Locust Cuticle Research Articles

Role of Cuticle-Degrading Proteases in the Virulence ofMetarhiziumspp. for the Desert Locust,Schistocerca gregaria

The production of the subtilisin proteinase, PR1, and the trypsin-like enzyme, PR2, in liquid cultures of 19 isolates ofMetarhiziumspp. was investigated. PR1 activity was generally observed 72 h after inoculation whilst PR2 activity was detected in most strains 48 h after inoculation. PR1 was partially purified from each isolate ofMetarhiziumspp. The ability of this enzyme from each isolate to degrade different types of insect cuticle was investigated. The cuticle types used wereManduca sextapupal cuticle, adult desert locust (Schistocerca gregaria) wing cuticle, abdominal cuticle, and pharate adult locust abdominal cuticle. These cuticle types differ in their protein composition and degree of sclerotization. PR1 hydrolyzed the locust cuticle to different degrees. The data suggest a hierarchy for the susceptibility to hydrolysis in the order pharate adult abdominal > adult abdominal > wing. There was no significant correlation between the ability of the enzymes to degrade any of the cuticle types and median lethal time of the isolates against the desert locust. However, a correlation was found between the ability of enzymes to hydrolyze different locust cuticles. Such correlations were not apparent to the same extent betweenM. sextacuticle and the locust cuticles. These results are discussed in terms of a possible contribution of PR1 to host specificity.

Effects of Rehydration on the Conidial Viability of Metarhizium flavoviride Mycopesticide Formulations

The rehydration of dried conidia of Metarhizium flavoviride was investigated in an attempt to increase speed of kill of locusts and grasshoppers by formulations of this fungus. Conidia were dried to 4-5% moisture content with no apparent adverse effects on viability, but rapid rehydration (by putting dried conidia directly in free water) reduced viability. Rehydration in an atmosphere of high humidity allowed dry conidia to absorb sufficient moisture to avoid imbibition damage. Rehydrating and pre-germinating conidia prior to spraying (in an oil-based formulation) on to the desert locust, Schistocerca gregaria, did not decrease the time to death, suggesting that moisture uptake by dry conidia on the desert locust cuticle is easily achieved.

Sequence studies of proteins from larval and pupal cuticle of the yellow meal worm, Tenebrio molitor

Complete amino acid sequences have been determined for six larval-pupal cuticular proteins from Tenebrio molitor. The sequenced proteins are major components in both larval and pupal cuticle, and both basic and slightly acidic proteins are represented. The proteins show pronounced similarities to some of the proteins sequenced from other insect cuticles. Three slightly acidic larval-pupal Tenebrio cuticular proteins contain a 66-residue central, hydrophilic region, resembling regions in cuticular proteins from insect species of four different orders (Coleoptera, Diptera, Lepidoptera and Orthoptera), and three basic proteins from larval-pupal Tenebrio cuticle have a 51-residue hydrophilic region in common with two proteins from cuticle of pharate adult locusts ( Locusta migratoria). The Tenebrio larval-pupal cuticular proteins are also similar to locust adult cuticular proteins, by frequent occurrence of the short sequence motif Ala-Ala-Pro-Ala/Val. The pronounced sequence similarities between cuticular proteins from different insect orders indicate that the conserved regions are functionally important.

The effects of the duration of exposure on the toxicity of diflubenzuron, hexaflumuron and teflubenzuron to various stages of II instarSchistocerca gregaria

Second-instar (II) nymphs of the Desert Locust, Schistocerca gregaria (Forsk.) were exposed to three benzoylphenyl ureas (BPUs), diflubenzuron, hexaflumuron and teflubenzuron. Nymphs were treated with precise doses by allowing them to ingest treated barley leaves at varying stages of the II instar. They were exposed to the same total quantity of active ingredient over one (Days 1, 2, 3 or 4), two (Days 1-2, 2-3 or 3-4) or four days (Days 1-4) of the four-day inter-moult period. The total amounts applied were 60 μg per nymph of diflubenzuron, 30 μg per nymph of hexaflumuron or 0.25 μg per nymph of teflubenzuron. The nymphs were then monitored for two moults after treatment until they reached the fourth (IV) instar, to observe both the acute and chronic effects of treatment. The timing of the exposure during the inter-moult period and the duration of exposure were both found to result in significantly different acute responses for each BPU. Treatment over one or two days showed that the closer to the moult II instars were treated, the greater the mortality. This indicated that locust pharate cuticle is predominantly synthesised late in the instar. Treatment over four days resulted in higher mortality than exposure to the same quantity of active ingredient over one or two days, suggesting that BPUs are highly toxic to locust nymphs but non-cumulative within their bodies. The timing of death was also significantly affected by both the timing and duration of treatment. A significant proportion of the mortality occurred after the first moult following treatment when nymphs were dosed on Day 1 and Day 2 with hexaflumuron and diflubenzuron respectively. Mortality following all other treatments occurred during the first moult after treatment. The duration of the II and third (III) instars were significantly prolonged in many cases following treatment with BPUs. The implications of increased mortality following prolonged exposure to BPUs and extended development periods are discussed in relation to the use of BPUs as barrier-sprayed insecticides for the control of mobile locust nymph populations.

