Comparative transcriptomics reveal host-specific nucleotide variation in entomophthoralean fungi.

Obligate parasites are under strong selection to increase exploitation of their host to survive while evading detection by host immune defences. This has often led to elaborate pathogen adaptations and extreme host specificity. Specialization on one host, however, often incurs a trade-off influencing the capacity to infect alternate hosts. Here, we investigate host adaptation in two morphologically indistinguishable and closely related obligate specialist insect-pathogenic fungi from the phylum Entomophthoromycota, Entomophthora muscae sensu stricto and E. muscae sensu lato, pathogens of houseflies (Musca domestica) and cabbage flies (Delia radicum), respectively. We compared single nucleotide polymorphisms within and between these two E. muscae species using 12 RNA-seq transcriptomes from five biological samples. All five isolates contained intra-isolate polymorphisms that segregate in 50:50 ratios, indicative of genetic duplication events or functional diploidy. Comparative analysis of dN/dS ratios between the multinucleate E. muscae s.str. and E. muscae s.l. revealed molecular signatures of positive selection in transcripts related to utilization of host lipids and the potential secretion of toxins that interfere with the host immune response. Phylogenetic comparison with the nonobligate generalist insect-pathogenic fungus Conidiobolus coronatus revealed a gene-family expansion of trehalase enzymes in E. muscae. The main sugar in insect haemolymph is trehalose, and efficient sugar utilization was probably important for the evolutionary transition to obligate insect pathogenicity in E. muscae. These results support the hypothesis that genetically based host specialization in specialist pathogens evolves in response to the challenge of using resources and dealing with the immune system of different hosts.

Effect of light quality and light-dark cycle on sporulation patterns of the mite pathogenic fungus Neozygites floridana (Neozygitales: Entomophthoromycota), a natural enemy of Tetranychus urticae.

A controlled climatic chamber microcosm experiment was conducted to examine how light affects the hourly sporulation pattern of the beneficial mite pathogenic fungus Neozygites floridana during a 24h cyclus over a period of eight consecutive days. This was done by inoculating two-spotted spider mites (Tetranychus urticae) with N. floridana and placing them on strawberry plants for death and sporulation. Spore (primary conidia) discharge was observed by using a spore trap. Two light regimes were tested: Plant growth light of 150µmolm(-2)s(-1) for 12h supplied by high pressure sodium lamps (HPS), followed by either; (i) 4h of 50µmolm(-2)s(-1) light with similar HPS lamps followed by 8h darkness (full HPS light+reduced HPS light+darkness) or (ii) 4h of 50µmolm(-2)s(-1) red light followed by 8h darkness (full HPS light+red light+darkness). A clear difference in hourly primary conidia discharge pattern between the two different light treatments was seen and a significant interaction effect between light treatment and hour in day during the 24h cycle was observed. The primary conidia discharge peak for treatment (ii) that included red light was mainly reached within the red light hours (19:00-23:00) and the dark hours (23:00-07:00). The primary conidia discharge peak for treatment (i) with HPS light only was mainly reached within the dark hours (23:00-07:00).

Phylogenetic placement of two species known only from resting spores: Zoophthora independentia sp. nov. and Z. porteri comb nov. (Entomophthorales: Entomophthoraceae).

Molecular methods were used to determine the generic placement of two species of Entomophthorales known only from resting spores. Historically, these species would belong in the form-genus Tarichium, but this classification provides no information about phylogenetic relationships. Using DNA from resting spores, Zoophthora independentia, infecting Tipula (Lunatipula) submaculata in New York State, is now described as a new species and Tarichium porteri, described in 1942, which infects Tipula (Triplicitipula) colei in Tennessee, is transferred to the genus Zoophthora. We have shown that use of molecular methods can assist with determination of the phylogenetic relations of specimens within the form-genus Tarichium for an already described species and a new species for which only resting spores are available.

Natural occurrence of entomophthoroid fungi of aphid pests on Medicago sativa L. in Argentina.

Four species of entomophthoroid fungi, Pandora neoaphidis (Entomophthorales: Entomophthoraceae), Zoophthora radicans (Entomophthorales: Entomophthoraceae), Entomophthora planchoniana (Entomophthorales: Entomophthoraceae) and Neozygites fresenii (Neozygitales: Neozygitaceae) were found to infect Aphis craccivora, Therioaphis trifolii, and Acyrthosiphon pisum and unidentified species of Acyrthosiphon on lucerne in Argentina. Samples were collected from five sites (Ceres, Rafaela, Sarmiento, Monte Vera and Bernardo de Irigoyen) in the province of Santa Fe. In this study, Zoophthora radicans was the most important pathogen and was recorded mainly on Acyrthosiphon sp. Zoophthora radicans was successfully isolated and maintained in pure cultures. This study is the first report of entomophthoroid fungi infecting lucerne (Medicago sativa L.) aphids in Argentina.

