55 ENTOMOPATHOGENIC FUNGI OF THE WINTER TICK IN MOOSE WALLOWS: A POSSIBLE BIO-CONTROL FOR ADULT MOOSE? Jay A. Yoder1, Cameron J. Dobrotka1, Kelli A. Fisher1, Anthony P. LeBarge1, Peter J. Pekins2, and Scott McLellan3 1Department of Biology, Wittenberg University, Springfield, Ohio 45501, USA; 2Department of Natural Resources and the Environment, University of New Hampshire, Durham, New Hampshire, 03824, USA; 3Maine Department of Inland Fisheries & Wildlife, Greenville, Maine, 04441, USA ABSTRACT: Soil fungi were cultured from 24 wallows and proximal control sites in Maine and New Hampshire, USA during the autumn moose (Alces alces) breeding season of 2016 to investigate the presence of soil fungi pathogenic to winter tick larvae (Dermacentor albipictus). Twenty genera of fungi were isolated, and all are considered common in a forested ecosystem. The predominant genera isolated in wallows were pathogenic to winter tick larvae and included Aspergillus spp. (in particular A. flavus), Beauveria bassiana, Mortierella spp., Mucor spp., Paecilomyces spp., Penicillium spp., and Trichoderma spp. Wallow soils had specific characteristics and differed from proximal control sites by having: 1) lower fungal diversity, 2) a higher frequency of primary colonizers including Mortierella spp., Mucor spp., Penicillium spp., and Trichoderma spp., and 3) a more variable total amount of fungi indicative of changing (disturbed) soil conditions. We conclude that wallows are sites of soil disturbance that concentrate fungi known to be pathogenic to larval winter ticks. Fungi acquired by breeding moose using wallows might subsequently act as an on-host mechanism of tick control. ALCES VOL. 54: 55–70 (2018) Key words: Behavior, Dermacentor albipictus, fungi, tick control, Alces alces, Maine, New Hampshire, winter tick. INTRODUCTION The breeding season and wallowing behavior of moose (Alces alces) coincide directly with the questing season of larval winter ticks (Dermacentor albipictus) as they seek a moose host (Samuel 2004). As a one-host species, the winter tick is unique in that it remains and feeds on the same moose for its 3 parasitic life stages: larva, nymph, and adult. Excessive blood loss and other complicating factors associated with heavy infestations of > 30,000 ticks cause periodic, high mortality of calf moose in early spring when adult winter ticks take a blood meal. For example, average annual mortality of radio-collared calf moose with high tick abundance was >70% in New Hampshire and Maine in 3 consecutive win- ters in 2014-2016 (Jones et al. 2017). Wallowing is believed to reduce exter- nal parasites in several programmed groom- ing animals including bison (Bison bison), deer (Odocoileus virginianus), and pigs (Sus spp.) (Espmark and Langvatn 1979, McMillan et al. 2000, Bracke 2011). The mechanism would largely seem mechanical via rubbing, abrading, and damaging para- sites while an animal rolls vigorously in the soil. In addition to possibly reducing tick infestations, wallowing behavior of moose may have the added benefit of exposing lar- vae to pathogenic soil fungi. ENTOMOPATHOGENIC FUNGI IN MOOSE WALLOWS – YODER ET AL. ALCES VOL. 54, 2018 56 Soil contains an abundance of spore-pro- ducing ascomycetes that function as agents of decay and include a group of fungi that are the greatest cause of tick mortality in nature (Fernandes et al. 2012, Cafarchia et al. 2015). Infection of ticks occurs by spores, which upon contact, germinate and produce hyphae that penetrate the tick via mouth, anus, glandular openings, and soft membranes between leg segments, killing it in the process. Beauveria and Metarhizium spp. are the most noteworthy entomopatho- genic fungi used in biological control of ticks (Samish et al. 2004, Fernandes et al. 2012). Indeed, Beauveria bassiana, B. cale­ donica, and Metarhizium anisopliae (now M. robertsii) are known pathogens of winter tick larvae (Yoder et al. 2017b). Depending upon their relative concentration, these fungi may possibly serve as natural regulators of winter tick abundance on moose. The pur- pose of this study was to identify whether entomopathogenic fungi are present in wal- low soil, and to test the hypothesis that fungi isolated from wallow soils may be infective to winter tick larvae. MATERIALS AND METHODS All laboratory materials and instruments were obtained sterile from the manufacturer (Fisher Scientific, Pittsburgh, Pennsylvania, USA) or were sterilized by autoclave (121 °C, 19 psi, 15 min), Bunsen burner flame, or 95% ethanol; all methods followed standard aseptic