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Scopulariopsis brevicaulis, the anamorph of Microascus brevicaulis (Microascaceae, Ascomycota), has been identified in the body contents of the tick Dermacentor variabilis. After topical application of the fungal inoculum, tick mortality was marked. This is the first account describing the internal mycoflora of D. variabilis with a novel technique used to recover potential biological control agents.
Obligate parasitic entomogenous fungi, most notably Beauveria bassiana (8), which is used in biological control (12), represent only a small fraction of the diversity of microscopic fungi found naturally on the body surfaces of ticks. The majority of these fungi are anamorphs of ascomycetes capable of producing copious numbers of conidia (asexual spores). In nature, these fungi usually exist as harmless saprophytes or biotrophs that, on occasion, come into contact with terrestrial arthropods inhabiting soil debris (13). Under certain environmental conditions, saprophytic fungi may become parasitic, and it is assumed that some of the external tick mycoflora may, in fact, harbor these potential parasites that are believed to be responsible for natural reductions in tick populations (13). As such, this mycoflora may represent an understudied resource with considerable potential for biological control. To date, the identity and whereabouts of fungi present internally in ticks remain unstudied and no technique to isolate such fungi in pure culture exists. Such a technique represents an urgently needed, practical tool for studies aimed at acquiring tick fungi as potential biological control agents.
Under conditions of high relative humidity, fungi are known to produce conidia over a tick's body surface, and this feature may lead to the effective spread of the fungus through the tick population. Fungal hyphae from germinating conidia may be capable of penetrating the cuticle directly or may invade via the genital pore, anus, or spiracles, thereby preventing the hatching of eggs or causing tick death (12). The application of such fungi has potential for use against Ixodes ricinus (and other members of this species complex), Boophilus microplus, Rhipicephalus appendiculatus, Hyalomma detritum, Dermacentor reticulatus, and Dermacentor marginatus (12, 13). Although the American dog tick, Dermacentor variabilis, a vector of Rocky Mountain spotted fever and tularemia, is ubiquitous in the United States, no studies have examined this species for the presence of naturally occurring fungi. The discovery and development of fungi for the biological control of D. variabilis would be of particular value considering that pheromone-assisted techniques are limited because there are few known attractants for this tick (7, 17).
It was the central objective of this study to examine the mycoflora of nonfed D. variabilis females, with the goal of isolating, culturing, and identifying naturally occurring fungi on the tick's body surface. To screen more effectively for parasitic fungi, a technique modified from plant pathology (18) was used to detect and identify fungi from internal tick body contents. We anticipated that these isolates would be particularly effective for biological control because it is assumed that they penetrate the tick's cuticle.
D. variabilis ticks were obtained from field collections (Clark Co., Springfield, Ohio) and 10-year established laboratory colonies (Oklahoma State University, Stillwater) and stored at 22 to 24°C, with a 14- and 10-h light-dark cycle, at 93% rH over a saturated solution of KNO3 (15) prior to use. Only nonfed female adults (age, 2 to 4 weeks postecdysis for the laboratory population and unknown for field material) were used in the experiment. In all cases, ticks were handled with sterile forceps and all materials were sterilized before use.
Ticks (n = 12) were sampled for the presence of fungi on body surfaces by a standard aseptic technique (5). Briefly, the ticks were shaken vigorously in deionized (DI) water in capped vials (up to four ticks per vial) for 1 min, the water was discarded, and the process was repeated. The ticks were transferred to a petri plate (inside diameter [i.d.], 9 cm; three ticks per plate) and covered with 20 ml of molten modified Melin-Norkrans agar (9). The plate contents were gently swirled prior to solidification of the agar to separate the ticks and allowed to cool. Plates were incubated at 22 to 24°C for 1 week, with periodic observations for fungal growth. With the aid of a dissection microscope, pure fungal cultures were obtained by using a sterile scalpel to remove hyphal tips from actively growing mycelia emerging from the tick exoskeletons. The hyphal tips were subcultured onto potato dextrose agar (Fisher Scientific, St. Louis, Mo.) and incubated at 22 to 24°C until cultural characteristics could be induced (2 weeks). Fungi were identified according to the keys described by Barnett (2).
Next, using a method described previously but with modifications (5), we determined whether ticks (n = 13) harbored active fungi internally. First, external fungi were killed by sterilizing the tick surfaces by shaking the ticks vigorously for 1 min in a capped sterile vial (up to four ticks per vial) in a solution of DI water-absolute ethanol-5.25% NaOCl (18:1:1, vol/vol/vol). The surface sterilization solution was removed, and the ticks were rinsed twice (with shaking) for 1 min with fresh sterile DI water. The ticks were transferred to a petri plate (i.d., 9 cm) and cut into fourths with a sterile scalpel. The scalpel was sterilized after each tick was sectioned to ensure that each tick harbored its own specific fungal isolate(s). Tick sections were placed into fresh petri plates, molten modified Melin-Norkrans medium was added, and the plate contents were gently swirled and allowed to cool. Pure cultures were obtained from hyphal tips that were excised only from hyphae originating from within the tick.
