Fungal dimorphism in the entomopathogenic fungus Metarhizium rileyi: Detection of an in vivo quorum-sensing system.

This investigation documents the expression of the in vivo dimorphic program exhibited by the insect mycopathogen Metarhizium rileyi. This insect mycopathogen represents the key mortality factor regulating various caterpillar populations in legumes, including subtropical and tropical soybeans. Using two hosts and M. rileyi isolates, we have measured M. rileyi growth rates under in vivo and in vitro conditions and have assessed the pathogen's impact on host fitness. Significantly, the hyphal bodies-to-mycelia transition that occurs at the late infection stage is regulated by a quorum-sensing molecule(s) (QSM) that triggers hyphal bodies (Hb) to synchronously switch to the tissue-invasive mycelia. Within hours of this transition, the host insect succumbs to mycosis. The production of the QS chemical(s) occurs when a quorum of Hb is produced in the hemolymph (late-stage infection). Furthermore, the QS activity detected in late-stage infected sera is unique and is unrelated to any known fungal QSM. The lack of similar QS activity from conditioned media of M. rileyi suggests that the chemical signal(s) that mediates the dimorphic switch is produced by host tissues in response to a quorum of hyphal bodies produced in the host hemolymph. The serum-based QS activity is retained after lyophilization, mild heat treatment, and proteinase digestion. However, attempts to extract/identify the QSM have been unsuccessful. Results suggest that the observed hyphal body-to-mycelia transition is a multi-step process involving more than one chemical signal.

Enhancement of the Musca domestica hytrosavirus infection with orally delivered reducing agents.

House flies (Musca domestica L.) throughout the world are infected with the salivary gland hypertrophy virus MdSGHV (Hytrosaviridae). Although the primary route of infection is thought to be via ingestion, flies that are old enough to feed normally are resistant to infection per os, suggesting that the peritrophic matrix (PM) is a barrier to virus transmission. Histological examination of the peritrophic matrix of healthy flies revealed a multilaminate structure produced by midgut cells located near the proventriculus. SEM revealed the PM to form a confluent sheet surrounding the food bolus with pores/openings less than 10nm in diameter. TEM revealed the PM to be multilayered, varying in width from 350 to 900 nm, and generally thinner in male than in female flies. When flies were fed on the reducing agents dithiothetriol (DTT) or tris (2-caboxyethyl)phosphine hydrochloride (TCEP) for 48 h before per os exposure to the virus, infection rates increased 10- to 20-fold compared with flies that did not receive the reducing agent treatments. PM's from flies treated with DTT and TCEP displayed varying degrees of disruption, particularly in the outer layer, and lacked the electron-dense inner layer facing the ectoperitrophic space. Both drugs were somewhat toxic to the flies, resulting in>40% mortality at doses greater than 10mM (DTT) or 5 mM (TCEP). DTT increased male fly susceptibility (55.1% infected) more than that of females (7.8%), whereas TCEP increased susceptibility of females (42.9%) more than that of males (26.2%). The cause for the sex differences in response to oral exposure the reducing agents is unclear. Exposing flies to food treated with virus and the reducing agents at the same time, rather than pretreating flies with the drugs, had no effect on susceptibility to the virus. Presumably, the reducing agent disrupted the enveloped virus and acted as a viricidal agent. In summary, it is proposed that the reducing agents influence integrity of the PM barrier and increase the susceptibility of flies to infection by MdSGHV.

Comparative metatranscriptomic signatures of wood and paper feeding in the gut of the termite Reticulitermes flavipes (Isoptera: Rhinotermitidae).