techniques. Powder-free gloves (Microlex Co., Reno, Nevada, USA) were worn in the field and laboratory. All work in the laboratory was performed in a vertical laminar flow hood (Cole-Palmer, Vernon Hills, Illinois, USA) sterilized daily. Methods for isolating and identifying the fungi, as well as testing pathogenicity to lar- val ticks, conformed with standard practice (Tuininga et al. 2009, Suleiman et al. 2013, Cafarchia et al. 2015, Yoder et al. 2017a, b). Soil collection Soil samples were collected from wal- lows and proximate control sites located in typical moose habitat (forest description in Jones et al. 2017) in northern New Hampshire and central Maine, USA from 2 October – 1 November 2016. The wallows (n = 24) were located in 3 distinct geographic locations: 1) Mount Katahdin, Maine (Katahdin, 2-8 October, n = 4); Greenville, Maine (Greenville, 5 October – 1 November, n = 13); and Milan, New Hampshire (Milan, 7-17 October, n = 7). The Universal Transverse Mercator (UTM) coordinates of the wallow locations are held by P. J. Pekins (University of New Hampshire, Durham, New Hampshire) and S. McLellan (Maine Department of Inland Fisheries & Wildlife, Greenville, Maine). Samples were collected during the moose breeding period (generally the month of October) coinciding with when wallows were active and winter tick larvae were questing. Samples were scooped from surface soil (~8-10 cm depth) with 50 mL polypropylene centrifuge tubes and stored in Whirl-Pak bags (Nasco, Salida, California, USA). The samples were held at approximately 15 °C in a 5 L cooler contain- ing cold packs (Koolit; FDC Packaging, Medfield, Massachusetts, USA) for transport to the laboratory where they were stored at 4 °C (frost-free refrigerator; Fisher) and pro- cessed within 24-36 h. Eight soil samples were taken (filling 50 mL tubes) from each wallow site: 4 ran- domly collected from within the wallow (designated “wallow”), and 4 control sam- ples collected in opposite directions 1–2 m from the edge of the wallow (designated “proximate”). These proximal samples were assumed close enough to the wallow to be representative of the same site location. An estimate of soil pH was measured by placing 20 g of soil into 40 mL of distilled, deionized (DI) water. The soil samples (5 g from each of the 4 tubes per site) were ALCES VOL. 54, 2018 YODER ET AL. – ENTOMOPATHOGENIC FUNGI IN MOOSE WALLOWS 57 weighed to 0.1 g with a Light Balance (Mettler Toledo, Columbus, Ohio, USA). The soil-water mixture was stirred periodi- cally for 30 min (Kalra 1995), and pH was measured (Oakton pH meter, Vernon Hills, Illinois, USA) at 1, 2, and 3 h; data were combined (mean ± SE). The pH of the fungal agar growth media was adjusted to approxi- mate that of the wallow soil sample. Fungus isolation and identification A 1 g sample from each soil sample (equalized to dry weight) was weighed to 0.01 g using a microbalance (Ventron Co., Cerritos, California, USA) according to standard practice (Kumpula et al. 2000, Brown 2007, Shubina et al. 2013). This sam- ple was placed into a 9.0 mL DI water blank mixed with a vortex (Scientific Industries, Bohemia, New York, USA), and used as the stock solution for serial dilution with DI water (Brown 2007). An optimum dilution was used for all subsequent platings based on the standard counting plate method of 30 – 300 colonies per plate (Brown 2007). Aliquots of 0.1 and 1.0 mL were each plated using calibrated glass micropipettes. The DI water was used as a control to gauge the extent of contamination during the experi- mental manipulations. Potato dextrose agar (PDA + lactic acid to pH 5.5 + 0.05% chlo- ramphenicol) in 100 × 15 mm Petri plates was used as the growth media for plating. Selection of this mildly acidic PDA was based on the soil pH values (see RESULTS); mildly acidic PDA supplemented with chlo- ramphenicol as a bacterial growth regulator is a common agar for enumerating soil fungi (Brown 2007). Incubation was at 25 ± 0.5 °C in darkness in a programmable incubator. Colonies were counted after 7 days with an automatic colony counter (Bantex Co., Burlingame, California, USA). Each indi- vidual fungal colony was considered an iso- late. A 1 cm3 block from the advancing edge of the fungus mycelium was removed using a scalpel under a stereoscopic microscope at 40X for subculturing, and each isolate was then purified by 3 successive rounds of sub- culturing hyphal tips. Fungal identification was based on macro- scopic colony characteristics, and microscopic spore and phialide characteristics under oil emmersion