Carbohydrate Storage in the Entomopathogenic Fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana was grown in 1% (wt/vol) gelatin-liquid media singly supplemented with a monosaccharide (glucose or fructose), a disaccharide (maltose or trehalose), a polyol (glycerol, mannitol, or sorbitol), or the amino sugar N-acetyl-d-glucosamine. The relative contributions of the carbohydrate, protein, and water contents in the fungal biomass were determined. Carbohydrates composed 18 to 42% of the mycelial dry weight, and this value was lowest in unsupplemented medium and highest in medium supplemented with glucose, glycerol, or trehalose. Biomass production was highest in liquid cultures supplemented with trehalose. When liquid cultures were grown in medium supplemented with 0 to 1% (wt/vol) glucose, trehalose, or N-acetyl-d-glucosamine, there was an increase in the biomass production and the contribution of carbohydrate to mycelial dry weight. Regardless of the glucose concentration in the culture, water content of the mycelia remained about 77.5% (wt/wt). Mycelial storage carbohydrates were determined by capillary gas chromatography. In gelatin-liquid medium supplemented with 1% (wt/vol) glucose, B. bassiana stored glycogen (12.0%, wt/dry wt) and the polyols mannitol (2.2%), erythritol (1.6%), glycerol (0.4%), and arabitol (0.1%). Without glucose, B. bassiana stored glycogen (5.4%), mannitol (0.8%), glycerol (0.6%), and erythritol (0.6%) but not arabitol. To our knowledge, this is the first report of carbohydrate storage in an entomopathogenic fungus, and the results are discussed in relation to other fungi and the potential implications to commercial formulation and insect-fungus interactions.     

Prevalence and Diversity of Viruses in the Entomopathogenic Fungus Beauveria bassiana

Viruses have been discovered in numerous fungal species, but unlike most known animal or plant viruses, they are rarely associated with deleterious effects on their hosts. The knowledge about viruses among entomopathogenic fungi is very limited, although their existence is suspected because of the presence of virus-like double-stranded RNA (dsRNA) in isolates of several species. Beauveria bassiana is one of the most-studied species of entomopathogenic fungi; it has a cosmopolitan distribution and is used as a biological control agent against invertebrates in agriculture. We analyzed a collection of 73 isolates obtained at different locations and from different habitats in Spain and Portugal, searching for dsRNA elements indicative of viral infections. The results revealed that the prevalence of viral infections is high; 54.8% of the isolates contained dsRNA elements with viral characteristics. The dsRNA electropherotypes of infected isolates indicated that virus diversity was high in the collection analyzed and that mixed virus infections occurred in fungal isolates. However, a hybridization experiment indicated that dsRNA bands that are similar in size do not always have similar sequences. Particular virus species or dsRNA profiles were not associated with locations or types of habitats, probably because of the ubiquity and efficient dispersion of this fungus as an airborne species. The sequence of one of the most common dsRNA elements corresponded to the 5.2-kbp genome of a previously undescribed member of the Totiviridae family, termed B. bassiana RNA virus 1 (BbRV1).     

N-Acetyl-d-Glucosamine-Mediated Regulation of Extracellular Protease in the Entomopathogenic Fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana GK2016 grown in a liquid medium incorporating gelatin as the sole carbon and nitrogen source produced an extracellular serine protease (molecular weight, 35,000; pI ca. 10). Without gelatin, B. bassiana could utilize N-acetyl-d-glucosamine (GlcNAc; 2-acetamido-2-deoxy-d-glucose) as the sole source of carbon and nitrogen, and GlcNAc availability increased the storage carbohydrate content in mycelia. Synthesis of protease was repressed in gelatin medium containing GlcNAc at levels of >1.07 mumol mg of fungal dry weight. At levels below this, protease synthesis was initiated; subsequently, free amino nitrogen appeared in the medium and diauxic growth was observed. Slow feeding with GlcNAc (35.34 mug ml h) did not repress protease synthesis nor did GlcNAc accumulate in the medium above 0.5 mg ml. Increasing the rate of release of GlcNAc (83.51 mug ml h) resulted in the accumulation of GlcNAc in the medium to 2.0 mg ml, a 45% increase in growth and a decrease in protease synthesis by about 81%. Free amino acids generated from the hydrolysis of gelatin did not repress protease synthesis. These data are interpreted in terms of known interaction of B. bassiana with insect cuticular components. We suggest that the action of extracellular chitinases synthesized by B. bassiana on insect cuticle, and pursuant release of GlcNAc, may have important consequences on the regulation of other extracellular catabolic enzymes such as the protease.     