Termites are highly eusocial insects that thrive on recalcitrant materials like wood and soil and thus play important roles in global carbon recycling and also in damaging wooden structures. Termites, such as Reticulitermes flavipes (Rhinotermitidae), owe their success to their ability to extract nutrients from lignocellulose (a major component of wood) with the help of gut-dwelling symbionts. With the aim to gain new insights into this enzymatic process we provided R. flavipes with a complex lignocellulose (wood) or pure cellulose (paper) diet and followed the resulting differential gene expression on a custom oligonucleotide-microarray platform. We identified a set of expressed sequence tags (ESTs) with differential abundance between the two diet treatments and demonstrated the source (host/symbiont) of these genes, providing novel information on termite nutritional symbiosis. Our results reveal: (1) the majority of responsive wood- and paper-abundant ESTs are from host and symbionts, respectively; (2) distinct pathways are associated with lignocellulose and cellulose feeding in both host and symbionts; and (3) sets of diet-responsive ESTs encode putative digestive and wood-related detoxification enzymes. Thus, this study illuminates the dynamics of termite nutritional symbiosis and reveals a pool of genes as potential targets for termite control and functional studies of termite-symbiont interactions.

Analysis of the structural proteins from the Musca domestica hytrosavirus with an emphasis on the major envelope protein.

The Musca domestica hytrosavirus (MdHV), a member of the family Hyrosaviridae, is a large, dsDNA, enveloped virus that infects adult house flies and causes a diagnostic hypertrophy of the salivary gland. Herein, studies were directed at identifying key structural components of the viral envelope and nucleocapsid. SDS-PAGE of detergent-treated virus fractions identified protein bands unique to the envelope and nucleocapsid components. Using prior LC-MSMS data we identified the viral ORF associated with the major envelope band, cloned and expressed recombinant viral antigens, and prepared a series of polyclonal sera. Western blots confirmed that antibodies recognized the target viral antigen and provided evidence that the viral protein MdHV96 underwent post-translational processing; antibodies bound to the target high molecular weight parent molecule as well as distinct sets of smaller bands. Immuno gold electron microscopy demonstrated that the anti-MdHV96 sera recognized target antigens associated with the envelope. The nucleocapsids migrated from the virogenic stroma in the nucleus through the nuclear membrane into the cytoplasm, where they acquired an initial envelope that contained MdHV96. This major envelope protein, appeared to incorporate into intracellular membranes of both the caniculi and rough endoplasmic reticulum membranes and mediate binding to the nucleocapsids. Oral infection bioassays demonstrated that the anti-HV96 polyclonal sera acted as neutralizing agents in suppressing the levels of orally acquired infections.

Impact of house fly salivary gland hypertrophy virus (MdSGHV) on a heterologous host, Stomoxys calcitrans.

The effect of Musca domestica salivary gland hypertrophy virus (MdSGHV) on selected fitness parameters of stable flies, Stomoxys calcitrans (L.), was examined in the laboratory. Virus-injected stable flies of both genders suffered substantially higher mortality than control flies. By day 9, female mortality was 59.3 +/- 10.1% in the virus group compared with 23.7 +/- 3.7% in the controls; mortality in virus-injected males was 78.1 +/- 3.1% compared with 33.3 +/- 9.3% for controls. Fecundity of control flies on days 6-9 was 49-54 eggs deposited per live female per day (total, 8,996 eggs deposited), whereas virus-injected flies produced four to five eggs per female on days 6-7 and less then one egg per female per day thereafter (total, 251 eggs). Fecal spot deposition by virus-injected flies was comparable to controls initially but decreased to approximately 50% of control levels by day 4 after injection; infected flies produced only 26% as many fecal spots as healthy flies on days 6 and 7. None of the virus-injected stable flies developed symptoms of salivary gland hypertrophy. Quantitative real-time polymerase chain reaction demonstrated virus replication in injected stable flies, with increasing titers of virus genome copies from one to four days after injection. MdSGHV in stable flies displayed tissue tropism similar to that observed in house fly hosts, with higher viral copy numbers in fat body and salivary glands compared with ovaries. Virus titers were approximately 2 orders of magnitude higher in house fly than in stable fly hosts, and this difference was probably due to the absence of salivary gland hypertrophy in the latter species.