Primary structure of proteins from the wing cuticle of the migratory locust, Locusta migratoria

Wing cuticle from pharate adult locusts, Locusta migratoria, contains several prominent proteins which occur as minor components or are completely absent in other cuticular regions. Six of the wing-specific proteins have been purified and their amino acid sequences determined by combined use of mass spectrometry and automated Edman degradation. During the sequence determination very long sequence runs (90–121 residues) were necessary in order to establish the primary structure. All the wing-specific cuticular proteins from locusts contain the repeated short sequence motif -Ala-Ala-Pro-Ala/Val-, which is common for all hitherto sequenced cuticular proteins from pharate locusts. Several of the wing-specific proteins also possess an N-terminal region rich in glycine, tyrosine and leucine, characteristic for many locust cuticular proteins. Two of the analysed proteins have a conserved 61-residue sequence in common with a previously sequenced protein from locust wing cuticle and with two proteins from the pharate cuticle of adult Tenebrio molitor. Possible roles for the various sequence motifs are discussed.

Comparison of larval and pupal cuticular proteins in Tenebrio molitor

Protein extracts from pupal and larval pharate cuticle from the meal worm, Tenebrio molitor, gave nearly identical patterns by two-dimensional electrophoresis and by ion-exchange chromatography. The main components in the cuticular extracts from the two metamorphic stages were also identical with respect to molecular mass according to electrospray ionization mass spectrometry. The complete amino acid sequence for one of the pupal cuticular proteins was determined; according to partial amino acid sequences and the mass spectrometric peptide map for the corresponding larval cuticular protein, it was concluded that the larval protein has the same amino acid sequence as the pupal protein. The sequence is characterized by a high content of alanine, proline, valine, and tyrosine and the complete absence of acidic amino acid residues, the sulphur containing amino acids and tryptophan. The sequence is further characterized by a high frequency of repeated sequence motifs, among which the Ala-Ala-Pro-Ala motif is the most abundant, but also longer sequence motifs are repeated. The sequence shows striking resemblance to sequences of proteins isolated from pharate locust cuticle.

Specific induction of a cuticle-degrading protease of the insect pathogenic fungus Metarhizium anisopliae

Summary: The insect pathogenic fungus Metarhizium anisopliae produces several extracellular cuticle-degrading proteases and evidence is consistent that one of these, a chymoelastase PR1, is a determinant of pathogenicity. We have shown previously that the wide-domain regulatory circuits of carbon and nitrogen derepression regulate PR1 production. In the present work we have established in addition that PR1 is specifically induced by insect cuticle, but not by other soluble or insoluble proteinaceous substrates. The feeding of elastin or collagen to derepressed established mycelium (starved for carbon and nitrogen) did not enhance PR1 production significantly and the soluble proteins BSA and gelatin rapidly and completely repressed PR1. The carbohydrate polymers cellulose and xylan gave derepressed basal levels of PR1. However, addition of locust cuticle enhanced PR1 production to a level approximately 10-fold that of derepressed mycelium. In order to establish if the enhancing effect of insect cuticle on PR1 production was due to specific induction or merely a reflection of enhanced growth on this insoluble dual carbon and nitrogen source, ergosterol was used as a measure of fungal growth. Expressing enzyme activity per mg dry weight showed that PR1 production in cuticle cultures increased approximately five- and ninefold after 12 and 24 h growth compared with elastin-grown cultures. Thus, the substantial increase in PR1 production on cuticle was shown not to be a function of fungal growth and this confirms that PR1 is induced by a component of insect cuticle; we believe this is the first report of induction by a specific substrate for any microbial protease.

