JOURNAL OF INVERTEBRATE PATHOLOGY 56, 91-97 (1990)

Decline in Virulence of Entomophaga maimaiga (Zygomycetes: Entomophthorales) with Repeated in Vitro Subculture

ANNE. HAJEK,RICHARD A. HUMBER,AND MICHAEL H. GRIGGS

U.S. Department of Agriculture, Agricultural Research Service, Plant Protection Research Unit, U.S. Plant, Soil, Nutrition Laboratory, Tower Road, Ithaca, New York 14853

Received March 20, 1989; accepted December 6, 1989

Repeated subculture of Entomophaga maimaiga protoplasts in liquid media resulted in aberrant morphology after 50 passages. Bioassays demonstrated an increase in disease incubation time after 15 in vitro passages. Associated with increased incubation time, percentage mortality caused by E. maimaiga declined with repeated subculture (3 = 0.62). After prolonged subculture, E. maimaiga produced only resting spores in many cadavers, while fungal lines that had not been repeatedly subcultured produced conidia or both conidia and resting spores after host death. After 2 months of in vitro culture, the rate of subculturing had an impact on virulence, while the absohrte length of time in axenic culture did not. Q 1990 Academic RUSS, IN.

KEY WORDS: Entomophaga maimaiga; entomopathogen; biocontrol; fungal culture.

INTRODUCTION

Maintenance of consistent levels of viru- lence is an essential element for studies of invertebrate pathogens. Frequently, the simplest method for maintaining pathogenic microorganisms is in vitro culture. How- ever, prolonged in vitro culture of fungal pathogens has been shown to decrease the virulence of six species of Hyphomycetes: Beauveria bassiana, B. densa, Metarhi- zium anisopliae, Nomuraea rileyi, Paecilo- myces farinosus, and Verticillium lecanii (see Ignoffo et al., 1982; Morrow et al., 1989). On the contrary, no change in viru- lence after repeated in vitro subculture has been reported for seven species of Hypho- mycetes: Aspergillus jlavus, B. bassiana, B. brongniartii, Culicinomyces clavis- porus, Metarhizium brunneum, P. farino- sus, and V. lecanii (see Ignoffo et al., 1982; Sweeney, 1981). Among these reports, B. bassiana, P. farinosus, and V. lecanii were reported by some studies as losing viru- lence with repeated subculture (Ganhao, 1956; Kerner, 1959; Kawakami, 1960; Schaerffenberg, 1964; Fargues, 1972; Naga- ich, 1973), while other studies found no change (Arnaud, 1927; Lefebvre, 1931; Toumanoff, 1933; Hall, 1980). Discrepan-

cies in results may partially be due to dif- ferences in experimental methodology. However, changes in the virulence of fun- gal lines may be due to initial lack of genetic stability of individual isolates (C. M. Ig- noffo, pers. commun.). In one extreme ex- ample of stable virulence among the Hy- phomycetes, C. clavisporus has been re- ported as remaining highly virulent to mosquitoes after ~8 years of continuous culture on nutrient agar (Sweeney, 1981). Within a more primitive group, the Ento- mophthorales, the only report of the effects of in vitro culture states that extended sub- culturing caused decreased virulence in Pandora neoaphidis (as Entomophthora uphidis) (Rockwood, 1950).

The mechanisms for decreased virulence have seldom been explored among those fungi with decreased virulence after subcul- turing. Hall (1980) described altered mor- phology of V. lecanii colonies but noted no parallel decline in virulence against aphids. Ignoffo et al. (1982) studied the electropho- retie banding patterns of N. rileyi after 18 in vitro passages and found few alterations compared with the controls. He also noted a trend toward decreased virulence in noc- tuid larvae after 18 passages, although this trend was not statistically significant.

91 0022-2011190 $1.50 Copyright 0 1990 hy Academic Press, Inc. AU rights of reproduction in any form reserved.

92 HAJEK, HUMBER, AND GRIGGS

In the present study, we explore the in- fluence of successive subculture on Ento- mophaga maimaiga, a Japanese isolate of a pathogen of the gypsy moth, Lymantria dis- par (Soper et al., 1988). We report here growth of protoplasts in vitro and infection of larvae by conidia and by protoplast in- jection for E. maimaiga cultures grown for up to 50 passages in vitro over 4 months.

