Harman and norharman, metabolites of entomopathogenic fungus Conidiobolus coronatus (Entomopthorales), disorganize development of Galleria mellonella (Lepidoptera) and affect serotonin-regulating enzymes.

Naturally occurring entomopathogenic fungi such as Conidiobolus coronatus are important regulatory factors of insect populations. GC-MS analysis of fungal cell-free filtrates showed that C. coronatus synthesizes two ß- carboline alkaloids: harman and norharman. Significantly higher levels of both alkaloids are produced by C. coronatus in minimal postincubation medium than in rich medium. The beta-carboline alkaloids may have an effect on the nervous system of insects and their behavior. Harman and norharman were applied to Galleria mellonella larvae (a parasite of honeybees) either topically or mixed with food. Larvae received alkaloids in three concentrations: 750, 1000 or 1250 ppm. The effect on the survival and further development of larvae was examined. Both harman and norharman delayed pupation and adult eclosion, and inhibit total monoamine oxidase activity. In addition, they increased the serotonin concentration and decreased the monoamine oxidase A level in the heads of the moths. It is likely that the alkaloids were metabolized by the insects, as their effect wore off 24 hours after topical application. This is the first study to show that C. coronatus produces alkaloids. Its aim was to identify the actions of ß-carboline alkaloids on insect development and serotonin-regulating enzymes. Knowledge of the potential role of harman and norharman in the process of fungal infection might lead to the development of more effective and environmentally-friendly means of controlling insect pests.

Hyphal tip growth and cytoplasmic characters of Conidiobolus coronatus (Zoopagomycota, Entomophthoromycotina).

Characteristics of hyphal structure and growth can provide insights into the mechanisms of polarized growth and support investigations of fungal phylogeny. To assist with the resolution of evolutionary relationships of the zygomycetes, the authors used comparative bioimaging methods (light [LM] and transmission electron [TEM] microscopy) to describe selected subcellular characters of hyphal tips of Conidiobolus coronatus. Growing hyphae of C. coronatus contain Spitzenkörper (Spk). Spk are most commonly present in hyphae of Dikarya (Ascomycota and Basidiomycota) and are rarely reported in zygomycete hyphae, which possess an apical vesicle crescent (AVC). Such findings raise questions regarding the evolution of the Spk and its relationship with the AVC. Descriptions of additional subcellular characters (e.g., mitotic-phase spindle pole bodies, cytoplasmic behavior, organelle structure) are also presented.

Biotechnological Production of Methyl-Branched Aldehydes.

A number of methyl-branched aldehydes impart interesting flavor impressions, and especially 12-methyltridecanal is a highly sought after flavoring compound for savory foods. Its smell is reminiscent of cooked meat and tallow. For the biotechnological production of 12-methyltridecanal, the literature was screened for fungi forming iso-fatty acids. Suitable organisms were identified and successfully grown in submerged cultures. The culture medium was optimized to increase the yields of branched fatty acids. A recombinant carboxylic acid reductase was used to reduce 12-methyltridecanoic acid to 12-methyltridecanal. The efficiency of whole-cell catalysis was compared to that of the purified enzyme preparation. After lipase-catalyzed hydrolysis of the fungal lipid extracts, the released fatty acids were converted to the corresponding aldehydes, including 12-methyltridecanal and 12-methyltetradecanal.

Experimental infection in gerbils by Conidiobolus lamprauges.

Conidiobolomycosis is an emerging entomophthoramycosis caused by fungi Conidiobolus spp. Animal models are essential for the study of infectious disease in various areas such as pathogenesis, diagnostic methods, treatment and prevention. There is not currently an animal model for conidiobolomycosis. The aim of this study was to create an experimental infection protocol for Conidiobolus lamprauges in gerbils (Meriones unguiculatus). The study animals were randomly divided into four groups of four animals: immunosuppressed with cyclophosphamide (CPA) and infected with C. lamprauges (G1), immunocompetent and infected with C. lamprauges (G2), immunosuppressed with CPA (G3), and an immunocompetent control group (G4). Clinical signs were observed only in G1 animals, where the mortality rate reached 75% by day 7 after infection (AI) with a median survival of 2 days. C. lamprauges was detected only in G1, both by PCR and by isolation. Necropsies of the G1 animals showed lesions in the nasal cavity and lung tissue. These lesions were characterized by polymorphonuclear infiltrate cells and by the presence of hyphal structures under silver staining. This animal model will be useful for further investigation of diseases caused by C. lamprauges, particularly of those associated with immunosuppression factors in naturally occurring animal infections.

Free fatty acids in the cuticular and internal lipids of Calliphora vomitoria and their antimicrobial activity.