at 1000x (Barnett and Hunter 2003). Further confirmation of identification was done by comparison with previously iden- tified, authentic cultures (nucITS identifica- tion) from 1) the University of Alberta Microfungus Collection and Herbarium (UAMH) Centre for Global Microfungal Biodiversity (Toronto, Ontario, Canada), 2) the Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (ARSEF), United States Department of Agriculture - Agricultural Research Service (USDA-ARS; Ithaca, New York, USA), and 3) the University of Cincinnati Microfungus Collection, Department of Biological Sciences, University of Cincinnati (Cincinnati, Ohio, USA). We analyzed 3, 1-g samples (dry weight equivalent) from each tube of soil at the wal- low; that is, 4 tubes of soil within the wallow and 4 tubes of soil that were proximal (control). Data from the 2 groups of 4 tubes at each wallow site were pooled and represented as a mean ± SE. Data were compared using an analysis of covariance (ANCOVA; P = 0.05; JMP, SAS Institute, Cary, North Carolina, USA). Fungus diversity was measured with the reciprocal Simpson diversity index (1/D; Simpson 1949) by treating all members of the same genus as a category. The total amount of fungus from the enumeration was expressed as colony forming units (CFU), and values were compared using a least significant differ- ence (LSD) test. Fungus inoculum Spores from 1-month-old cultures of individual fungal isolates were scraped from ENTOMOPATHOGENIC FUNGI IN MOOSE WALLOWS – YODER ET AL. ALCES VOL. 54, 2018 58 the plates with a scalpel and placed into a 1 mL phosphate buffered saline (PBS, pH 7.5) + 0.05% Tween 20 (emulsifier) to form an aqueous inoculum, 1 inoculum for each iso- late. Isolates of the same genus were pooled to reflect the diversity (mixture of strains) of fungi that ticks would be exposed to natu- rally from soil. The spore count of the pooled inoculum was adjusted to 1.6 × 108 spores/ mL using a hemocytometer (AO Spencer Bright-Line hemocytometer, St. Louis, Missouri, USA); the control inoculum was PBS + 0.05% Tween. Topical application of this concentration of spores/tick conforms to standard practice in pathogenicity tests (Tuininga et al. 2009, Suleiman et al. 2013, Cafarchia et al. 2015, Yoder et al. 2017a, b). Three different inocula representing a partic- ular treatment group were prepared such that treatments to ticks were not made from a single tube of spores. Only fungal isolates cultured from the wallow samples were used for the pathogenicity experiments. Presumably, adult moose would acquire these fungi on their fur during wallowing, and these fungi would have the greatest like- lihood of making contact with ticks on the moose. Lethal testing Larvae are one of two stages potentially exposed to fungi via wallowing (the other being nymphs; Addison and McLaughlin 1988), but the only stage available for test- ing. Larvae were obtained from hatched egg masses that had been laid by 42 blood- engorged females of winter ticks collected from 3 dead calf moose in Milan and Berlin, New Hampshire (P. Pekins holds permits). The fed females were transported to the lab- oratory in Whirl-Pak bags in 5 L coolers containing cold packs (Koolit). In the labo- ratory, a single, fed, female tick was placed into a mesh-covered, 50 mL polypropylene tube in an incubator held at 93% RH (Winston and Bates 1960) in a 3000 mL desiccator at 25 ± 0.5 °C at 10 h L:14 h D. After the female laid eggs, the egg mass was removed, placed into a clean 50 mL polypro- pylene tube and kept at 93% RH, 25 °C, and 10 h L:14 h D for hatching. The females and eggs were not surface sterilized because they were stored in sterile tubes for oviposition and hatching. Any differences in mortality would be the result of fungus treatment because the larvae originated from the same batch of ticks. Keys were used to confirm the identity of adult ticks as D. albipictus (Lindquist et al. 2016). The larvae were ~4 months of age when exposed to treatment with spore inocula to match the age at which they quest for a host. An aspirator was used to handle larvae, one larva at a time. The aspirator was made by attaching a mesh-covered pipette tip to the end of a piece of Tygon tubing. Larvae were selected randomly from 42 tubes of hatched egg masses; thus, all larvae did not originate from the same egg mass for a par- ticular treatment group. Prior to experimen- tation, larvae were examined at 40x using a stereoscopic microscope and checked for their ability to self-right and crawl 5 body lengths (health threshold); only healthy lar- vae