Susceptibility of North-American and European crickets to Acheta domesticus densovirus (AdDNV) and associated epizootics.

The European house cricket, Acheta domesticus L., is highly susceptible to A. domesticus densovirus (AdDNV). Commercial rearings of crickets in Europe are frequently decimated by this pathogen. Mortality was predominant in the last larval stage and young adults. Infected A. domesticus were smaller, less active, did not jump as high, and the adult females seldom lived more than 10-14 days. The most obvious pathological change was the completely empty digestive caecae. Infected tissues included adipose tissue, midgut, epidermis, and Malpighian tubules. Sudden AdDNV epizootics have decimated commercial mass rearings in widely separated parts of North America since the autumn of 2009. Facilities that are producing disease-free crickets have avoided the importation of crickets and other non-cricket species (or nonliving material). Five isolates from different areas in North America contained identical sequences as did AdDNV present in non-cricket species collected from these facilities. The North American AdDNVs differed slightly from sequences of European AdDNV isolates obtained in 1977, 2004, 2006, 2007 and 2009 and an American isolate from 1988. The substitution rate of the 1977 AdDNV 5kb genome was about two nucleotides per year, about half of the substitutions being synonymous. The American and European AdDNV strains are estimated to have diverged in 2006. The lepidopterans Spodoptera littoralis and Galleria mellonella could not be infected with AdDNV. The Jamaican cricket, Gryllus assimilis, and the European field cricket, Gryllus bimaculatus, were also found to be resistant to AdDNV.

Hytrosaviridae: a proposal for classification and nomenclature of a new insect virus family.

Salivary gland hypertrophy viruses (SGHVs) have been identified from different dipteran species, such as the tsetse fly Glossina pallidipes (GpSGHV), the housefly Musca domestica (MdSGHV) and the narcissus bulbfly Merodon equestris (MeSGHV). These viruses share the following characteristics: (i) they produce non-occluded, enveloped, rod-shaped virions that measure 500-1,000 nm in length and 50-100 nm in diameter; (ii) they possess a large circular double-stranded DNA (dsDNA) genome ranging in size from 120 to 190 kbp and having G + C ratios ranging from 28 to 44%; (iii) they cause overt salivary gland hypertrophy (SGH) symptoms in dipteran adults and partial to complete sterility. The available information on the complete genome sequence of GpSGHV and MdSGHV indicates significant co-linearity between the two viral genomes, whereas no co-linearity was observed with baculoviruses, ascoviruses, entomopoxviruses, iridoviruses and nudiviruses, other large invertebrate DNA viruses. The DNA polymerases encoded by the SGHVs are of the type B and closely related, but they are phylogenetically distant from DNA polymerases encoded by other large dsDNA viruses. The great majority of SGHV ORFs could not be assigned by sequence comparison. Phylogenetic analysis of conserved genes clustered both SGHVs, but distantly from the nudiviruses and baculoviruses. On the basis of the available morphological, (patho)biological, genomic and phylogenetic data, we propose that the two viruses are members of a new virus family named Hytrosaviridae. This proposed family currently comprises two unassigned species, G. pallidipes salivary gland hypertrophy virus and M. domestica salivary gland hypertrophy virus, and a tentative unassigned species, M. equestris salivary gland hypertrophy virus. Here, we present the characteristics and the justification for establishing this new virus family.

Serine proteases identified from a Costelytra zealandica (White) (Coleoptera: Scarabaeidae) midgut EST library and their expression through insect development.

Costelytra zealandica larvae are pests of New Zealand pastures causing damage by feeding on the roots of grasses and clovers. The major larval protein digestive enzymes are serine proteases (SPs), which are targets for disruption in pest control. An expressed sequence tag (EST) library from healthy, third instar larval midgut tissue was constructed and analysed to determine the composition and regulation of proteases in the C. zealandica larval midgut. Gene mining identified three trypsin-like and 11 chymotrypsin-like SPs spread among four major subgroups. Representative SPs were examined by quantitative PCR and enzyme activity assayed across developmental stages. The serine protease genes examined were expressed throughout feeding stages and downregulated in nonfeeding stages. The study will improve targeting of protease inhibitors and bacterial disruptors of SP synthesis.