MATERIALS AND METHODS

Source and Growth Conditions

The isolate of E. maimaiga used for these studies was collected in June 1984 at Tori- goe, Ishikawa Prefecture, Japan. This fun- gus was initially isolated on egg yolk media supplemented with Sabouraud maltose agar (EYSMA; Soper et al., 1975). E. maimaiga also grows readily as fusoid to catenate pro- toplasts in Grace’s insect tissue culture me- dium (GM) (GIBCO, Grand Island, New York) supplemented with 5% (v/v) fetal bo- vine serum. The isolate was brought to our laboratory where it was catalogued (AR- SEF 1400; Agricultural Research Service Collection of Entomopathogenic Fungi) and stored under liquid nitrogen in the pro- toplast stage. Before the present study had begun, AFSEF 1400 was thawed, subcul- tured, and refrozen one time.

At the onset of this study, the culture was unfrozen and a large quantity of protoplasts were grown. From this time, aliquots of this large protoplast culture were evaluated inde- pendently (referred to as fungal lines) be- cause of the potential for genetic variability among these aliquots. One aliquot of this initial culture was used for the first experi- ments (month 0), and this line of the fungus was subcultured every 2-4 days for 4 months. The majority of the initial large culture was frozen in individual aliquots and, every month for 4 months, an aliquot was unfrozen for experimental studies. Each thawed aliquot was subcultured for 1 month and then used for bioassays (as a l-month-old culture). During months 24, comparisons were made between a newly

unfrozen culture (l-2 transfers), a l- month-old culture (12-15 transfers), and a culture 2-4 months old (2 months: 25 trans- fers; 3 months: 40 transfers; 4 months: 50 transfers). Comparisons were also made between the fungus unfrozen at month 0 and a culture that had been subcultured as protoplasts every 2-4 days for 1.5 years.

The interaction between the length of time an aliquot was in axenic culture and the number of passages was also investi- gated. One thawed aliquot was split into two treatments: transfers every 7 days or transfers every 2-3 days. After 2 months, cultures undergoing the first treatment (9 transfers) were compared with cultures un- dergoing the second treatment (25 trans- fers) .

Protoplast development was compared among a new culture and cultures having undergone 13 and 50 transfers. Cultures were microscopically observed for 8 days after subculturing. Protoplasts were grown in the dark at 20-22°C.

Bioassays

Bioassays were conducted using L. dis- par larvae obtained as eggs from the U.S. Department of Agriculture, APHIS Meth- ods Development Center, Otis Air National Guard Base, Massachusetts. Larvae were reared in the laboratory on a high wheat germ diet (Bell et al., 1981). All laboratory bioassays were conducted at 25°C with a 1ight:dark cycle of 14: 10. During months O- 4, fourth instar larvae were injected intra- hemocoelically with protoplasts at the base of a proleg using a microinjector (Micro- Metric Instrument, Cleveland, Ohio) and a 23-gauge hypodermic needle. Each larva was injected with ca. 500 protoplasts in 5 ~1 GM from actively growing protoplasts (sub- cultured l-2 days previously).

Bioassays using conidia were conducted during months 34. EYSMA plates were in- oculated with 0.5 ml of actively growing protoplasts per plate. After 10 days, the mature mycelia was cut from the surface of the medium and placed on filter paper on

DECLINE IN VIRULENCE OF E. maimaign 93

the surface of a water agar plate. Moist pa- per towelling was added to the bottom of each container to maintain high humidity. Mycelial mats were allowed to shower on larvae for 4 hr once conidial discharge was abundant. After showering, larvae were placed individually in Petri dishes contain- ing moistened filter paper for 2 days and then transferred to cups containing artificial diet.

Twenty larvae were used per treatment and all larvae were checked daily. Dead lar- vae were placed on water agar and exam- ined daily for conidial production. After 34 days, the cadavers were dissected to deter- mine if resting spores had been produced. Virulence of a fungal line was quantified using the mean disease incubation time, de- fined as the mean number of days from co- nidial inoculation or injection until larval death.

Statistical Analysis

Comparisons were made among bioas- says begun on the same day and among re- sults from the same original aliquot. For in- jection bioassays, results for all newly thawed and all l-month samples of E. mai- maiga were compared across all trials. For these multiple comparisons, multiple t tests were used with the Sidak inequality to dis- tribute the cx of 0.05 (Jones, 1984).

RESULTS

Zn Vitro Liquid Culture

Recently thawed cultures of E. maimaiga contained many long chains of catenate protoplasts (Fig. la). After 4 days of incu- bation, the chains were shorter and many individual fusoid protoplasts were present. By day 7, the culture contained an even higher proportion of individual protoplasts. By day 8, the predominant form of the fun- gus was sphaeroplastic (spherical proto- plasts with granular cytoplasm); fusoid pro- toplasts were not present. A culture that had been subcultured 13 times followed this same progression.