were used in the experiment. Larvae were treated in groups of 10 with 1 mL of a particular spore inoculum, or PBS + 0.05% Tween control, in a 1.5 mL micro- centrifuge tube. The tube containing ticks was gently agitated for 2 min after which the contents were poured on a piece of filter paper (No. 3, Whatman, Hillsboro, Oregon, USA). Individual larvae that could self-right and crawl 5 body lengths were collected off the filter paper, and each was placed into a clean, 1.5 mL mesh-covered microcentrifuge tube (1 larva per tube). Larvae were stored at 80% RH (Winston and Bates 1960), 25 °C, and 10h:14h L:D cycle in a sealed glass des- iccator placed in an incubator. Larvae were ALCES VOL. 54, 2018 YODER ET AL. – ENTOMOPATHOGENIC FUNGI IN MOOSE WALLOWS 59 examined daily at 40-45X using stereoscopic microscopy and the number of dead larvae was counted 10 days post-treatment; indica- tion of death included lack of movement, curled legs, and deflated opisthosoma. Each spore inoculum experiment was replicated 10 times on 10 tubes of larvae (n = 100 larvae for each treatment). Data are expressed as the mean ± SE. An Abbott correction and log- it-transformation, and ANCOVA for number of survivors (P = 0.05; JMP, SAS Institute, Cary, North Carolina, USA) were used to compare mortality between treatment and control groups. Larvae were exposed to those fungi isolated most frequently from the wal- low soil samples. Reisolation of fungi Dead larvae were rinsed in a solution of DI water:absolute ethanol:5.25% NaOCl (18:1:1 v/v/v) for 1 min, followed by 2, 1-min rinses in DI water. This treatment in mild bleach solution was to externally remove fungi from the tick’s body surface. After the final rinse, each larva was sec- tioned in half with a scalpel, and each por- tion was embedded separately into PDA growth medium in a Petri plate and incu- bated at 25 °C in darkness. Plates were examined daily at 45X for the appearance of hyphae emerging from within the tick’s internal body contents. A 1 cm3 block of agar containing the hyphal tip was removed from the plate for subculturing on a fresh plate of PDA (25 °C, darkness). Three additional subcultures were performed to purify the fungus for identification. The fungus that was isolated internally from within the larval tissues was compared to the fungus that was used to prepare the treatment inoculum as confirmation of infection (Koch’s postu- lates). All dead larvae were analyzed for internal fungi within a particular treatment group and separated into statistical repli- cates. The number of dead larvae that tested positive for the treatment fungus was expressed as the mean ± SE and compared using an analysis of covariance (ANCOVA - Tukey’s Test; significance at P < 0.05). RESULTS Sampling locations Soil pH of samples from wallow and proximal samples ranged, respectively, from 5.6 – 6.3 and 5.1 – 5.8 at Katahdin, 5.0 – 6.4 and 5.4 – 6.0 at Greenville, and 5.8 – 6.6 and 5.1 – 6.0 at Milan. To match these soil condi- tions, we used slightly acidified potato dex- trose agar (PDA + lactic acid to pH 5.5) as the agar growth medium. Preliminary observa- tions indicated that wallow sample cultures contained a larger number of bacterial colo- nies than the proximal samples. Bacteria appeared prior to the appearance of the major- ity of fungal colonies that were discerned by the presence of hyphae, a mycelium, and capacity to produce spores. Accordingly, the media were supplemented with 0.05% chlo- ramphenicol to limit bacterial growth. Mycoflora profile Tables 1-3 list the genera that were iso- lated. Water-treated controls showed 1 Trichoderma spp. colony/45 plates. Large variability existed in the amount of fungi (CFU/g dry soil) between wallow and proxi- mal samples. The fungal diversity index was lower in all 4 wallow (versus proximal) sam- ples at Katahdin, in 9 of 13 wallow samples at Greenville, and 5 of 7 wallow samples at Milan (Tables 1-3). There were 1-3 samples at each geographic location that had either much higher or lower (orders of magnitude) relative amounts of fungi (Fig. 1). Aspergillus flavus was present at each geographic location, with 75% abundance at Katahdin, 62% at Greenville, and 29% at Milan (Tables 1-3). Abundance of A. fumig­ atus was 50% Katahdin, 38% at Greenville, and 14% at Milan. There was a lower ENTOMOPATHOGENIC FUNGI IN MOOSE WALLOWS – YODER ET AL. ALCES VOL. 54, 2018 60 Table 1. Percent occurrence of fungus genera in soil from moose wallows and adjacent control sites from Katahdin, Maine, USA. Fungi % mean # isolates K1 K2 K3 K4 P W P W P W P W