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Cross-transmission of Entomophthora muscae (Zygomycetes: Entomophthoraceae) among naturally infected muscoid fly (Diptera: Muscidae) hosts
JOURNAL
OF
INVERTEBRATE
PATHOLOGY
53, 272-215 (1989)
Cross-Transmission of Entomophthora muscae (Zygomycetes: Entomophthoraceae) among Naturally Infected Muscoid Fly (Diptera: Muscidae) Hosts Several families and species of Diptera have been reported as hosts of the fungal pathogen Entomophthora muscae, and morphological characteristics, such as size of conidia, vary considerably (D. M. MacLeod, E. Muller-Kogler, and N. Wilding, Mycologia 68, l-29, 1976). Recently, it was suggested that E. muscae is a species complex (S. Keller, Mitt. Schweiz. Entomol. Ges. 57, 131-132, 1984). Host associations may be useful in resolving the current state of taxonomic confusion. Considerable variability in susceptibility to a single E. muscue strain among species and families of Diptera has been shown (J. P. Kramer and D. C. Steinkraus, Mycopathologia 76, 139143, 1981; D. C. Steinkraus and J. P. Kramer, Mycopathologia 100, 55-63, 1987). E. muscae infects several species of manure-breeding flies (Muscidae) on poultry facilities in southern California: Musca domestica, Ophyra aenescens, Fannia canicularis, and Fannia femoralis (B. A. Mullens, J. L. Rodriguez, and J. A. Meyer, Hilgardia 55, l-41, 1987). In order better to understand the dynamics of E. muscae in these hosts, cross-transmission experiments were conducted in the laboratory. Flies were collected by sweep net at a site (Lohr) known to have E. muscae in the four hosts. Infected 0. aenescens were collected in April, infected F. canicularis and F. femoralis were collected in June, and the infected M. domestica were from an in vivo laboratory culture of the fungus originally obtained in November from wild M. domestica at a dairy nearby and maintained in M. domestica since 1982 (B. A. Mullens, J. Med. Entomol. 23, 457458, 1986). After field collection, individuals were segregated by species and held with access to a
water wick and dried milk and sugar in 237ml cardboard containers in the laboratory at 21 ? 2°C. Infected flies died in late afternoon and were used to inoculate unexposed, colony-reared flies of the same species (J. P. Kramer and D. C. Steinkraus, Mycopathologia 76, 139-143, 1981). The pathogen was passaged one or more times to build numbers of infected hosts, which then were used in the transmission trials. From Cl0 cadavers were used as inoculum for each cage of exposed flies. Young (<5 days old), colony-reared flies were placed into clean cardboard containers as described above. Uninfected members of all four hosts normally were confined together in the same container. In some cases, all four hosts of the proper age were not available, but in every case the container held flies of the same species as the cadavers (to serve as positive controls). The fly species used for inoculum was referred to as the “donor” host, while the uninfected flies were referred to as “receptor” hosts. Each container normally held 20-30 receptor flies of each species, though the exact numbers occasionally varied with availability. A moist paper towel was placed on top of the donor hosts to assure a high level of humidity. A Petri dish then was placed on top of the container, and donor hosts were allowed to sporulate for 36-48 hr, when they and the moist paper towel were removed. Levels of conidial exposure were not monitored, but were assumed to be similar for the flies in each container (including positive controls). After exposure (day 0) receptor flies were checked every morning at 0800 hr for 10 days. Dead flies were removed. Flies with obvious external signs of E. muscae mycosis (outstretched wings, legs, and pro-
272 0022-201 l/89 $1.50 Copyright AlI rights
0 1989 by Academic Press, Inc. of reproduction in any form reserved.
