fungal biology 121 (2017) 561e572 journal homepage: www.elsevier.com/locate/funbio Ultrastructural characterization of the hosteparasite interface between Allomyces anomalus (Blastocladiomycota) and Rozella allomycis (Cryptomycota) Martha J. POWELLa, Peter M. LETCHERa,*, Timothy Y. JAMESb aDepartment of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487, USA bDepartment of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI 48109, USA article info abstract Article history: Rozella allomycis is an obligate endoparasite of the water mold Allomyces and a member of Received 16 December 2016 a clade (¼ Opisthosporidia) sister to the traditional Fungi. Gaining insights into Rozella’s de- Received in revised form velopment as a phylogenetically pivotal endoparasite can aid our understanding of struc- 8 March 2017 tural adaptations and evolution of the Opisthosporidia clade, especially within the context Accepted 13 March 2017 of genomic information. The purpose of this study is to characterize the interface between Available online 21 March 2017 R. allomycis and Allomyces anomalus. Electron microscopy of developing plasmodia of R. al- Corresponding Editor: lomycis in host hyphae shows that the interface consists of three-membrane layers, inter- Gordon William Beakes preted as the parasite’s plasma membrane (inner one layer) and a host cisterna (outer two layers). As sporangial and resting spore plasmodia develop, host mitochondria typically Keywords: cluster at the surface of the parasite and eventually align parallel to the three-membrane Evolution layered interface. The parasite’s mitochondria have only a few cristae and the mitochon- Interface drial matrix is sparse, clearly distinguishing parasite mitochondria from those of the Mitochondrial recruitment host. Consistent with the expected organellar topology if the parasite plasmodia phagocy- Parasitism tize host cytoplasm, phagocytic vacuoles are at first bounded by three-membrane layers Phagocytosis with host-type mitochondria lining the inner membrane. Thus, Rozella’s nutrition, at least in part, is phagotrophic in contrast to osmotrophic nutrition of traditional fungi. ª 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction plasmodium uses the host wall as its own as it cleaves into nu- merous posteriorly uniflagellated zoospores, which are dis- Species of Rozella Cornu are obligate endoparasites of Oomy- charged through one to multiple discharge pores or tubes. cetes, Chytridiomycetes, Monoblepharidomycetes, Blastocla- Thick-walled resting spores may also be produced, and they diomycetes, and green algae (Sparrow 1960) in which the lie loosely within the host. unwalled, multinucleate sporangial plasmodium eventually Because Rozella reproduces asexually with the formation of completely fills the host. At maturity the sporangial posteriorly uniflagellate zoospores, this genus has in general * Corresponding author. Department of Biological Sciences, 1332 SEC, Box 870344, The University of Alabama, Tuscaloosa, AL 35487, USA. Tel.: þ1 205 348 8208; fax: þ1 205 348 1786. E-mail addresses: [email protected] (M.J. Powell), [email protected] (P.M. Letcher), [email protected] (T.Y. James). http://dx.doi.org/10.1016/j.funbio.2017.03.002 1878-6146/ª 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved. 562 M. J. Powell et al. been considered a member of the Chytridiomycetes (Barr in Hattiesburg, MS. Cultures were maintained on 1/8 strength 1980; Held 1975; Sparrow 1938, 1960), but its placement has Emerson YPSS agar (Difco 273910). been controversial (reviewed in Karling 1942). Based on zoo- spore ultrastructure of Rozella allomycis Foust (Held 1975), Transmission electron microscopy Barr (1980) classified Rozella in the Spizellomycetales. How- ever, the shape, flagellar apparatus organization, and organel- Infected hyphae were collected at 2e6 d after inoculation of lar arrangements of R. allomycis zoospores are quite distinct host with zoospores of the parasite. Host hyphae infected from these features in zoospores of core Spizellomycetales with the parasite were prepared for electron microscopy as members, and Barr (1980) in fact questioned the validity of described (Letcher & Powell 2005) and thin embedded so that classifying it in the Spizellomycetales. infected regions could be selected, excised, and mounted on The discovery that R. allomycis placed in a clade sister to all plastic stubs for sectioning. Thin sections were cut with a Leica other Fungi was an unexpected result of the first broad-based ultramicrotome and mounted on 300 mesh grids. Serial sec- phylogenetic analysis of zoosporic Fungi (James et al. 2006). tions were mounted on 30 nm coated slot grids. Sections Following this study, phylogenetic analyses