Variations in the ability of galactose and mannose-specific lectins to bind to cell wall surfaces during growth of the insect pathogenic fungus Paecilomyces farinosus.

The arrangement of carbohydrate molecules on surfaces of fungal cells may play an important role in nonself recognition of these microorganisms by potential invertebrate hosts. Changes in the ability of various galactose and mannose-specific lectins to bind to surface components on cell walls of the insect pathogen Paecilomyces farinosus were therefore examined during growth and differentiation of the fungus. Fluorescein isothiocyanate conjugates of concanavalin A (Con A, specific for alpha-D-mannose) and peanut agglutinin (PNA, beta-D-galactose) bound inconsistently to blastospores and weakly to mycelia except at apical regions where strong fluorescence was observed. Labeling patterns were similar on cells tested with a galactose-specific lectin purified from Spodoptera exigua (beet armyworm) hemolymph, but Bandeiraea simplicifolia lectin (BS-I alpha-D-galactose) bound only to mycelia. Electron microscopy using ferritin and gold probes showed that the galactomannans are located in a loosely bound coating on the cell wall surface. Variations in lectin binding patterns are apparently due to absence (e.g., by shedding) of the coat or to rearrangement of carbohydrate components in the coat. Staining of Western blots of dithiothreitol (DTT) cell wall extracts further indicated that the BS-I-binding entity is a unique component of the mycelial surface since, as in the fluorescence studies, blastospore preparations were not labeled. Staining of blastospore blots with other galactose-specific probes (e.g., PNA) was comparable to staining of mycelial blots.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of monoclonal antibodies against cell wall epitopes of the insect pathogenic fungus, Nomuraea rileyi: differential binding to fungal surfaces and cross-reactivity with host hemocytes and basement membrane components.

Monoclonal antibodies (MAbs) were generated against epitopes on yeast-like hyphal bodies and hyphae of the entomopathogenic hyphomycete, Nomuraea rileyi. Two MAbs (4C10, 2H4) bind to epitopes common to both hyphal bodies and hyphae, whereas MAb 4E9 binds only to hyphal surfaces. 4C10 and 2H4 appear to be directed towards carbohydrate portions of cell surface mannoproteins, as evidenced by similarities in staining patterns between these MAbs and Concanavalin A on Western blots of N. rileyi cell wall extracts. These MAbs cross-react with antigens on blastospore and hyphal surfaces of two other entomopathogenic fungi, Beauveria bassiana and Paecilomyces farinosus in fluorescence microscopy assays, but do not cross-react with a non-entomopathogenic strain of Candida albicans or with Saccharomyces cerevisiae yeasts. MAb 4C10 also cross-reacts with immunocompetent granular hemocytes from Spodoptera exigua (beet armyworm) and Trichoplusia ni (cabbage looper) larvae and with S. exigua plasmatocytes. Electron microscopy revealed that this MAb binds to a component in cytoplasmic granules in the hemocytes, and that surface labeling may be due to the release of this MAb-positive component upon degranulation. MAb 2H4 does not cross-react with granular hemocytes, but does bind to plasmatocytes and hemocytes that tightly adhere to the substrate in monolayer assays. Additionally, MAb 4C10 specifically labels a basement membrane epitope on S. exigua fat body, suggesting that this antibody binds to mannose residues on extracellular matrix glycoproteins. Cross-reactivity of these N. rileyi MAbs with insect hemocyte and tissue components indicates that fungal surface epitopes can mimic host surface molecules, which could explain why N. rileyi hyphal bodies are not recognized by granulocytes and are able to circulate freely in the hemolymph without binding to basement membranes lining the hemocoel.

Comparative analysis of phagocytosis of fungal cells by insect hemocytes versus horse neutrophils.