The development of E. maimaiga proto- plasts was different after 50 passages. After only 1 day of subculturing, protoplasts be- came spherical (Fig. lb) and contained a large vacuole (Fig. lc); nuclei could be seen in the cytoplasm at the edges of the large sphaeroplasts (Fig. lc). By day 3, very few fusoid cells remained; nuclei were no longer readily observed in the cytoplasm of many of the large sphaeroplasts (Fig. Id). The diameter of the sphaeroplasts contin- ued to enlarge from days 3-8, and the pe- ripheral cytoplasmic layer became progres- sively thinner.

Bioassays

Injection studies. For the fungal line thawed (begun) at month 0, the incubation time for E. maimaiga increased signifi- cantly after 1 month of subculture (15 pas- sages) (P G 0.05) (Fig. 2). From 1 month to 4 months later (U-50 passages), there were no statistically significant differences in in- cubation time (P > 0.05), although at 1 month, a mean of 7.15 days (SE = 0.15) was required before larval death and at 4 months, a mean of 8.50 days (SE = 1.04) was required.

Only two of the four fungal lines tested demonstrated a significant initial increase in disease incubation time (decline in viru- lence) after 1 month of subculture (12-15 passages) (Fig. 2). Disease incubation time did not significantly increase after 12 and 13 passages (fungal lines begun months 1 and 3) (P > 0.05), while in both trials receiving 15 transfers (fungal lines begun months 0 and 2), incubation time increased (P G 0.05). In all cases, however, the trend was toward increased incubation time and an overall analysis demonstrated that E. mai- maiga virulence changed after 12-15 pas- sages (t = 2.46 > t,, 0.02s; P s 0.05). No differences in incubation time were de- tected among the different E. maimaiga lines after l-2 passages (range: 4.9-5.3 days to death).

An inverse relationship existed between incubation time and mortality (y = 154.68

FIG. 1. Protoplasts of Entomophaga maimaiga in Grace’s insect tissue culture medium + 5% fetal bovine serum. Catenate protoplasts (a) typical of vegetative growth found in cultures soon after isolation from the host or soon after transfer of relatively young cultures to fresh medium. Surface view (b) and optical section (c) of enlarged spherical cells typically found in aged cultures. Nuclei are seen in (b) as “holes” in the cytoplasm and in (c) as lumps in the very thin peripheral layer of cytoplasm surrounding the large central vacuole. Optical section of a sphaeroplast in an aged culture (d); note that nuclei are not readily apparent, even though this sort of cell continues to grow in diameter (a, phase contrast, 1200x; b-d, Hoffman modulation contrast, 660x).

- 15.32~; 2 = 0.62; P 6 0.05). We inter- pret this to be a decline in the overall viru- lence with the increasing age of a culture. Mortality declined from a mean of 85.0% (SE = 5.45) after l-2 passages to 43.8% (SE = 9.45) after 12-15 passages (P s 0.05). After 50 passages, only 20% of in- jected larvae died. Another line of ARSEF 1400, continually subcultured for 1.5 years, declined only to 50% mortality.

An unusual pattern of spore production was noted among those cadavers from lar- vae injected with the 2- to 4-month-old cul- tures: 33.3% (25 passages) and 50.0% (50 passages) produced resting spores but not conidia. Cadavers from larvae inoculated with E. maimaiga cultures passaged 1-15

times uniformly produced only conidia or both conidia and resting spores.

An aliquot undergoing 9 transfers caused larval death more quickly than another sample of the same original aliquot subcul- tured 25 times within the same 2 months (P G 0.05). After 9 transfers, incubation time did not differ from the incubation time when the aliquot was initially thawed (P > 0.05). In addition, after 25 transfers, 80.0% of infections yielded only resting spores while after 9 transfers, no infections re- sulted in only resting spores. After 2 months, the length of time in axenic culture had little influence on pathogenicity, while the rate of subculturing did have an impact.

Conidial showers. Differential rates of

DECLINE IN VIRULENCE OF E. maimaiga 95

0 10 20 30 40 50

#TRANSFERS

FIG. 2. Disease incubation time as a function of number of transfers for Lymantria dispa; injected with Entomophaga maimaiga. *, for the same fimgal line, incubation time significantly different than incubation time after one to two transfers (multiple t tests using the Sidak inequality; P G 0.05).

spore production by different ages of cul- tures on EYSMA prohibited quantitative analysis of disease prevalence and incuba- tion times. Types of spores produced by older cultures after conidial infection of lar- vae were similar to those noted in injection bioassays. Only resting spores were pro- duced in 28.6% of cadavers of larvae in- fected using cultures passaged 40 times. Cadavers from larvae infected by conidia from cultures passaged 1-15 times in vitro always produced conidia, with or without resting spores.