27:
NOTES
boscis; conidiophores emerging from swollen abdomen) were noted. In some trials abdomens of remaining dead hosts (without external signs) were dissected and examined microscopically (wet smear) for internal signs of mycosis. Some cadavers from field-collected flies were placed on clean glass microscope slides for collection of primary conidia. These were stained with aceto-orcein and mounted in lactophenol to make preliminary observations on size of conidia and number and size of nuclei. Length and width of 20 primary conidia were measured for each of five individuals for each host species. The results of the cross-transmission experiments are summarized in Table 1. The E. muscae from M. domestica donors had excellent infectivity for that host (82%), but relatively few receptor 0. aenescens died with external signs (26%). Neither of the receptor Fannia spp. died with external signs, though on dissection a few F. canicularis had low numbers of threadlike mycelial strands inside the abdomen. Most of the 0. aenescens receptors were killed TABLE INFECTIVITY
OF Entomophthora
domestica
0. aenescens
F. canicularis
1
muscae FOR MUSCOID FLY SPECIES NATURALLY CALIFORNIA POULTY FACILITIES
Donor host Musca
by the M. domestica pathogen, but did not produce conidia. Instead, cadavers contained internal mycelial growth and numerous spherical fungal forms of various sizes (most ca. 20 km in diameter). Due to the prolonged period of culture of the M. domestica strain of E. muscae in the laboratory before the trials were done, one must acknowledge the fact that some of the host specificity observed may have been influenced by laboratory culture. A very similar trend, however, was noted for flies exposed to E. muscae from 0. aenescens donors. Neither of the Fannia spp. was found to be susceptible, but infectivity was good (66%) for 0. aenescens receptors and fair (32%) for M. domestica receptors. Whereas most 0. aenescens exposed to the M. domestica pathogen were killed without showing external signs of mycosis, few M. domestica receptors exposed to the 0. arnescens strain of E. muscae died without patent signs of mycosis. Though the absolute level of transmission efficiency was generally only 25-50%, relative transmission was good between the two Fannia spp. E. muscae from either
Receptor host Muscu Ophyra Fannia Fannia
domestica
aenescens canicularis femoralis
0. M. F. F.
aenescens domestica canicularis femoralis
F. F. M. 0.
canicularis femoralis domestica aenescens
Flies at risk (Reps)
F. F. M. 0.
femoralis canicularis domestica aenescens
ON SOUTHERW
‘% with E. muscu/’
265 (13) 175 (9) 150 (7) 150 (7)
82 26 0 0
29(2) (2)
66 32 0 0
60
60 (2)
60(2) 222 (7) 156 (6) 159 (6) 146 (4)
16 (1) 19 (1) 20(1) Not tested __~ ~_.. n Includes flies with obvious external signs of mycosis only.
F. femoralis
INFECTED
25 39 II 0 50 37 5
274
NOTES
Funniu donor species was less infective for 44. domestica receptors, but some patent cases were observed, and conidial production appeared to be excellent in these individuals. 0. aenescens receptors were not susceptible to E. muscae from F. caniculark donors, while 0. aenescens was not checked for susceptibility to E. muscae from F. femoralis donors. Individuals of 0. aenescens or M. domestica exposed to E. muscae from the alternate Muscinae donor host died with external signs more slowly and asynchronously than did individuals of the donor species (Fig. la, b). The mean time of death for M. domestica receptors exposed to E. muscae from M. domestica donors (6.1 days) was significantly (P < 0.001, t test) sooner than 0. aenescens (7.5 days) exposed in the same containers. Likewise, patent cases in M. domestica receptors exposed to E. muscae from 0. aenescens donors (8.5 days) were significantly (P < 0.001, t test) later than 0. aenescens receptors (7.1 days). This suggests that 0. aenescens was a relatively poor host for the M. domestica pathogen, and vice versa. The incubation period of E. muscae in the two Funniu spp. was similar regardless of the donor host (Fig. lc). For E. muscue from F. canicufaris donors, the mean incubation period was 6.9 days in F. canicularis receptors and 7.1 days in F. femoralis receptors (P > 0.05, t test). Though the sample size was small, the same trend held for the two Fannia species exposed to E. muscue from F. femorulis donors (incubation 7.3 days in F. femoralis and 6.7 days in F. canicularis, P > 0.05). Interestingly, though few M. domestica succumbed to the pathogen from F. caniculuris donors, the incubation period was the same (6.9 days) as that of the two Fannia spp. Several factors influence the incubation period of the M. domestica strain of E. muscae in M. domestica, including level of pathogen exposure, host age, and host size (B. A. Mullens, Entomol. Exp. Appl. 37, 33-39, 1985). Host age presumably reflects
100 1
4
100 -
P 2
80-
i
60-
g
40-
z z-
2O-
Donor:
5
a.