In this study, the phagocytic ability of Spodoptera exigua hemocytes was compared to horse neutrophils. In vitro assays showed that the insect granulocytes and horse neutrophils actively phagocytose FITC-labeled Paecilomyces farinosus blastospores opsonized with S. exigua hemolymph lectin or horse serum, respectively. Killing of fungal cells by the neutrophils and hemocytes was analyzed under in vitro conditions. Neutrophils reduced the growth of P. farinosus up to 65% whereas no fungicidal activity was observed with hemocyte monolayers. The production of oxygen metabolites by both phagocytic cells incubated with various elicitors (fungal cells, bacteria, phorbol myristate acetate) was examined using luminol-enhanced chemiluminescence. Phagocytosis of opsonized microbes by horse neutrophils resulted in marked increase of chemiluminescence activity whereas no chemiluminescence was detected in similarly challenged phagocytic insect hemocytes. Electron microscopy was used to examine phagocytic events and confirmed that insect phagocytes were unable to kill tested microbes.

Characteristics of a galactose-binding hemagglutinin (lectin) from hemolymph of Spodoptera exigua larvae.

Hemolymph from Spodoptera exigua larvae agglutinates rabbit and human O erythrocytes. The agglutinin appears to be naturally occurring i.e., injury (by injection) or injection of larvae with fungal cells does not induce an increase in titer of hemolymph samples. Hemagglutinin activity is destroyed by heat or EDTA, and galactosidic carbohydrates inhibit agglutination of both red blood cell types. The agglutinin was purified by affinity chromatography using an Affi-Gel ovalbumin column. Binding to the column is calcium (cation) dependent. SDS gel electrophoresis shows that the agglutinin is a minor component of whole hemolymph represented by two bands with molecular weights of 30,500 and 31,000 daltons. Fluorescence microscopy using rhodamine-labeled agglutinin indicates that the agglutinin binds to fungal cell wall surfaces known to have galactose residues (e.g., Paecilomyces ochraceus), and that binding is specifically inhibited by galactose. There is no specific binding to fungal walls known to lack galactose residues (e.g., Paecilomyces ochraceus, Nomuraea rileyi). The agglutinin may be involved in the immune response of the insect e.g., by opsonization of microbial (fungal) surfaces which render the invading cells more susceptible to phagocytosis or agglutination.

Hemagglutinin activity in the hemolymph of Anticarsia gemmatalis larvae infected with the fungus Nomuraea rileyi.

Hemolymph samples from larvae of 3 lepidopteran species (Anticarsia gemmatalis, Trichoplusia ni and Spodoptera frugiperda) were tested for hemagglutination activity. Samples from A. gemmatalis larvae which had been injected 12-24 hrs previously with hyphal bodies of the entomopathogenic fungus Nomuraea rileyi showed agglutination activity against human 0, rabbit and sheep erythrocytes. Little or no activity was detected in samples from the other 2 larval species. Low titers (approximately 1:2) were observed when rabbit and sheep erythrocytes were tested with hemolymph from non-injected or water-injected A. gemmatalis control larvae. Higher titers (1:256-1:1024) were obtained when human erythrocytes were incubated with control hemolymph, but values were greater in the hyphal body-injected samples (1:2048-1: greater than 32,000). These results indicate a direct correlation between agglutinin production and the presence of fungal cells in the larval hemolymph. Injection with heat-killed or homogenated hyphal bodies did not cause increased activity. Decreases in titer values after these injections and for 1-12 hrs after injection with viable hyphal bodies suggest that the agglutinin(s) may function in immune surveillance. Agglutination of rabbit erythrocytes was inhibited by lactose, galactose and L-fucose. N-acetylneuraminic acid inhibited agglutination of human erythrocytes.

The galactose binding lectin from the beet armyworm, Spodoptera exigua: distribution and site of synthesis.