Carboxylate Transporter Gene JEN1 from the Entomopathogenic Fungus Beauveria bassiana Is Involved in Conidiation and Virulence

Beauveria bassiana is an important entomopathogenic fungus widely used as a biological agent to control insect pests. A gene (B. bassiana JEN1 [BbJEN1]) homologous to JEN1 encoding a carboxylate transporter in Saccharomyces cerevisiae was identified in a B. bassiana transfer DNA (T-DNA) insertional mutant. Disruption of the gene decreased the carboxylate contents in hyphae, while increasing the conidial yield. However, overexpression of this transporter resulted in significant increases in carboxylates and decreased the conidial yield. BbJEN1 was strongly induced by insect cuticles and highly expressed in the hyphae penetrating insect cuticles not in hyphal bodies, suggesting that this gene is involved in the early stage of pathogenesis of B. bassiana. The bioassay results indicated that disruption of BbJEN1 significantly reduced the virulence of B. bassiana to aphids. Compared to the wild type, ΔBbJEN1 alkalinized the insect cuticle to a reduced extent. The alkalinization of the cuticle is a physiological signal triggering the production of pathogenicity. Therefore, we identified a new factor influencing virulence, which is responsible for the alkalinization of the insect cuticle and the initiation of fungal pathogenesis in insects.Mycoinsecticides are considered promising biological control agents and alternatives or supplements to chemical pesticides (15). However, the dearth of physiological, genetic, and molecular knowledge of entomopathogenic fungi has retarded their widespread application.For mycoinsecticide improvement, greater attention and effort have been given to elucidate the mechanisms of fungal pathogenesis (13, 14, 18, 20, 29, 49, 50, 51, 52, 53). Entomopathogenic fungi, e.g., Metarhizium anisopliae and Beauveria bassiana, invade their hosts by direct penetration of the host exoskeleton or cuticle. M. anisopliae and B. bassiana produce hydrophobic spores which contact and adhere to the insect cuticle (12). Once attached, the conidium germinates and the germ tubes differentiate into swollen infection structures called appressoria. The appressoria produce penetration pegs which penetrate the insect cuticle via cuticle-degrading enzymes (11, 19, 46) as well as mechanical pressure (24, 53). Hyphae proliferate within the hemocoel, emerge from inside the insect, and subsequently conidiate on the cadaver (15). However, much remains to be elucidated regarding the mechanisms of insect fungal pathogenesis.To obtain detailed knowledge of the mechanisms of fungal pathogenesis, a pool of B. bassiana transfer DNA (T-DNA) insertional mutants had been generated through an Agrobacterium-mediated-transformation method (21). A mutant, designated T12, characterized by the presence of more conidia, was isolated, and its flanking sequence was obtained by T-DNA tagging. The flanking fragment contained an open reading frame (ORF), which corresponded to a gene termed JEN1, encoding a transporter of carboxylates (http://www.ncbi.nlm.nih.gov/Blast.cgi). Organic acid transportation is important for the metabolism of almost all cells of multicellular organisms and unicellular microorganisms (17, 25, 26). Transport across the plasma membrane is the first step in the metabolism of these substrates, which may affect many aspects of the organism, including regulation of energy metabolism (9, 34) and acid-base equilibrium status (10).JEN1p has been identified in several fungal species, e.g., Saccharomyces cerevisiae, Candida albicans, and Kluyveromyces lactis (9, 35, 45), which is a lactate/pyruvate symporter (1, 9, 34). The enzyme imports lactate or some short-chain monocarboxylates across the plasma membrane into cells. Then, the lactate is stereo-specifically oxidized to pyruvate. This reaction is performed by ferricytochrome c oxidoreductase in mitochondria (23, 33) and is tightly connected to the respiratory chain (34). JEN1 was induced by lactic, pyruvic, acetic, and propionic acids and repressed by glucose (2, 9, 35, 45). Nevertheless, for entomopathogenic fungi, the characterization of JEN1p has not been investigated, and its role in infection is still a mystery.For this paper, we studied the functions of a putative carboxylate transport gene, JEN1, in B. bassiana (BbJEN1). Our results demonstrated that BbJEN1 is involved in conidiation of B. bassiana and that the gene is a new factor influencing virulence in entomopathogenic fungi.     

