Biological Control 39 (2006) 218–224

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Concentration-response and temperature-related susceptibility of the forest tent caterpillar (Lepidoptera: Lasiocampidae)

to the entomopathogenic fungus Furia gastropachae (Zygomycetes: Entomophthorales)

M.J. Filotas a,¤, J.D. Vandenberg b, A.E. Hajek a

a Department of Entomology, Cornell University, Ithaca, NY 14853, USAb US Plant, Soil and Nutrition Laboratory, USDA Agricultural Research Service, Tower Road, Ithaca, NY 14853, USA

Received 17 February 2006; accepted 17 May 2006Available online 25 May 2006

Abstract

Furia gastropachae (Raciborski) Filotas, Hajek & Humber is an important but little studied fungal pathogen of the forest tent caterpil-lar, Malacosoma disstria Hübner, a hardwood forest pest. As a means of assessing its potential as a biological control agent, a bioassaymethodology was developed to estimate virulence of two isolates of F. gastropachae (ARSEF 5544 and 5545 from NY and MD, USA,respectively) and to investigate the eVect of temperature on fungal virulence and activity. To assess virulence, fourth instar M. disstria lar-vae were exposed to conidial showers, with duration of exposure varied to produce diVerent concentrations. Resulting LC50 estimateswere low for both isolates (0.5§ 0.4 and 36.5§ 16.9 conidia/mm2 for the NY and MD isolate, respectively), though the NY isolate wasmore virulent. Both isolates had a relatively rapid rate of kill (ca. 5 days). To study the eVect of temperature on fungal activity, M. disstrialarvae were exposed to a single concentration of the NY or MD isolates, and then incubated at 10, 15, 20, 25, or 30 °C. Both isolatescaused mycoses at all temperatures, however, percent infection by the NY isolate was maximized at cooler temperatures (620 °C). Per-cent infection by the more southerly MD isolate also tended to decline with increasing temperature, however a signiWcant diVerence wasobserved only between 15 and 30 °C. Results suggest that F. gastropachae is well adapted to the early spring temperatures in which its hostis most likely to be active. The virulence of this fungus suggests that it may be a good candidate for biological control of the forest tentcaterpillar.© 2006 Elsevier Inc. All rights reserved.

Keywords: Entomophthorales; Furia gastropachae; Malacosoma disstria; Forest tent caterpillar; Virulence; Bioassay; Biological control

1. Introduction

The forest tent caterpillar, Malacosoma disstria Hübner(Lepidoptera: Lasiocampidae), is one of North America’smost common forest pests. Populations of this univoltineinsect increase to outbreak densities in a cyclic fashion,causing extensive defoliation of numerous hardwood spe-cies during early spring (Fitzgerald, 1995). Prolonged defo-liation can have severe impacts on aVected trees, including

* Corresponding author. Fax: +1 607 255 1132.E-mail address: mjf23@cornell.edu (M.J. Filotas).

1049-9644/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.biocontrol.2006.05.006

reduced radial growth, increased susceptibility to disease,economically signiWcant decreases in sugar maple sap yieldand, in extreme cases, tree mortality (Gross, 1991; Ander-son and Martin, 1981; Witter, 1979). High density popula-tions also represent a serious public nuisance when theyoccur near populated areas (Fitzgerald, 1995).

Among the array of naturally occurring pathogens ofM. disstria is the fungus Furia gastropachae (Raciborski)Filotas, Hajek & Humber [previously Furia crustosa(MacLeod & Tyrrell) Humber] (Zygomycetes: Entomoph-thorales). F. gastropachae has been isolated from tent cater-pillars (Malacosoma spp.) in North America and Europe,

M.J. Filotas et al. / Biological Control 39 (2006) 218–224 219

and epizootics of this fungus have long been associatedwith rapid declines in forest tent caterpillar populations(Filotas et al., 2003 and references cited therein). In contrastto other major pathogens of M. disstria, F. gastropachae’senvironmentally resistant resting spores and infectiousconidia are readily produced in vitro (MacLeod and Tyr-rell, 1979), facilitating the manipulation of this fungus forbiological control.

