that the highly negatively charged surface may have anticomplementary and anticoagulation effects.     

The chemical nature of the outer epicuticle from Lucilia cuprina larvae

Nuclear magnetic resonance, i.r. absorption and u.v. absorption studies show the outer epicuticle from Lucilia cuprina larvae, as isolated from puparia by acidic hydrolysis, to be composed essentially of methylene (CH₂) groups together with a small number of aromatic and carbonyl groups. It has a complex, stabilized cyclic or crosslinked structure and more than one high molecular weight, molecular species may be present.     

The Structure and Mineralization of the Carapace of the Crab (Cancer pagurus L.) 3. The epicuticle

Decalcified and undecalcified preparations of the crab Cancer pagurus in the intermoult condition were studied to examine the mineralization and structure of the epicuticle, using light microscopic, electron microscopic, and microradiographic methods. The epicuticle was found to be composed of two layers, one superficial membrane, and one thicker layer, measuring 1-2 μm. From the base layer spines or microtrichia projected. These were approximately 18 μm long and built like the remainder of the epicuticle. The spines and the base layer of the epicuticle contained vertical canals which in undecalcified sections accomodated columns of crystals. These canals were the only location in which minerals occurred in the epicuticle. In decalcified preparations filamentous strands were observed in the canals. Elsewhere in the epicuticular tissue no fibrillar structure was observed. The canals and their contents seemed to extend across the junctional zone between the epicuticle and the exocuticle.     

Fine structure of the epicuticle of the desert scorpion, Hadrurus arizonensis, with reference to location of lipids.

The surface and transverse sections of the epicuticle of the desert scorpion, Hadrurus arizonensis, were examined by scanning and transmission electron microscopy, respectively. Sclerite cuticle that was untreated prior to normal EM preparative procedures was compared to cuticle subjected to lipid solvents, high temperature, and concentrated alkali. Surface morphology of untreated intersegmental cuticle was also examined. The epicuticle is composed of four sublayers: outer membrane, outer epicuticle, cuticulin, and the dense homogeneous layer. Lipid solvents did not significantly alter the morphology of any of these layers or the contents of the wax canals that penetrate the cuticulin layer even though the solvents effectively remove lipids from the epicuticle for chemical analysis. The surface of the sclerite cuticle contains amorphous particles, crystalline projections, and scattered openings to dermal gland ducts. Perforations that correspond to the opening of wax canals were faintly visible after extraction of surface waxes and clearly visible after KOH treatment. No openings to dermal gland ducts or wax canals were observed in untreated intersegmental cuticle. However, wax canals are likely obscured by surface waxes similar to those present in sclerite cuticle.     

Procuticle proteins and chitin-like material in the inner epicuticle of the Drosophila pupal cuticle

The inner (protein) epicuticle of the pupal cuticle of Drosophila is shown to contain at least two hydrophobic proteins (19 and 21 kD) that are also present in the outer procuticle lamellae. An N-acetylglucosamine-containing carbohydrate is also present in the inner epicuticle. This represents the first attempt to characterize the non-lipid components of an insect epicuticle.     

Puparium formation in Calliphora vomitoria: A hitherto unsuspected function of the epicuticle

Removal of the epicuticle from larvae of Calliphora vomitoria causes the underlying procuticle to become deeply coloured owing to the oxidation of its dihydroxyphenols. This oxidation is non-enzymatic. In the intact larva the epicuticle protects the phenols from spontaneous oxidation so that they are available later for use in puparium formation. The changes which take place in the procuticle as puparium formation is approached are described, and their significance in relation to the onset of phenolic tanning is discussed.

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Zusammenfassung Die Entfernung der Epikutikula von Larven von Calliphora vomitoria bewirkt, daß die darunter liegende Prokutikula infolge Oxydation ihrer Dioxyphenole eine dunkle Färbung annimmt. Diese Oxydation erfolgt nicht enzymatisch. In der unverletztent Larve schützt die Epikutikula die Phenole vor spontaner Oxydation, so daß sie später bei der Bildung des Pupariums zur Verfügung stehen. Die Umwandlungen, welche während des Herannahens der Pupariumbildung in der Prokutikula stattfinden, werden beschrieben und ihre Bedeutung in Bezug auf den Beginn der Gerbung besprochen.