MD
6 9 6 7 Days Post Exposure
Donor:
10
11
b.
OA 0A receptors
MD receptors
0l’T.T.l.l’l’T.l 4 5
1001
6 Days
Donor:
fj8 8 z 05
7 6 9 Post Exposure
1011
FC
C.
60 40
E *
20
4
6 Days
7 6 9 Post Exposure
1011
FIG. 1. Incubation period (days to death with patent signs of mycosis) of four species of muscoid flies exposed to three Entomophrhoru mu~cae host strains in the laboratory. Donor host is fly species from which inoculum originated. Hosts as follows: MD = Musca domestica, OA = Ophyra aenescens, FC = Fannia canicularis, FF = Fannia femoralis.
the fact that older flies are suboptimal hosts for the pathogen. Such a prolonged incubation period in unusual (suboptimal) host species has been observed for E. muscae (D. C. Steinkraus and J. P. Kramer, Mycopathologia 100, 55-63, 1987). Length and width of primary conidia did not differ among these four hosts (P > 0.05,
775
NOTES
analysis of variance F test). The length and width of conidia (3 + SD in pm) were: M. domestica (22.7 +- 1.8 X 18.1 IT 2.3), 0. aenescens (22.0 2 2.1 x 18.2 rt 2.0), F. canicufaris (21.4 + 2.3 x 17.7 ? 2.4), and F. femoralis (22.8 + 2.1 X 18.2 + 2.0). For each host the size range was 18-28 pm long x 14-22 p,rn wide. From 12-18 nuclei/ conidium could be seen in conidia from each of the E. muscae strains from each of these four hosts. Size of nuclei was approximately 3 pm diameter for E. muscae from each of these four hosts. An additional group of M. domestica cadavers was sent to Dr. R. A. Humber, who counted an average of 16.3 nuclei per primary conidium (n = 18 from 3 flies). Thus, though the average conidial size is smaller, these strains seem to fit best in Keller’s (lot. cit.) group c. The same strain of pathogen probably is operating in the two Fannia spp. However, E. muscae from Fanniinae donors was very low in infectivity for Muscinae (0. aenescens and M. domestica) receptors, and E. muscae from Muscinae donors was not infective for Fanniinae. The seasonal pattern of cases in nature (spring for Fanniinae; broadly distributed, but mostly fall, for Muscinae) (B. A. Mullens, J. L. Rodriguez, and J. A. Meyer, Hilgardia 55, l-41, 1987) supports the premise that E. muscae
transmission between flies in the two subfamilies does not occur in nature. There is some potential for cross-transmission of E. muscae between Muscinae, yet susceptibility of flies in the alternate species is reduced. Different activity and spatial distribution patterns probably tend to minimize contact in nature. While E. muscae probably is a complex, strain differences could account for the marked variability in infectivity among strains from these four hosts as, for example, also noted for Zoophthora radicans (B. Papierok, B. V. L. Torres, and M. Arnault, Entomophaga 29, 109-I 19, 1984). KEY WORDS: Entomophthora muscar; Fannia; Ophyra; Musca; susceptibility; incubation; transmission; taxonomy. 1 am indebted to Dr. R. A. Humber (U. S. Department of Agriculture Insect Pathology Research Unit, Boyce Thompson Institute, Ithaca, NY) for examining material and to Dr. Humber and Dr. D. C. Steinkraus (Department of Entomology, Cornell University, Ithaca, NY) for reviewing the manuscript. 1 also thank Dr. E. Fisher (California Department of Food and Agriculture, Sacramento, CA) for confirmation of host identifications.
