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.

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.

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.

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.

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).

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.

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

Sequential utilization of hosts from different fly families by genetically distinct, sympatric populations within the Entomophthora muscae species complex.

The fungus Entomophthora muscae (Entomophthoromycota, Entomophthorales, Entomophthoraceae) is a widespread insect pathogen responsible for fatal epizootic events in many dipteran fly hosts. During epizootics in 2011 and 2012 in Durham, North Carolina, we observed a transition of fungal infections from one host, the plant-feeding fly Delia radicum, to a second host, the predatory fly Coenosia tigrina. Infections first appeared on Delia in the middle of March, but by the end of May, Coenosia comprised 100% of infected hosts. Multilocus sequence typing revealed that E. muscae in Durham comprises two distinct subpopulations (clades) with several haplotypes in each. Fungi from either clade are able to infect both fly species, but vary in their infection phenologies and host-specificities. Individuals of the more phylogenetically diverse clade I predominated during the beginning of the spring epizootic, infecting mostly phytophagous Delia flies. Clade II dominated in late April and May and affected mostly predatory Coenosia flies. Analysis of population structure revealed two subpopulations within E. muscae with limited gene exchange. This study provides the first evidence of recombination and population structure within the E. muscae species complex, and illustrates the complexity of insect-fungus relationships that should be considered for development of biological control methods.

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).

Biocontrol of pigeon tick Argas reflexus (Acari: Argasidae) by entomopathogenic fungus Metarhizium Anisopliae (Ascomycota: Hypocreales)

The pigeon tick Argas reflexus is a pathogen-transmitting soft tick that typically feeds on pigeons, but can also attack humans causing local and systemic reactions. Chemical control is made difficult due to environmental contamination and resistance development. As a result, there is much interest in increasing the role of other strategies like biological control. In this study, the efficacy of three strains (V245, 685 and 715C) of entomopathogenic fungus Metarhizium anisopliae for biological control of three life stages of pigeon tick A. reflexus including eggs, larvae, engorged and unfed adults was investigated under laboratory conditions. Five concentrations of different strains of M. anisopliae ranging from 10³ to 10(7) conidia/ml were used. All fungal strains significantly decreased hatchability of A. reflexus eggs. Strain V245 was the most effective strain on the mortality of larval stage with nearly 100 percent mortality at the lowest concentration (10³ conidia/ml) at 10 days post-inoculation. The mortality rate of both engorged and unfed adult ticks were also increased significantly exposed to different conidial concentrations compared to the control groups (P < 0.05) making this fungus a potential biological control agent of pigeon tick reducing the use of chemical acaricides.

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.

Differential divergences of obligately insect-pathogenic Entomophthora species from fly and aphid hosts.

Three DNA regions (ITS 1, LSU rRNA and GPD) of isolates from the insect-pathogenic fungus genus Entomophthora originating from different fly (Diptera) and aphid (Hemiptera) host taxa were sequenced. The results documented a large genetic diversity among the fly-pathogenic Entomophthora and only minor differences among aphid-pathogenic Entomophthora. The evolutionary time of divergence of the fly and the aphid host taxa included cannot account for this difference. The host-driven divergence of Entomophthora, therefore, has been much greater in flies than in aphids. Host-range differences or a recent host shift to aphid are possible explanations.

Variability in thermal responses among Furia gastropachae isolates from different geographic origins.

The effect of temperature ranging from 5-30 degrees C on in vitro vegetative growth and conidial germination of isolates of the entomophthoralean fungus Furia gastropachae was investigated. Eleven isolates were used for growth studies; two from Maryland, six from New York, and three from Ontario. A subset of four isolates, one each from Maryland and New York and two from Ontario, were used in conidial germination experiments. Growth and germination were significantly associated with temperature for all isolates, occurring throughout the range 5-30 degrees C, though both processes were inhibited to varying degrees at upper and lower extremes. Temperature optima for growth ranged from 20 to 27 degrees C, and for germination from 20 to 25 degrees C. Although significant variability was observed among isolates in growth at temperatures above 13 degrees C, temperature optima were not significantly different among isolates, and variability did not appear to relate to the geoclimatic origins of the isolates. In contrast, germination responses to temperature did appear to be related to geographic origin. Furia gastropachae isolates from New York and Maryland germinated more slowly at 10 degrees C than did Ontario isolates, although the percentage of conidia ultimately germinating at each temperature was the same for all isolates. The New York and Maryland isolates performed much better at 30 degrees C, with significantly greater overall germination and secondary conidial discharge, than the Ontario isolates. Compared with other isolates at 30 degrees C, Ontario isolates were the least active, often failing to successfully discharge any secondary conidia.

Status of Eldana saccharina (Lepidoptera: Pyralidae), its host plants and natural enemies in Ethiopia.

Surveys for sugarcane stem borers were undertaken in Ethiopia to determine the prevalence and distribution of these and their natural enemies in crops and indigenous host plants. Eldana saccharina Walker was not recovered from sugarcane, but was present in three indigenous wetland sedges, Cyperus papyrus, C. fastigiatus and C. dives in the southern, central and northern part of the country. The latter indigenous host plant was present in waterways adjacent to sugarcane on the commercial sugar estates. The tachinids Schembria eldanae Barraclough and Actia sp. were common parasitoids of E. saccharina larvae in these indigenous sedges. The braconid Dolichogenidea sp. was recovered from E. saccharina larvae in C. dives. Pathogens comprising Beauveria bassiana, Bacillus thuringiensis and Entomophthora sp., were found to be important mortality factors of E. saccharina larvae in the indigenous sedges. The occurrence of E. saccharina in Ethiopia is reported for the first time, and the host plant preferences of the borer and its indigenous natural enemies found during the surveys are recorded. In addition, its potential threat to sugarcane production in Ethiopia is discussed.

Value of host range, morphological, and genetic characteristics within the Entomophthora muscae species complex.

Entomopthora muscae sensu lato is a complex of morphologically similar fungal species pathogenic to evolutionarily advanced flies (Cyclorrhapha). To reach an operational species definition and recognition of species within this complex, the values of host range, morphological and genetic characteristics are reconsidered. Within the E. muscae species complex morphological and nuclear characteristics of the primary conidia are taxonomically important. In this study we compared the dimensions and nuclear numbers of the primary conidia of isolates from their original (natural) hosts and after being transferred to alternative hosts (cross-transmission) in order to check the stability of these characteristics. The conidial characteristics change substantially when produced in alternative host species, but their overall range in variability still fit within the traditional morphological species circumscriptions. The phylogenetic analyses of the ITS II and LSU rRNA gene sequences, revealed three distinct lineages within the complex: E. schizophorae, E. muscae and E. syrphi. Within each of these lineages sequence divergence was seen between isolates originating from different host species. Our studies on the physiological host range showed that several isolates were able to infect alternative dipteran species. Musca domestica was a particularly good receptor. The ecological host range of any individual isolate seems, however, to be limited to one host species evidenced by the occurrence of distinct genotypes within each natural infected host species shown by RAPD. The high host specificity of these fungi emphasizes the importance of identifying the host taxon at species level in the recognition of Entomophthora species. We recommend that morphological characteristics of fungal structures and host taxon, together with molecular data, serve as criteria for species determination in future studies on members of the E. muscae complex.

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.