     

The formation of the epicuticle and associated structures inOniscus asellus (Crustacea,Isopoda)

Summary The integument of the woodlouse,Oniscus asellus, consists of a two-layered epicuticle, a largely lamellate procuticle — itself divided into two regions (pre-and postecdysial cuticles), and the epidermis. At the initiation of new cuticle production the epidermal cells become vacuolated and retract away from the cuticle. Apolysis occurs immediately after the cessation of postecdysial cuticle production. The formation of the epicuticle is unique among the arthropods since material aggregates along the distal epidermal membrane. By indenting, doubling back on itself, and incorporating septa, the epicuticle forms surface structures such as plaques and tricorns.The innervation, and so the receptive function of the tricorns is confirmed, but since there is no connection between the old and new receptors during premoult, sensory information from these exoreceptors must be severely curtailed. This may explain the biphasic moult in all isopods since it ensures that only half the body experiences this sensory deprivation at any one time. In terrestrial species there is the additional advantage of restricting the area of permeable new cuticle. The frequency of moulting may be due to the need to renew disrupted receptor surfaces.Tricorns do not appear to be the mechanoreceptors involved in the marked thigmotactic response of woodlice since they do not have the typical internal structure of such receptors; rather, the dendrite —which extends into the lumen of the tricorn —is protected from deformation by the previously unreported combination of a dendritic sheath and a cuticular tube. The modality of tricorns is possibly one of hygro-perception. One of the behavioural responses of woodlice to desiccation is aggregation. The numerical distribution of tricorns over the body surface is admirably suited to assist in the formation and maintenance of such aggregates during desiccation and to their observed dispersal when the relative humidity rises.     

渗透剂对高效氯氰菊酯毒力和三种鳞翅目害虫上表皮超微结构的影响

以甜菜夜蛾Spodoptera exigua (Hübner)、小菜蛾Plutella xylostella (L.)和棉铃虫Helicoverpa armigera (Hübner)为试虫,通过室内生物测定技术,研究了氮酮、噻酮和N-甲基-2-吡咯烷酮等3种渗透剂对高效氯氰菊酯毒力的影响;利用扫描电镜观察了甜菜夜蛾和小菜蛾4龄幼虫对照组和渗透剂处理组间上表皮超微结构的变化。结果表明：含3.0% N-甲基-2-吡咯烷酮的高效氯氰菊酯对小菜蛾幼虫LD₅₀为0.0074 μg/头、毒力系数为2.0,对高效氯氰菊酯增效作用明显,而对其他两种试虫没有增效作用;氮酮和噻酮对高效氯氰菊酯对3种鳞翅目幼虫毒力均无增效作用。甜菜夜蛾对照组上表皮蜡质层和护蜡层清晰可见,渗透剂处理组上表皮护蜡层有不同程度的消失;而小菜蛾对照组和N-甲基-2-吡咯烷酮处理组上表皮护蜡层没有明显变化,只有氮酮处理组上表皮的护蜡层消失。以上结果提示,渗透剂对上表皮护蜡层的影响不是其对高效氯氰菊酯增效作用的机制。     

Fine structure and morphogenesis of the sclerite epicuticle in the Atlantic shore crab Carcinus maenas

Three basic sublayers are identified in the epicuticle of the mineralised sclerites of the Atlantic shore crab Carcinus maenas (Crustacea, Decapoda): the surface coat, the cuticulin layer, and the inner epicuticle. Their morphogenesis and subsequent changes are described throughout the moulting cycle in the normal cuticle and the cuticular structures, namely the sensory bristles and epicuticular spines. At first, the cuticulin layer begins to form just after apolysis. This layer is built directly over the plasma membrane and immediately appears as a membrane-like structure 40 nm thick, composed of five symmetrically arranged laminae: two inner electron-lucent leaflets sandwiched between two thick electron-dense leaflets and separated by a thin dense median stratum. Elaboration of the inner epicuticle below the cuticulin layer is thought to occur via an intussusceptive process involving the pore canal cell extensions as transport routes. The inner epicuticle is made of vertically oriented microfibres embedded in an electron-dense matrix material. During the second half of the premoult period, the surface coat is deposited on the upper side of the cuticulin layer.     

