Entomophthora muscae (Entomophthorales: Entomophthoracae) mycosis in the onion fly, Delia antiqua (Diptera: Anthomyiidae)

JOURNAL

OF INVERTEBRATE

PATHOLOGY

45, 81-93 (1985)

Entomophthora muscae (Entomophthorales: Entomophthoracae) Mycosis in the Onion Fly, Delia antiqua (Diptera: Anthomyiidae)’ RAYMOND Department

I. CARRUTHERS,~ of Entomology,

DEAN Michigan

L. HAYNES, AND DONALD M. MACLEOD~ Stare

University,

East Lansing.

Michigan

48824

Received March 6, 1984; accepted June 25. 1984 muscae was identified as a common fungal pathogen of the onion fly, Delia the adult seed corn maggot, D. platura. Low infection levels also were found in populations of the cluster fly, Pollenia rudis (Diptera: Muscidae), and the tiger fly. Coenosia tigrina (Diptera: Muscidae). The disease cycle, as it affects D. antiqua in the onion agroecosystem, is described, including the etiology, symptomatology, and phenology. Natural infection levels approaching 100% were noted early in the spring and in late fall, impacting the 1st and 3rd generations of the D. antiqua population significantly. A lagged density-dependent disease response was noted at the gross population level. although more specific biological interactions may be involved in regulating the disease intensity. 0 1985 Academic Press. Inc. KEY WORDS: Entomophthoru mucue; Delia antiqua; onion maggot: Delia platura; seed corn maggot: etiology: epizootiology; fungal pathogen; disease life system; onion agroecosystem; Pollenia rudis; cluster fly: Coenosia tigrina; tiger fly. Entomophthora anriqua, and

Characterization of natural disease cycles, including identification of host and pathogen complexes, their basic life stages, and modes of interaction in the environment, are important in understanding the dynamics behind disease epizootiology. Few investigators have clearly detailed host-pathogen interactions as they occur under natural field conditions (Matanmi and Libby, 1975). Such has been the case with Entomophthora muscae, a common fungal pathogen of several adult Dipterans (MacLeod et al., 1976). Since its first description (Cohn, 1855), E. muscae has been noted inducing epizootics in numerous host species including the onion fly, Delia antiqua (Thaxter, 1888; Yeager, 1939; Baird, 1957; Miller and McClanahan, 1959; Kramer, 1971; Berisford and Tsao, 1974; Wilding and Lauckner,

1974). Despite the documented importance of E. muscae mycosis as a major mortality factor of the onion fly (Miller and MaClanahan, 1959; Perron and Crete, 1960: Kramer, 1971; Loosjes, 1976), virtually no research has been conducted on its field biology or its impact on the host population. In this paper, we will detail this hostpathogen life system using laboratory and field observations to provide a structural base for reporting concurrent epizootiological studies (Carruthers, 1981). MATERIALS

AND METHODS

Etiology. D. antiqua adults, naturally infected with the fungal pathogen E. muscae, were first collected from an onion field near Grant, Michigan (Grant Township, Newaygo County), in the spring of 1977. Microscopical examination of the cadavers revealed masses of hyaline, multinucleate ellipsoidal conidia (II- 12 nuclei/conidia), with a papillate apex and flattened base, protruding from the abdomen of the host flies. Conidiophore penetration was limited to the membranous ventral aspect of the abdomen and dorsally in the intersegmental

’ Michigan State Agricultural Experiment Station Journal Article Number 11287. ? Present address: Department of Entomology, Cornell University, Ithaca, N.Y. 14850. 3 Present address: Forest Pest Management Institute, Canadian Forest Service, Sault Saint Marie, Ontario, Canada. 81

0022-2011185 $1.50 Copyright All rights

0 1985 by Academic Press. Inc. of reproduction in any form reserved.