Composition (± SE ≤ 3.2) Absidia spp. 11 0 14 0 0 0 0 0 Alternaria spp. 0 0 0 0 0 0 2 0 Aspergillus spp. 0 0 14 0 0 0 0 0 Aspergillus flavus 0 16 0 0 8 0 7 0 Aspergillus fumigatus 0 0 0 0 8 0 2 0 Beauveria bassiana 0 0 14 8 0 0 4 0 Cladosporium spp. 11 0 0 0 15 11 4 0 Fusarium spp. 0 16 0 0 8 0 7 11 Mortierella spp. 11 0 14 25 23 22 20 11 Mucor spp. 34 16 14 33 8 22 13 22 Paecilomyces spp. 0 0 0 0 0 11 9 0 Penicillium spp. 11 0 0 8 23 11 16 43 Scopulariopsis spp. 11 0 0 8 8 0 4 0 Trichoderma spp. 11 48 29 17 0 22 11 11 Simpson’s index, 1/D 5.5 3.1* 5.6 4.5* 6.7 5.7* 9.0 3.7* SE 0.1 0.2 0.1 0.2 0.1 0.2 0.2 0.1 Isolate, 1 cm3 block of an individual fungus colony on a culture plate. P = proximal soil sample; and W = wallow soil sample. * denotes a significant difference between the proximal-wallow pair at the geographic site. Table 2. Percent occurrence of fungus genera in soil from moose wallows and adjacent control sites from Greenville, Maine, USA. Fungi % mean # isolates G1 G2 G3 G4 G5 G6 G7 P W P W P W P W P W P W P W Composition (± SE ≤ 3.6) Absidia spp. 7 8 17 0 17 13 0 0 2 3 0 13 5 4 Acremonium spp. 0 0 0 0 0 0 0 0 2 0 0 0 0 0 Alternaria spp. 4 0 0 0 0 0 7 0 0 6 10 0 3 9 Aspergillus spp. 7 4 0 14 0 13 0 0 0 0 10 0 0 9 Aspergillus flavus 2 4 0 0 0 0 7 0 4 3 0 0 5 0 Aspergillus fumigatus 4 0 0 0 0 0 0 0 4 0 0 0 3 0 Aspergillus niger 0 0 0 0 0 0 0 0 0 3 0 0 0 0 Beauveria bassiana 4 0 17 0 0 0 0 0 2 5 0 0 3 0 Cladosporium spp. 7 13 0 0 0 0 0 0 4 3 10 0 3 9 Table 2 Continued ... ALCES VOL. 54, 2018 YODER ET AL. – ENTOMOPATHOGENIC FUNGI IN MOOSE WALLOWS 61 Table 2. continued Fungi % mean # isolates G1 G2 G3 G4 G5 G6 G7 P W P W P W P W P W P W P W Epicoccum spp. 2 0 0 0 17 0 0 0 4 0 0 0 0 4 Fusarium spp. 0 0 0 0 0 13 10 0 6 8 20 0 8 4 Mortierella spp. 16 4 33 14 0 13 19 14 14 21 10 13 19 9 Mucor spp. 9 33 17 29 17 25 10 29 10 10 10 25 5 13 Paecilomyces spp. 0 4 0 0 0 0 16 0 12 0 0 0 11 4 Penicillium spp. 13 17 17 14 0 0 16 14 8 19 20 13 11 13 Rhizopus spp. 7 0 0 0 0 0 0 0 0 0 0 0 0 0 Scopulariopsis spp. 0 0 0 0 0 0 0 0 2 2 0 0 5 0 Trichoderma spp. 13 13 0 29 33 25 16 43 16 21 10 38 16 22 Umbelopsis spp. 4 0 0 0 17 0 3 0 8 0 0 0 5 0 Simpson’s index, 1/D 11.2 5.7* 4.7 4.6 4.7 5.6* 8.0 3.3* 11.3 7.6* 7.6 4.2* 10.5 8.9* SE 0.2 0.1 0.2 0.2 0.2 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.2 0.2 Fungi % mean # isolates G8 G9 G10 G11 G12 G13 P W P W P W P W P W P W Composition (± SE ≤ 3.1) Absidia spp. 0 3 0 5 5 2 3 15 0 14 0 9 Acremonium spp. 0 0 0 0 0 0 0 0 0 0 4 0 Alternaria spp. 3 0 0 3 0 0 3 0 0 0 0 0 Aspergillus spp. 3 0 5 0 16 7 6 0 0 14 7 0 Aspergillus flavus 5 7 0 3 0 0 0 0 6 0 2 0 Aspergillus fumigatus 0 0 0 0 0 0 6 0 6 0 0 0 Aspergillus niger 0 0 5 0 0 0 0 0 0 0 0 0 Beauveria bassiana 3 3 0 8 0 0 3 0 0 0 2 4 Cladosporium spp. 11 7 0 5 0 9 19 23 6 29 15 4 Fusarium spp. 11 14 0 0 11 11 0 4 0 0 2 0 Mortierella spp. 5 17 19 10 16 7 13 15 25 14 13 22 Mucor spp. 11 14 10 18 16 17 6 23 13 0 11 9 Mycelia sterilia 3 0 0 0 0 0 0 0 0 0 0 0 Nigrospora spp. 3 0 0 3 5 0 0 0 0 0 0 0 Paecilomyces spp. 8 0 14 13 0 0 3 4 0 0 7 4 Penicillium spp. 8 17 19 13 16 19 10 12 13 14 22 22 Rhizopus spp. 0 0 0 0 0 0 3 0 13 0 0 0 Scopulariopsis spp. 5 0 0 0 5 0 6 0 0 0 0 0 Trichoderma spp. 16 21 24 18 11 17 10 8 19 14 15 26 Umbelopsis spp. 5 0 5 5 0 11 6 0 0 0 0 0 Table 2 Continued ... ENTOMOPATHOGENIC FUNGI IN MOOSE WALLOWS – YODER ET AL. ALCES VOL. 54, 2018 62 Table 2. continued Fungi % mean # isolates G8 G9 G10 G11 G12 G13 P W P W P W P W P W P W Verticillium spp. 3 0 0 0 0 0 0 0 0 0 0 0 Simpson’s index, 1/D 13.2 7.4* 6.4 9.3* 8.1 7.8 10.9 6.5* 6.7 5.6* 7.9 5.6* SE 0.2 0.1 0.2 0.1 0.2 0.2 0.1 0.2 0.1 0.1 0.1 0.2 Isolate, 1 cm3 block of an individual fungus colony on a culture plate. P = proximal soil sample and W = wallow soil sample. * denotes a significant difference between the proximal-wallow pair at the geographic site. Table 3. Percent occurrence of fungus genera in soil from moose wallows and adjacent control sites from Milan, New Hampshire, USA. Fungi % mean # isolates M1 M2 M3 M4 M5 M6 M7 P W P W P W P W P W P W P W Composition (± SE ≤ 3.3) Absidia spp. 2 0 3 0 5 11 0 0 3 2 9 5 0 10 Alternaria spp. 0 0 0 0 0 0 0 0 0 6 8 0 0 0 Aspergillus spp. 0 0 11 0 5 9 13 0 6 0 9 3 0 0 Aspergillus flavus 0 0 0 0 2 0 0 0 6 0 0 0 0 0 Aspergillus fumigatus 0 0 0 0 0 0 0 0 0 0 0 3 0 0 Beauveria bassiana 0 0 0 0 6 0 0 0 0 0 4 8 0 0 Cladosporium spp. 10 9 6 6 7 2 13 0 11 0 8 10 0 0 Epicoccum spp. 2 0 0 0 0 0 0 9 11 6 0 0 0 10 Fusarium spp. 12 1 4 0 7 7 0 0 6 6 0 0 0 0 Geotrichum spp. 0 0 0 0 0 0 0 0 3 0 0 8 0 0 Mortierella spp. 21 