Adhesion of the Entomopathogenic Fungus Beauveria (Cordyceps) bassiana to Substrata

The entomopathogenic fungus Beauveria bassiana produces at least three distinct single-cell propagules, aerial conidia, vegetative cells termed blastospores, and submerged conidia, which can be isolated from agar plates, from rich broth liquid cultures, and under nutrient limitation conditions in submerged cultures, respectively. Fluorescently labeled fungal cells were used to quantify the kinetics of adhesion of these cell types to surfaces having various hydrophobic or hydrophilic properties. Aerial conidia adhered poorly to weakly polar surfaces and rapidly to both hydrophobic and hydrophilic surfaces but could be readily washed off the latter surfaces. In contrast, blastospores bound poorly to hydrophobic surfaces, forming small aggregates, bound rapidly to hydrophilic surfaces, and required a longer incubation time to bind to weakly polar surfaces than to hydrophilic surfaces. Submerged conidia displayed the broadest binding specificity, adhering to hydrophobic, weakly polar, and hydrophilic surfaces. The adhesion of the B. bassiana cell types also differed in sensitivity to glycosidase and protease treatments, pH, and addition of various carbohydrate competitors and detergents. The outer cell wall layer of aerial conidia contained sodium dodecyl sulfate-insoluble, trifluoroacetic acid-soluble proteins (presumably hydrophobins) that were not present on either blastospores or submerged conidia. The variations in the cell surface properties leading to the different adhesion qualities of B. bassiana aerial conidia, blastospores, and submerged conidia could lead to rational design decisions for improving the efficacy and possibly the specificity of entomopathogenic fungi for host targets.     

Colonization of Corn, Zea mays, by the Entomopathogenic Fungus Beauveria bassiana

Light and electron microscopy were used to describe the mode of penetration by the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin into corn, Zea mays L. After inoculation with a foliar spray of conidia, germinating hyphae grew randomly across the leaf surface. Often a germ tube formed from a conidium and elongated only a short distance before terminating its growth. Not all developing hyphae on the leaf surface penetrated the cuticle. However, when penetration did occur, the penetration site(s) was randomly located, indicating that B. bassiana does not require specific topographic signals at an appropriate entry site as do some phytopathogenic fungi. Long hyphal structures were observed to follow the leaf apoplast in any direction from the point of penetration. A few hyphae were observed within xylem elements. Because vascular bundles are interconnected throughout the corn plant, this may explain how B. bassiana travels within the plant and ultimately provides overall insecticidal protection. Virulency bioassays demonstrate that B. bassiana does not lose virulence toward the European corn borer, Ostrinia nubilalis (Hübner), once it colonizes corn. This endophytic relationship between an entomopathogenic fungus and a plant suggests possibilities for biological control, including the use of indigenous fungal inocula as insecticides.     

Reduction of Banana Weevil Populations Using Different Formulations of the Entomopathogenic Fungus Beauveria bassiana

One of the major constraints for banana production in Uganda is the banana weevil, Cosmopolites sordidus (Germar), (Coleoptera: Curculionidae). Investigations were carried out to evaluate the efficacy of maize, soil-based and oil formulations of an indigenous isolate of Beauveria bassiana for the control of the banana weevil. Weekly trapping of weevils over a 9-month monitoring period showed significant reduction in unmarked and marked weevil population in B. bassiana treated plots. Application of maize formulation at 2 ×10 15 conidia ha -1 proved most effective, reducing the weevil populations by 63-72% within 8 weeks after a single application. The soil based formulation at 2 ×10 14 conidia ha -1 was intermediate while the oil formulation at 6 ×10 15 conidial ha -1 was least effective. Trapping efficiency declined in B. bassiana treated and untreated banana plots but was greatest in the latter.     

