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

<i>Entomophthora</i>-infected <i>Delia</i> flies from Durham, North Carolina, USA.

<p>A. Fly cadavers on wild onions (a) and grasses (b). B. Fresh fly firmly attached to wild onion; the yellow color of the abdomen indicates active sporulating of conidia. C. Sporulation from an infected fly. D. Proboscis of the fly attached to the plant. E. Fly abdomen, sporulating with conidia (a) on comparatively young “hymenial” zones separated by dorsal cuticular tergites (b). F. Conidiophores (a) breaking through mechanically folded cuticle (b).</p

Phylogenetic analysis of <i>E. muscae</i> from Durham, NC, USA.

<p>Del_ indicate fungal sequences extracted from fly <i>Delia</i> (grey rectangles), Coe_ – from <i>Coenosia</i> (yellowish rectangles). Number after each sample (−71) indicates the number of identical sequences. Thick lines indicate branches with strong bootstrap support (>70%). A. ML tree of ITS sequences of genus <i>Entomophthora</i> with dominate genotypes from Durham, NC. B. ML tree of 120 ITS sequences of <i>Entomophthora</i>, isolated from <i>Coenosia</i> and <i>Delia</i> flies, representing all ITS sequences which differ in one or more bp positions or by host.</p

Percentage of rDNA identity and gap lengths for gene fragments from <i>E. muscae</i> specimens from Durham compared to other published <i>Entomophthora</i> and outgroup species sequences.

<p>99–100/0 - here 99–100% of identity and/0% of gaps to the whole length of fragment, bp.</p>*<p>also includes genotypes KC404070–71, KC404078–88.</p>**<p>also includes genotypes KC404090–103.</p>***<p>variability in different isolates.</p

Conidiophores and hymenial layer of <i>Entomophthora</i>-infected flies from Durham, North Carolina, USA.

<p>A. Conidiophore layer between sclerites of fly cuticle, SEM. B. Conidiophores breaking through the cuticle of the fly, SEM. C. Conidiophore layer with young conidia, SEM. D. Conidiophores with young conidia stained with cotton blue. E. Nuclei in conidiophores stained with DAPI. F. Hypha (a) and young zygospore stained with cotton blue (b).</p

Unrooted trees of 22 randomly selected <i>E. muscae</i> samples using <i>RPB2</i> and <i>EFL</i>.

<p>Del_ indicate fungal sequences extracted from <i>Delia</i> (grey rectangles), Coe_ – from <i>Coenosia</i> (yellowish rectangles). Thick lines indicate branches with strong bootstrap support (>70%).</p

Summary of reported primary conidial sizes and numbers of nuclei per conidium (mean ± SE and range of means) of <i>E. muscae</i> isolates from different hosts and in species of the genus <i>Entomophthora</i>[4]–[6], [26], [29], [47].

<p>//indicates morphological characteristics of collections from <i>E. muscae</i> species complex affecting these different muscoid fly hosts.</p

The Early Terrestrial Fungal Lineage of Conidiobolus&mdash;Transition from Saprotroph to Parasitic Lifestyle

Fungi of the Conidiobolus group belong to the family Ancylistaceae (Entomophthorales, Entomophthoromycotina, Zoopagomycota) and include over 70 predominantly saprotrophic species in four similar and closely related genera, that were separated phylogenetically recently. Entomopathogenic fungi of the genus Batkoa are very close morphologically to the Conidiobolus species. Their thalli share similar morphology, and they produce ballistic conidia like closely related entomopathogenic Entomophthoraceae. Ballistic conidia are traditionally considered as an efficient tool in the pathogenic process and an important adaptation to the parasitic lifestyle. Our study aims to reconstruct the phylogeny of this fungal group using molecular and genomic data, ancestral lifestyle and morphological features of the conidiobolus-like group and the direction of their evolution. Based on phylogenetic analysis, some species previously in the family Conidiobolaceae are placed in the new families Capillidiaceae and Neoconidiobolaceae, which each include one genus, and the Conidiobolaceae now includes three genera. Intermediate between the conidiobolus-like groups and Entomophthoraceae, species in the distinct Batkoa clade now belong in the family Batkoaceae. Parasitism evolved several times in the Conidiobolus group and Ancestral State Reconstruction suggests that the evolution of ballistic conidia preceded the evolution of the parasitic lifestyle