Alain Vey

Are there any risks in using entomopathogenic fungi for pest control, with particular reference to the bioactive metabolites of Metarhizium, Tolypocladium and Beauveria species?

Entomopathogenic fungi are promising alternatives to chemical insecticides. However, a major hurdle concerning the registration of these fungi as plant protection agents is the possible toxicity of secreted metabolites, especially secondary metabolites. This review summarizes data on specific secondary metabolites (destruxins, efrapeptins, oosporein, beauvericin and beauveriolides) produced by the important genera Beauveria, Metarhizium and Tolypocladium . The quantities of secondary metabolites produced by these fungi in vivo are usually much less than those secreted in nutrient rich liquid media. Methods and strategies are suggested which could standardize the risk assessment of fungal biological control agents.

Histological and ultrastructural studies of Beauveria bassiana infection in Leptinotarsa decemlineta larvae during ecdysis

Abstract Studies of Leptinotarsa decemlineata larvae infected by Beauveria bassiana during ecdysis have enabled us to define the modes of fungal penetration employed to enter the ecdysial cuticle. We have observed the mechanically active passage of the penetrant hyphae and have followed the growth of the filaments and blastospore formation in the molting fluid. The attack of the new integument and its consequent alteration and the entry into the body cavity have also been studied. The infection develops rapidly in some of the larvae which die in premolt, while others are able to molt. Conditions rendered abnormal due to the presence of the fungus cause integumentary injuries which serve as an important factor in pathogenesis since they enhance the entry of fungal elements and bacteria thereby inducing septicemia. Contaminated larvae are able to molt, showing no signs of injury or disease, and survive for a long time, until the fungus finally invades the organism and causes death. This postponement of mortality shows that molting and hemocytic reactions are, to a certain extent, an effective defense mechanism. These last observations can be useful in the understanding of pathological processes associated with a hidden phase of fungal infection.

Bassiacridin, a protein toxic for locusts secreted by the entomopathogenic fungus Beauveria bassiana

A toxic protein, bassiacridin, was purified from a strain of the entomopathogenic fungus Beauveria bassiana isolated from a locust, using chromatographic methods. The final toxic fraction contained between 0.1 and 0.3% of the proteins of the crude extract. Bassiacridin showed no affinity for ion exchangers, was characterised as a monomer with a mol. wt of 60 kDa and an isoelectric point of 9.5, and exhibited beta-glucosidase, beta-galactosidase and N-acetylglucosaminidase activities. Injection of fourth instar nymphs of Locusta migratoria with the pure protein at relatively low dosage (3.3 microg toxin g body wt(-1)) caused a rate of mortality near to 50%. The effects of the crude and pure fractions were characterized at tissular and cellular levels. The formation of melanised spots on tracheae and air sacs and of melanised nodules in contact with the fat body was observed in injected locusts. Alterations of the fine structure of epithelial cells of tracheae, air bags, and integument were also revealed. The insecticidal protein showed a specific activity against locusts. Bassiacridin is different from the other macromolecular toxins of entomopathogenic fungi already described. Microsequencing of peptides generated by trypsic digestion of bassiacridin confirmed that it is a novel molecule and showed that it exhibits a probably limited similarity with a chitin binding protein from yeast.

Effects of the peptide mycotoxin destruxin E on insect haemocytes and on dynamics and efficiency of the multicellular immune reaction

Destruxins (DTXs) are cyclic peptide toxins secreted by the entomopathogenic fungus Metarhizium anisopliae var. anisopliae. The effects of DTX E, the most active compound of this family on haemocytes, the immunocompetent insect cells, and on the dynamics and efficacy of the multicellular defense of insect hosts have been investigated. Ultrastructural alterations have been observed in circulating plasmatocytes and granular haemocytes, and in attached haemocytes of Galleria mellonella larvae treated with a toxic dose of DTX E (LC50). These changes appear as a consequence of disturbances induced in the cellular calcium balance. An effect on the cell surface of granulocytes was also noted in cells incubated with the toxin and FITC-Con A, even when the concentration of DTX was as low as 0.005 microg/ml. Morphological studies of haemocytic capsules formed in vivo revealed disturbances of the multicellular defense mechanism after toxin treatment However, an attempt to establish if these changes were significant was unsuccessful. In contrast, comparative assays regarding the effect of toxin treatment on the efficacy of the antifungal effect of encapsulation has given conclusive results. The germination of injected Aspergillus niger spores became slightly but significantly increased, and when the granuloma were incubated the fungus escaped more easily from the haemocytic envelope. These results are discussed in terms of significance of the contribution of DTXs to the fungal infection process. It is suggested that the fungal peptides may intervene during the disease by a true immune-inhibitory effect occurring at doses which do not cause paralysis or any general sign of toxicity (e.g., 0.8 microg/g of body weight).