82

CARRUTHERS,

HAYNES.

areas (Fig. 1). One hundred conidia and 100 resting spores were randomly selected from each of 20 (10 containing each spore type) different D. antiqua cadavers. These conidia were measured microscopically for length and width, which were in turn used to identify the pathogen (MacLeod et al., 1976). A second collection of D. antiqua adults from Grant were transported live to the Forest Pest Management Institute (FPMI), Sault Saint Marie, Ontario, Canada. The flies were caged in the laboratory and maintained at ca. 21°C. After sporulation, the pathogen was isolated from these flies and maintained in liquid culture medium (Welton and Tyrrell, 1975). Isolated conidia were examined throughout their germination and early developmental periods. Cultures were maintained in liquid tissue culture medium (supplemented with 5%, v/v. heat-denatured fetal bovine serum and a wide-spectrum antibiotic, gentamycin, 50 Fl/ml). The culture flasks were incubated at laboratory temperature (ca. 2 1’C) under ambient lighting. At 2-week intervals, 5-ml portions of the isolate were added to IO ml of fresh tissue culture medium to maintain a continuous supply of the pathogen in vegetative stages. The cultures were examined under light microscopy throughout the in vitro developmental period of the pathogen. To ensure a stable, long-term source of the isolate, a subculture was placed in liquid nitrogen storage. One hundred laboratory-reared D. antiqzra adults (I: 1 sex ratio) were anesthetized with CO, and injected with 2 ~1 of the above isolate (5 days after culture transfer). Two control treatments (n = IOO), one with 2 ~1 of sterile tissue culture medium injected and the other with no injections, also were maintained. All injections were made in the ventral aspect of the abdomen using glass-drawn needles. The flies (held at 21°C under ambient lighting) were monitored daily for mortality, and were examined both macro- and microscopically for the presence of E. muscae infection.

AND

MAC

LEOD

Live-catch emergence traps were placed in the field directly over areas of known D. aruiqun larval occurrence, allowing teneral adults to be collected daily. These flies, along with others emerging from surfacesterilized pupae (sifted from adjacent soil sites and treated with a 10% bleach solution for 30 set), were held in the laboratory and evaluated for primary E. mucue infection. Microscopical examination (both SEM and light) was used to characterize the fungal stages in vivo. Regional distribution and field impucts. To evaluate the natural distribution of E. muscne, a statewide detection survey was conducted in the spring of 1978 throughout most major vegetable-producing areas within Michigan. Pest management field assistants (Bird et al., 1975) were trained to recognize and collect adult dipteran cadavers exhibiting the characteristic death patterns associated with E. musccle mycosis (Berisford and Tsao, 1974). The specimens collected in the survey were preserved in 70% alcohol for subsequent identification of host and pathogen. Host identification was accomplished with assistance from Dr. Dirk Spillemaeckers from the Department of Entomology, Michigan State University. The keys provided by MacLeod et al. (1976) were used to identify the pathogen. Field-level sampling was conducted in three Michigan onion-production regions during the 1978-79 growing seasons to determine the incidence of E. muscae infection in known host populations. The primary study site was the Rice Lake vegetable-production region (Grant Township, Newaygo County), where commercially maintained fields, adjacent natural areas, and specific research plots were monitored. Due to heavy pesticide use in the commercial onion fields, one research site, a I .6-ha onion field, was maintained free of insecticides and fungicides throughout the season. These study sites were used by numerous researchers and evaluated for pest populations, natural enemies. onion plant

Entomophthora

rnuxae

IN

Delia

antiqua

FIG. 1. Conidial life stages of Entomophthora muscae, parasitic on the onion fly (Delia antiqun). (A) Female fly infected with E. mu~cae in the process of sporulation. Note the conidiophore penetration only on the membranous ventral aspect of the abdomen and dorsally between the tergites (fungal growth, whitish area on abdomen). (B-C) Lateral views of a male fly infected with E. muscae in the process of sporulation. The area of fungal penetration (darkened area in the SEM) is again restricted to the membranous areas of the abdomen. (D) Conidiophore penetration in the intersegmental area of the abdomen. Note the singular born conidia in various stages of development from the immature fingerlike conidiophore to the mature bell-shaped conidia. (E) Single E. rnu~cae conidium attached to the thoracic region of an onion fly. The conidia are forcibly ejected from the conidiophores and adhere readily to most substrates. Note the mucilaginous material attached to the base of the conidium and the point of separation from the parent conidiophore. The SEM gives a different perspective, showing a lower separation point, than is seen in light micrographs (Fig. 5A). (F) Two E. rrt~~cae conidia attached to an abdominal setae of a host fly showing the adhesive nature of the mucilaginous protoplasm attached to the conidia.

