The inhibitory effect of the fungal toxin, destruxin A, on behavioural fever in the desert locust.

During an infection locusts behaviourally fever by seeking out higher environmental temperatures. This behaviour places the pathogen at sub-optimal growth temperatures while improving the efficiency of the immune system, thereby prolonging the lifespan of the host. It is therefore in the interest of the pathogen to either adapt to fever-like temperatures or to evolve mechanisms to interfere with, or inhibit fever. We investigated the behavioural fever response of desert locusts to two fungal pathogens. A prolonged fever was observed in locusts infected with Metarhizium acridum. However, fever was comparatively short-lived during infection with Metarhizium robertsii. In both cases restriction of thermoregulation reduced lifespan. Destruxin A (dtx A) produced by M. robertsii, but not M. acridum has previously been associated with the inhibition of the insect immune system. Injection of dtx A during infection with the fever-causing M. acridum inhibited fever and was particularly effective when administered early on in infection. Furthermore, locusts injected with dtx A were more susceptible to M. acridum infection. Therefore engineering M. acridum isolates currently used for locust biocontrol, to express dtx A may improve efficiency of control by interfering with fever.

Composition of Acridid gut bacterial communities as revealed by 16S rRNA gene analysis.

The gut bacterial community from four species of feral locusts and grasshoppers was determined by denaturing gradient gel electrophoresis (DGGE) analysis of bacterial 16S rRNA gene fragments. The study revealed an effect of phase polymorphism on gut bacterial diversity in brown locusts from South Africa. A single bacterial phylotype, consistent with Citrobacter sp. dominated the gut microbiota of two sympatric populations of Moroccan and Italian locusts in Spain. There was evidence for Wollbachia sp. in the meadow grasshopper caught locally in the UK. Sequence analysis of DGGE products did not reveal evidence for unculturable bacteria and homologies suggested that bacterial species were principally Gammaproteobacteria from the family Enterobacteriaceae similar to those recorded previously in laboratory reared locusts.

Hyperphagocytic haemocytes in Manduca sexta.

We have discovered a new type of haemocyte in the larval stage of the tobacco hornworm moth Manduca sexta that has extreme phagocytic ability; each cell can engulf up to 500 bacteria. This level of phagocytosis may be unprecedented among animal cells. Although these hyperphagocytic cells (HP) only represent about 1% of the circulating haemocytes, they are responsible for sequestering the majority of the bacteria by circulating haemocytes when non-pathogenic, heat-killed Escherichia coli are injected into the haemolymph. Extreme phagocytosis by HP is not limited to Gram-negative bacteria since heat-killed Staphylococcus aureus as well as positively and negatively charged microspheres are also highly phagocytosed. Evidence is presented to show that phagocytosis by HP is involved in the early stages of nodule formation in infected insects. In addition, HP are also present in non-infected insects, characterised by their distinctive spreading morphology, which becomes impaired following hyperphagocytosis of bacteria. This is the first time that a dedicated "professional" phagocytic class of haemocyte has been reported for an invertebrate. The importance of these specialised cell types in the M. sexta immune response and their role in nodule formation is discussed.

Eicosanoid involvement in the regulation of behavioral fever in the desert locust, Schistocerca gregaria.

The desert locust Schistocerca gregaria behaviorally thermoregulates in order to try and maintain a favoured "set point" body temperature. Locusts infected with the deuteromycete fungal pathogen Metarhizium anisopliae var acridumchoose a significantly elevated temperature. This "behavioral fever" greatly delays the progress of mycosis. We have confirmed this phenomenon and shown that desert locusts also fever when infected with the bacterial pathogen Serratia marcescens. Elevation in the prefered environmental temperature occurs also upon injection with laminarin and lipopolysaccharide (microbial cell wall components). Since such treatments also stimulate the immune system it would appear that "behavioral fever" is probably a feature of the immune response. The eicosanoid biosynthesis inhibitor dexamethasone prevented laminarin invoked fever. This effect was reversable by arachidonic acid. Therefore in common with the febrile response in mammals behavioral fever in insects may be mediated locally by circulating eicosanoids.

Trehalose-hydrolysing enzymes of Metarhizium anisopliae and their role in pathogenesis of the tobacco hornworm, Manduca sexta.

Trehalose is the main haemolymph sugar in most insects including the tobacco hornworm, Manduca sexta, and is potentially a prime target for an invading pathogenic fungus. There was considerably more trehalose-hydrolysing activity in the haemolymph of caterpillars infected with Metarhizium anisopliae than in controls. This appeared to be due primarily to additional isoforms; one of which could also hydrolyse maltose and was designated an alpha-glucosidase. A comparable isoform was identified in in vitro culture of the fungus, supporting a fungal origin for the in vivo enzyme. The in vitro fungal enzyme, alpha-glucosidase-1 (alpha-gluc-1), was purified to homogeneity and partially characterised. A study with the trehalase inhibitor trehazolin and C14 trehalose suggested that extracellular hydrolysis is important for fungal mobilisation of trehalose. Haemolymph glucose increases significantly during mycosis of tobacco hornworm larvae by M. anisopliae, consistent with the hydrolysis of trehalose by extracellular fungal enzymes. The implications for the host insect are discussed.

A note: gut bacteria produce components of a locust cohesion pheromone.

