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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 unpublished), but most of them derived from well referenced strains of the ARSEF, ATCC and The genus Basidiobolus was erected by Eidam (1886) for two NRRL culture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 microscope 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 placed in the center. Living springtails (Collembola) served as (1993), and oribatid mites were determined by F. Horak (Karls- food. The animals were checked daily for signs of fungal infec- ruhe). Leptogamasus obesus Holzmann (Gamasina) and Oppiella tions and dead mites were removed immediately for micro- subpectinata Oudemans (Oribatida) were the only mite species scopic analysis. occurring at the sampling site throughout the whole sampling To test whether prey of L. obesus serves as vector, spring- period. Individuals of the two species were pooled into sam- tails were incubated for 2 d on agar plates completely colo- ples of five and ten, respectively. The different sample sizes nized by B. haptosporus and then offered as food to L. obesus. compensated for the different body sizes of the species and Five individuals of these mites were kept as described above thus for the amount of potentially enclosed fungal material. and fed exclusively with B. haptosporus-exposed springtails. Author's personal copy

92 S. Werner et al.

Freshly killed mites of the species L. obesus (ten individuals) To ensure that negative results (absence of PCR product) and Oppiella subpectinata (ten individuals) were placed on agar were not caused by technical failure, positive controls were plates with B. haptosporus to observe saprobic colonization of carried out, with DNA isolated from culture material of B. hap- the mites. tosporus added to the DNA extracts from soil, mites, and springtails in dilutions of up to 1:100 000. The controls also Microscopical analyses served to determine the minimum amount of DNA necessary to detect B. haptosporus in environmental samples. Mites were briefly washed in a water drop and transferred Sub-cloning, RFLP-analysis and sequencing onto glass slides with a central spherical depression, and the The TOPO TA Cloning Kit for Sequencing (Invitrogen) was used surface was examined by light microscopy without further for sub-cloning the fungal PCR products (900e1200 bp) ob- treatment. Subsequently, the cuticles were carefully perfo- tained from the 49 (29 þ 20) mite and 7 soil samples. Plasmids rated with an insect needle and the mite samples were stained of 12 and 96 clones each from mite and soil samples were pu- with Lactophenol Cotton Blue for a second examination. Glass rified using the Charge Switch Plasmid ER Mini Kit (Invitrogen). slide cultures of Basidiobolus haptosporus (see above) were Both kits were applied according to the manufacturer’s in- examined under the microscope without any additional structions. The inserts within the plasmids were amplified us- treatment. ing the M13 primers enclosed in the cloning kit. The restriction enzymes MspI and AluI (Fermentas) were Molecular analyses used to digest the 1347 inserts obtained by amplification. The polymorphism of the restriction fragment lengths (RFLP) DNA isolation and amplification was registered by analyses of ethidium bromide-stained aga- Total DNA was extracted from mite samples (29 samples of rose gels. Identical RFLP patterns were treated as belonging Leptogamasus obesus and 20 of Oppiella subpectinata) and fungal to one RFLP type. For each RFLP type detected multiple times, cultures using the Charge Switch gDNA Plant Kit (Invitrogen) at least two inserts originating from different samples were as recommended by the manufacturer, but using 0.2 ml tubes, sequenced using a Beckman-Coulter CEQ 8000 capillary se- and with all volumes reduced to 10 %. DNA from 19 soil sam- quencer. The ITS sequences assignable to Basidiobolus hapto- ples (0.5 g) was isolated according to Persoh et al. (2008). The sporus were deposited in GenBank (www.ncbi.nlm.nih.gov) ITS rRNA gene (ITS1, 5.8S, and ITS2 region) was amplified in under the accession numbers GU324663eGU324667. The ITS a total volume of 25 ml containing 0.1 ml Taq polymerase (5 U/ sequence of strain JMRC 10633 was deposited under accession m m m l, Invitrogen), 2.5 l10 PCR buffer, 0.75 l MgCl2 (50 mM), number JF317357. 2.5 ml dNTP mix (2 mM), and 1.25 ml forward and reverse primers (10 mM) each, as well as 1 ml DNA extract. Newly Phylogenetic analyses designed primers SSUh35-F (50-TTA GAT GTT CTG GGC The most similar sequences among those deposited in Gen- CG-30) and LSUh11-F (50-TAT GCT TAA GTT CAG CGG-30) Bank, when searched in February 2011, were determined us- were applied to differentiate fungal from mite DNA, because ing ‘Mega BLAST’ (Zhang et al. 2000) for the ITS region (ITS1, the commonly applied fungal ITS primers ITS1F (Gardes & 5.8S, and ITS2 rRNA gene) and separately for the ITS1 and Bruns 1993) and ITS4 (White et al. 1990) were shown to match ITS2 regions to account for short sequences as usually ob- to some of the publicly available mite rRNA sequences (data tained from massive parallel sequencing approaches. The se- not shown). [Detailed information on primer specificity is quences found were aligned with the respective fungal available from the authors on request.] Following an initial de- sequences from the mite samples. Phylogenetic trees were naturation step for 2 min at 95 C, 35 cycles (20 s denaturation reconstructed for the ITS region and for the ITS1 fragment us- at 94 C, 1 min primer annealing at 58 C, and 2 min elongation ing RAxML 7.0.3 (Stamatakis 2006) and applying the GTRCAT at 72 C) were conducted. The PCR ended with an elongation approximation of nucleotide substitution. To assess support step for 15 min at 72 C. The amplified ITS regions of different of the respective branches, 500 bootstrap replicates were cal- fungal taxa in the obtained PCR products were separated by culated. Only unambiguously alignable positions were in- sub-cloning (see below). cluded in the phylogenetic analyses, which correspond to Based on the sequences obtained from Basidiobolus hapto- base pairs 51e110, 123e386, and 401e556 of sequence sporus (see below), primers which selectively amplify a frag- EF392528 (Basidiobolus haptosporus). ment of its ITS region were designed. The forward primer (fBh1: 50-TTG GCA GGG AGT GAA ATG-30) targets the ITS1 region and the reverse primer (rBh1: 50-CCA TGC TAC ACG CTT Results CCG-30) binds to the ITS2 region. Both primers bind to sites which are highly variable among fungal taxa and show at least Morphological analyses three mismatches to any other known fungal species. The B. haptosporus-specific primers were used to screen DNA ex- While no fungi could be isolated from soil by using the canopy tractions from soil and mites for the presence of B. haptospo- plating technique, pure cultures of Basidiobolus haptosporus rus. In addition to the samples used in the sub-cloning were easily isolated from Leptogamasus obesus, since its hy- approach, 12 samples of soil and 16 of springtails (2e5 individ- phae readily emerged from the inoculated mites. uals, each), the predominant food of L. obesus, were exclu- The fungus grew rapidly at room temperature after inocu- sively screened for B. haptosporus with the specific primers. lation onto fresh media and produced no noticeable odour. On Author's personal copy