The Spodoptera exigua galactose binding lectin, extracted from hemolymph by affinity chromatography, was comprised of large molecular weight aggregates (100-700 kDa). SDS-PAGE of this lectin preparation revealed it to contain 2 subunits of 33.2 and 34.4 kDa in equimolar concentrations. These subunits had similar amino acid profiles, possessed identical N-terminal sequences and reacted equally to a bank of antilectin monoclonal antibodies. By staining Western blots with various lectin conjugate probes, we demonstrated that the 34.4 kDa subunit contains complex mannose residues, suggesting that this subunit is the glycosylated form of the 33.2 kDa subunit. The N-terminal sequence of the S. exigua lectin was distinct from other invertebrate galactose binding lectins. Light microscopy in combination with immunoelectron microscopy was used to localize the S. exigua lectin in the granules of the granulocyte class of hemocytes. Degranulation of these cells resulted in the release of the lectin. Isotope incorporation studies followed by immunoprecipitation with a S. exigua monoclonal antibody demonstrated that the fat body was the major site of lectin synthesis. Similar studies with hemocyte monolayers did not result in the production of detectable levels of 35S-labeled S. exigua lectin.

Salivary gland hypertrophy virus of house flies in Denmark: prevalence, host range, and comparison with a Florida isolate.

House flies (Musca domestica) infected with Musca domestica salivary gland hypertrophy virus (MdSGHV) were found in fly populations collected from 12 out of 18 Danish livestock farms that were surveyed in 2007 and 2008. Infection rates ranged from 0.5% to 5% and averaged 1.2%. None of the stable flies (Stomoxys calcitrans), rat-tail maggot flies (Eristalis tenax) or yellow dung flies (Scathophaga stercoraria) collected from MdSGHV-positive farms displayed characteristic salivary gland hypertrophy (SGH). In laboratory transmission tests, SGH symptoms were not observed in stable flies, flesh flies (Sarcophaga bullata), black dump flies (Hydrotaea aenescens), or face flies (Musca autumnalis) that were injected with MdSGHV from Danish house flies. However, in two species (stable fly and black dump fly), virus injection resulted in suppression of ovarian development similar to that observed in infected house flies, and injection of house flies with homogenates prepared from the salivary glands or ovaries of these species resulted in MdSGHV infection of the challenged house flies. Mortality of virus-injected stable flies was the highest among the five species tested. Virulence of Danish and Florida isolates of MdSGHV was similar with three virus delivery protocols, as a liquid food bait (in sucrose, milk, or blood), sprayed onto the flies in a Potter spray tower, or by immersiion in a crude homogenate of infected house flies. The most effective delivery system was immersion in a homogenate of ten infected flies/ml of water, resulting in 56.2% and 49.6% infection of the house flies challenged with the Danish and Florida strains, respectively.

Phenol-oxidizing laccases from the termite gut.

cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Laccase transcripts and phenoloxidase activity were most abundant in symbiont-free salivary gland and foregut tissue, verifying that the genes and activities are host-derived. Using a baculovirus-insect expression system, the two isoforms were functionally expressed with histidine tags and purified to near homogeneity. ICP-MS (inductively coupled plasma - mass spectrometry) analysis of RfLacA identified bound metals consisting mainly of copper (∼4 copper molecules per laccase protein molecule and ∼3 per histidine tag) with lesser amounts of calcium, manganese and zinc. Both recombinant enzyme preparations showed strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by the recombinant RfLacA preparation was also observed. These findings provide evidence that R. flavipes gut laccases are evolutionarily distinct, host-derived, produced in the salivary gland, secreted into the foregut, bind copper, and play a role in lignocellulose digestion. These findings contribute to a better understanding of termite digestion and gut physiology, and will assist future translational studies that examine the contributions of individual termite enzymes in lignocellulose digestion.

Characterization of an Indonesian isolate of Paecilomyces reniformis.