Signatures of transposon-mediated genome inflation, host specialization, and photoentrainment in Entomophthora muscae and allied entomophthoralean fungi.

Despite over a century of observations, the obligate insect parasites within the order Entomophthorales remain poorly characterized at the genetic level. In this manuscript, we present a genome for a laboratory-tractable Entomophthora muscae isolate that infects fruit flies. Our E. muscae assembly is 1.03 Gb, consists of 7810 contigs and contains 81.3% complete fungal BUSCOs. Using a comparative approach with recent datasets from entomophthoralean fungi, we show that giant genomes are the norm within Entomophthoraceae owing to extensive, but not recent, Ty3 retrotransposon activity. In addition, we find that E. muscae and its closest allies possess genes that are likely homologs to the blue-light sensor white-collar 1, a Neurospora crassa gene that has a well-established role in maintaining circadian rhythms. We uncover evidence that E. muscae diverged from other entomophthoralean fungi by expansion of existing families, rather than loss of particular domains, and possesses a potentially unique suite of secreted catabolic enzymes, consistent with E. muscae's species-specific, biotrophic lifestyle. Finally, we offer a head-to-head comparison of morphological and molecular data for species within the E. muscae species complex that support the need for taxonomic revision within this group. Altogether, we provide a genetic and molecular foundation that we hope will provide a platform for the continued study of the unique biology of entomophthoralean fungi.

Entomophthovirus: an insect-derived iflavirus that infects a behavior-manipulating fungal pathogen of dipterans.

We report a virus infecting Entomophthora muscae, a behavior-manipulating fungal pathogen of dipterans. The virus, which we name Berkeley Entomophthovirus, is a positive-strand RNA virus in the iflaviridae family of capsid-forming viruses, which are mostly known to infect insects. The viral RNA is expressed at high levels in fungal cells in vitro and during in vivo infections of Drosophila melanogaster, and virus particles can be seen intracellularly in E. muscae. This virus, of which we find two closely related variants in our culture of E. muscae, is also closely related to three different viruses reported from metagenomic surveys, two of which were isolated from wild dipterans, and a third isolated from wild ticks. By analyzing sequencing data from these earlier reports, we find abundant reads aligning to E. muscae specifically in the samples from which viral reads were sequenced. These data establish a wide and perhaps obligate association with E. muscae in the wild, consistent with our laboratory data that E. muscae is the host for these closely related viruses. Because of this, we propose the name Entomophthovirus (EV) for this group of highly related virus variants. As other members of the iflaviridae have been reported to cause behavioral changes in insects, we speculate on the possibility that EV plays a role in the behavioral manipulation of flies infected with E. muscae.

A comparative characterization of indigenous keratinase enzymes from district Khairpur, Sindh, Pakistan.

To isolate and characterize keratinolytic fungi and bacteria from indigenous soils, a total of 80 samples were collected from Ghari Mori District. Khairpur, and these organisms were isolated using standard microbiological technique. The isolated keratinolytic microorganisms comprised: Absidia sp., Chrysosporium asperatum, Chrysosporium keratinophilum, Entomophthora coronata, Bacillus subtilis and Staphylococcus aureus and their keratinolytic properties were distinguished from the production of keratinase by measurement of zone of hydrolysis on skimmed milk agar (p<0.05). C.keratinophylum and B. subtilis produced largest zone among all the isolated species. The crude keratinase revealed that the optimum time for production of the enzyme was seven days, optimum temperature 30°C and optimum pH 9 for C.keratinophylum but for B. subtilis, the optimum time was three days, optimum temperature 37°C and optimum pH 7. The enzyme activity of C. keratinophylum and B. subtilis were determined to be 220 U/ml and 260 U/ml respectively (P<0.05).

Rhinoentomophthoromycosis with mutilation.

Rhinoentomophthoromycosis is a rare subcutaneous zygomycosis. We report a case of rhinoentomophthoromycosis with mutilation, which is an even more unusual phenomenon, and discuss the clinical features and management of this rare dermatosis.

Genetic variation for resistance to the specific fly pathogen Entomophthora muscae.