83

84

CARRUTHERS,

HAYNES.

AND

MAC

LEOD

Entomophthora muscae IN Delia antiquu

development and damage, and several other biological parameters of interest to the overall research project (Haynes et al., 1980). Detailed environmental data including air and soil temperature, rainfall, solar radiation, wind velocities, and leaf wetness were recorded at each site. E. muscae samples were collected from each study site approximately twice per week, using a tractor-mounted suction sampler (Cobb and Ruppel, 1976). The suction sampler was operated at a speed sufficient to catch adult muscoid flies in onion and grassy border canopies without causing physical injury. Known host flies (100 per species) were placed in isolation and held under laboratory conditions 21 + 1°C 80% RH, and a 16-8 light-dark photoperiod throughout the incubation period of the disease (Carruthers, 1981). Water and food (honey and brewer’s yeast) were provided in conjunction with daily observations for fly mortality. Dead flies were removed from the containers and examined for the presence of E. muscae infection (conidia and resting spores). Data were recorded as to host species, sex, date of death, and presence of E. muscae. Further population assessments were conducted to estimate the number of conidia producing cadavers present in the field at each sampling date. At each sample location, the number of D. antiqua cadavers attached to sporulation substrates (onion leaf tips, grass leaf tips, etc.) were recorded over 10, 30-m sections of row or border habitat.

85

Host fly populations were monitored approximately twice per week in each of the study areas using emergence traps, activity traps, flight interception traps, and sweepnet samples. Species of the host monitoring techniques and resulting population estimates are given by Whitfield (1981). RESULTS AND DISCUSSION

Etiology. Based on resting spore diameter (mean diameter + 95% CL, 22.4 f 1.2 pm), conidial dimensions (mean length 295% CL, 22.7 ? 1.4 km; mean width +95% CL, 17.8 2 1.1 km), and conidial shape (Figs. lE, 2A), the pathogen was identified as E. muscae (MacLeod et al., 1976). Rhizoids were not apparent on any of the host flies examined. Germination of the conidia released into the insect tissue culture medium was noted approximately 9 hr after transfer to the culture flasks. A variety of germination responses were seen although the initial production of secondary conidia was the principal developmental path noted in liquid tissue culture media. Elongate, branched germ tubes developed directly from the secondary conidia (Fig. 2B), but occasional germ tubes developed directly from primary conidia (Fig. 2C). Vegetative growth developed readily in tissue culture medium, producing small mycelial-like colonies (Figs. 2D; 3A, E, F). Conidial formation was never noted in the tissue culture media although, several months after isolation, hyphal fragments divided and formed

FIG. 2. Phase-contrast micrographs of Enromophthora muscae in Grace’s insect tissue culture medium. (A) Single conidium just after ejection from conidiophore. Note the mucilaginous protoplasmic material surrounding the conidium and the apparent separation point from the conidiophore (contrast with Figs. I,E-F). (B-C) Conidia in the process of germination in tissue culture medium. Note the long singular germ tube arising from the secondary conidium in (B). The formation of secondary conidia seem to be the normal mode of germination although some conidia produce germ tubes directly, as in (C). The germ tubes are found both singularly and branched, independent of secondary conidia formation. (D) Initial hyphal growth develops from branchlets radiating from the germination point. Hyphal fragments or buds are broken off of the original colony and released into the medium. (E-F) Several months after initial isolation, numerous fragments divided, forming spherical bodies, possible precursors of the thick-walled resting spores.

CARRUTHERS,

HAYNES,

AND

MAC

LEOD

FIG. 3. Enfomophfhora WZUSC~P in Graces insect tissue culture medium. (A. E-F) Typical mode of E. tnu~cae growth in tissue culture flasks. The pathogen grows in colonies that are mycelial-like although it tends to form spheres (hyphal bodies) which continue to bud. (B-D) E. IWSC’UC held under slight pressure induced by a coverslip tends to develop thread-like forms similar to the protoplast phase of other Entomophthora spp.

FIG. 4. Resting spore life stage of Enfomophthora 171uscaefrom natural mycosis of the onion fly, D. antiqua. (A) Female fly containing E. muscae resting spores internally in the abdomen. Note the dark coloration and the shriveled nature of the abdomen. (B-C) Resting spores of E. muscae embedded in the internal abdominal tissue of the host fly. (D) Resting spore isolated from inoculated muck soil via centrifugation-flotation techniques.

s

88

CARRUTHERS.

