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Yoder et al. The entomopathogenic fungus Beauveria caledonica, a newly identified pathogen of cave crickets, Hadenoecus spp. (Orthoptera: Rhaphidophoridae) Jay A. Yoder1,2, Joshua B. Benoit3, Horton H. Hobbs III1,4, Blake W. Nelson1,5, Leighanne R. Main1,6 & Connie Fe C. Gibas7 1 Department of Biology, Wittenberg University, Springfield, Ohio 45501, USA 2 [email protected] 3 Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221 USA [email protected] (corresponding author) 4 [email protected] 5 [email protected] 6 [email protected] 7 University of Alberta Microfungus Collection and Herbarium, Devonian Botanic Garden, T6G 2E1, Edmonton, Alberta, Canada, [email protected] Key Words: Insecta, Orthoptera, Rhaphidophoridae, Hadenoecus cumberlandicus, Hadenoecus opilionoides, Hadenoecus jonesi, Sordariomycetes, Hypocreales, Cordycipitaceae, Beauveria caledonica, Sloan's Valley Cave System, Pulaski County, Kentucky, United States, Horton Hobbs Cave, Carter County, Kentucky, United States, Laurel Cave, Carter County, Kentucky, United States, Gap Cave, Claiborne County, Tennessee, United States, Russell Cave, Jackson County, Alabama, United States, fungal pathogen, phylogenetic analysis, population ecology. We report an additional species of infected cave cricket (family Rhaphidophoridae) by a fungus resulting in cricket mummification (Figure 1A), otherwise known as the 'cricket marshmallow' (a term coined by Lavoie et al. 2007). This conspicuous white, pillowy fungus is sometimes observed fastening dead crickets (Hadenoecus spp.) to the walls and ceilings of caves. We have found mummified crickets in caves throughout northern Kentucky infecting H. cumberlandicus Hubbell and Norton, 1978 central Tennessee infecting H. opilionoides Hubbell, 1978 and northern Alabama infecting H. jonesi Hubbell, 1978. The sites where mummies are collected do not seem to be associated with any special, restricting conditions, as they are found occurring in vertical shafts (> 67 m deep) and horizontal passages with or without streams (H.H. Hobbs III, unpublished data; B.W. Nelson, unpublished data). Previous investigations have identified the well-known entomopathogenic fungus Beauveria bassiana (as Isaria densa Cali, 1897; cited in Lavoie et al. 2007), used in biological control (synonymy in Zimmerman 2007), from infected mummies of cave crickets (H. subterraneus) in the Mammoth Cave system (Lavoie et al. 2007). In mummies of H. cumberlandicus, we have recovered and identified the fungus Beauveria caledonica as the primary isolate 2015 Speleobiology Notes 7: 1–9 1 Yoder et al. (Table 1), accession number UAMH 11821 (molecular systematics Blast match 99% ITS similarity to B. caledonica; Rehner and Buckley 2005; Rehner et al. 2011). This cricket mummy fungal isolate matched with 99% similarity to several B. caledonica species (Figure 1B), specifically B. caledonica strain ARSEF 2251 (GenBank HQ880819) from beetles (adult [Coleoptera], unknown species, Brazil, Pará). We have deposited this cricket mummy fungal isolate of B. caledonica at the University of Alberta Microfungus Collection and Herbarium (UAMH), Canada (accession number UAMH 11821). The mummy consists of a higher percentage of B. caledonica that greatly outnumbers other fungal components, Mucor, Aspergillus, Cladosporium, Penicillium, and Verticillium (p < 0.05 in each pairwise comparison; Table 1). As a measure of diversity, the Simpson's reciprocal index for the cricket mummy approaches 1; i.e., this signifies a low diversity and is essentially a single fungus community. Beauveria caledonica is undetectable, or recovered at low frequency, on each cricket species that do not show signs of disease compared to the mummy (Table 1; p < 0.05 in each pairwise comparison). In contrast to the mummy, Aspergillus followed by Penicillium were the primary fungi recovered in healthy H. cumberlandicus (p < 0.05; Table 1). Aspergillus and Penicillium were also the predominant fungi of healthy individuals in H. opilionoides and H. jonesi (Table 1; p > 0.05 in each species-wise comparison). Simpson's reciprocal index was higher for healthy crickets than the mummy (p < 0.05; Table 1), which reflected a greater diversity of fungal components for healthy crickets. Diversity of fungal components at the generic level, however, did not vary much among healthy cricket species (p > 0.05). Topical application of the B. caledonica mummy isolate (1.1 x 108 spores/insect in 0.05% Tween 20) to the dorsum of healthy crickets resulted in an approximate three- fold increase in mortality of H. cumberlandicus than topically-applied saline controls (Table 2). Subsequently, nearly all the dead crickets having received a topical application of spores tested positive for B. caledonica (Table 2). High mortality, and subsequent recovery of B. caledonica from dead individuals, were noted for crickets that crawled over dried, B. caledonica spore-treated filter papers for 2–3 minutes (1.1 