B. haptosporus is associated with L. obesus 93

maize meal agar the colonies expanded at a rate of approxi- Basidiobolales, it may not be assigned unambiguously to any mately 0.5 cm d 1. The colonies appeared as a slightly opaque certain species. and radially expanding area but were nearly invisible. Neither Fungal structures were not observed on or inside the 20 development of aerial hyphae nor the formation of conidia Leptogamasus obesus individuals having been incubated with was observed during the whole growth period. Following Basidiobolus haptosporus cultures for 3 weeks. This also applied interhyphal conjugations, Zygospores began to be produced for the three mites that died during the experiment. L. obesus in great quantities approximately 4 d after inoculation. individuals fed with springtails that had been exposed to At the initial stage (days 24), the hyphae grew linearly B. haptosporus cultures before, were still alive after 3 weeks with few branches (Fig 1A). Non-branching hyphae were sep- of incubation; no fungal structures were found inside the tate at more or less regular intervals and measured between 9 mites or on their cuticles. Dead mites (ten L. obesus and ten and 20 mm in diameter. Each cell contained a single nucleus. Oppiella subpectinata individuals) were rapidly colonized by More mature hyphae, mainly during zygospore formation B. haptosporus when placed onto fungal cultures (Fig 2B, C). (day 4 onwards), tended to branch frequently and were of di- The hyphae preferably entered the bodies through mouth verse shapes. At that stage no further radial growth was ob- and anus. An active penetration of the cuticle could not be servable. With proceeding spore development most parts of observed, and only few hyphae grew on the surface. Interhy- the mycelium started to die off, and only cells contributing phal conjugations were observed within the haemocoel, and to the development of zygospores remained active (Fig 1B). Zy- after several days the body cavities were completely filled gospores (Fig 1C) were between 30 and 45 mm in diameter, and with zygospores (Fig 2D), but the cuticles still remained intact. exhibited a thick and smooth wall. The colourless wall is mostly of solid structure, but its stratification into two layers was recognizable at the two poles (Fig 1C). Spore contents Molecular analyses are characterized by a central oil droplet and faintly brownish pigmented elements appearing granular. Squash preparations Analyses of the ITS sequence of Basidiobolus haptosporus revealed these elements to be mostly separate oil droplets. Four of the sequenced clones obtained in the course of the initial screening for fungi associated with Leptogamasus obesus Experimental approaches were identical to EF392528 (B. haptosporus), and one differed at a single position. The ITS rRNA sequence (JF317357) of the The microscopic screening of 60 freshly captured gamasid fungal strain described above was also identical to EF392528. mites did not reveal any fungal structures like hyphae, zygo- BLAST search revealed no significant matches among envi- spores or conidia inside the bodies or on the cuticle, with ronmental samples for the ITS region as a whole and only one exception in which an adhesive conidium was found at- three for the ITS1 region. tached to the leg of one mite individual (Fig 2A). While this co- In the phylogenetic analysis, the five sequences clustered nidium is morphologically in agreement with those in exclusively with four sequences of B. haptosporus (EF392520,