Uptake, retention, metabolism and excretion of [22,23- 3H 2]dihydroazadirachtin in Schistocerca gregaria

[22,23- 3H 2]dihydroazadirachtin was used as a tracer of azadirachtin to follow the distribution and metabolism of the tertranortripenoid in the locust Schistocerca gregaria. Injected tracer was rapidly removed from the haemolymph into many of the locust tissues. This uptake was inhibited by a large excess of injected dihydroazadirachtin, suggesting that the uptake was by a specific mechanism. High concentrations of injected unlabelled derivatives of azadirachtin inhibited to different extents the clearance of the tracer from the haemolymph. The results suggest that azadirachtin and its derivatives have different affinities for the uptake mechanism. Radiolabelled dihydroazadirachtin applied topically was shown to penetrate the locust cuticle quite slowly. A large fraction of the tracer was present in the fat body as well as gut and nervous tissue. Binding was persistent and not easily displaced. There was no evidence that dihydroazadirachtin was secreted into the primary urine by an active pumping mechanism. Metabolism of the dihydroazadirachtin was largely restricted to fat body and crop.

Combined plasma-desorption mass spectrometry and Edman degradation applied to simultaneous sequence determination of isoforms of structural proteins from the cuticle of Locusta migratoria.

The primary structures of two basic low-molecular-mass proteins, Lm-67 and Lm-70 from the pharate cuticle of the migratory locust, Locusta migratoria, were determined. The sequencing strategy was based on combined use of plasma--desorption mass spectrometry (PDMS) and automatic Edman degradation of the proteins and their enzymically derived peptides. The mass-spectral data showed the presence of two proteins in each preparation. For protein preparation Lm-67, this was indicated by the mass spectrum of the intact protein. For protein preparation Lm-70, the presence of two variants only became evident by mass-spectrometric analysis of the enzymically derived peptides. Both proteins show strong similarity to other exocuticular proteins from L. migratoria.

Purification and partial characterisation of a novel trypsin-like cysteine protease fromMetarhizium anisopliae

Trypsin-like enzymes from the entomopathogenic fungus Metarhizium anisopliae have been characterised. Two proteases with tryptic activity were purified by narrow range isoelectric focussing and affinity chromatography. One of these proteases, with an isoelectric point of 5.4 and a molecular mass of 28.8 kDa is a ‘classical’ trypsin belonging to the serine protease class. The other protease, with an isoelectric point of 4.6 and a molecular mass of 26.7 kDa, demonstrates trypsin-like specificity but, on the basis of inhibition and activation studies, belongs to the cysteine protease family and as such is the first fungal protease to be found of this type. The amino acid composition, kinetic constants and activity against proteinaceous substrates, including locust cuticle have been determined.

Induction of vitellogenin synthesis in Locusta migratoria by the juvenile hormone analog, pyriproxyfen

Several aspects of the activity of the juvenile hormone mimic, pyriproxyfen, particularly related to the induction of vitellogenin synthesis in the fat body, have been studied in Locusta migratoria. After topical application in acetone to the neck cuticle of adult locusts, pyriproxyfen is taken up rapidly, reaching roughly constant levels in hemolymph and fat body in 6 h. For induction of vitellogenin synthesis in fat body, and for accumulation of vitellogenin in hemolymph, the ED 50 in adult females is 2.3 μg, whereas adult males produce no vitellogenin with doses up to 300 μg. In fifth-instar larvae, in contrast, the ED 50 in females is about 30 μg, and males can also be induced to produce vitellogenin, but in a smaller amount which may be related to gene dosage. With pyriproxyfen, as with methoprene, there is a lag of about 24 h before vitellogenin appears in hemolymph after primary stimulation and a reduced lag time after secondary stimulation. The accumulation of vitellogenin mRNA in fat body, assayed by hybridization with a DNA probe, shows a lag of nearly 24 h, which is extended when protein synthesis is temporarily blocked with cycloheximide. The parallel responses to pyriproxyfen and to methoprene in several different tests suggest that, despite their different structures, both compounds share the same primary mode of action, which is believed to correspond to that of natural juvenile hormone.

Primary structure of a 14 kDa basic structural protein (Lm-76) from the cuticle of the migratory locust, Locusta migratoria

The complete amino acid sequence of a 14 kDa structural protein (LM-76) isolated from pharate cuticle of the locust, Locusta migratoria, was determined by Edman degradation of the intact protein and enzymatically derived peptides. Plasma desorption and electrospray mass spectrometry was used as an integrated part of the structure determination. Protein Lm-76 has characteristics similar to proteins previously isolated from the pharate locust. The amino acid composition shows a high content of alanine (32%) and absence of the amino acids Glu, Cys, Met, Phe and Trp. The sequence has a central hydrophilic region surrounded by two hydrophobic regions with 7 repeats of a (Tyr)-Ala-Ala-Pro-Ala/Val motif. The conservation around the prolyl residues within this sequence motif is demonstrated for the hitherto presumptive exocuticle proteins from L. migratoria. The N-terminal region of protein Lm-76 is enriched in the amino acids Gly, Leu and Tyr located in the conserved sequence NH 2-Gly-Tyr-Leu-Gly-Gly-(Tyr)-.