Impact of a Nematode-parasitic Fungus on the Effectiveness of Entomopathogenic Nematodes

The impact of the nematode-parasitic fungus Hirsutella rhossiliensis on the effectiveness of Steinernema carpocapsae, S. glaseri, and Heterorhabditis bacteriophora against Galleria mellonella larvae was assessed in the laboratory. The presence of Hirsutella conidia on the third-stage (J3) cuticle of S. carpocapsae and H. bacteriophora interfered with infection of insect larvae. Conidia on the J3 cuticle of S. glaseri and on the ensheathing second-stage cuticle of H. bacteriophora did not reduce the nematodes'' ability to infect larvae. The LD₅₀ values for S. carpocapsae, S. glaseri, and H. bacteriophora in sand containing H. rhossiliensis were not different from those in sterilized sand when Galleria larvae were added at the same time as the nematodes. However, when Galleria larvae were added 3 days after the nematodes, the LD₅₀ of S. glaseri was higher in Hirsutella-infested sand than in sterilized sand, whereas the LD₅₀ of H. bacteriophora was the same in infested and sterilized sand. Although the LD₅₀ of S. carpocapsae was much higher in Hirsutella-infested sand than in sterilized sand, the data were too variable to detect a significant difference. These data suggest that H. bacteriophora may be more effective than Steinernema species at reducing insect pests in habitats with abundant nematode-parasitic fungi.     

A High-Throughput Gene Disruption Methodology for the Entomopathogenic Fungus Metarhizium robertsii

Systematic gene disruption is a direct way to interrogate a fungal genome to functionally characterize the full suite of genes involved in various biological processes. Metarhizium robertsii is extraordinarily versatile, and it is a pathogen of arthropods, a saprophyte and a beneficial colonizer of rhizospheres. Thus, M. robertsii can be used as a representative to simultaneously study several major lifestyles that are not shared by the “model” fungi Saccharomyces cerevisiae and Neurospora crassa; a systematic genetic analysis of M. robertsii will benefit studies in other fungi. In order to systematically disrupt genes in M. robertsii, we developed a high-throughput gene disruption methodology, which includes two technologies. One is the modified OSCAR-based, high-throughput construction of gene disruption plasmids. This technology involves two donor plasmids (pA-Bar-OSCAR with the herbicide resistance genes Bar and pA-Sur-OSCAR with another herbicide resistance gene Sur) and a recipient binary plasmid pPK2-OSCAR-GFP that was produced by replacing the Bar cassette in pPK2-bar-GFP with a ccdB cassette and recombination recognition sites. Using this technology, a gene disruption plasmid can be constructed in one cloning step in two days. The other is a highly efficient gene disruption technology based on homologous recombination using a Ku70 deletion mutant (ΔMrKu70) as the recipient strain. The deletion of MrKu70, a gene encoding a key component involved in nonhomologous end-joining DNA repair in fungi, dramatically increases the gene disruption efficiency. The frequency of disrupting the conidiation-associated gene Cag8 in ΔMrKu70 was 93% compared to 7% in the wild-type strain. Since ΔMrKu70 is not different from the wild-type strain in development, pathogenicity and tolerance to various abiotic stresses, it can be used as a recipient strain for a systematic gene disruption project to characterize the whole suite of genes involved in the biological processes of M. robertsii.     

Purification and Properties of an Extracellular Protease Produced by the Entomopathogenic Fungus Beauveria bassiana

Beauveria bassiana GK2016 grown in a medium with gelatin as the sole carbon and nitrogen source produced an extracellular protease. The protease production was highest when the fungus was grown on a semiliquid medium and was purified about 18-fold, with a recovery of 21%. The protease molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 35,000. It had an optimum activity at pH 8.5 and 37 degrees C and was rapidly inactivated at 50 degrees C. Its enzymatic activity was that of an endopeptidase which hydrolyzed elastin, casein, and gelatin but was much less active on bovine serum albumin and collagen. No trypsinlike activity was detected on N-alpha-benzoyl-dl-arginine-p-nitroanilide. It was, however, inhibited by phenylmethylsulfonyl fluoride, indicating that a serine residue is present in the active site. The protease was unaffected by metal-chelating agents, sulfhydryl reagents, trypsin inhibitor, and chymotrypsin inhibitor.     