AIMS: Faecal pellets from germ-free locusts were used as culture media to determine the ability of locust gut bacteria to synthesize phenolic components of the locust cohesion pheromone. METHODS AND RESULTS: Inoculation of germ-free faecal pellets with Pantoea agglomerans, a species commonly isolated from locusts, resulted in the release of large amounts of guaiacol and small amounts of phenol, both of which are components of the locust cohesion pheromone. Two other locust-derived species, Klebsiella pneumoniae pneumoniae and Enterobacter cloacae, also produced guaiacol from germ-free faecal pellets, but the opportunistic locust pathogen, Serratia marcescens, did not. The most likely precursor for guaiacol is the plant-derived vanillic acid, which is present in large amounts in the faeces of both conventional and germ-free locusts. CONCLUSIONS: These observations are consistent with previous ones, that locust gut bacteria are responsible for the production of components of the locust cohesion pheromone. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings illustrate how an insect can adapt to make use of a common bacterial metabolite produced by one or more of its indigenous gut bacterial species. This observation has implications for our appreciation of insect gut microbiota interactions.

Acid phosphatases of Metarhizium anisopliae during infection of the tobacco hornworm Manduca sexta.

Three acid phosphatase (AcP) isozymes, pI 8.1, 8.0 and 7.8, were isolated, purified and partially characterised from optimised cultures of the entomopathogenic fungus Metarhizium anisopliae. The enzymes had similar molecular masses (approximately 44.0 kDa), and could degrade sugar phosphates found in the haemolymph of a host insect, the tobacco hornworm Manduca sexta. The AcP activity in haemolymph of mycosed insects increased significantly over controls, and some new isozymes were present. The infection-related isoforms were similar in molecular mass and pI to some of the in vitro AcP isozymes of M. anisopliae. Results of dot blot and Western blot analyses using anti-AcP antibodies suggested that at least one Metarhizium phosphatase isoform was present in haemolymph of infected caterpillars. Antibodies did not cross-react with immune (chemically stimulated) or non-immune haemolymph from Manduca sexta. Consistent with the appearance of highly active fungal phosphatase in caterpillar blood, free phosphate concentration increased dramatically during the late stages of infection to a level two to five times that of controls. Phosphate was limiting to growth of the fungus at the concentration found in control haemolymph and supplementation of phosphate significantly increased fungal growth in vitro in haemolymph. These results suggest that Metarhizium AcP may play a key role in providing phosphorus for fungal growth at the expense of the insect.

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.

Cloning and sequence analysis of an intron-containing domain from a peptide synthetase-encoding gene of the entomopathogenic fungus Metarhizium anisopliae.

A gene encoding a putative peptide synthetase has been cloned and partially sequenced from the filamentous fungus, Metarhizium anisopliae. The deduced amino acid sequence of one entire domain and the following spacer is typical of fungal peptide synthetases, showing good conservation of the six expected core sequences. There are two introns within this region, the first interrupting core 5 (RLDLTDIE) of the domain and the second in a conserved area of the spacer region.

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.

Cloning and characterisation of a gene encoding a cuticle-degrading protease from the insect pathogenic fungus Metarhizium anisopliae.

Metarhizium anisophilae (Ma) secretes a range of proteases when grown in vitro on insect cuticle. A trypsin-like serine protease, PR2, was purified from culture filtrates by anion exchange chromatography and the N-terminal sequence determined. Using oligodeoxyribonucleotide probes based on this sequence and that of the highly conserved trypsin active site, a gene was isolated from a lambda EMBL3 genomic library of Ma isolate ME1. Sequencing of the gene and RT-PCR revealed that the gene contains two introns which are 94 and 40 bp long. The deduced protein consists of 254 amino acids, has a putative signal sequence to allow transport into the endoplasmic reticulum and probably undergoes a second proteolytic processing step at its N terminus to yield the mature enzyme. The putative mature enzyme has extensive homology with other serine proteases of the trypsin subclass and, in particular, with the trypsin characterised from Fusarium oxysporum.

Partial characterization of specific inducers of a cuticle-degrading protease from the insect pathogenic fungus Metarhizium anisopliae.

The insect pathogenic fungus Metarhizium anisopliae produces several extracellular cuticle-degrading proteases and evidence is consistent with one of these, PR1, which is a chymoelastase, being a determinant of pathogenicity. We have shown previously that PR1 production is regulated by both carbon catabolite and nitrogen metabolite repression and also by specific induction under derepressed conditions by insect cuticle. In the present work we have established that an enzymically released proteinaceous component(s) of insect cuticle is capable of inducing PR1 (based on appearance of extracellular activity). Cuticle of the desert locust Schistocerca gregaria treated with KOH to remove protein failed to induce PR1 production, whereas cuticle treated with either chloroform or ether to remove lipids still induced PR1. Cuticle digested with either PR1 or the trypsin-like PR2 of M. anisopliae released peptides mainly in the range 150-2000 Da; addition of these peptides generated by PR1 or PR2 at 3 micrograms alanine equivalents ml-1 induced PR1 production to a level similar (75%) to that obtained with untreated insect cuticle. Several amino acids and peptides which are abundant in insect cuticular protein (Ala, Gly, Ala-Ala, Ala-Ala-Ala, Ala-Pro and Pro-Ala) were tested at a range of concentrations and in restricted cultures for their ability to induce PR1. None induced the protease to the levels seen with cuticle or peptides enzymically released from cuticle, although some dimers and notably the monomers Ala and Gly gave 2-2.7-fold enhanced PR1 activity above depressed basal levels (up to 48-57% of that achieved with induced synthesis on cuticle).(ABSTRACT TRUNCATED AT 250 WORDS)

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 enzyme's 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.

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.

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

Nine isolates of the entomopathogenic deuteromycetes Metarhizium anisopliae, Beauveria bassiana, Verticillium lecanii, Nomuraea rileyi, and Aschersonia aleyrodis produced basic (pI greater than 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) (pI 10.3 Mr 25,000) and trypsin (Pr2) (pI 4.42, Mr 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 S5 and S4 subsites show a preference for negatively charged succinyl and hydrophobic acetyl groups, respectively. The S3 and S2 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.