An entomopathogenic fungus (IndGH 96), identified as Paecilomyces reniformis, was isolated from long-horned grasshoppers (Orthoptera: Tettigoniidae) in Sulawesi, Indonesia. The phenotypic and molecular data identified the IndGH 96 as a P. reniformis. We present the first comprehensive characterization of this species using morphological features, sequencing of the ITS1-5.8s-ITS2 region, D1/D2 region of 28S of rDNA, and a portion of the tubulin gene, and laboratory bioassays. Distinguishing features include a hyphal body stage during vegetative growth and the production of distinctly curved, light-green conidia. High dosage bioassays showed that IndGH 96 was infectious to both long-horned and short-horned grasshoppers but not to the house cricket, Acheta domestica, or to the lepidopterans velvetbean caterpillar, Anticarsia gemmatalis or fall armyworm, Spodoptera frugiperda. Phenotypic and genetic analyses suggest that IndGH 96 and other isolates of P. reniformis are more closely related to Nomuraea rileyi than to other species of Paecilomyces.

Comparative analysis of the binding of antibodies prepared against the insect Spodoptera exigua and against the mycopathogen Nomuraea rileyi.

Polyclonal antibodies were produced in mice against Spodoptera exigua (beet armyworm) larval hemolymph and hemocytes and against cell wall surfaces of hyphal bodies and hyphae of the entomopathogenic hyphomycete Nomuraea rileyi. In addition to exhibiting strong activity against their original antigenic substrates, all of the antibodies cross-react extensively with other substrates. The hemolymph antibody binds to hemocytes and vice versa, and both antibodies cross-react to the insect fat body basement membrane (extracellular matrix (ECM) and to N. rileyi and Beauveria bassiana (another entomopathogenic fungus) cell wall surfaces (ECM). Likewise, the anti-fungal antibodies cross-react with S. exigua hemolymph and hemocytes, especially the granules that may contain ECM components, and with fat body basement membrane. These cross-reactivities are specific as indicated by negative controls in the microscopy and Western blotting assays. Parallel labeling experiments using Con A suggest that the reactive epitopes contain mannose; however, none of the antibodies bind to mannose residues of nonentomopathogenic Candida albicans or Saccharomyces cerevisiae yeast cells. Thus, these cross-reactivities suggest that the host mimicry expressed by surface components of entomopathogenic fungi represents an important pathogenic determinant.

Isozyme differentiation among three pathotypes of the entomogenous fungus Nomuraea rileyi.

The zymogram technique has been applied to three pathotypes of the entomopathogenic fungus Nomuraea rileyi. Isozyme profiles of isolates from Heliothis zea, Pseudoplusia includens, and Anticarsia gemmatalis were compared for 17 enzymes of known metabolic function. Electrophoretic data supported the taxonomic differences inferred for the three pathotypes based on host specificity. The isolate from A. gemmatalis was found consistently to be the most distinct. This study demonstrates that isozyme analysis may be used to distinguish closely related fungal isolates of N. rileyi.

Extraction and characterization of the insecticidal toxin hirsutellin A produced by Hirsutella thompsonii var. thompsonii.

Hirsutellin A (HtA) produced by Hirsutella thompsonii var. thompsonii (strain JAB-04) was extracted and purified using a combination of ion-exchange, gel-permeation, and immunoaffinity chromatography. The identity of the purified HtA was confirmed by amino acid analysis and N-terminal sequencing. Monoclonal antibodies prepared against HtA were capable of detecting 25-50 ng of HtA by direct sandwich ELISA. In addition, utilizing Western blot methods, the antibodies were shown to be specific to HtA. The production of HtA was monitored during submerged fermentation. The peak level of exocellular HtA (13-14 micrograms/ml) was during the late exponential growth phase (39-45 h), determined by utilizing a combination of densitometric analysis of the 16.3-kDa bands on SDS-PAGE gels and ELISA. HtA production was directly correlated with mycelial growth. Twenty-one-hour culture filtrates were highly toxic to larvae of the greater wax moth. Pure HtA at a final concentration of 40 pmol was highly toxic to Galleria mellonella larvae.