of molecular envi- were stained with uranyl acetate and lead citrate and ob- ronmental surveys from diverse habitats have revealed nu- served on a Hitachi 7650 transmission electron microscope merous phylotypes that form a clade with R. allomycis, at 60 kV. Rozella ex. Rhizoclosmatium JEL 347, Rozella ex Pythium, and Rozella rhizoclosmatii Letcher and Longcore (Corsaro et al. Results 2016; James & Berbee 2012; Jones et al. 2011; Lara et al. 2010; Lazarus & James 2015; Letcher et al. 2016b). To recognize the Light microscopy of Rozella allomycis development distinctive nature of the clade containing Rozella species and environmental phylotypes, Jones et al. (2011) erected the new Uninfected hyphae of Allomyces are typically aseptate, except phylum, Cryptomycota (Jones et al. 2011; Karpov et al. 2014) for the septal delimitation of their reproductive structures (¼ Rozellomycota, Corsaro et al. 2014). (vegetative sporangia, resting sporangia or gametangia). How- Cryptomycota is phylogenetically related to two other ever, the parasite induces host hyphae to produce septa, which groups of plasmodial endoparasites, which once were consid- compartmentalize the parasite plasmodia as they develop into ered disparate groups: Aphelida, parasites of algae which unwalled sporangial plasmodia (Fig 1A and B) or walled resting phagocytize host cytoplasm (Karpov et al. 2013; Letcher et al. spores (Fig 1CeE). Often times these host compartments that 2013, 2015; Schnepf et al. 1971), and Microsporidia, parasites result from the parasite’s induction of septation are swollen of animal cells which exhibit an osmotrophic mode of nutri- (Fig 1D). At maturity, sporangial plasmodia and resting spores tion (Corradi 2015). Because these three groups share charac- occupy separate host compartments (Fig 1E). teristics with both fungi and protists, there is debate as to In the infection process, a single parasite sporangial plas- which kingdom they belong (Grossart et al. 2016). Suggesting modium completely fills a host compartment (Fig 1A). In basip- that they share characteristics with both kingdoms, Karpov etal progression, each sporangial plasmodium cleaves into et al. (2014) erected the superphylum Opisthosporidia for these numerous zoospores (Fig 1A). Zoospores undergo a character- three groups. Because of their pivotal phylogenetic position in istic period of roiling before leaving the host compartment as the evolution of fungi and divergence from protists, it is im- a mass through discharge papillae in the host wall (Fig 1A). Af- portant to characterize this group more fully. Characterizing ter zoospore discharge, host compartments are empty, and the the nature of the interaction between R. allomycis and its blas- irregular configuration of septa is visible (Fig 1B). tocladialean host, Allomyces anomalus R. Emers., at the cellular Unlike sporangial plasmodium development, during rest- level is vital to an understanding of fungal evolution (Stajich ing spore development multiple plasmodia occupy a host et al. 2009). compartment (Fig 1C). Plasmodia developing into resting Foust (1937) demonstrated that R. allomycis induces its host spores are irregular in outline at first but eventually become hyphae to produce septa across their coenocytic hyphae and spherical (Fig 1C). The spherical plasmodia then become to compartmentalize infection, which suggests a great degree more compact, and walls appear around the resting spore of host manipulation in which the parasite maintains major plasmodia (Fig 1D). Resting spore wall formation is not syn- host function solely for its own reproduction. Held (Held chronous within a compartment (Fig 1D) and some rounded 1973, 1974, 1975, 1980, 1981) made extensive developmental plasmodia abort before maturing into resting spores (Fig 1E). studies of R. allomycis in Allomyces sp. but never described de- Remnants of host cytoplasm remain in host compartments af- tails of the hosteparasite interface. The purpose of this study ter resting spores are formed (Fig 1CeE). is to explore the ultrastructure of the interface between R. allo- mycis and A. anomalus. Transmission electron microscopy of hosteparasite interface Materials and methods Sporangial plasmodial stage The developing sporangial plasmodium is lobed and irregular Culture in outline (Fig 2A). There are numerous nuclei which are dis- tinctive from host nuclei because the nucleolus is crescent- Rozella allomycis (CSF 55) on Allomyces anomalus was isolated by shaped and adpressed to the inner surface of the nuclear en- Timothy James from a sample collected from a roadside ditch velope (Fig 2A), rather than oval and peripherally located in Ultrastructural characterization of the hosteparasite interface 563 Fig 1 e