19 19 56 17 13 0 36 8 23 13 8 17 20 Mucor spp. 10 42 19 11 7 18 25 9 8 17 6 18 17 10 Nigrospora spp. 2 0 0 0 0 2 0 0 0 0 9 0 0 0 Paecilomyces spp. 0 0 3 0 5 0 0 0 3 0 0 0 0 0 Penicillium spp. 21 13 13 11 12 18 40 18 17 17 11 18 33 20 Rhizopus spp. 0 0 1 0 0 0 0 0 0 0 0 0 0 0 Scopulariopsis spp. 0 0 3 0 7 2 0 9 3 0 0 3 0 0 Trichoderma spp. 14 13 12 17 13 13 13 18 14 23 15 18 17 30 Umbelopsis spp. 5 3 6 0 7 0 0 0 3 0 6 0 17 0 Verticillium spp. 0 0 3 0 0 0 0 0 0 0 2 0 0 0 Simpson’s index, 1/D 7.1 4.0* 8.9 2.8* 11.5 7.2* 4.1 4.6 11.4 6.0* 11.3 8.5* 4.7 5.2 SE 0.1 0.1 0.2 0.2 0.2 0.1 0.1 0.2 0.1 0.1 0.2 0.1 0.1 0.1 Isolate, 1 cm3 block of an individual fungus colony on a culture plate. P = proximal soil sample; and W = wallow soil sample. * denotes a significant difference between the proximal-wallow pair at the geographic site. ALCES VOL. 54, 2018 YODER ET AL. – ENTOMOPATHOGENIC FUNGI IN MOOSE WALLOWS 63 presence of A. niger: 0% at Katahdin, 15% at Greenville, and 0% at Milan. Occurrence of B. bassiana was 50% at Katahdin, 62% at Greenville, and 29% at Milan. Species detectability and abundance was higher in proximal than wallow samples, except for A. niger at Greenville. Tick mortality The most common fungi at each wallow (n = 24) were tested for pathogenicity against winter tick larvae. Higher mortality (P < 0.05) compared to the PBS + Tween- treated control (5-16% mortality) occurred consistently for 8 genera that were isolated at all 3 geographic locations (Aspergillus spp., Beauveria bassiana, Fusarium spp., Mortierella spp., Mucor spp., Paecilomyces spp., Penicillium spp., and Trichoderma spp.). The mean proportion killed by these genera ranged from ~30-95%, with Beauveria bassiana, Mortierella spp., Mucor spp., and Paecilomyces spp. the most lethal (>50%; Tables 4-6). These 4 genera were also recovered from larval cadavers at a rate higher than controls. Trichoderma spp. was recovered from cadavers for 20 of 24 wal- lows, and Penicillium spp. at 18 of 22 wal- lows; both were less lethal (typically <50%) than the other 4 genera (Tables 4-6). Aspergillus flavus was the sole Aspergillus isolate at 4 wallows and occurred within a test mixture of Aspergillus at another. It induced mortality (23-78%) and was recov- ered from larval cadavers at these specific wallows; otherwise, it did not cause signifi- cant larval mortality. Mortality associated with Fusarium spp. was mixed and recovery Fig. 1. Amount of fungi (colony forming unit, CFU/g dry soil) in soil samples collected in Katahdin (K) and Greenville (G), Maine, and Milan (M), New Hampshire, USA. The * above a bar denotes a significant difference (P < 0.05) between proximal and wallow soil samples at that geographic site. Note the variation in fungus amounts: sometimes proximal > wallow, proximal < wallow, and proximal = wallow. ENTOMOPATHOGENIC FUNGI IN MOOSE WALLOWS – YODER ET AL. ALCES VOL. 54, 2018 64 from larval cadavers was irregular. Isolates of Cladosporium spp. and Umbelopsis spp. did not cause larval mortality. DISCUSSION The 8 genera of fungi found patho- genic against winter tick larvae have been previously identified as entomopathogenic against ticks (Chandler et al. 2000, Samish et al. 2004, Greengarten et al. 2011; Trichoderma spp. listed as its teleomorph Hypocrea in Greengarten et al. 2011; Yoder et al. 2017a). When artificially infected by topical application, each of these fungi was Table 4. Mortality of healthy larvae of Dermacentor albipictus after 10 days exposed to spores (1.6 × 108 spores/larva) from wallow soil fungi from Katahdin, Maine, USA. Treatment # of larvae 10 days post-treatment Control K1 K2 K3 K4 Dead/100 larvae 11 ± 2.8 % positive for fungus 27 ± 1.2 Aspergillus spp. Dead/100 76 ± 2.7* - - - % positive for fungus 82 ± 1.4* - - - Beauveria bassiana Dead/100 - 86 ± 4.1* - - % positive for fungus - 93 ± 2.7* - - Cladosporium spp. Dead/100 - - 6 ± 0.7 - % positive for fungus - - 0 - Fusarium spp. Dead/100 11 ± 0.5 - - 16 ± 1.6* % positive for fungus 9 ± 0.3 - - 19 ± 2.1 Mortierella spp. Dead/100 - 67 ± 3.1* 89 ± 2.4* 66 ± 2.4* % positive for fungus - 93 ± 2.0* 95 ± 1.9* 86 ± 1.9* Mucor spp Dead/100 58 ± 4.0* 36 ± 3.4* 73 ± 2.2* 84 ± 3.7* % positive for fungus 86 ± 3.2* 61 ± 1.7* 90 ± 1.1* 95 ± 2.9* Paecilomyces spp Dead/100 - - 86 ± 3.7* - % positive for fungus - - 85 ± 2.4* - Penicillium spp. Dead/100 - 65 ± 2.0* 21 ± 2.9* 41 ± 3.6* % positive for fungus - 49 ± 1.8* 33 ± 2.1 66 ± 3.2* Trichoderma spp. Dead/100 63 ± 2.4* 44 ± 3.1* 71 ± 4.5* 39 ± 2.3* % positive for fungus 78 ± 2.8* 68 ± 1.9* 58 ± 2.2* 79 ± 3.1* Control, PBS + 0.05% Tween. - indicates not determined, isolate not present in wallow soil. Data are the mean ± SE. * indicates significant greater difference from respective control (P < 0.05). ALCES VOL. 54, 2018 YODER ET AL. – ENTOMOPATHOGENIC FUNGI IN MOOSE WALLOWS 65 Table 5. Mortality of healthy larvae of Dermacentor albipictus after 10 days exposed to spores (1.6 × 108 spores/larva) from wallow soil fungi from Greenville, Maine, USA. Treatment # of larvae 10 days post-treatment G1 G2 G3 G4 G5 G6 G7 Aspergillus spp. Dead/100 81 ± 3.2* 42 ± 4.3* 21 ± 1.7* - 68 ± 2.1* - 28 ± 3.1* % positive for fungus 91 ± 2.2* 21 ± 2.8 38 ± 2.4 - 94 ± 1.3* - 21 ± 2.1 Beauveria bassiana Dead/100 - - - - 91 ± 2.7* - - % positive for fungus - - - - 97 ± 2.1* - - Cladosporium spp. Dead/100 11 ± 0.6 - - - 7 ± 0.9 - 5 ± 1.4 % positive for fungus 18 ± 1.1 - - - 14 ± 0.8 - 0 Fusarium spp. Dead/100 - - 16 ± 2.5* - 20 ± 1.9* - 11 ± 2.0 % positive for fungus - - 25 ± 1.5 - 40 ± 2.2* - 27 ± 1.8 Mortierella spp. Dead/100 43 ± 2.8* 71 ± 3.3* 78 ± 3.1* 61 ± 3.3* 39 ± 2.2* 42 ± 1.2* 72 ± 2.8* % positive for fungus 84 ± 1.7* 76 ± 2.0* 90 ± 2.2* 82 ± 2.4* 77 ± 1.7* 90 ± 2.5* 88 ± 3.1* Mucor spp. Dead/100 60 ± 2.9* 54 ± 3.1* 56 ± 1.9* 46 ± 3.1* 52 ± 1.9* 71 ± 1.3* 60 ± 3.4* % positive for fungus 82 ± 3.1* 91 ± 2.7* 82 ± 2.3* 70 ± 2.6* 85 ± 1.4* 83 ± 1.4* 68 ± 2.1* Paecilomyces spp. Dead/100 57 ± 2.8* - - - - - 61 ± 2.9* % positive for fungus 65 ± 1.4* - - - - - 90 ± 2.1* Penicillium spp. Dead/100 21 ± 2.7* 49 ± 3.5* - 24 ± 2.0* 47 ± 2.9* 31 ± 2.2* 45 ± 3.3* % positive for fungus 38 ± 2.1 63 ± 2.4* - 71 ± 1.7* 83 ± 1.9* 55 ± 2.3* 64 ± 3.1* Trichoderma spp. Dead/100 29 ± 3.4* 43 ± 2.6* 19 ± 3.1* 34 ± 2.6* 41 ± 3.1* 22 ± 3.0* 42 ± 2.4* % positive for fungus 58 ± 4.2* 70 ± 3.1* 37 ± 2.1 76 ± 3.1* 85 ± 2.2* 32 ± 3.0 79 ± 1.3* Treatment # of larvae 10 days post-treatment G8 G9 G10 G11 G12 G13 Aspergillus spp. Dead/100 73 ± 3.1* 78 ± 2.2* 15 ± 1.8 - 26 ± 3.1* - % positive for fungus 84 ± 2.1* 83 ± 1.6* 27 ± 2.0 - 31 ± 2.2 - Beauveria bassiana Dead/100 83 ± 2.1* 91 ± 2.6* - - - 87 ± 3.2* % positive for fungus 93 ± 1.8* 92 ± 2.5* - - - 84 ± 2.7* Cladosporium spp. Dead/100 14 ± 1.5 12 ± 1.7 16 ± 0.8 6 ± 1.1 10 ± 1.1 5 ± 0.9 Table 5 Continued ... ENTOMOPATHOGENIC FUNGI IN MOOSE WALLOWS – YODER ET AL. ALCES VOL. 54, 2018 66 recovered from within the tissues of larval cadavers by internal fungus culture as con- firmation of infection. Changes in moisture level, relative humidity, salts, and extent of aeration quan- titatively alter the fungal activity and amount in disturbed soil (Miller and Lodge 2007, Morris et al. 2007). Such changes could be attributed to moose activity: 1) removal of surface habitat by digging that changes the drainage patterns and moisture level within the wallow, 2) concentration and frequency of salts from urination in the wallow, 3) changes in aeration from loosening and compacting soil from the digging, and 4) the overall frequency and intensity of distur- bance of the soil. This contrasts to the rela- tively stable, undisturbed conditions of the proximal soil samples where fungi were typ- ically more abundant and diverse. Fungi that were not cultured with our methods certainly exist in the soil samples. Whether the amount and composition of soil fungi were measurably influenced by stand type, age, and canopy cover associ- ated with the wallows cannot be answered given our somewhat random approach in locating active wallows. Clearly, moose can Table 5. Continued Treatment # of larvae 10 days post-treatment G8 G9 G10 G11 G12 G13 % positive for fungus 7 ± 1.8 33 ± 0.7 19 ± 1.1 0 20 ± 1.3 0 Fusarium spp. Dead/100 22 ± 3.1* - 26 ± 3.1* 14 ± 1.9 - - % positive for fungus 64 ± 2.3* - 65 ± 2.4* 36 ± 2.1 - - Mortierella spp. Dead/100 55 ± 2.4* 66 ± 2.5* 49 ± 2.2* 75 ± 2.7* 81 ± 2.3* 56 ± 3.1* % positive for fungus 85 ± 3.0* 80 ± 1.2* 82 ± 1.9* 76 ± 1.7* 83 ± 2.7* 88 ± 2.6* Mucor spp. Dead/100 81 ± 2.4* 74 ± 2.2* 59 ± 2.1* 47 ± 3.4* - 63 ± 2.4* % positive for fungus 95 ± 2.0* 93 ± 2.0* 93 ± 2.7* 83 ± 3.2* - 90 ± 2.4* Paecilomyces spp. Dead/100 - 70 ± 2.0* - 68 ± 3.5* - 71 ± 3.6* % positive for fungus - 81 ± 1.7* - 88 ± 2.6* - 73 ± 2.0* Penicillium spp. Dead/100 52 ± 2.4* 26 ± 2.1* 38 ± 2.4* 44 ± 1.9* 51 ± 2.0* 29 ± 2.2* % positive for fungus 79 ± 2.8* 31 ± 3.0 59 ± 1.9* 75 ± 2.2* 61 ± 2.5* 28 ± 1.9 Trichoderma spp. Dead/100 61 ± 3.0* 37 ± 2.1c* 26 ± 2.7* 31 ± 2.8* 46 ± 3.4* 31 ± 3.1* % positive for fungus 85 ± 2.4* 64 ± 3.2* 31 ± 2.3 77 ± 2.6* 72 ± 2.6* 35 ± 2.1 Umbelopsis spp. Dead/100 - 11 ± 1.4 4 ± 0.5 - - - % positive for fungus - 0 