A. Keith Charnley

New insights into the mechanisms of fungal pathogenesis in insects

Entomopathogenic fungi are attracting attention as potential biological control agents of insect pests. The mechanisms of pathogenesis have parallels with those of some plant-pathogenic fungi, particularly in the areas of formation of an infection structure, entry into the host and toxin-mediated host death. Understanding these processes will provide a rational basis for strain selection and improvement.

Cuticle-degrading Enzymes of Entomopathogenic Fungi: Regulation of Production of Chitinolytic Enzymes

Summary: Synthesis of chitinase and chitosanase by the entomopathogenic fungus Metarhizium anisopliae is regulated by products of chitin and chitosan degradation through an inducer-repressor mechanism. Slow-feeding with sugars or alanine (about 20 μg ml−1 h−1) in a carbon deficient medium to prevent catabolite repression (restricted cultures) demonstrated that the most effective inducers of chitinase and chitosanase were the principal monomeric constituents of chitin (N-acetylglucosamine) and chitosan (glucosamine) respectively. Increasing the rate of release of N-acetylglucosamine decreased chitinase synthesis by about 87% while causing a sevenfold increase in growth. In batch cultures high chitinase activities were present only in chitin-containing medium. There was a negative correlation between accessibility and amount of chitin substrates, levels of free N-acetylglucosamine in culture fluids and chitinase production. Addition of carbohydrates, lipid or proteins to chitin-grown cultures repressed chitinase production. Basal levels of chitinase were produced in non-inducing media. Production of chitobiase (N-acetylglucosaminidase) was enhanced from high basal levels by amino sugars, but was less inducible and less susceptible to catabolite repression than chitinase.

Production of cuticle-degrading enzymes by the entomopathogen Metarhizium anisopliae during infection of cuticles from Calliphora vomitoria and Manduca sexta

SUMMARY: A biochemical and histochemical investigation with specific substrates and inhibitors was used to visualize protease, esterase and aminopeptidase activities produced in situ during penetration of Calliphora vomitoria and Manduca sexta cuticles by hyphae of the entomopathogenic fungus Metarhizium anisopliae. Two endoproteases, and aminopeptidase and esterase activities, were mainly localized in simple and complex appressoria and germinating conidia. The effect of inhibitors on two characterized proteases (Pr1 and Pr2) and aminopeptidase activity in appressorial plates was quantified by microdensitometric measurement of reaction products. Pr1 and Pr2 activities were differentially inhibited by various protease inhibitors. Pr1, Pr2, esterase, aminopeptidase and N-acetylglucosaminidase (exochitinase) activities were present during penetration as detected directly following desorption from fungal and cuticle components. The proteases produced in situ were fractionated, and were shown by immunological and enzymological criteria to be the same as those produced in culture media. The sequence of enzyme appearance in situ showed that production of proteolytic enzymes precedes exochitinase production. No production of endochitinase was found before or during hyphal penetration of the cuticle.

Pheromones: Exploitation of gut bacteria in the locust

The congregation of locusts into vast swarms can cause crop devastation of biblical proportions. Here we show that guaiacol, a key component of a pheromone derived from locust faecal pellets that promotes the aggregation of locusts, is produced by bacteria in the locust gut. This adaptation by an insect to exploit a common metabolite produced by indigenous gut bacteria has wide implications for our appreciation of the role of the gut microbiota in insects.

The immune response of the desert locust Schistocerca gregaria during mycosis of the entomopathogenic fungus, Metarhizium anisopliae var acridum.

Topical application of Metarhizium anisopliae var acridum to the desert locust Schistocerca gregaria resulted in changes in the biochemistry and antimicrobial defenses of the haemolymph. M. anisopliae var acridum colonized the host haemolymph from day two post application. The haemocytes did not attach to, phagocytose or nodulate elements of the fungus. However, the presence of the fungus appeared to stimulate hemocyte aggregation over the first few days of mycosis though the number of aggregates declined subsequently. The total hemocyte count increased two days after application, indicating an overall stimulation of the immune system, but declined to a value below that for uninoculated controls by day four. The differential haemocyte count showed that the initial increase in total haemocyte count was primarily due to a larger number of coagulocytes. After day two consistent declines in cell number were observed for all haemocyte classes in mycosed insects. The activity of the enzyme, phenoloxidase, decreased during the course of infection. However, the converse was true for prophenoloxidase. Lysozyme levels were significantly smaller in infected than control locusts. There was a significant correlation between lysozyme and PO activities when data from mycosed and control insects were combined. The total protein content of the haemolymph decreased during the course of infection.

