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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy fungal biology 116 (2012) 90e97 journal homepage: www.elsevier.com/locate/funbio Basidiobolus haptosporus is frequently associated with the gamasid mite Leptogamasus obesus Sebastian WERNER, Derek PERSOH*, Gerhard RAMBOLD University of Bayreuth, Dept. of Mycology, Universitatsstraße€ 30, D-95447 Bayreuth, Germany article info abstract Article history: Two species of mites inhabiting a pine forest soil were screened for associated fungi. The Received 23 February 2011 fungal community composition was assessed in 49 mite and 19 soil samples by environ- Received in revised form mental PCR with a focus on fungi of the genus Basidiobolus. PCR products of the fungal 11 October 2011 ITS rRNA gene were analyzed by sub-cloning, RFLP-analysis, and sequencing. Thereby Ba- Accepted 12 October 2011 sidiobolus haptosporus was found for the first time to be frequently associated with the ga- Available online 20 October 2011 masid mite species Leptogamasus obesus, while being absent from the oribatid mite Oppiella Corresponding Editor: subpectinata, and from the surrounding soil. The fungus was isolated in pure culture for Richard A. Humber a detailed morphological characterization and experimental approaches concerning the nature of this fungusemite association. The experiments and a supporting microscopic Keywords: screening of freshly captured gamasid mites revealed no indications for the fungus being Acari localized in the mites’ gut or haemocoel, but a single spore was found attached to an indi- Arthropoda vidual of L. obesus. However, an exclusive phoretic association does not satisfactorily ex- Basidiobolus haptosporus plain the frequent detection of B. haptosporus DNA on or in L. obesus, and the absence of Fungi the fungus from soil samples seems not to be in line with its assumed ecology as a wide- Leptogamasus obesus spread saprobic soil fungus. Therefore, a second host species in the life cycle of B. haptospo- Mites rus is discussed as a working hypothesis. Molecular analyses ª 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved. Oppiella subpectinata Introduction GenBank (e.g., Huang et al. unpublished), but most of them de- rived from well referenced strains of the ARSEF, ATCC and The genus Basidiobolus was erected by Eidam (1886) for two NRRL culture collections. species, with Basidiobolus ranarum Eidam as generic type. An Members of Basidiobolus have been shown to be distributed extensive monographic treatment of the genus comprising worldwide (Cannon & Kirk 2007). They have been isolated eight taxa dates back to Drechsler (e.g., 1952a, 1953, 1955, from a wide range of different substrates like soils, decaying 1964). Formerly placed in the order Entomophthorales (Zygomy- fruits or rotten vegetation (Drechsler 1947; Coremans- cota), the Basidiobolales are currently treated as an order of its Pelseneer 1974). However, its representatives also occur in own, as molecular studies indicated a closer relationship to the guts and feces of amphibians, reptiles, and other verte- the Chytridiomycota (Nagayama et al. 1995; Tanabe et al. 2004; brates, which mainly feed on arthropods (Benjamin 1962; White et al. 2006). However, a precise taxonomic assignment Coremans-Pelseneer 1974). Certain Basidiobolus species are of the genus is not yet possible. Currently only few ITS rRNA even capable of infecting mammalian (Joe et al. 1956; Greer & sequences of Basidiobolus spp. are publicly available in Friedman 1966) and reptilian (Taylor et al. 1999) dermal tissue. * Corresponding author. Tel.: þ49 (0) 921 55 2456; fax: þ49 (0) 921 55 2567. E-mail address: [email protected] 1878-6146/$ e see front matter ª 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2011.10.004 Author's personal copy B. haptosporus is associated with L. obesus 91 Additionally, members of various groups of insects are known Additional mites were extracted for isolation of associated to be hosts of or to carry Basidiobolus spp. (Levisohn 1927; fungi and for experimental studies (see below). The presence Zahari & Shipton 1988; Gugnani 1999). A saprobic life habit of adhesive conidia formed by entomophthoralean fungi (Coremans-Pelseneer 1974) has been suggested as well as was studied by microscopic examination of 60 freshly cap- a commensalistic association with its hosts (Manning et al. tured gamasid mites (40 L. obesus,20Gamasina spp.) as de- 2007) and an antagonistic (parasitic) relationship (Taylor scribed below. et al. 1999). Springtails (Collembola) coextracted with the mites were While most entomophthoralean fungi are known as true or screened for the presence of Basidiobolus haptosporus (see at least opportunistic insect pathogens (Humber 2008), the na- Molecular analyses) and served as food for L. obesus in the ex- ture of the interaction between Basidiobolus spp. and arthro- perimental approaches (see Experimental approaches). The pods remains unclear. During a survey on mite-associated five different morphotypes of springtails coextracted from fungi in soil we detected for the first time an association of the soil samples, were treated as a single taxon, i.e., they Basidiobolus haptosporus Drechsler with a gamasid mite spe- were not identified any further and individuals were selected cies, Leptogamasus obesus Holzmann. To find evidence for the randomly for the molecular surveys, regardless of the nature of this association, follow-up experiments and mor- morphotypes. phological studies were conducted. Fungal cultivation Materials and methods For the isolation of associated fungi, mite individuals were washed in a water drop under sterile conditions and subse- Conceptual design of the study quently fragmented and dispersed on a Petri dish containing yeast malt agar (1 % malt extract, 0.4 % yeast extract, 0.4 % glu- The presented study emerged from a descriptive molecular in- cose, and 1.2 % agar). The agar plates were stored at room tem- vestigation on mite-associated fungi in soil, in the course of perature and observed at daily time intervals. Emerging which an association between Basidiobolus haptosporus and mycelia were removed and inoculated onto fresh agar plates. the mite Leptogamasus obesus was observed. These findings For microscopic analyses, pure cultures of Basidiobolus hapto- initiated closer analyses including the microscopic screening sporus were grown on maize meal agar (Drechsler 1956) under of L. obesus for fungi as well as the isolation and cultivation the same conditions. For isolating and cultivating soil- of B. haptosporus for morphological studies and experiments. dwelling entomophthoralean fungi able to form ballistospores The latter include coincubation with living or dead mites (like Basidiobolus spp.), the canopy plating technique by and also address the propagation by food animals. Drechsler (1952b) was applied. For tracing hyphal growth and zygospore formation in vitro, Sampling site and sample collection liquid maize meal agar was dropped on a sterilized micro- scope slide and covered by a sterilized coverslip. Subse- The investigation site of about 4 m2 in size is located in quently, a small fragment of fungal mycelium was placed in a mixed forest with predominant Pinus sylvestris L. on the contact with the agar drop. The slide was incubated in a closed hill ‘Hohe Warte’ close to the city of Bayreuth, Germany sterile Petri dish, with some moistened paper added to supply (495801600N, 113405100E, 460 m alt.). The understory is domi- humidity. nated by Calluna vulgaris (L.) Hull and two moss species (Poly- The strain of B. haptosporus examined morphologically here trichum formosum Hedw., Pleurozium schreberi (Brid.) Mitt.). was deposited in the Jena Microbial Resource Collection as The soil is a podsolized ranker and covered by an organic layer JMRC 10633. of 5e10 cm in depth. Four replicates of soil samples (250 cm3 each) were drawn Experimental approaches every month from August to December 2008 and from January to December 2010 from the uppermost 5 cm of the organic Oe To investigate Basidiobolus haptosporus for its potential to in- layer, consisting of fragmented litter. The covering layer of fest living mites, 20 individuals of Leptogamasus obesus were fresh and undecomposed litter was removed before taking coincubated with JMRC 10633. Groups of five mites were the samples. kept in snap-cap vials. The vials were filled with a moistened Three samples were used for the mite extraction and substrate consisting of active carbon and plaster of Paris as a fourth to analyze the soil-inhabiting fungal community. described by Karg (1993) and covered with a fine nylon Mites were extracted by gradual heating treatment (Berlese mesh. A small piece of agar colonized by the fungus was 1905). Gamasid mites were identified using the key of Karg