x 108 spores/insect). There was also high recovery of B. caledonica from dead crickets that had ingested blocks of agarose containing B. caledonica spores after a 12-hr exposure (1.1 x 108 spores/insect; Table 2). All fungi that were identified from healthy crickets are common in cave soil, particularly Aspergillus, Penicillium, and Mucor (Cubbon 1976; Vanderwolf et al. 2013). All of these fungi grow between 5–37 °C (Koilraj et al. 1999), and none are exclusively psychrophilic. This temperature range of mesophilic growth is within the range of temperatures that these crickets experience either inside or outside the cave. Some fungi may be unculturable with our methods, suggesting that other fungi are present on the cricket's body surface that remain to be examined. The large percentages of Aspergillus and Penicillium were a consistent feature of the mycoflora of H. cumberlandicus, H. opilionoides, and H. jonesi in spite of differences among cricket species, locality of origin, and foraging habitat. Some of the fungi that are isolated from 2015 Speleobiology Notes 7: 1–9 2 Yoder et al. healthy crickets can be entomopathogenic, such as Beauveria (including B. caledonica), Paecilomyces, and Verticillium (Gunde-Cimerman et al. 1998). Although present, these potential entomopathogenic fungi are apparently not sufficiently high in spore count to be virulent or these fungal strains do not readily kill cave crickets. Figure 1. (A) Female of Hadenoecus cumberlandicus and cricket mummy that were collected from the same southern Kentucky cave, with (B) phylogenetic confirmation using a Neighbor-Joining tree based on18S rRNA internal transcribed spacer to confirm the identity of the white fungus making the mummy as Beauveria caledonica. Only bootstrap values (1,000 total replicates) over 50% are shown. Photo credit (A): K.M. Gribbins, Biology Department, University of Indianapolis, Indiana, USA, by permission. 2015 Speleobiology Notes 7: 1–9 3 Yoder et al. Table 1. External mycoflora of live, healthy females of Hadenoecus cumberlandicus (HC), H. opilionoides (HO), and H. jonesi (HJ). A mummy of H. cumberlandicus is shown in Figure 1A. No mummies of H. opilionoides and H. jonesi were available for analysis. Untreated control, sterile water that was processed in the cave without using a cricket. Data are the mean of n = 30 live crickets for each species (SE was less than or equal to 2.7) and n = 9 mummies (SE was less than or equal to 1.4). % Mean no. isolates/ml surface extract from: Mummy Fungus HC Live HC Live HO Live HJ Control Aspergillus sp. 3 16 5 10 0 Aspergillus flavus 0 2 4 8 0 Aspergillus fumigatus 0 7 7 0 0 Aspergillus niger 0 8 11 9 0 Beauveria bassiana 0 3 0 1 0 Beauveria caledonica 87 0 0 2 0 Cladosporium cladosporioides 2 10 8 6 0 Fusarium sp. 0 1 9 8 0 Geotrichum candidum 0 2 6 0 0 Mucor sp. 5 9 16 14 0 Mortierella sp. 0 3 0 1 0 Mycelia sterilia 0 1 0 2 0 Paecilomyces sp. 0 0 0 9 0 Penicillium sp. 2 22 15 17 0 Rhizopus sp. 0 4 11 2 0 Scopulariopsis brevicaulis 0 0 0 3 0 Trichoderma sp. 0 5 0 3 3 Verticillium sp. 1 0 8 2 0 Simpson's reciprocal index 0.70 2.90 3.40 3.70 - SE 0.04 0.07 0.06 0.06 - Beauveria caledonica is not novel to caves or to cave crickets. There is prior record of B. caledonica from crickets from a cave in Georgia, United States, W. K. Reeves, [Orthoptera: Gryllacrididae], unknown species, 2003 (listed as B. caledonica strain ARSEF 7117 by the USDA-ARS Collection of Entomopathogenic Fungal Cultures, Ithaca, New York, USA). Use of different genes in identifying the cricket isolate B. caledonica from Georgia prevented us from including it on our identification tree phylogeny (Figure 1B). Although occasionally recovered from earwigs (Dermaptera) and cicadas (Orthoptera), the typical host-association for B. caledonica appears to be with beetles (Glare et al. 2008; Cummings 2009; but not in a beetle survey by Takov et al. 2012). Virulence properties of different strains of Beauveria vary considerably (Zimmerman 2007). Results of this study indicate that B. caledonica strain UAMH 11821 2015 Speleobiology Notes 7: 1–9 4 Yoder et al. is a pathogenic strain to the cave cricket H. cumberlandicus, occurring by ingestion or direct contact. Table 2. Mortality of healthy females of Hadenoecus cumberlandicus within five days after treatment with spores from the original identified isolate of Beauveria caledonica from the cricket mummies. Dead crickets were subsequently tested for presence of B. caledonica by fungus culture in an attempt to recover B. caledonica as the infectious agent (Koch's postulates). Data (mean ± SE) that are followed by the same superscript letter within a column are not significantly different (Abbott correction; p < 0.05). n = 3 replicates of five crickets each. % Crickets after B. caledonica exposure by: Application Crawling on Ingestion of Experimenta l group topically treated surface agar block Control (PBS + 0.05% Tween) Dead out of 15 crickets 33.3 ± 3.2a 40.0 ± 4.7a 33.3 ± 4.1a Positive for B.