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.

Coupling reactions between amino compounds and N-acetyldopamine catalyzed by cuticular enzymes

Incubation of larval cuticle from the American silkmoth, Hyalophora cecropia with N-acetyldopamine (NADA) and amino acid derivatives results in a number of products. Some of the products obtained with α- N-acetyllysine and with β-alanine have been purified and identified. Two types of adducts are formed from both amino acids: one in which the amino group is linked to the 1-carbon atom (β-carbon) in the aliphatic side chain in NADA, and one in which the amino group is linked to the 6-position of the aromatic ring, followed by oxidation to a quinoid structure. Both types of NADA-adducts have also been obtained in experiments where adult pharate locust femur cuticle was incubated with NADA and either N-acetyllysine or β-alanine, demonstrating that products similar to those obtained by means of soft larval cuticle may also be formed in vitro by sclerotizing cuticle. Together with previous observations on cuticle-catalysed adduct formation between N-acetylhistidine and NADA the results indicate that amino groups as well as histidine residues in cuticular proteins can take part in the sclerotization process. The adducts involving the side chain of NADA have an intact o-diphenolic structure and are potential participants in formation of benzodioxan-type (also called dihydrobenzodioxin-type) NADA-oligomers. Co-incubation of NADA with the β-alanyl NADA adduct and locust cuticle gave products containing both the benzodioxan-structure and covalently attached β-alanine, indicating that NADA linked to cuticular amino groups may participate in polymerization reactions during natural sclerotization.

Cuticle-catalyzed coupling between N-acetylhistidine and N-acetyldopamine

Several types of insect cuticle contain enzymes catalyzing the formation ofof adducts between N-acetyldopamine (NADA) and N-acetylhistidine (NAH). Two such adducts, NAH-NADA-I and NAH NADA-II, have been isolated and their structures determined. In one of the adducts the link connecting the two residues occurs between the I-position (ß-position) in the NADA side chain and the 1-N atom (τ-N) in the imidazole ring of histidine. Diphenoloxidase activity alone is not sufficient for formation of this adduct, whereas extracts containing both diphenoloxidase and o-quinone- p-quinone methide isomerase activities catalyze the coupling reaction. The adduct consists of a mixture of two diastereomers and they are presumably formed by spontaneous reaction between enzymatically produced NADA- p-quinone methide and N-acetylhistidine. The other adduct has been identified as a ring addition product of N-acetylhistidine and NADA. In contrast to the former adduct it can be formed by incubation of the two substrates with mushroom tyrosinase alone. An adduct between N-acetylhistidine and the benzodioxan-type NADA-dimer is produced in vitro, when the N-acetylhistidine-NADA adduct is incubated with NADA and locust cuticle containing a 1,2-dehydro-NADA generating enzyme system. Trimeric NADA-polymerization products of the substituted benzodioxan-type have been obtained from in vivo sclerotized locust cuticle, confirming the ability of cuticle to produce NADA-oligomers. The results indicate that some insect cuticles contain enzymes promoting linkage of oxidized NADA to histidine residues. It is suggested that histidine residues in the cuticular proteins can serve as acceptors for oxidized NADA and that further addition of NADA-residues to the phenolic groups of bound NADA can occur, resulting in formation of protein-linked NADA-oligomers. The coupling reactions identified may be an important step in natural cuticular sclerotization.

Characterization of chitinase and chitobiase produced by the entomopathogenic fungus Metarhizium anisopliae

Extracellular fluids from Metarhizium anisopliae grown on chitin as the sole carbon source contained distinct chitinase ( pH optimum 5.3, MW 33 kDa) and chitobiase ( pH optimum 5, MW 110 kDa, pI 6.4) activities. Chitinase activity was stabilized against extremes of pH and temperature by the presence of its chitin substrate. Chitinase fractions eluted from a sephadex column had activity against chitosan as well as locust cuticle chitin, crystalline chitin, and colloidal chitin. Products of enzymolysis were analyzed by descending paper chromatography. Chitinase had no activity against chitobiose, only trace activity against chitotriose, but preferentially cleaved chitotetraose in the middle bond releasing chitobiose. N-Acetyglucosamine was the major (vs colloidal chitin) or only (vs crystalline chitin) product detected following chitin hydrolysis. The chitobiase (also called β- N-acetylglucosaminidase) possessed simple glycosidase activity against p-nitrophenyl-acetylglucosaminide and hydrolyzed di-, tri-, and tetrasaccharides to N-acetylglucosamine. Chitobiase activity was significantly inhibited by its reaction product, N-acetylglucosamine. The mechanism of action of chitinolytic enzymes is discussed.