3 ± 0.2 - - - - indicates not determined, isolate not present in wallow soil. Data are the mean ± SE. * indicates significant greater difference from control in Table 4 (P < 0.05). ALCES VOL. 54, 2018 YODER ET AL. – ENTOMOPATHOGENIC FUNGI IN MOOSE WALLOWS 67 acquire fungi at bedding sites, in open, regenerating forest habitats, and in muddy locales – especially on bare soil. Aspects of wallowing behavior and any moose-winter tick-fungal relationship is likely not univer- sal given the wide geographic range, diverse use of habitats and forest types, and variable seasonal and microclimatic ground condi- tions found throughout North American moose range. From a fungal ecology perspective, the mycoflora of wallow soil can best be explained relative to soil disturbance, in par- ticular digging and burrowing (Kumpula et al. 2000, Shubina et al. 2013). The proximal soil samples can be considered as undis- turbed soils due to the absence of direct dig- ging/activity. The qualitative differences between the wallow and proximal samples presumably reflect the disturbance to wallow Table 6. Mortality of healthy larvae of Dermacentor albipictus after ten days exposed to spores (1.6 × 108 spores/larva) from wallow soil fungi from Milan, New Hampshire, USA. Treatment # of larvae 10 days post-treatment: M1 M2 M3 M4 M5 M6 M7 Aspergillus spp. Dead/100 - - 23 ± 2.9* - - 35 ± 3.4* - % positive for fungus - - 35 ± 3.2 - - 63 ± 2.6* - Beauveria bassiana Dead/100 - - - - - 91 ± 2.3* - % positive for fungus - - - - - 80 ± 2.2* - Cladosporium spp Dead/100 7 ± 0.4 8 ± 0.5 6 ± 1.1 - - 15 ± 0.9 - % positive for fungus 0 13 ± 1.2 0 - - 13 ± 0.4 - Fusarium spp. Dead/100 21 ± 2.0* - 25 ± 3.4* - 31 ± 2.2* - - % positive for fungus 19 ± 2.4 - 44 ± 2.8* - 65 ± 3.1* - - Mortierella spp. Dead/100 74 ± 3.1* 79 ± 3.4* 64 ± 2.2* 72 ± 2.4* 58 ± 2.5* 74 ± 2.3* 68 ± 3.3* % positive for fungus 77 ± 2.9* 77 ± 3.5* 91 ± 3.1* 92 ± 2.1* 84 ± 2.5* 93 ± 2.8* 78 ± 2.6* Mucor spp. Dead/100 81 ± 2.2* 54 ± 3.2* 40 ± 2.8* 66 ± 2.0* 66 ± 2.1* 80 ± 3.1* 59 ± 2.7* % positive for fungus 93 ± 1.8* 91 ± 2.6* 78 ± 2.6* 86 ± 2.4* 91 ± 3.4* 84 ± 2.2* 85 ± 2.2* Penicillium spp. Dead/100 34 ± 2.5* 45 ± 2.7* 31 ± 3.3* 73 ± 3.1* 38 ± 2.1* 47 ± 2.8* 54 ± 2.9* % positive for fungus 44 ± 2.2 87 ± 2.4* 26 ± 2.0 90 ± 2.9* 79 ± 2.7* 68 ± 2.4* 84 ± 3.0* Trichoderma spp. Dead/100 57 ± 3.0* 53 ± 2.2* 26 ± 2.8* 33 ± 2.4* 41 ± 1.9* 57 ± 2.5* 53 ± 2.6* % positive for fungus 81 ± 3.0* 83 ± 1.9d* 73 ± 2.4* 58 ± 2.3* 66 ± 2.2* 68 ± 2.3* 75 ± 2.4* Umbelopsis spp. Dead/100 15 ± 1.2 - - - - - - % positive for fungus 27 ± 1.1 - - - - - - -, not determined, isolate not present in wallow soil. Data are the mean ± SE. *, significant greater difference from control in Table 4 (P < 0.05). ENTOMOPATHOGENIC FUNGI IN MOOSE WALLOWS – YODER ET AL. ALCES VOL. 54, 2018 68 soil that modifies the fungal community structure (criteria from Miller and Lodge 2007, Morris et al. 2007): 1) there was lower fungal diversity in wallow soil than proximal soil, 2) a group of fungi (Mortierella spp., Penicillium spp., Mucor spp., and Trichoderma spp.) that function as primary colonizers (i.e., the first to grow on roots) predominates in wallow soil, and 3) the amount of total fungi was highly variable exhibiting no consistent pattern in CFU/g soil between wallow and proximal samples. Qualitatively, each time a moose visits a wal- low and disturbs the soil, the fungi subse- quently recolonize in response, indicating why Mortierella spp., Penicillium spp., Mucor spp., Trichoderma spp., and other genera were isolated most frequently. The extent that male urine alters the soil environ- ment, in positive or negative ways, is not known and invites further inquiry. Fur, hooves, and horns of animals harbor numerous saprobic fungi acquired from forest soils (Shubina et al. 2013), including many fungi taxa identified here. We conclude that through soil disturbance, wallowing behavior makes spores readily available in infections (Mortierella spp., Penicillium spp., Mucor spp., Trichoderma spp.) in wallow soil. We emphasize that some pathogenic genera (i.e., Aspergillus flavus, Beauveria bassiana, Paecilomyces spp., Scopulariopsis spp.) were even more abundant in proximal soil samples. Thus, areas outside of wallows are not neces- sarily less important as potential sources of infection. All of these fungi are heavily spore-producing genera, and our study further identifies that their fungal strains are indeed pathogenic to winter tick larvae, many with a lethal rate >50%. 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