Fig 1 e B. haptosporus (JMRC 10633) on maize meal agar. [A] Young and still expanding mycelium, 3 d after inoculation (bright ﬁeld, 4003). [B] Progressing stages of zygospore formation following interhyphal conjugations, 10 d after inoculation (DIC, 1603). [C] Zygospore, 15 d after inoculation (bright ﬁeld, 4003). Scale bars: 20 mm. Author's personal copy

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Fig 2 e B. haptosporus on living [A] and in dead individuals of L. obesus [BeD]. [A] Adhesive conidium, probably originating from a species of Basidiobolus (bright field, 4003). [B] Hyphae and interhyphal connections stained with Lactophenol Cotton Blue in L. obesus, overview (bright field, 1003). [C] Detail of [B] (bright field, 4003). [D] Zygospores formed inside L. obesus (bright field, 1003). Scale bars: 20 mm. Selected zygospores (z), hyphae (h), and conjugating hyphae (ch) are indicated.

EF392528, EF392529, EF392531) in a well supported clade (96 % 50 pg B. haptosporus DNA yielded PCR products with all DNA bootstrap support, Fig 3A). Three partial sequences (ITS1 re- extracts from mites, soil, and springtails. gion only) from environmental samples clustered within this clade in analyses restricted to the ITS1 region (Fig 3B). Se- quences within this clade differed at two positions at most. Discussion The five other species of Basidiobolus (B. ranarum, B. meristospo- rus, B. heterosporus, B. magnus, B. microsporus) clustered outside Methodology the clade, as, however, did one sequence (EF392535) assigned to B. haptosporus var. minor. The fungal diversity in soil and soil-inhabiting mites was screened by applying newly designed primers. These are less se- Screening for Basidiobolus haptosporus in environmental lective among fungal taxa (especially among zygomycetes) and samples more distinct from sequences of Acari than the commonly ap- RFLP patterns corresponding to the sequences assigned to plied primers ITS1F and ITS4. Sub-cloning and RFLP analyses fre- B. haptosporus were detected in 13 (45 %) of the 29 analyzed Lep- quently revealed Basidiobolus haptosporus in samples of togamasus obesus samples (351 clones). Nine PCR reactions Leptogamasus obesus, but not from other mites or soil. The design with the B. haptosporus-specific primers were positive (31 %). of primers selectively amplifying B. haptosporus enabled the Neither the cloning approach, nor the PCR reactions with time- and cost-effective screening of additional samples for B. haptosporus-specific primers revealed the presence of B. hap- the fungus. This second approach was less sensitive since tosporus in Oppiella subpectinata (215 clones and 20 samples, re- B. haptosporus was detected in 31 % of 29 samples, while the fun- spectively), soil (781 clones and 19 samples, respectively) or gus was detected in 45 % of the same samples by cloning. How- springtail samples (16 samples). A minimum of 50 pg DNA ever, this loss seemed acceptable considering the higher number (1:10 000 dilutions of the DNA extracts from pure cultures of of samples which could be processed using the specific primers. B. haptosporus) was necessary to obtain PCR products with A major obstacle for screening samples exclusively by the the B. haptosporus-specific primers. Likewise, addition of success of PCR amplification is the verification of true negative Author's personal copy

B. haptosporus is associated with L. obesus 95

Fig 3 e Phylogenetic relationship among Basidiobolus spp. as inferred from ITS rRNA sequence data. Most likely trees found by RAxML including the whole ITS region [A] and the ITS1 fragment [B]. Taxon names are preceded by the GenBank accession number. Support values from 500 bootstrap replicates are noted above the respective branches. Sequences obtained from this study are marked with an asterisk.