A modification of a standard technique (6) was followed to examine pathogenicity. A fungus suspension was prepared from pure fungal isolates in sterile water and shaken overnight at 37°C. Viable spores (conidia) were counted with a hemacytometer (AO Spencer Bright-Line, St. Louis, Mo.) by the trypan blue (0.1%) exclusion test and expressed as numbers of conidia per milliliter. The preparation was diluted to reflect the concentrations used in previous studies of ticks (6, 8) to permit comparisons with the literature. Ticks were coated with 5 μl of the fungus suspension, placed into individual mesh-covered vials (one tick per chamber), and stored at 93% rH, with a 14- and 10-h light-dark cycle, and at 22 to 24°C. A diluted preparation (1/2 dilution) was also tested; water served as a control. No oil formulations were tested.
Prior to treatment with the fungus, the ticks were stripped of all external fungi and bacterial contaminants by a 10-s shake in water-ethanol-5% NaOCl (for surface sterilization) to be certain that mortality was due to fungal treatment and not to a fungus already present. For confirmation of our surface sterilization technique, treated ticks were embedded in nutrient agar (Fisher Scientific) in a petri plate (i.d., 9 cm), incubated at 37°C, and observed after 2 days; no bacterial or fungal growth was evident (data not shown). Surface-sterilized ticks that were not treated with the fungus served as controls. Ticks were observed daily for 25 days. Ticks were considered dead when they would not respond to stimuli, would not crawl one body length when prodded, and had their legs tightly curled under their bodies. Percent mortality was calculated using Abbott's equation (10), which corrects for mortality in the controls: [(number surviving in control − number surviving treatment)/number surviving in control] × 100.
Chi-square test statistics were used to compare data, with 100% mortality as the expected value. The level of significance was at a P of 0.05 with 1 df.
To determine whether fungi isolated from the ticks displayed antibactericidal properties, pure fungal cultures were subcultured on nutrient agar in a petri plate (i.d., 9 cm) that was subsequently flooded with pure broth cultures of Staphylococcus aureus and Escherichia coli (Fisher Scientific) and incubated at 37°C for 2 days. An Enterotube II (BBL, Becton Dickinson, Sparks, Md.) was used to confirm the identity of these bacteria and certain naturally occurring unknown bacteria; after inoculation, the Enterotube was incubated at 37°C and the results were read after 2 days.
Upon macroscopic and microscopic examination, the tick surfaces had no visible fungal structures. One fungus, identified as Scopulariopsis brevicaulis, was recovered from the internal tick contents (n = 25 ticks). The presence of this fungus was especially pronounced in all of the tick body sections of all of the ticks sampled. On potato dextrose agar, the colony growth rate was moderate (0.9 mm/day), yielding copious numbers of conidia that provided the culture with a tan, powdery appearance on the colony surface when incubation lasted >10 days at 21°C. Colony margins were slightly submerged, entire, and creamy white. Scopulariopsis, in addition to species from two additional anamorphic genera (Fusarium and Penicillium), was isolated from the external surfaces of the ticks. Because only a single fungus, S. brevicaulis, was present internally, all subsequent studies focused on this particular species. Our conclusion was that only S. brevicaulis breached the tick by means of direct hyphal penetration; our remaining studies focused on this particular fungus because of the novelty of these results.
An aqueous formulation, comparable to one used in previous studies of ticks (6, 8), of 1.5 × 106 conidia/ml (based on 20 separate counts with a hemacytometer) was used to examine the pathogenicity of S. brevicaulis. Under conditions of 93% rH and 22 to 24°C, 100% mortality was observed within 15 (±1) days after a 5-μl topical application. Only a single tick was found dead in the water-treated controls during this 15-day period, and the remaining ticks were still living after the 25-day course of this study (replicates of 10; n = 3) (χ2, P < 0.05). In treatment groups, ticks began dying about 10 days after treatment (1 to 2 ticks/day) and mortality peaked (78%) on day 15; no ticks were living 16 days posttreatment. No statistical differences were noted when less inoculum was used (7.5 × 103 versus 1.5 × 106 conidia/ml of fungus treatment) or between results for males and females (χ2, P > 0.05). We concluded that topical treatment with S. brevicaulis at 93% rH is lethal to ticks, killing ticks in about 1/2 month.