HAYNES.

spherical bodies, possibly precursors of thick-walled resting spores (Figs. 2E, F). When vegetative hyphal material was placed under slight pressure (induced by the addition of a cover-slip) the fungal hyphae tended to develop thread-like forms (Figs. 3B-D) similar to the protoplast phase of other Entomophthova spp. (Tyrrell and MacLeod, 1972; MacLeod et al., 1980). In vivo development of E. mnscae is still not well understood, and the presence of protoplasts in the hemolymph of D. rzntiqun has not yet been verified, although our microscopical examination of infected hosts revealed no identifiable hyphal material 1 and 2 days postinfection. The lack of hyphal bodies or fragments within the abdomen of the host suggests that such a protoplasmic stage exists during the early stages of E. muscae infection. Recently, E. muscae protoplasts have been identified from other dipteran host species (Humber, pers. commun.), but further research is

not

AND

MAC

LEOD

necessary to clarify the exact mode of in vivo development in D. utztiqlrrr. Injecting D. antique adults with tissue culture medium containing the above isolate in a vegetative state resulted in only six flies (n = 100) dying of fungal mycosis, four in the conidial state and two in the resting spore state (Figs. 4A-D). No mycosis was found in either control: the survival rate over the incubation period remained well above 90%. This mycosis, although low in percentage. was highly significant as it completed the requirements associated with Koch’s postulates. We cannot presently explain the low success rate of infection through injection; however, subtle changes in osmotic pressure, pH, and nutrient content of the microhabitat may have played a distinct role. Infection of teneral adults at emergence is common, although the habitat and microclimate exert major effects on the observed infection levels (Carruthers, 1981). Surface-

sampled

& L

antigua

p-

p1atura rudis

& P. -

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FIG. 5. Distribution sampling of infected

of Entornophthorrr flies.

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peninsula

of Michigan

based on detection

Entomophthora

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probably present throughout Michigan at low numbers or in habitats not sampled in this study. Fifteen sets of environmental and E. muscae infection data were collected over the 1978-79 sampling seasons from populations of D. antiqua and D. platura adults associated with the specific research and production onion fields described above. Conidia were the predominant spore type collected and reared from both D. antiqua and D. platura populations. Flies dying of E. muscae mycosis were found with conidia over 95% of the time. Resting spore production was found throughout the season at levels always below 10% of the total diseased cadavers. No apparent pattern in spore type, conidia vs resting spore, was noted during the growing season that would indicate abiotic control of spore differentiation. Further laboratory experimentation (Carruthers, 1981) found no evidence of temperature dependent control of spore differentiation. These data were used with host population estimates (Whitfield, 1981) to correlate gross population trends and environmental conditions. Visual examination of the data suggest a lagged, density-dependent response of infection level with host number. This response is reflected in the data sets from the 1978-79 Grant research field (Figs. 6, A-F). Increases in D. antiqua

sterilized host pupae, obtained by collecting pupae from areas of known E. muscae presence, never became infected when allowed to emerge through laboratory-sterilized soil (n = 512). Parallel collections and rearing from naturally emerging flies produced infection levels between 10 and 33% (Carruthers, 1981). These data suggest that primary host infection occurs in the soil at the time of emergence. Presumably resting spores or germ conidia originating from resting spores infect the fly in the soil between the pupal case and the soil surface. Regional distribution and field impact. Twenty-six Michigan counties were sampled during 1978 in the southern Lower Peninsula. E. muscae was found naturally occurring in every county sampled, including the 10 counties where most of Michigan’s onions are produced (Fig. 5). In onion-production areas, E. muscae was found primarily with the onion maggot, D. antiqua, and the seed corn maggot, D. platura. From counties where the onion fly was not collected, D. platura was the primary host. The cluster fly, Pollenia rudis, was an occasional host in a few counties (Fig. 5). The tiger fly, Coenosia tigrina, was found only in Newago and Eaton counties. Numerous other host species are associated with E. muscae, but none were detected in this survey. Other host species are TABLE MULTIVARIABLE

REGRESSION

AND MAC LEOD

1

STATISTICS AND VARIABLES EXAMINED Entomophthora muscae INFECTION

FOR PREDICTION

OF LAGGED

Variables examined DEN, Total host density CAD, Cadaver density ATEMP, Average temperature MAXT, Maximum temperature MINT, Minimum temperature Variable

F

Value

LOGlO(DEN) 28.05* CAD 24.23* (Constant) y = 17.79 + 13.79LoglO(DEN) + 0.49CAD *P

s 0.001.

