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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., (© 2016 John Wiley & Sons Ltd.)

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.

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.

Neozygites fresenii-infected Aphis gossypii cadavers, containing dormant hyphal bodies of N. fresenii, were stored in 4 ml glass vials at -14 degrees C in a standard consumer-type refrigerator/freezer for 1, 21, 30, 43, 51, and 68 months to determine the effect of storage on fungal survival. When the cadavers were removed from the freezer and placed in 25+/-1 degrees C, 100% relative humidity, and 12:12 (L:D) conditions, N. fresenii survival, as shown by fungal sporulation from the cadavers, was high at all storage periods. The average percentage of cadavers from which the fungus sporulated were 93, 47, 100, 100, 80, and 60% from 1, 21, 30, 43, 51, and 68 months storage periods, respectively. The number of primary conidia discharged from each sporulating cadaver was estimated using a scale of 1 (low, ca. 1000 primary conidia), 2 (medium, ca. 2000 primary conidia) and 3 (high, ca. 3000 primary conidia). The median scores for the number of primary conidia produced per sporulating cadaver were 3, 2, 3, 3, 2.5, and 1 for 1, 21, 30, 43, 51, and 68 months, respectively. Therefore, except for the longest storage period, most cadavers produced medium to high numbers of primary conidia. Mean germination of primary conidia produced from N. fresenii-infected-aphid cadavers from each time period varied significantly from 66.3 to 86.1% in the 21 and 43 months categories, respectively. Infectivity of capilliconidia, produced from frozen N. fresenii, to live healthy cotton aphids varied significantly from 16.7 to 68.7% from cadavers stored 68 months and 1 month, respectively. Overall N. fresenii survived well in dried frozen cotton aphid cadavers for up to 6 years with little reduction in sporulation, numbers of spores produced, germination of primary conidia, or infectivity.

In temperate regions, insect pathogenic fungi face the challenge of surviving through the winter. Winter is a time when hosts are immobile, low in number or are present in a stage which is not susceptible to infection. Fungi from Entomophthoromycota have so far been known to survive the winter in two ways: either as (1) thick-walled resting spores released into environment from dead hosts, or as (2) structures inside the dead host (e.g., hyphal bodies). Here we report, from the Danish environment, a third way to survive the winter, namely a slow progression and transmission of Entomophthora schizophorae in adult dipteran Pollenia hosts that hibernate in clusters in unheated attics, sheltered areas outdoors (under bark etc.). Fungus-killed sporulating flies were observed outside very early and very late in the season. By sampling adults at the time of their emergence from hibernation in late winter/early spring we documented that the fungus was naturally prevalent and killed flies after a period of incubation. Experimentally we documented that even at the low temperature of 5 °C, the fungus was able to maintain itself in Pollenia cohorts for up to 90 days. From these observations the full winter cycle of this fungus is elucidated. The three types of winter survival are discussed in relation to fungus epidemic development. [ABSTRACT FROM AUTHOR]

Three species within the Entomophthora muscae (Cohn) Fresenius complex (Entomophthora schizophorae Keller & Wilding, E. muscae s.str., and E. muscae "group B") were investigated for resting spore formation in vivo in the house fly (Musca domestica L.). Resting spores of E. muscae group B were experimentally induced from August to the beginning of February, while no resting spores were ever observed in E. schizophorae infected M. domestica or in flies infected by E. muscae s.str. originating from M. domestica. When newly dead fly cadavers containing E. muscae group B resting spores were kept moist, cystidia emerged from the abdomen; this is the first report of cystidia in the genus Entomophthora. Resting spore production was significantly affected by both temperature and E. muscae group B strain. More infected flies formed resting spores when kept 1 week at 10°C compared with constant exposure at 22°C, but the tendency of the different E. muscae group B strains to form resting spores persisted with shifting temperatures. After 4 months of incubation under natural winter conditions in Denmark, E. muscae group B resting spores germinated on water agar at 20°C with a 16 h light : 8 h dark photoperiod within 1 week, but no germ conidia were observed.Key words: Entomophthorales, Entomophthora muscae complex, Diptera, Musca domestica, resting spore formation, resting spore germination.Les auteurs ont examiné, chez la mouche de maison (Musca domestica L.), la formation in vivo des spores de repos de trois espèces du complexe Entomophthora muscae (Cohn) Fresenius (Entomophthora schizophorae Keller & Wilding, E. muscae, s.str., et E. muscae "groupe B"). Les auteurs ont induit expérimentalement la formation de spores de repos de l'E. muscae groupe B, à partir du mois d'août jusqu'en février, alors qu'aucune spore de repos n'a jamais été observée chez les M. domestica infectées par l'E. schizophorae, ou chez des mouches infectées par l'E. muscae s.str., provenant de M. domestica. Lorsque qu'on maintient humide des cadavres de mort récente, contenant des spores de repos de l'E. muscae groupe B, des cystides émergent de l'abdomen; il s'agit de la première mention de cystides pour le genre Entomophthora. La production des spores de repos est significativement affectée à la fois par la température et par la souche d'E. muscae groupe B. Un plus grand nombre de mouches infectées forment des spores de repos lorsqu'on les maintient à 10°C pendant une semaine comparativement à une exposition constante à 22°C, mais la tendance des différentes souches de l'E. muscae groupe B à former des spores de repos persiste avec des températures changeantes. Après 4 mois d'incubation sous les conditions hivernales naturelles du Danemark, les spores de l'E. muscae groupe B germent sur eau gélosée à 20°C et sous une photopériode de 16 h : 8 h L:O, en moins d'une semaine, sans qu'on puisse observer de conidies de germination.Mots clés : Entomophthorales, complexe Entomophthora muscae, Diptères, Musca domestica, formation de spores de repos, germination de spores de repos.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]

Pandora neoaphidis and Entomophthora planchoniana (phylum Entomophthoromycota) are important fungal pathogens on cereal aphids, Sitobion avenae and Rhopalosiphum padi. Here, we evaluated and compared for the first time the virulence of these two fungi, both produced in S. avenae cadavers, against the two aphid species subjected to the same exposure. Two laboratory bioassays were carried out using a method imitating entomophthoralean transmission in the field. Healthy colonies of the two aphid species were exposed to the same conidial shower of P. neoaphidis or E. planchoniana, in both cases from a cadaver of S. avenae. The experiments were performed under LD 18:6 h at 21 °C and a successful transmission was monitored for a period of nine days after initial exposure. Susceptibility of both S. avenae and R. padi to fungal infection showed a sigmoid trend. The fitted nonlinear model showed that the conspecific host, S. avenae, was more susceptible to E. planchoniana infection than the heterospecific host R. padi, was. In the case of P. neoaphidis, LT50 for S. avenae was 5.0 days compared to 5.9 days for R. padi. For E. planchoniana, the LT50 for S. avenae was 4.9 days, while the measured infection level in R. padi was always below 50 percent. Our results suggest that transmission from conspecific aphid host to heterospecific aphid host can occur in the field, but with expected highest transmission success to the conspecific host. [ABSTRACT FROM AUTHOR]