Enzymatic activities in locust cuticle involved in sclerotization

Insect cuticle contains enzymes catalyzing the oxidation of N- acetyldopamine ( N ADA ). The reaction products obtained on incubation of N ADA with locust femur cuticle or with soluble phenoloxidases have been studied with reverse-phase high pressure liquid chromatography (RP-HPLC). The effects of various inhibitors and of various pretreatments of the samples were compared. Soluble phenoloxidases give N- acetylnorepinephrine ( N ANE ) and a yet unidentified product X 1 as main products. Short term incubations of N ADA with pieces of locust cuticle give several dimeric compounds as main products together with some N ANE AND X 1 . Nucleophilic inhibitors favour the formation of an unsaturated derivative of N ADA , α,β- dehydro-N ADA , and pretreatments of the cuticle at high or low pH-values or at 80°C suppress formation of the unsaturated derivative and of dimeric compounds while stimulating formation of N ANE and of X 1 . The results are interpreted in favour of the two-enzyme model (“desaturase” plus laccase) for β-sclerotization suggested previously (Andersen and Roepstorff, 1982; Andersen, 1985), and it is concluded that the quinone methide model (Sugumaran, 1987a,b) is insufficient to explain the formation of the various products.

Characterization of proteins from pharate adult wing cuticle of the migratory locust, Locusta migratoria

Thirteen of the more abundant proteins in pharate adult wing cuticle of the migratory locust, Locusta migratoria, have been purified to near homogeneity. Their amino acid compositions and electrophoretic titration curves were determined, showing that these proteins resemble the proteins from pharate adult locust body cuticle in being rich in alanine and/or glycine, containing few charged or polar groups, and having mainly alkaline isoelectric points. Glycine-rich proteins tend to be more dominating in wing cuticle than in sclerites, and one wing protein has a very high content of proline and valine. The specific pattern of proteins in locust wing cuticle is suggested to be important for giving the wings optimal mechanical properties.

Distribution of chymoelastases and trypsin-like enzymes in five species of entomopathogenic deuteromycetes

Nine isolates of the entomopathogenic deuteromycetes Metarhizium anisopliae, Beauveria bassiana, Vertidllium lecanii, Nomuraea rileyi, and Aschersonia aleyrodis produced basic ( pI > 7.0) chymoelastases that possessed extended binding sites, comprising at least four or five subsites, with preference for hydrophobic residues at the primary binding site. Most isolates also produced additional acidic enzymes with similar specificities against ester and amide substrates but which lacked activity against elastin. Both acidic and basic enzymes degraded high protein azure or locust cuticle and, as shown by inhibition studies, possessed essential serine and histidine residues in the active site. In spite of similarities in catalytic properties antibodies generated against a Metarhizium chymoelastase cross-reacted only with enzymes from two (out of four) Metarhizium isolates; enzymes from all other isolates did not cross-react. Two isolates of Metarhizium produced a third class of protease which degraded Bz-AA-AA-Arg-NA substrates (AA, various amino acids) and hide protein azure. Analogous peptidases were produced by other isolates but they were specific for Bz-Phe-Val-Arg-NA and showed less sensitivity to trypsin inhibitors. The possible significance to pathology of the presence of diverse yet similar protease forms in five genera of entomopathogens is discussed.

Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae

Two chymoelastases and three trypsinlike proteases were separated from culture filtrates of the entomopathogen Metarhizium anisopliae. A chymoelastase (Pr1) (p I 10.3 M r 25,000) and trypsin (Pr2) (p I 4.42, M r 28,500) were purified to homogeneity by ammonium sulphate precipitation, isoelectric focusing, and affinity chromatography. Inhibition studies showed that both enzymes possessed essential serine and histidine residues in the active site. Pr1 shows greater activity than Pr2 or mammalian enzymes against locust cuticle and also possesses activity vs elastin. Pr1 shows a broad primary specificity toward amino acids with hydrophobic side groups in synthetic ester and amide substrates. The kinetic properties of Pr1 demonstrate a preference for extended peptide chains with the active site recognising at least five substrate residues. The S 5 and S 4 subsites show a preference for negatively charged succinyl and hydrophobic acetyl groups, respectively. The S 3 and S 2 subsites both discriminated in favor of alanine and against proline. Pr2 rapidly hydrolyzed casein and synthetic substrates containing arginine or lysine. It possessed little or no activity vs cuticle, elastin, or synthetic substrates for chymotrypsin and elastase. Specific active site inhibitors confirmed the similarities between Pr2 and trypsin.