AHUM, Average relative humidity HOURS, Consecutive hours above 95% RH MAXH. Maximum relative humidity MINH, Minimum relative humidity Y, Arcsine proportion infection Multiple r? 0.5557 0.6508

Model coefficients (b) 13.79 0.49

17.79

Entomophthora

PATHOGEN DEVELOPING WITHIN THE

muscae

IN Delia antiqua

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FIG. 7. Disease cycle associated with Entomophrhora

adult numbers, primarily in the second and third generations, are followed by a lagged increase in E. mu~cae infection (Figs. 6, A, D). This effect was not noted in the first generation of D. antiqua until the D. platura adult population was considered (Figs. 6, B, E). Total host density (II. antiqua + D. platura) clearly suggests that the two

muscae

*

’

mycosis of Delia antiqua.

populations are responding to a common pathogen population in a lagged, densitydependent manner (Figs. 6, C, F). Laboratory experimentation (Carruthers, 198 1) further suggests that these two hosts are reacting to a common pathogen population. Multivariable techniques were used to regress several independent variables (Table

92

CARRUTHERS.

HAYNES,

l), including total host density, inoculum densities (estimated number of sporulating cadavers per sample period), and several abiotic parameters against the arcsin of the lagged proportion infection. The percentage infection was lagged by half the length of the disease incubation period (105 DD base 4.8 “C, Carruthers, 1981), thus correlating the independent variables with the percentage infection during the time interval when spore germination and infection was estimated to have occurred. Host density and inoculum density were the only variables eliciting a significant response (P d 0.05). Abiotic variables, including temperature and moisture levels, had no significance in the variability associated with population infection levels during the 197879 growing season. The effect of abiotic variables on E. muscae infection and development probably was important, but was not the limiting or controlling factor because conditions conducive to infection were almost always present in the microenvironment inhabitated by these populations (Carruthers, 1981). Host and pathogen density seem to be the predominant stimulus variables in this disease system and many other infections caused by Entomophthora spp. (Perron and Crete, 1960; Wilding and Lauckner, 1976; Soper and MacLeod, 1980). This host-pathogen relationship is much more complex than discussed in the above regression model, which gives only slight insight into the dynamics of this life system. Detailed population studies of specific components are necessary to better understand the actual population interactions, epizootiology, and the implications of E. muscae mycosis for regulating host populations. More detailed analyses of specific system components and the population level implications are given by Carruthers (1981). The overall disease cycle (Fig. 7) consists of initial host emergence in the spring, with primary infection occurring as some flies are infected via overwintering resting spores or germ conidia in the soil. Following the temperature-dependent incuba-

AND MAC LEOD

tion period, host death occurs. and either conidia or resting spores result. With conidial production, flies attach thenselves to an elevated loci in late afternoon: conidia are then produced and released throughout the night, when environmental conditions are typically optimal for germination (Carruthers, 198 1). If successful, secondary host infection occurs within or between each of the three D. antiqua generations. If resting spore production occurs, the flies die on the soil surface, and their abdomens blacken, become brittle, and then fracture open, releasing resting spores back into the soil. Both methods of spore production and infection seem to operate simultaneously throughout the entire season, although sometimes E. muscae infection may be below detectable levels. ACKNOWLEDGMENTS We thank Ms. M. Welton of the Forest Pest Management Institute, Environment Canada, for helping to isolate this pathogen; Dr. R. Humber of the USDA Insect Palhology Research Unit for nuclear number determination; and S. Flegler of the Michigan State University Electron Microscopy Laboratory for assistance with the scanning electron micrographs. Thanks also go to Ms. S. Battenfield, Mr. T. Ellis. and Dr. R. Soper for their critical review of this manuscript and subsequent editorial comments. This research was supported by EPA Grant R806065.