results, i.e., the absence of a fungus from a certain sample. To A detailed morphological examination of the corresponding exclude false negative results due to coextracted substances strain seems therefore necessary before drawing conclusions from soil and mite samples inhibiting the PCR, positive control on the possibility of identifying all representatives of B. hapto- reactions were conducted by adding 50 pg of pure B. haptospo- sporus based exclusively on ITS sequence data. Against the rus DNA to the samples. This amount of DNA was previously current data background, phylogenetic analyses based only ascertained as being the minimum amount necessary to ob- on ITS rRNA genes may not resolve the existing controversies tain PCR products from pure B. haptosporus DNA. about species concepts within Basidiobolus (Hutchison et al. 1972; Yangco et al. 1986). Morphology and phylogeny of Basidiobolus haptosporus Nature of the association of Basidiobolus haptosporus with The morphological characteristics of strain JMRC 10633 iso- Leptogamasus obesus lated from the predatory mite Leptogamasus obesus matched the original circumscription of B. haptosporus by Drechsler The inconsistent species concepts applied in various studies (1947, 1956). Most notably, the lack of aerial hyphae in combi- (e.g., Emmons et al. 1957; Drechsler 1958; Srinivasan & nation with the absence of conidia formation in established Thirumalachar 1965; Greer & Friedman 1966) obscure a clear cultures while producing large numbers of smooth-walled zy- picture on the biology and ecology of the different species of gospores unambiguously separates the fungus from other Basidiobolus. Srinivasan & Thirumalachar (1965) as well as species of the genus. Coremans-Pelseneer (1974) concluded that certain species do Phylogenetic analyses revealed the corresponding ITS se- not differ in their habitat preferences. The described biological quence to cluster in a well supported clade (Fig 3), including and ecological traits of B. haptosporus, B. ranarum, and B. heter- otherwise only sequences obtained from strains identified as osporus appear notably to be rather similar. Thus, previous B. haptosporus and from unidentified environmental samples. studies provide no evidence on the nature of the association One sequence (EF392535) assigned to B. haptosporus var. minor between B. haptosporus and Leptogamasus obesus. Theoretically, clustered outside the B. haptosporus-clade. In case the correct the fungus could colonize the cuticle, the gut or the haemo- identification of the strain is verified by morphological stud- coel of the mite. It may also be ingested with prey animals ies, phylogenetic analyses including multiple loci are needed or just adhere to the mites’ surface. The plausibility of each to justify a separation of the variety from B. haptosporus. How- of these possibilities is discussed in the following against the ever, all sequences within the ‘B. haptosporus-clade’ are likely background of the obtained results. to represent B. haptosporus, and five sequences obtained While no hyphae could be found colonizing the body sur- from L. obesus also cluster within this clade. It can, therefore, face of L. obesus, the conidium attached to its leg (Fig 2A) indi- be concluded that B. haptosporus has been present in the corre- cates a phoretic association between an unidentifiable species sponding mite samples, which is the first evidence for an as- of Basidiobolus and the mite. Conidia of Basidiobolus spp. are sociation between mites and fungi of the genus Basidiobolus. known to be very adhesive to arthropod cuticles (Drechsler The other five species of Basidiobolus, for which sequence 1956; Coremans-Pelseneer 1974), and the attached capilliconi- data are available are either represented by a single sequence dia of Basidiobolus spp. were reported from several collection or appear to be paraphyletic in the phylogenetic trees (Fig 3). specimens of mites (Blackwell & Malloch 1989). However, Author's personal copy