BRADLEYA.MULL.ENS Department University Riverside, Received
of Entomology of California California 92521 January
25, 1988; accepted
May
18, 1988
OF
INVERTEBRATE
PATHOLOGY
53, 272-215 (1989)
Cross-Transmission of Entomophthora muscae (Zygomycetes: Entomophthoraceae) among Naturally Infected Muscoid Fly (Diptera: Muscidae) Hosts Several families and species of Diptera have been reported as hosts of the fungal pathogen Entomophthora muscae, and morphological characteristics, such as size of conidia, vary considerably (D. M. MacLeod, E. Muller-Kogler, and N. Wilding, Mycologia 68, l-29, 1976). Recently, it was suggested that E. muscae is a species complex (S. Keller, Mitt. Schweiz. Entomol. Ges. 57, 131-132, 1984). Host associations may be useful in resolving the current state of taxonomic confusion. Considerable variability in susceptibility to a single E. muscue strain among species and families of Diptera has been shown (J. P. Kramer and D. C. Steinkraus, Mycopathologia 76, 139143, 1981; D. C. Steinkraus and J. P. Kramer, Mycopathologia 100, 55-63, 1987). E. muscae infects several species of manure-breeding flies (Muscidae) on poultry facilities in southern California: Musca domestica, Ophyra aenescens, Fannia canicularis, and Fannia femoralis (B. A. Mullens, J. L. Rodriguez, and J. A. Meyer, Hilgardia 55, l-41, 1987). In order better to understand the dynamics of E. muscae in these hosts, cross-transmission experiments were conducted in the laboratory. Flies were collected by sweep net at a site (Lohr) known to have E. muscae in the four hosts. Infected 0. aenescens were collected in April, infected F. canicularis and F. femoralis were collected in June, and the infected M. domestica were from an in vivo laboratory culture of the fungus originally obtained in November from wild M. domestica at a dairy nearby and maintained in M. domestica since 1982 (B. A. Mullens, J. Med. Entomol. 23, 457458, 1986). After field collection, individuals were segregated by species and held with access to a
water wick and dried milk and sugar in 237ml cardboard containers in the laboratory at 21 ? 2°C. Infected flies died in late afternoon and were used to inoculate unexposed, colony-reared flies of the same species (J. P. Kramer and D. C. Steinkraus, Mycopathologia 76, 139-143, 1981). The pathogen was passaged one or more times to build numbers of infected hosts, which then were used in the transmission trials. From Cl0 cadavers were used as inoculum for each cage of exposed flies. Young (<5 days old), colony-reared flies were placed into clean cardboard containers as described above. Uninfected members of all four hosts normally were confined together in the same container. In some cases, all four hosts of the proper age were not available, but in every case the container held flies of the same species as the cadavers (to serve as positive controls). The fly species used for inoculum was referred to as the “donor” host, while the uninfected flies were referred to as “receptor” hosts. Each container normally held 20-30 receptor flies of each species, though the exact numbers occasionally varied with availability. A moist paper towel was placed on top of the donor hosts to assure a high level of humidity. A Petri dish then was placed on top of the container, and donor hosts were allowed to sporulate for 36-48 hr, when they and the moist paper towel were removed. Levels of conidial exposure were not monitored, but were assumed to be similar for the flies in each container (including positive controls). After exposure (day 0) receptor flies were checked every morning at 0800 hr for 10 days. Dead flies were removed. Flies with obvious external signs of E. muscae mycosis (outstretched wings, legs, and pro-
272 0022-201 l/89 $1.50 Copyright AlI rights
0 1989 by Academic Press, Inc. of reproduction in any form reserved.