We found substantial variation in resistance to the fly-specific pathogen Entomophthora muscae 'Berkeley' (Entomophthoromycota), in 20 lines from the Drosophila melanogaster Genetic Reference Panel (DGRP). Resistance to E. muscae is positively (r = 0.55) correlated with resistance to the broad host range ascomycete entomopathogen Metarhizium anisopliae (Ma549), indicative of generalist (non-specific) defenses. Most of the lines showing above average resistance to Ma549 showed cross-resistance to E. muscae. However, lines that succumbed quickly to Ma549 exhibited the full range of resistance to E. muscae. This suggests fly populations differ in E. muscae-specific resistance mechanisms as well as generic defences effective against both Ma549 and E. muscae. We looked for trade-offs that could account for inter-line variation, but increases (decreases) in disease resistance to E. muscae are not consistently associated with increases (decreases) of resistance to oxidative stress, starvation stress and sleep indices. That these pathogens are dynamic agents of selection on hosts is reflected in this genetic variation for resistance in lines derived from wild populations.

Fungal artillery of zombie flies: infectious spore dispersal using a soft water cannon.

Dead sporulating female fly cadavers infected by the house fly-pathogenic fungus Entomophthora muscae are attractive to healthy male flies, which by their physical inspection may mechanically trigger spore release and by their movement create whirlwind airflows that covers them in infectious conidia. The fungal artillery of E. muscae protrudes outward from the fly cadaver, and consists of a plethora of micrometric stalks that each uses a liquid-based turgor pressure build-up to eject a jet of protoplasm and the initially attached spore. The biophysical processes that regulate the release and range of spores, however, are unknown. To study the physics of ejection, we design a biomimetic 'soft cannon' that consists of a millimetric elastomeric barrel filled with fluid and plugged with a projectile. We precisely control the maximum pressure leading up to the ejection, and study the cannon efficiency as a function of its geometry and wall elasticity. In particular, we predict that ejection velocity decreases with spore size. The calculated flight trajectories under aerodynamic drag predict that the minimum spore size required to traverse a quiescent layer of a few millimetres around the fly cadaver is approximately 10 µm. This corroborates with the natural size of E. muscae conidia (approx. 27 µm) being large enough to traverse the boundary layer but small enough (less than 40 µm) to be lifted by air currents. Based on this understanding, we show how the fungal spores are able to reach a new host.

Mitovirus and Mitochondrial Coding Sequences from Basal Fungus Entomophthora muscae.

Fungi constituting the Entomophthora muscae species complex (members of subphylum Entomophthoromycotina, phylum Zoopagamycota) commonly kill their insect hosts and manipulate host behaviors in the process. In this study, we made use of public transcriptome data to identify and characterize eight new species of mitoviruses associated with several different E. muscae isolates. Mitoviruses are simple RNA viruses that replicate in host mitochondria and are frequently found in more phylogenetically apical fungi (members of subphylum Glomeromyoctina, phylum Mucoromycota, phylum Basidiomycota and phylum Ascomycota) as well as in plants. E. muscae is the first fungus from phylum Zoopagomycota, and thereby the most phylogenetically basal fungus, found to harbor mitoviruses to date. Multiple UGA (Trp) codons are found not only in each of the new mitovirus sequences from E. muscae but also in mitochondrial core-gene coding sequences newly assembled from E. muscae transcriptome data, suggesting that UGA (Trp) is not a rarely used codon in the mitochondria of this fungus. The presence of mitoviruses in these basal fungi has possible implications for the evolution of these viruses.

Elephantine nose due to rhinoentomophthoromycosis.

Rhinoentomophthoromycosis in an immunocompetent Indian male due to Basidiobolus species resulting in a huge (elephantine) nasal deformity, is reported. The diagnosis was done by demonstration of hyphae in direct tissue smear examination in potassium hydroxide, histopathological examination and by cultural characteristics. He showed an excellent response to oral potassium iodide solution.

Epizootics of an entomopathogenic fungus Entomophthora aphidis Hoggman on the pomegranate aphid aphispunicae passerini (Homoptera: Aphididae).