DISCUSSION

Morphological Changes after Prolonged Subculture

The shift in the morphology of vegetative protoplasts from small, fusoid cells and bead-like chains of wall-less cells to en- larged spherical protoplasts represents a developmental anomaly. These giant sphaeroplasts (which can be several hun- dred micrometers in diameter) are derived from the hypertrophy of smaller proto- plasts. It is not clear why the normal con- trol of cell size and cell division breaks down, but these giant cells have cytoplas- mic and vacuolar volumes as well as nu- clear numbers that are vastly greater than

those of “normal” protoplasts and catenate filaments. During their relatively early de- velopment, the giant cells may be multivac- uolate and rather irregular in shape. Even- tually, however, the vacuoles fuse to form a single, rapidly growing spherical vacuole over which the cytoplasm is spread in a pro- gressively thinner layer. Associated with morphological changes, Glare (1989) has shown differences in enzyme composition. After 7 days of subculturing, this same iso- late (ARSEF 1400) demonstrated different band mobility for malic enzyme during starch gel electrophoresis.

Giant sphaeroplasts cannot be derived by repeated fusions of smaller cells because fusions between protoplasmic (or walled) cells of entomophthoralean fungi have never been observed and confirmed except those between gametangia during the initi- ation of zygosporogenesis. Aberrant struc- tures similar to various developmental stages of the giant sphaeroplasts discussed here have been reported for Entomophaga aulicae as “fusion spheres” (Dunphy and Nolan, 1977; Nolan, 1987), but these au- thors provided no evidence that these sphaeroplasts were, in fact, derived from cellular fusions. It should also be noted that IPRU 458, the strain from which the “fu- sion spheres” were reported, has been in continuous in vitro culture since its original isolation in 1970 (Tyrrell and MacLeod, 1972). The production of giant sphaero- plasts after protracted periods in axenic culture has also been observed in members of the E. grylli species complex (Humber, unpubl. data) and E. aulicae (Humber, un- publ. data; D. Tyrrell, pers. commun.).

Bioassays of Virulence

Among bioassays using injection of pro- toplasts as a means for inoculation, incuba- tion time significantly increased after 15 in vitro passages in two trials. Trials of 12 and 13 passages did not demonstrate a signifi- cant decrease in virulence, although data showed the same trend of increased incu- bation time. Overall analyses from injection

96 HAJEK, HUMBER, AND GRIGGS

studies, therefore, suggest that the nature of E. maimaiga does change after approxi- mately 1 month of subculturing every 2 to 3 days. Despite a clear trend toward in- creased in vivo incubation times in aging cultures of protoplasts, no statistically sig- nificant increase in the time required for host mortality was noted until after at least 15 passages in culture, when there ap- peared to be a marked decline in the rapid- ity of fungal development in vivo. How- ever, repeated bioassays would be neces- sary to detect the actual commencement of culture decline. Based on results from 2 months of subculture, the absolute length of time protoplasts are in axenic culture does not inIluence pathogenicity, while the number of times cultures are subcultured can change fungal virulence. In agreement with other studies (see Ignoffo et al., 1982), we have found that in vivo passages of fun- gal lines that have been repeatedly subcul- tured restore virulence to E. maimaiga cul- tures (A. E. Hajek, unpubl. data).

It is of interest that variability exists in the virulence of samples originally taken from one protoplast culture; cultures trans- ferred 15 times demonstrated in vivo incu- bation times of 5.63 and 7.15 days before causing mortality. Therefore, each fungal aliquot may have a different character and a different absolute response to repeated sub- culturing.

Culture decline affects conidial produc- tion since some cadavers from samples sub- cultured for 225 passages produced only resting spores in larvae infected by either injection or conidial showering. This type of spore production (resting spores only) was previously reported for fifth instar L. dispar at 25°C (Shimazu and Soper, 1986), although the length of time that the culture that was used had been in axenic culture

ACKNOWLEDGMENTS

We thank L. Marold and L. Strelow for help con- ducting these studies. T. M. Butt assisted with pho- tography. C. M. Ignoffo and S. T. Jaronski reviewed this manuscript. This research was partially funded by USDA Competitive Grant 87-CRCR-l-2503.

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