27:
NOTES
boscis; conidiophores emerging from swollen abdomen) were noted. In some trials abdomens of remaining dead hosts (without external signs) were dissected and examined microscopically (wet smear) for internal signs of mycosis. Some cadavers from field-collected flies were placed on clean glass microscope slides for collection of primary conidia. These were stained with aceto-orcein and mounted in lactophenol to make preliminary observations on size of conidia and number and size of nuclei. Length and width of 20 primary conidia were measured for each of five individuals for each host species. The results of the cross-transmission experiments are summarized in Table 1. The E. muscae from M. domestica donors had excellent infectivity for that host (82%), but relatively few receptor 0. aenescens died with external signs (26%). Neither of the receptor Fannia spp. died with external signs, though on dissection a few F. canicularis had low numbers of threadlike mycelial strands inside the abdomen. Most of the 0. aenescens receptors were killed TABLE INFECTIVITY
OF Entomophthora
domestica
0. aenescens
F. canicularis
1
muscae FOR MUSCOID FLY SPECIES NATURALLY CALIFORNIA POULTY FACILITIES
Donor host Musca
by the M. domestica pathogen, but did not produce conidia. Instead, cadavers contained internal mycelial growth and numerous spherical fungal forms of various sizes (most ca. 20 km in diameter). Due to the prolonged period of culture of the M. domestica strain of E. muscae in the laboratory before the trials were done, one must acknowledge the fact that some of the host specificity observed may have been influenced by laboratory culture. A very similar trend, however, was noted for flies exposed to E. muscae from 0. aenescens donors. Neither of the Fannia spp. was found to be susceptible, but infectivity was good (66%) for 0. aenescens receptors and fair (32%) for M. domestica receptors. Whereas most 0. aenescens exposed to the M. domestica pathogen were killed without showing external signs of mycosis, few M. domestica receptors exposed to the 0. arnescens strain of E. muscae died without patent signs of mycosis. Though the absolute level of transmission efficiency was generally only 25-50%, relative transmission was good between the two Fannia spp. E. muscae from either
Receptor host Muscu Ophyra Fannia Fannia
domestica
aenescens canicularis femoralis
0. M. F. F.
aenescens domestica canicularis femoralis
F. F. M. 0.
canicularis femoralis domestica aenescens
Flies at risk (Reps)
F. F. M. 0.
femoralis canicularis domestica aenescens
ON SOUTHERW
‘% with E. muscu/’
265 (13) 175 (9) 150 (7) 150 (7)
82 26 0 0
29(2) (2)
66 32 0 0
60
60 (2)
60(2) 222 (7) 156 (6) 159 (6) 146 (4)
16 (1) 19 (1) 20(1) Not tested __~ ~_.. n Includes flies with obvious external signs of mycosis only.
F. femoralis
INFECTED
25 39 II 0 50 37 5
274
NOTES
Funniu donor species was less infective for 44. domestica receptors, but some patent cases were observed, and conidial production appeared to be excellent in these individuals. 0. aenescens receptors were not susceptible to E. muscae from F. caniculark donors, while 0. aenescens was not checked for susceptibility to E. muscae from F. femoralis donors. Individuals of 0. aenescens or M. domestica exposed to E. muscae from the alternate Muscinae donor host died with external signs more slowly and asynchronously than did individuals of the donor species (Fig. la, b). The mean time of death for M. domestica receptors exposed to E. muscae from M. domestica donors (6.1 days) was significantly (P < 0.001, t test) sooner than 0. aenescens (7.5 days) exposed in the same containers. Likewise, patent cases in M. domestica receptors exposed to E. muscae from 0. aenescens donors (8.5 days) were significantly (P < 0.001, t test) later than 0. aenescens receptors (7.1 days). This suggests that 0. aenescens was a relatively poor host for the M. domestica pathogen, and vice versa. The incubation period of E. muscae in the two Funniu spp. was similar regardless of the donor host (Fig. lc). For E. muscue from F. canicufaris donors, the mean incubation period was 6.9 days in F. canicularis receptors and 7.1 days in F. femoralis receptors (P > 0.05, t test). Though the sample size was small, the same trend held for the two Fannia species exposed to E. muscue from F. femorulis donors (incubation 7.3 days in F. femoralis and 6.7 days in F. canicularis, P > 0.05). Interestingly, though few M. domestica succumbed to the pathogen from F. caniculuris donors, the incubation period was the same (6.9 days) as that of the two Fannia spp. Several factors influence the incubation period of the M. domestica strain of E. muscae in M. domestica, including level of pathogen exposure, host age, and host size (B. A. Mullens, Entomol. Exp. Appl. 37, 33-39, 1985). Host age presumably reflects
100 1
4
100 -
P 2
80-
i
60-
g
40-
z z-
2O-
Donor:
5
a.