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only one conidium was found attached to one individual of would explain the data from the molecular analyses best, the 60 freshly captured gamasid mites microscopically exam- even though we found no microscopic evidence to support ined in this study. This is not fully in accordance with the re- this hypothesis. sults of the molecular studies, which revealed B. haptosporus in The results clearly indicate that further studies are re- 45 % of the L. obesus samples, i.e., in 9 % of the individuals, at quired to ascertain the nature of the association of B. haptospo- the very least. Furthermore, host selectivity, as found in this rus with L. obesus. Evidence may be obtained by keeping the study, is rather unlikely in the case of coincidental associa- mites without food until their intestines are completely tions. Therefore, while we found evidence for a phoretic asso- empty. Subsequently, microscopic examination should verify ciation between Basidiobolus and L. obesus, such an association that only mites are selected for DNA extraction, the surfaces does not satisfactorily explain the results from the molecular of which are also free of any fungal spores. Whether or not analyses. Nevertheless, a patchy distribution of B. haptosporus B. haptosporus would be detected in such prepared samples, in soil in conjunction with a preference of L. obesus for these the results would further narrow the possibilities for the na- patches would not entirely contradict the hypothesis of a pho- ture of the association of B. haptosporus with L. obesus. retic association between the two species, as discussed below (see Basidiobolus haptosporus). Basidiobolus haptosporus in soil Considering the feeding preferences of L. obesus, it is not surprising that the microscopic examination did not reveal B. haptosporus is considered to be widely distributed in soil any fungal structures inside its gut. L. obesus is a real predator (Drechsler 1947; Drechsler 1956; Coremans-Pelseneer 1974). that does not feed on fungi or litter but ingests only externally These conclusions derived from cultivation-based studies digested food by sucking out its prey. The cuticles and every- are not supported by surveys using molecular methods, be- thing that may be attached to the surfaces of the mites’ prey cause only three potential Basidiobolus individuals (cf. Fig 3B) are not ingested. In any event, fungal material possibly pres- have so far been detected in a single study (Jumpponen et al. ent inside in the prey animals would be largely destroyed dur- 2010) among numerous molecular studies screening for soil ing passage through the mite’s gut (Van der Drift 1965; Ponge fungi. The discrepancy may be either explained by a heavy & Charpentie 1981). These feeding habits already argue bias of molecular approaches against the detection of Basidio- against the ingestion of considerable amounts of intact fungal bolus, or by an overestimation of its abundance based on con- e material. Additionally, DNA fragments of 900 1200 bp in ventional, cultivation-based methods. The former possibility length, as amplified by the applied primers, are unlikely to seems rather unlikely because we verified that the commonly be detected from gut contents (Zaidi et al. 1999). Finally, the ab- used ITS primers (i.e., ITS1F, ITS2, and ITS4) perfectly match sence of B. haptosporus from the springtails that appear to be with the rRNA sequences of B. haptosporus, and we applied the mites’ mostly preferred food, renders a fungal ingestion two different and newly designed sets of primers. Further- with food even more unlikely, although B. haptosporus may more, we assured that inhibitory effects of coextracted sub- still be present in other prey organisms. stances may not be responsible for the negative results. The microscopic examination of L. obesus also revealed no Hence, everything indicates that B. haptosporus should have fungal structures to be present inside the haemocoel or intes- been detectable if it were present in the soil samples. tinal system. However, it is conceivable that B. haptosporus However, B. haptosporus may have been missed by molecu- forms much smaller and more slowly growing structures in- lar studies in case of a patchy distribution of its hyphae or di- side the potential hosts than in culture. Such tiny structures aspores in soil. Its association with Leptogamasus obesus would might be hard to visualize by light microscopy. While this is indicate that such patches are preferentially visited by preda- just a hypothetical scenario due to lacking evidence from tory mites. As a working hypothesis for future studies we any entomopathogenic fungi, it would be in line with the ob- therefore propose the following scenario: Amphibians or in- servation that the mites were obviously not negatively af- sects are additional hosts of B. haptosporus, and the fungus fected by the association. Because the ingestion of intact produces infectious conidia on their feces or cadavers. Be- fungal structures seems to be rather unlikely for L. obesus, cause dung and carrion represent a rich source of energy, B. haptosporus would have to be able to penetrate the cuticle these substrates attract a high diversity and quantity of for an internal colonization of the mites. The capability to di- carrion-feeding animals (Putman 1983; Galante & Marcos- gest chitin-rich substrates, thereby possibly allowing the pen- Garcia 2008). These may include prey organisms of L. obesus, etration of arthropod cuticles, seems to be a common trait of causing the mite to preferentially visit these substrates. L. obe- all entomopathogenic fungi (Humber 2008), and Basidiobolus sus may either distribute the fungal spores phoretically spp. are known to be capable of synthesizing the necessary or serve as an intermediate or even primary host for enzymes like chitinases, lipases, and proteases (Manning B. haptosporus. et al. 2007; Mishra et al. 2011). Our experimental approaches revealed no indication for penetration of the mites’ cuticles by B. haptosporus. Even the cuticles of dead mite bodies remained intact, while the fungus completely exploited the Acknowledgements body contents as a saprobe. These findings do not support an entomopathogenic potential of B. haptosporus against L. obe- We appreciate the advice of R. A. Humber (Ithaca) which sus, but the possibility that the fungus may be parasitic on allowed a significant improvement of an earlier version of other arthropods, especially insects, cannot be excluded. Nev- the manuscript. We further thank F. Horak (Karlsruhe) and ertheless, an internal colonization of L. obesus by B. haptosporus A. Christian (Gorlitz)€ for the identification of mite species. Author's personal copy

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