Ticks harbor numerous bacteria, which are regulated by their immune systems (14), and it is conceivable that additional benefits relating to the production of antibiotics are garnered by the presence of S. brevicaulis within the tick. When S. brevicaulis was incubated together with S. aureus, E. coli, and Enterobacter aerogenes, no clear zone around the fungus, denoting inhibition of bacterial growth, was apparent (5 per replicate; n = 3). Filter paper disks (no 3; Whatman, Hillsboro, Oreg.) treated with 5 μl of an aqueous formulation of S. brevicaulis similarly produced no clear zone when placed onto a bacterial lawn (5 per replicate; n = 3). These observations indicate that S. brevicaulis displays no particular bactericidal properties toward certain common gram-negative and -positive bacteria. Thus, the presence of S. brevicaulis within the tick provides no mutual benefit.
The occurrence of Penicillium spp., Fusarium spp., and S. brevicaulis appears to be fairly widespread in ticks, all having been identified from the external surfaces of field-collected I. ricinus, D. reticulatus, D. marginatus (13), and now D. variabilis ticks. All three fungal genera produce copious numbers of conidia and are common saprophytes, particularly Penicillium. Because of the apparent consistency in the mycoflora among ticks from both wild and laboratory populations, it seems reasonable that these fungi originated from and persisted in the original natural populations and are not merely laboratory contaminants. The fact that S. brevicaulis is a facultative parasite that can switch from saprophyte to parasite under certain environmental conditions (e.g., probably high relative humidity) makes it unique among the fungi identified on D. variabilis ticks, as both Penicillium spp. and Fusarium spp. are saprophytic (13). Our isolation of fungal hyphae from inside the ticks combined with significant tick mortality following inoculation with conidia provides evidence of successful parasitism by S. brevicaulis. S. brevicaulis has also been isolated from the gut contents of certain Collembola organisms (4) and honeybees (3), possibly also as a result of parasitism. The present study is the first record of the occurrence in ticks.
In nature, it is conceivable that ticks encounter a wide range of fungi, given that >90% of their lifetime is spent off of their hosts between blood meals (14). Ticks frequently take shelter in soil where conditions, especially relative humidity, favor fungal development (8). In general, most obligate and facultative parasitic fungi originate in forested areas, whereas the saprophytic species are associated with meadows (13). Meadows and forests, areas where tick hosts (mammals, namely, rabbits, raccoons, dogs, and deer) frequently migrate, are likewise natural habitats for D. variabilis (14). Higher rates of fungal infection in ticks would be expected to occur during the summer months, when ticks are most active, when vegetation is abundant, and when conditions (e.g., warm temperatures) are more favorable to fungi. Fungi on one tick may be spread to other ticks, especially since these arthropods are known to form clusters to conserve water (16). If tick-to-tick transmission of fungi takes place, it is likely by means of conidia. Once in contact with the cuticle, the fungus is most likely to gain entry by way of emerging hyphae that invade the tick through small pores, between body segments, or through the anus or spiracles (12).
Only one fungus, identified as S. brevicaulis, was recovered from internal tick contents, and this fungus was prevalent in all of the body sections (in both laboratory populations and wild specimens) of the ticks. Members of this cosmopolitan genus have been recovered from a number of natural substrates, including soil, plant matter, feathers, and insects, and have been reported to cause disease and death in insects as well as infections in humans (11, 13). S. brevicaulis is the most common species within the genus Scopulariopsis and is regarded as a frequently occurring soil fungus known to produce one-celled conidia arising from simple or branched conidiophores. Recently, the species' teleomorph was identified as Microascus brevicaulis, a perithecium-forming ascomycete characterized by its small, black, ostiolate ascocarps (1), a finding which supports the placement of Scopulariopsis into the Microascaceae (1). Members of this family represent important saprophytic agents of decay, particularly on cellulose and protein-rich substrates. As anamorphs, the Microascaceae are especially common in human environments, where they exist as common molds. Teleomorphic stages are less frequently encountered, and their natural environments are not completely understood.
The occurrence of S. brevicaulis in the American dog tick, D. variabilis, remains puzzling and invites further inquiry. The fact that S. brevicaulis has been reported to cause infections in humans (11, 13) makes it unsafe for biocontrol applications. Our isolation of this fungus from the internal contents of D. variabilis represents a highlight of this study because it demonstrates a simple technique for extracting potential biological control agents. It is also conceivable that this technique could be used in related studies aimed at isolating fungi from other pestiferous arthropods.
We warmly thank Lynne Sigler of the University of Alberta (Canada) Microfungus Collection and Herbarium (UAMH) for her assistance with fungal identification and for providing helpful information. We also thank Jared Hartsock for his assistance with the recovery and storage of fungi in pure culture.
This work was supported in part by grants from Merck/AAAS (J.A.Y. and P.E.H.) and the McGregor Foundation (J.B.B. and F.G.).