MD
6 9 6 7 Days Post Exposure
Donor:
10
11
b.
OA 0A receptors
MD receptors
0l’T.T.l.l’l’T.l 4 5
1001
6 Days
Donor:
fj8 8 z 05
7 6 9 Post Exposure
1011
FC
C.
60 40
E *
20
4
6 Days
7 6 9 Post Exposure
1011
FIG. 1. Incubation period (days to death with patent signs of mycosis) of four species of muscoid flies exposed to three Entomophrhoru mu~cae host strains in the laboratory. Donor host is fly species from which inoculum originated. Hosts as follows: MD = Musca domestica, OA = Ophyra aenescens, FC = Fannia canicularis, FF = Fannia femoralis.
the fact that older flies are suboptimal hosts for the pathogen. Such a prolonged incubation period in unusual (suboptimal) host species has been observed for E. muscae (D. C. Steinkraus and J. P. Kramer, Mycopathologia 100, 55-63, 1987). Length and width of primary conidia did not differ among these four hosts (P > 0.05,
775
NOTES
analysis of variance F test). The length and width of conidia (3 + SD in pm) were: M. domestica (22.7 +- 1.8 X 18.1 IT 2.3), 0. aenescens (22.0 2 2.1 x 18.2 rt 2.0), F. canicufaris (21.4 + 2.3 x 17.7 ? 2.4), and F. femoralis (22.8 + 2.1 X 18.2 + 2.0). For each host the size range was 18-28 pm long x 14-22 p,rn wide. From 12-18 nuclei/ conidium could be seen in conidia from each of the E. muscae strains from each of these four hosts. Size of nuclei was approximately 3 pm diameter for E. muscae from each of these four hosts. An additional group of M. domestica cadavers was sent to Dr. R. A. Humber, who counted an average of 16.3 nuclei per primary conidium (n = 18 from 3 flies). Thus, though the average conidial size is smaller, these strains seem to fit best in Keller’s (lot. cit.) group c. The same strain of pathogen probably is operating in the two Fannia spp. However, E. muscae from Fanniinae donors was very low in infectivity for Muscinae (0. aenescens and M. domestica) receptors, and E. muscae from Muscinae donors was not infective for Fanniinae. The seasonal pattern of cases in nature (spring for Fanniinae; broadly distributed, but mostly fall, for Muscinae) (B. A. Mullens, J. L. Rodriguez, and J. A. Meyer, Hilgardia 55, l-41, 1987) supports the premise that E. muscae
transmission between flies in the two subfamilies does not occur in nature. There is some potential for cross-transmission of E. muscae between Muscinae, yet susceptibility of flies in the alternate species is reduced. Different activity and spatial distribution patterns probably tend to minimize contact in nature. While E. muscae probably is a complex, strain differences could account for the marked variability in infectivity among strains from these four hosts as, for example, also noted for Zoophthora radicans (B. Papierok, B. V. L. Torres, and M. Arnault, Entomophaga 29, 109-I 19, 1984). KEY WORDS: Entomophthora muscar; Fannia; Ophyra; Musca; susceptibility; incubation; transmission; taxonomy. 1 am indebted to Dr. R. A. Humber (U. S. Department of Agriculture Insect Pathology Research Unit, Boyce Thompson Institute, Ithaca, NY) for examining material and to Dr. Humber and Dr. D. C. Steinkraus (Department of Entomology, Cornell University, Ithaca, NY) for reviewing the manuscript. 1 also thank Dr. E. Fisher (California Department of Food and Agriculture, Sacramento, CA) for confirmation of host identifications.
BRADLEYA.MULL.ENS Department University Riverside, Received
of Entomology of California California 92521 January
25, 1988; accepted
May
18, 1988