Distribution of the Entomopathogenic Fungus Beauveria bassiana in Rice Ecosystems and Its Effect on Soil Enzymes

Fungal entomopathogens, especially Beauveria bassiana, are often studied within the context of their use in biological pest control; however, there is limited knowledge of their distributions in host plants and soil ecosystem. We examined the distribution of B. bassiana and its influence on rice plants and paddy soils. B. bassiana could only be detected on the foliar surfaces of rice plants within 15 days under Bb-4 (7.5 × 10⁴ conidia/mL) and Bb-7 (7.5 × 10⁷ conidia/mL) treatments. The endophytic colonization of B. bassiana could not be found in stems, roots, or seeds of rice plants under Bb-4 and Bb-7 treatments. The fungus was found only in the leaves of rice plants under Bb-4 and Bb-7 treatments at 15 days after inoculation. Moreover, B. bassiana was absent from paddy soils under Bb-4 and Bb-7 treatments at all times. Enzyme activity (urease and phosphatase) in the paddy soils of Bb-4 and Bb-7 treatments showed no significant difference from the control. It is possible that B. bassiana was not able to colonize paddy soil. Detailed understanding of distribution and ecological interactions of B. bassiana is helpful for understanding and predicting the effects of fungal entomopathogens on host populations, and the interactions among fungal entomopathogens and other organisms in the community.     

V-ATPase Is Involved in Silkworm Defense Response against Bombyx mori Nucleopolyhedrovirus

Silkworms are usually susceptible to the infection of Bombyx mori (B. mori) nucleopolyhedrovirus (BmNPV), which can cause significant economic loss. However, some silkworm strains are identified to be highly resistant to BmNPV. To explore the silkworm genes involved in this resistance in the present study, we performed comparative real-time PCR, ATPase assay, over-expression and sub-cellular localization experiments. We found that when inoculated with BmNPV both the expression and activity of V-ATPase were significantly up-regulated in the midgut column cells (not the goblet cells) of BmNPV-resistant strains (NB and BC8), the main sites for the first step of BmNPV invasion, but not in those of a BmNPV-susceptible strain 306. Furthermore, this up-regulation mainly took place during the first 24 hours post inoculation (hpi), the essential period required for establishment of virus infection, and then was down-regulated to normal levels. Amazingly, transient over-expression of V-ATPase c subunit in BmNPV-infected silkworm cells could significantly inhibit BmNPV proliferation. To our knowledge this is the first report demonstrating clearly that V-ATPase is indeed involved in the defense response against BmNPV. Our data further suggests that prompt and potent regulation of V-ATPase may be essential for execution of this response, which may enable fast acidification of endosomes and/or lysosomes to render them competent for degradation of invading viruses.     

Phenylalanine Metabolism Regulates Reproduction and Parasite Melanization in the Malaria Mosquito

The blood meal of the female malaria mosquito is a pre-requisite to egg production and also represents the transmission route for the malaria parasite. The proper and rapid assimilation of proteins and nutrients in the blood meal creates a significant metabolic challenge for the mosquito. To better understand this process we generated a global profile of metabolite changes in response to blood meal of Anopheles gambiae, using Gas Chromatography-Mass Spectrometry (GC-MS). To disrupt a key pathway of amino acid metabolism we silenced the gene phenylalanine hydroxylase (PAH) involved in the conversion of the amino acid phenylalanine into tyrosine. We observed increased levels of phenylalanine and the potentially toxic metabolites phenylpyruvate and phenyllactate as well as a reduction in the amount of tyrosine available for melanin synthesis. This in turn resulted in a significant impairment of the melanotic encapsulation response against the rodent malaria parasite Plasmodium berghei. Furthermore silencing of PAH resulted in a significant impairment of mosquito fertility associated with reduction of laid eggs, retarded vitellogenesis and impaired melanisation of the chorion. Carbidopa, an inhibitor of the downstream enzyme DOPA decarboxylase that coverts DOPA into dopamine, produced similar effects on egg melanization and hatching rate suggesting that egg chorion maturation is mainly regulated via dopamine. This study sheds new light on the role of amino acid metabolism in regulating reproduction and immunity.     

