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.

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.

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.

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.

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.

Intraspecific variation and host specificity of Entomophthora muscae sensu stricto isolates revealed by random amplified polymorphic DNA, universal primed PCR, PCR-restriction fragment length polymorphism, and conidial morphology.

The intraspecific variations of Entomophthora muscae s. str. associated with particular host species, Musca domestica and Delia radicum, sampled from different localities and different years in Denmark and the variation of E. muscae s. str. originating from different host taxa were investigated. The isolates were compared both by primary spore morphology and by three molecular methods: random amplified polymorphic DNA, universal primed PCR, and PCR-restriction fragment length polymorphism. Analyses of the different molecular data showed the same overall picture and separated E. muscae s. str. into two main groups with all the M. domestica isolates in one group and isolates from D. radicum, Coenosia tigrina, and Pegoplata infirma in the second group. E. muscae s. str. isolates from M. domestica also differ significantly from the rest of the E. muscae s. str. isolates with regard to the morphology of the primary conidia, which were bigger and contained significantly more nuclei per conidium. Several different E. muscae s. str. genotypes were documented and each type was restricted to a single host species, indicating a very high degree of host specificity at or below the level of the subfamily.

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.

Nucleosome DNA repeat length and histone complement in a fungus exhibiting condensed chromatin.

Fungal chromatins are reported to exhibit unusually short nucleosomal DNA repeat lengths. To test whether this is a phylogenetic feature of fungi or rather is correlated with an apparent absence of condensed chromatin in the organisms studied, we have examined the chromatin organization and the complement of basic nuclear proteins in the fungus Entomophthora, an organism which exhibits marked chromatin condensation. Micrococcal nuclease digestion of Entomophthora chromatin revealed a nucleosomal DNA repeat length of 197 +/- 1.2 base pairs (bp). This repeat length is 20-40 bp longer than that reported for any fungus. Entomophthora nucleosomes exhibited an HI-like protein which was much less basic than the HI histones reported for higher eukaryotes but which was similar in basicity to the HI histone reported for the fungus Neurospora. However, the nucleosomal DNA repeat length of Neurospora chromatin is reported to be unusually short, whereas that of Entomophthora was found to be typical of the repeat lengths observed for chromatins of higher eukaryotes. Thus, repeat length, at least in fungi, would not appear to be directly determined by the basicity of the fungal cognate of histone HI.