Encapsulation of foreign particles in vitro by separated blood cells from crayfish, Astacus leptodactylus

SummaryBlood cells from Astacus leptodactylus were separated by density gradient centrifugation and used to study in vitro encapsulation of different objects. Each of the three different populations of haemocytes responded differentially to the foreign particles tested (fungal spores of Aspergillus niger, or ion exchange particles with neutral, positive or negative surface charges). The semigranular cells encapsulated all foreign objects tested, regardless of origin or surface charge, and the capsules developed within 15 h to very dense and tight structures. The granular cells only aggregated around the fungal spores, but no dense capsules were formed. Only a very weak association between granular cells and the beads could be observed. The hyaline cells could not encapsulate any foreign particle, not even if incubated together with granular or semigranular cells. Capsule formation was associated with degranulation of the cells since encapsulation was either inhibited or delayed if the cells were incubated with compounds known to affect degranulation in crayfish granular and semigranular cells, such as A 23187, SITS, EDTA, LPS or laminaran.

Hemocyte lysate enhancement of fungal spore encapsulation by crayfish hemocytes.

Crayfish hemocytes exhibited a stronger encapsulation reaction to fungal blastospores of Beauveria bassiana coated with hemocyte lysate, than to blastospores treated with plasma or buffer, indicating an opsonic function of hemocyte lysate proteins. Five proteins of the prophenoloxidase activating system in the hemocytes were attached to foreign surfaces (including the blastospores) after activation and it is suggested that these attaching proteins (one being phenoloxidase) are responsible for the opsonic function of the hemocyte lysate on crayfish blood cells.

Hirsutellin A, a toxic protein produced in vitro by Hirsutella thompsonii

A toxic protein, hirsutellin A, has been purified from the mite fungal pathogen, Hirsutella thompsonii, using ammonium sulphate precipitation, ion exchange chromatography and gel filtration on Bio-Gel P-10. The protein has been characterized as a monomer with a molecular mass of 15 kDa and an isoelectric point of 10.5. The amino acid composition and the N-terminal sequence of hirsutellin A (34 amino acids) have been determined. From these results, the toxin appears to be distinct from other known proteins. It is not glycosylated, and does not show proteolytic activity. The toxin is also antigenic, thermostable and not inactivated by treatments with proteolytic enzymes. Toxicity bioassays showed that injection of larvae of the waxmoth, Galleria mellonella, with hirsutellin A at low dosages [1 microgr toxin (g body wt)-1] caused a high mortality rate. Hirsutellin A was also toxic per os to neonatal larvae of the mosquito Aedes aegypti.

Insecticidal and cytotoxic effects of natural and hemisynthetic destruxins

The insecticidal and cytotoxic effects of 13 natural and hemisynthetic destruxins have been studied. DE shows insecticidal effects similar to those of DA, while DE and DA are more active than all the other natural compounds and analogues tested. Brominated destruxin is a relatively active analogue displaying particular modalities of cytotoxic effects which reflect a certain originality of its mode of action. The linear molecule resulting from the opening of the DA cycle is not toxic. The most hydrophilic destruxins showing e.g. charged radicals (COO-) appear the least toxic probably because they do not penetrate easily the cellular membranes.

EFFECTS OF DESTRUXINS, CYCLIC DEPSIPEPTIDE MYCOTOXINS, ON CALCIUM BALANCE AND PHOSPHORYLATION OF INTRACELLULAR PROTEINS IN LEPIDOPTERAN CELL LINES

The influence of the destruxin E, cyclodepsipeptidic mycotoxin produced by the filamentous entomopathogenic fungus Metarhizium anisopliae, on calcium fluxes and protein phosphorylation of in vitro cultivated lepidopteran cell lines have been studied. The use of the fluorescent calcium indicator Fura 2 AM did not show a fast increase in the level of cytosolic calcium in lepidopteran and human cell lines treated with dtx E. In contrast, 45Ca+2 assays detected a late but significant calcium influx in insect cells exposed to dtx E and A for at least 30 min. This effect was not inhibited by calcium channel blockers with the exception of nifedipine 25 microM. A viral treatment potentiated the effect of dtx E on calcium balance. Dtx E also induced a strong, fast (10 min), transient phosphorylation of high molecular weight (250 kDa) cellular proteins. The activation of phosphorylation was only partially inhibited by EDTA. This study provides the first direct evidence of dtx E influence on calcium fluxes and protein phosphorylation.

EFFECTS OF MYCOTOXINS ON INVERTEBRATE CELLS IN VITRO

Publisher Summary This chapter discusses the effects of mycotoxins on invertebrate cells in vitro . The importance of cryptogamic toxins is not only because of their ability to cause lesions at the cellular level and to kill host animals but also because of their antimicrobial and antiviral effects. Mycotoxins act by altering cells, blocking multicellular defense reactions, disturbing macromolecular syntheses, and producing antiviral effects all at once. Among the defense reactions against microbial aggression or foreign agents, the formation of granulomas may be considered one of the most important. In a study described in the chapter, cell lesions were analyzed by phase microscopy, histology, and electron microscopy. To understand the mode of action of destruxins, especially at very weak doses, further studies were conducted on their effect on cell syntheses. These studies showed that research on microbial toxins seems particularly important in its relation to cell biology, yielding indispensable information for the comprehension of the mechanisms of action of pathogenic fungi and for the perspectives that the antiviral effect introduces at the comparative virology level.