Thrips of late are becoming more resurgent in agro-ecosystems especially in India and South Asian countries. One of the reasons attributed is the development of resistance in them to groups of insecticides of organo phosphates, carbamates, synthetic pyrethroids, etc., which form the core of recommendation for thrips management. The chilli thrips, Scirtothrips dorsalis Hood, is no exception and is tending to be an unmanageable pest. In S. dorsalis, besides evidence of resistance, elimination of natural enemies like Orius sp. is evident, which also probably explains the thrips resurgence. Scirtothrips dorsalis is highly polyphagous and of late has become serious on grapes. The damage is manifested as scab on fruit rind, affecting internal and export markets. Fortunately, it does not vector any virus on grapes in India. Farmers have been reporting inefficacy of established chemicals like acephate, dimethoate, monocrotophos, etc. As grapes is an important cash crop of horticulture, management through insecticides, require alternate or new insecticides. So two trials in 2005 on cv. Bangalore Blue following April and October prunings were conducted at the Indian Institute of Horticultural Research (IIHR), Bangalore, S. India to evaluate two doses of a newer molecule viz. Clothianidin [Dantop] on S. dorsalis. Clothianidin 0.006%, Clothianidin 0.008% were compared with monocrotophos 0.05%, dimethoate 0.06% and acephate 0.1125%. There was an untreated check. Each treatment was replicated five times; a vine constituted a replicate. These data were subjected to ANOVA, after arc sine transformation. The critical difference (CD) at p < 0.05 was the test criterion. In the first trial it was found that all the insecticides tried were superior to check and were on par at p < 0.05. However, Clothianidin 0.008% gave the best control with a low mean of 0.26% berry damage/bunch as compared to 4.42% in the unsprayed check. The trend was the same in the second trial also with Clothianidin 0.008% giving the best control with 0.64% berry damage/bunch as compared to 8.49% in the unsprayed check. It was on par with acephate and monocrotophos, but significantly superior to Clothianidin 0.006% and dimethoate.

Study of the long chain bases of sphingomyelin of Entomophthora coronata.

Sphingomyelin was isolated from a strain of Entomophthora coronata, a fungus pathogenic for humans. After hydrolysis, the long chain bases were converted to their dinitrophenyl (N2pH) derivatives and the aldehydes prepared by oxidizing these compounds with periodic acid. The aldehydes were studied by gas chromatography. Twelve different aldehydes were identified, the chain distributiion ranging from C14 to C17. The prominent chains were unsaturated. Straight and branched chains were found. The most abundant parent base which formed 52% of the total aldehyde was a 1,3-dihydroxy-2-aminohexadecene.

Laboratory and clinical assessment of ketoconazole in deep-seated mycoses.

Forty-eight cases of deep mycoses were studied and treated with ketoconazole, each with in vitro evaluation of the minimum inhibitory concentrations (MIC) of the causative fungi, in vivo pharmacokinetic, clinical, and mycologic evaluations, several months to two years after the treatment was stopped. Excellent results were obtained in six cases of chronic mucocutaneous candidiasis, with restoration of immunologic disturbances; 23 cases of systemic candidiasis, including new aspects of heroin addicts with cutaneous, ocular, or osteoarticular manifestations; eight cases of histoplasmosis, five due to Histoplasma capsulatum and three to Histoplasma duboisii, with cure in seven and remission in one; one case of African blastomycosis (Blastomyces dermatitidis); three cases of mycetoma, two due to Monosporium apiospermum, one due to a dematiacious fungus; three cases of entomophthoromycosis with cure; one case of fungal arthritis, due to new hyphomycete similar to M. apiospermum, pathogenic for laboratory animals; one case of Drechslera longirostrata causing vertebral arthritis, following a fungal endocarditis and cured by combination of ketoconazole with amphotericin B, each agent alone being ineffective; and other deep mycoses.

Visualization of exocellular lectins in the entomopathogenic fungus Conidiobolus obscurus.

We demonstrated the presence of lectins binding to glucose and N-acetylglucosamine on the surface of Conidiobolus obscurus spores by using glycosylated serum albumins substituted with fluorescent dyes and colloidal gold. The role of these exocellular lectins was examined in relation to the adhesion of the fungal spores to their host insect and the pathogenicity of the fungus.

Rhinofacial zygomycosis caused by Conidiobolus coronatus: a case report including in vitro sensitivity to antimycotic agents.

A case of zygomycosis caused by Conidiobolus coronatus, occurring in a 31-year-old priest from the Ivory Coast, is reported. Lesions eventually resolved after 2 1/2 years, during which the patient was variously treated with intravenous amphotericin B and miconazole as well as surgical resection. It is difficult to be certain which, if any, of these treatments contributed to resolution of lesions. A report of in vitro sensitivity data to a variety of antifungal agents is included.

Cloning of two chitin synthase gene fragments from a protoplastic entomophthorale.

Chitin synthase expression was studied in spontaneously produced protoplasts and in hyphal bodies of the Entomophthorale species Entomophaga aulicae. The absence of wall in protoplasts was correlated to an absence of chitin synthase. Two chitin synthase activities with different biochemical characteristics have been detected in the hyphal bodies. Two chitin synthase gene fragments EaCHS1 and EaCHS2 of 600 bp were obtained using PCR amplification of genomic DNA. Their amino acid sequences showed 75% identity. Compared with other fungal chitin synthases, they belong to class II. EaCHS1 and EaCHS2 were used to probe total RNA from E. aulicae hyphal bodies and protoplasts. A single transcript of 2.4 kb hybridized only with EaCHS1 in protoplasts and hyphal bodies.