Modulation by eicosanoid biosynthesis inhibitors of immune responses by the insect Manduca sexta to the pathogenic fungus Metarhizium anisopliae.

Metarhizium anisopliae conidia (spores) reduced weight gain and caused death when injected into Manduca sexta larvae. When the fungus was co-injected with the eicosanoid biosynthesis inhibitor dexamethasone, larval weight gain was further reduced and mortality increased. These effects were reversed when dexamethasone was given together with the eicosanoid precursor arachidonic acid (AA). Similarly, treatment with other eicosanoid biosynthesis inhibitors (esculetin, phenidone, ibuprofen, and indomethacin) with differing modes of action enhanced the reduction in weight gain caused by mycosis. Injection of M. anisopliae conidia induced nodule formation in vivo; nodule numbers were reduced by dexamethasone, and restored by AA. Incubation of hemocytes with conidia caused microaggregation of hemocytes (indicative of nodule formation) in vitro and this was inhibited by dexamethasone, suggesting that dexamethasone acts directly on hemocytes, although inhibition was only partially reversed by AA. We suggest that the M. sexta immune response to fungal pathogens is normally modulated by physiological systems that include eicosanoid biosynthesis. This is the first demonstration that the virulence of a fungal entomopathogen can be enhanced by compromising the insect hosts immune system.

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.

A cuticle-degrading proteinase from the moulting fluid of the tobacco hornworm, Manduca sexta.

Moulting fluid of pharate adult tobacco hornworm moths, Manduca sexta, contains a novel cuticle-degrading proteinase, designated as MFP-1. The enzyme has been purified using heparin affinity chromatography and partially characterized. Before purification MFP-1 is associated with a large complex having an apparent native molecular mass > 669 kDa. After purification MFP-1 has a molecular mass of 41 kDa. The pI of the enzyme is 5.54. MFP-1 can be classified as generally trypsin-like on the basis of its substrate specificity and inhibition by soybean trypsin inhibitor. The enzymes preferred substrate, Tos-Gly-Pro-Arg-pNA, its inhibition by hirudin, and its affinity for heparin, all indicate that MFP-1 has some characteristics in common with the vertebrate blood-clotting enzyme thrombin. MFP-1 is probably a serine protease, since it is inhibited by both DFP and PMSF (specific inhibitors of serine proteinases). However, the enzyme was also inhibited by a number of agents that affect cysteine proteinases. Purified MFP-1 degrades Manduca cuticle in vitro. We suggest that the enzyme may act as the first step in the degradation of the cuticle during the moulting process.

Diversity of gut microbiota increases with aging and starvation in the desert locust

Here we report the effects of starvation and insect age on the diversity of gut microbiota of adult desert locusts, Schistocerca gregaria, using denaturing gradient gel electrophoretic (DGGE) analysis of bacterial 16S rRNA genes. Sequencing of excised DGGE bands revealed the presence of only one potentially novel uncultured member of the Gammaproteobacteria in the guts of fed, starved, young or old locusts. Most of the 16S rRNA gene sequences were closely related to known cultured bacterial species. DGGE profiles suggested that bacterial diversity increased with insect age and did not provide evidence for a characteristic locust gut bacterial community. Starved insects are often more prone to disease, probably because they compromise on immune defence. However, the increased diversity of Gammaproteobacteria in starved locusts shown here may improve defence against enteric threats because of the role of gut bacteria in colonization resistance.

An aminopeptidase from the moulting fluid of the tobacco hornworm, Manduca sexta

Abstract A neutral metalloprotease (moulting fluid protease 2; MFP-2) from the moulting fluid of Manduca sexta (Lepidoptera: Sphingidae) pharate adults has been purified and partially characterized. The enzyme has a native molecular mass of 240 kDa as determined by HPLC gel filtration. SDS-PAGE of MFP-2 gave a single band of molecular mass 39.4 kDa indicating that the native enzyme probably exists as a hexamer. MFP-2 degrades a broad range of synthetic amino acid-β-naphthylamide substrates with a preference for methione-, leucine- or alamine β-naphthylamides. Activity is inhibited by amastatin, 1,10-phenanthroline and, to a lesser extent, ethylenediaminetetraacetic acid (EDTA). The inhibitory effects of divalent metal ion chelation are most effectively overcome by the addition of cobalt ions. MFP-2 alone has low cuticle degrading activity but acts with another moulting fluid enzyme, MFP-1, to degrade Manduca pupal cuticle.