The cuticular and internal lipid composition in Calliphora vomitoria larvae, pupae, and male and female adults was studied. The free fatty acid (FA) compositions of the lipids were chemically characterized using gas chromatography (GC) and gas chromatography-electron impact mass spectrometry (GC-MS). Analyses of cuticular extracts from larvae, pupae, and male and female adults revealed that the carbon numbers of the acids ranged from C7:0 to C22:0, from C8:0 to C24:0, from C7:0 to C24:0 and from C7:0 to C22:0 respectively. The internal lipids of C. vomitoria larvae, pupae, male and female adults contained FAs ranging from C8:0 to C20:0, from C9:0 to C22:0, from C8:0 to C24:0 and from C9:0 to C22:0 respectively. Nine FAs with odd-numbered carbon chains from C7:0 to C21:0 were identified in the cuticular lipids of the larvae. The internal lipids of C. vomitoria larvae contained 8 odd-numbered FAs ranging from C9:0 to C19:0. Eight odd-numbered FAs from C9:0 to C21:0 were identified in the cuticular and internal lipids of pupae, while nine such FAs were found in the cuticular lipids of male and female adults. The internal lipids of adult males and females respectively contained nine and seven odd-numbered FAs, while both larvae and pupae contained eight such compounds. Eight unsaturated FAs were identified in the cuticular lipids of larvae, adult males and females and also in the internal lipids of females. Seven unsaturated FAs were identified in the cuticular lipids of pupae. The internal lipids of larvae, pupae and males contained 10, 11 and 12 unsaturated FAs respectively. Developmental changes were found both in the amounts of extracted cuticular and internal FAs and in their profiles. Four cuticular FAs (C7:0, C9:0, C10:0 and C15:1), identified as being male-specific, were either absent in the female cuticle or present there only in trace amounts. Cuticular and internal extracts obtained from larvae, pupae, adult males and females were tested for their potential antimicrobial activity. The minimal inhibitory concentrations of extracts against reference strains of bacteria and fungi were determined. Antimicrobial activity was the strongest against Gram-positive bacteria; Gram-negative bacteria, on the other hand, turned out to be resistant to all the lipids tested. Overall, the activities of the internal lipids were stronger. All the lipid extracts were equally effective against all the fungal strains examined. In contrast, crude extracts containing both cuticular and internal lipids displayed no antifungal activity against the entomopathogenic fungus Conidiobolus coronatus, which efficiently killed adult flies, but not larvae or pupae.

Effects of insect cuticular fatty acids on in vitro growth and pathogenicity of the entomopathogenic fungus Conidiobolus coronatus.

Eighteen fatty acids identified in the cuticle of three insect species representing differing susceptibilities to C. coronatus infection, were tested for effects on the in vitro growth and pathogenicity of the parasitic fungus. At all applied concentrations (0.1-0.0001% w/v) growth was inhibited by C(16:0), C(16:1), C(18:0), C(18:1), C(18:2), C(18:3), C(20:0) and C(20:1). At high concentrations spore germination was inhibited by C(7:0), C(8:0), C(9:0), C(10:0), C(12:0), C(18:2) and C(18:3) and hyphal growth was merely retarded by C(5:0), C(6:0), C(6:2), C(14:0), C(16:0), C(16:1), C(18:0,) C(18:1), C(20:0) and C(20:1). The presence of C(15:0) at the 0.1% concentration stimulated growth of C. coronatus. Sporulation was inhibited by all concentrations of C(16:0) and C(18-20) fatty acids. Low concentrations of C(5:0), C(6:0), C(6:2) and C(7:0) enhanced sporulation. Fatty acids C(5-12) as well as C(18:3), C(20:0) and C(20:1) decreased the ability of fungal colonies to infect G. mellonella while C(16:1) elevated it thus suggesting that C(16:1) may stimulate production of enzymes involved in the host invasion. Toxicity of metabolites released into incubation medium decreased with varying degrees in the presence of C(6:0), C(6:2,) C(7:0), C(9:0), C(12:0), C(16:1), C(18:2), C(18:3), C(20:0) and C(20:1); other fatty acids had no effect. Further work is needed to analyse the effects of exogenous fatty acids on the C. coronatus enzymes implicated in fungal pathogenicity as well as on the production of insecticidal metabolites.

Rhinofacial conidiobolomycosis caused by Conidiobolus coronatus in a Chinese rice farmer.

We provide the first report of rhinofacial conidiobolomycosis caused by Conidiobolus coronatus in China. The patient was a 36-year-old male rice farmer from Jiang Xi province who presented with diffuse swelling with classical centrofacial distribution and involvement of the right paranasal sinuses. Histopathology showed granulomas with hyphae surrounded by an eosinophilic sheath (Splendore-Hoeppli phenomenon). Culture of biopsy specimens on Sabouraud's dextrose agar led to the growth of fungi with microscopically visible conidiophores and terminal spherical conidia (primary conidium), with multiple secondary conidia and villose conidia. The patient was successfully treated with combination therapy, primarily itraconazole and terbinafine. We conclude with a brief literature review of the epidemiology of conidiobolomycosis.

EST analysis of genes expressed by the zygomycete pathogen Conidiobolus coronatus during growth on insect cuticle.

Conidiobolus coronatus (Zygomycota) is a facultative saprobe that is a pathogen of many insect species. Almost 2000 expressed sequence tag (EST) cDNA clones were sequenced to analyse gene expression during growth on insect cuticle. Sixty percent of the ESTs that could be clustered into functional groups (E

[Influence of free fatty acids on growth, sporulation and virulence of the parasitic fungus Conidiobolus coronatus]. / Wplyw wolnych kwasów tluszczowych na wzrost, sporulacje i wirulencje pasozytniczego grzyba Conidiobolus coronatus.

Five free fatty acids (FFA): C16:0, C18:0, C18:1, C18:2 and C18:3 were introduced into culture media in order to investigate differential development of pathogenic fungus Conidiobolus coronatus as a function of FFA concentration. All tested FFA showed fungistatic action inhibiting hyphae growth and sporulation. Fungal colonies grown in the presence of FFA showed decreased virulence.