Serine Protease MP2 Activates Prophenoloxidase in the Melanization Immune Response of Drosophila melanogaster

In arthropods, melanization plays a major role in the innate immune response to encapsulate and kill the invasive organisms. It is mediated by a serine protease cascade and is regulated by serpins. The identification of the molecular components of melanization and the regulation of those components are still unclear in Drosophila melanogaster, although some genetic research on the activation of melanization has been reported. Here we report that Drosophila serine protease MP2 directly cleaves both recombinant and native prophenoloxidase-1. Overexpression or repression of MP2 in flies resulted in increased and decreased rates of cleavage, respectively, of prophenoloxidase-1. Moreover, serine protease inhibitor Spn27A formed SDS-stable complexes with MP2, both in vitro and in vivo. The amidase activity of MP2 was inhibited efficiently by Spn27A. Spn27A also prevented MP2 from cleaving prophenoloxidase-1. Taken together, these results indicate that under our experimental conditions MP2 functions as a prophenoloxidase-activating protease, and that this function is inhibited by Spn27A. MP2 and Spn27A thus constitute a regulatory unit in the prophenoloxidase activation cascade in Drosophila. The combination of genetic, molecular genetic and biochemical approaches should allow further advances in our understanding of the prophenoloxidase-activating cascade in insects and indirectly shed further light on protease-cascades in humans and other vertebrates.     

KDM2B Is Implicated in Bovine Lethal Multi-Organic Developmental Dysplasia

In the last decade breeders of Romagnola cattle observed an outbreak of a new congenital anomaly. This lethal multi-organ developmental dysplasia is mainly characterized by facial deformities, ascites and hepatic fibrosis. Affected stillborn calves were inbred to a single founder sire suggesting autosomal monogenic recessive inheritance. We localized the causative mutation to a 1.2 Mb interval on BTA 17 by genome-wide association and identical by descent mapping. A solution-based method for targeted DNA capture combined with massively parallel sequencing was used to analyze the entire critical region containing 24 genes. Homozygosity for two non-synonymous coding sequence variants affecting the RNF34 and KDM2B genes was detected by evaluating one affected calf. Here we show that the disease phenotype is associated with a KDM2B missense mutation (c.2503G>A) leading to an amino acid exchange (p.D835N) in an evolutionary strongly conserved domain. In addition, the genetic makeup of three inbred cattle strongly supports the causality of the KDM2B mutation. This report of a naturally-occurring spontaneous mutation of a JmjC domain containing histone demethylase gene provides evidence for their important role in the endo- and mesodermal organ development.     

The Major Yolk Protein Vitellogenin Interferes with the Anti-Plasmodium Response in the Malaria Mosquito Anopheles gambiae

When taking a blood meal on a person infected with malaria, female Anopheles gambiae mosquitoes, the major vector of human malaria, acquire nutrients that will activate egg development (oogenesis) in their ovaries. Simultaneously, they infect themselves with the malaria parasite. On traversing the mosquito midgut epithelium, invading Plasmodium ookinetes are met with a potent innate immune response predominantly controlled by mosquito blood cells. Whether the concomitant processes of mosquito reproduction and immunity affect each other remains controversial. Here, we show that proteins that deliver nutrients to maturing mosquito oocytes interfere with the antiparasitic response. Lipophorin (Lp) and vitellogenin (Vg), two nutrient transport proteins, reduce the parasite-killing efficiency of the antiparasitic factor TEP1. In the absence of either nutrient transport protein, TEP1 binding to the ookinete surface becomes more efficient. We also show that Lp is required for the normal expression of Vg, and for later Plasmodium development at the oocyst stage. Furthermore, our results uncover an inhibitory role of the Cactus/REL1/REL2 signaling cassette in the expression of Vg, but not of Lp. We reveal molecular links that connect reproduction and immunity at several levels and provide a molecular basis for a long-suspected trade-off between these two processes.