The structure of an epidermal cell during the development of the protein epicuticle and the uptake of molting fluid in an insect

A structure for a generalized insect epidermal cell during the formation of the epicuticle is proposed, based on studies of several different epidermal cell types. The protein epicuticle is defined as the dense homogeneous layer below the cuticulin. The formation of the protein epicuticle involves secretory vesicles arising in Golgi complexes, and marks an interlude in the involvement in cuticle formation of plasma membrane plaques. The plaques are concerned in cuticulin formation before and in fibrous cuticle formation after the deposition of the protein epicuticle. The epidermis is characterized by the possession of a cytoskeleton of microtubules and a matrix of microfibers. In the elongated cells forming bristles and spines, the microfibers are often oriented in bundles with an axial banding which repeats every 120 Å. The microtubules are also arranged in columns with a trigonal packing and center to center spacing of about 800 Å. These cytoskeletal structures separate the other organelles into channels which may restrict the pathways open for the movement of secretory and pinocytotic vesicles. The protein epicuticle arises from the secretory vesicles which discharge at the apical surface. The contents disperse and reaggregate below the cuticulin. The Golgi complexes in the basal and central regions have many secretory vesicles and a small saccular component, differing from those nearer the apex which are smaller and have fenestrated saccules. The small coated vesicles (800 Å in diameter) associated with both sorts of complex, probably move to the apical and basal faces of the cell where they may give rise to the large coated vesicles (2000 Å in diameter) inserted in the plasma membrane. Pinocytosis occurs from both apical and basal faces but most lytic activity is in the apical region. Plant peroxidase injected into the haemocoel is taken up basally and transported to the apical MVBs. The large coated vesicles on the apical face may be concerned in the control of the extracellular subcuticular environment. They appear to fill up and detach, fusing to become the apical MVBs.     

The structure and formation of the cuticulin layer in the epicuticle of an insect, Calpodes ethlius (Lepidoptera, Hesperiidae)

The cuticulin layer is defined as the dense lamina (120–175 Å thick in Calpodes larvae, depending upon the stage) forming the outer part of the epicuticle in insects. It completely invests an insect except for the gut and the openings of some sense organs. It is the first layer to be secreted during the formation of new cuticle. The formation of the cuticulin membrane may be a useful model for studying the origin of membranes in general. It arises as a triple layer de novo and is not a modified plasma membrane. Growth is by accretion at the edges of patches of cuticulin which increase in area until they cover the new surface. The triple layer (i.e. three dense laminae) may develop striations about 30 Å apart transverse to the membrane, which perhaps form a sieve allowing small molecules to pass while protecting the cell from enzymes in the molting fluid. A similar porous structure persists in the tracheoles. After the resorption of molting fluid the triple layered structure again becomes obvious and the outermost layer separates from the other two to become what may be the surface lipid monolayer. The surface patterns in cuticle of various sorts probably arise by buckling of the cuticulin layer as it increases in surface area.     

Food intake in Blattella germanica (L.) nymphs affects hydrocarbon synthesis and its allocation in adults between epicuticle and reproduction

The causal relationship between food intake and hydrocarbon synthesis was examined in vivo and in vitro. Fed Blattella germanica (L.) nymphs synthesized hydrocarbons in a stage‐specific manner, with high rates occurring in the first 6 days of a 13‐day last stadium, in relation to feeding. A similar pattern was exhibited in vitro by sternites and tergites from fed nymphs. In contrast, starved nymphs synthesized hydrocarbons at normal rates for the first 2 days, but then synthesis declined and ceased by day 6. Their abdominal sternites and tergites displayed a similar biosynthetic pattern in vitro, showing that starved tissues lost the capacity to synthesize hydrocarbons, even when provided appropriate nutrients. Synthesis resumed within 2 days of being fed on day 6, reaching a maximum rate 6 days later. Some hydrocarbon appeared on the nymphal cuticle, but almost 4‐fold more hydrocarbon was internal in hemolymph lipophorin, fat body, and the developing imaginal cuticle. Because most hydrocarbon synthesized in nymphs provisions the adult, and synthesis is related to food intake, we examined trade‐offs in allocations in food‐limited insects. Nymphs provided with insufficient quantities of food allocated normal amounts of hydrocarbons to the nymphal epicuticle, but molted into smaller adults with significantly less internal hydrocarbons. These cockroaches directed nearly normal amounts of hydrocarbons to their epicuticle, oocytes, and oothecae, at the cost of internal hydrocarbon reserves for repair and subsequent gonotrophic cycles. Hydrocarbons, thus, appear to serve an important cross‐stadial resource and the object of competition among several nymphal and adult tissues. Arch. Insect Biochem. Physiol. 41:214–224, 1999. © 1999 Wiley‐Liss, Inc.