REFERENCES BAIRD, R. 1957. Notes on a laboratory infection of Diptera caused by the fungus Empusa muscae Cohn. Canad. Entomol., 89, 432-435. BERISFORD,Y. C.. AND TSAO, C. H. 1974. Field and laboratory observations of an entomogenous infection of the adult seed corn maggot, Hylemya platura (Diptera: Anthomyiidae). J. Georgia Entomol. Sac., 9, 104-110. BIRD. G. W., CRESS, D., HAYNES, D.. LACY. M.. PUTNAM, A.. RUPPEL, R., AND WIESE, M. 1975. “On-line pest management of field and vegetable crops.” Pest Management Technical Report. Vol. 7. Michigan State University. CARRUTHERS,R. I. 1981. “The biology and ecology of Entomophthora muscae (Cohn) in the onion agroecosystem.” PhD thesis, Michigan State University, East Lansing, Mich. 234 pp. COBB, D. L.. AND RUPPEL, R. F. 1976. “A self-propelled machine for mass collection of insects.” Agric. Res. Ser. ARS-NC, 43.

Entomophthora

muscae

COHN, F. 1855. Empusa muscae und die krankheit der stubenfliegen. Nova Acta Leap. Carol.. 25, 301360. HAYNES, D. L., TUMMALA. R. L., AND ELLIS, T. L. 1980. Ecosystem management for pest control. BioScience,

30, 690-696.

KRAMER, J. P. 1971. Experimental studies on the phycomycosis of Muscoid flies caused by Entomophthora muscae (Cohn). N. Y. Entomol. Sot., 79, 4244. LOOSJES, M. 1976. “Ecology and genetic control of the onion fly, Hylemya anfiqua (Meig).” Ag. Res. Rept. 857. Pudoc, Wageningen. MACLEOD. D. M., MULLER-KOGLER, E., AND WILDING, N. 1976. Entomophthora species with E. mlrscae-like conidia. Mycologia, 68, l-29. MACLEOD. D. M., TYRRELL, D., AND WELTON, M. A. 1980. Isolation and growth of the grasshopper pathogen, Entomophthora grylli. .I. Invertehr. Pathol..

36, 85-89.

MATANMI, B. A., AND LIBBY, J. L. 1975. The life stages of Entomophthora virulenta. Mycopathologia,

56, 125-127.

MILLER, L. A., AND MCCLANAHAN. R. H. 1959. Note on occurrence of the fungus Empusa muscae (Cohn) on adults of the onion maggot, Hylemya antiqua (Meig.) (Diptera: Anthomyiidae). Canad. Entomol., 91, 525-526.

93

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PERRON, J. P., AND CRETE, R. 1960. Premieres observations surle champignon, Empusa muscae Cohn. (Phycomycetes: Entomophthoraceae) parasitant la mouche de I’oignon, Hylemya antiqua (Meig.) (Diptera: Anthomyiidae) dans le Quebec. Ann. Entomol. Sot.

Quebec,

5, 25-26.

SOPER, R. S., AND MACLEOD, D. M. 1981. “Descriptive epizootiology of an aphid mycosis.” USDA Tech. Bull. No. 1632. THAXTER, R. 1888. The Entomophthoraceae of the United States. Mem. Boston Sot. Nat. Hist.. 4, 133-201. TYRRELL, D.. AND MACLEOD. D. M. 1972. Spontaneous formation of protoplasts by a species of Entomophthora. J. Inb*ertebr. Pathol. 19, 354-360. WELTON, M. A., AND TYRRELL, D. 1975. A note on the isolation of Entomophthora species on artificial media. J. Invertebr. Pathol., 26, 405. WHITFIELD, G. 1981. “Spatial and temporal population analysis of the onion maggot, Hylemya antiqua. in Michigan.” PhD thesis. Michigan State University, East Lansing. WILDING. N., AND LAUCKNER, F. B. 1974. Entomophthora infecting wheat bulb fly at Rothamsted, Hertfordshire, 1967-71. Ann. Appl. Biol., 76, 161-170. YEAGER, C. C. 1939. Empzrsa infection of the housefly in relation to moisture conditions of northern Idaho. Mycologica. 31, 154-156.