Although some studies of the potential application of F.gastropachae for forest tent caterpillar control have alreadybeen conducted (Perry and Fleming, 1989; Abrahamsonand Harper, 1973), virulence of this fungus has never beenassessed, and the relative eYcacy of isolates has not beencompared. Furthermore, additional information is requiredon the conditions under which this fungus is likely to beeVective. The early spring period during which host andpathogen are active is often characterized by widely Xuctu-ating temperatures. Laboratory studies indicate that F. gas-tropachae resting spores can germinate between 8 and 28 °C(Perry and Fleming, 1989), but it is not known whetherconidia of this fungus are infective over a similarly broadtemperature range.

In the present study, a concentration-response bioassaywas designed to evaluate the virulence of F. gastropachae tolarvae of M. disstria. This technique was used to comparethe eYcacy of two isolates obtained from geographicallyseparated M. disstria populations. The inXuence of temper-ature on infectivity, survival time and resting spore andconidial production was also investigated.

2. Materials and methods

2.1. Experimental insects

Forest tent caterpillar egg masses were collected fromtrembling aspen, Populus tremuloides Michx., in northernMichigan and placed in cold storage at 4 °C until neededfor bioassays (ca. 1–4 months). Upon removal from coldstorage, egg masses were surface sterilized with a 10%sodium hypochlorite solution and reared as described inGrisdale (1985), with the following modiWcations. Hatch-ing egg masses were placed in 236.8 ml plastic cups con-taining approximately 80 ml of modiWed Addy (1969) diet(Filotas, 2002) and maintained at 23§ 2 °C with a 14 hlight: 10 h dark photoperiod. After 12–14 days, mid to latethird instars were transferred to fresh diet with 10–15 lar-vae per cup. To avoid contamination by saprophyticfungi, larvae were subsequently transferred to fresh dietweekly until used in bioassays. Fourth instar M. disstrialarvae that had molted within the past 24 h were used inall bioassays.

2.2. Fungal inoculum and infection methodology

Two F. gastropachae isolates, maintained under liquidnitrogen at the United States Department of Agriculture(USDA), Agricultural Research Service Collection of

Entomopathogenic Fungi (ARSEF), Ithaca, New York,were used in all bioassays. ARSEF 5544 was isolated fromlab-reared larvae exposed to resting spore-bearing soil col-lected in Oswego County, NY in 1997 and ARSEF 5545was isolated from an infected M. disstria larva collectednear Salisbury, Maryland in June 1997 (Filotas et al., 2003).Both isolates were maintained as mycelium in 95% Grace’sinsect tissue culture medium + 5% fetal bovine serum(Gibco/BRL, Gaithersburg, MD). Isolates were subcul-tured every 5–7 days, but not more than six times due topotential loss of virulence.

For all bioassays, larvae were inoculated by exposingthem to conidial showers from sporulating mats of F. gas-tropachae mycelia. To obtain conidia, fungal mycelia weregrown on egg yolk/Sabouraud maltose agar (EYSMA:Papierok and Hajek, 1997) for 7–10 days at 20 °C. Mycelialmats were then removed from the media to promote sporu-lation and suspended 10 cm above experimental larvae in aspecial showering box designed to ensure all larvae receivedan even distribution of conidia (see Filotas et al., 2003 fordetails on showering box construction). High humidity lev-els were maintained by lining the bottom of each box withmoistened paper toweling. Larvae were housed in 29.6 mlplastic cups coated internally with Sigmacote (SigmaChemical Co., St. Louis, MD) to prevent them from escap-ing during the shower. Because larvae were unable to climbon Sigmacote-coated surfaces, they generally remained onthe bottom of the cups throughout the exposure period.Cups were placed in a circle in the showering box on a plat-form rotating at 0.5 rpm, directly below the sporulatingmycelial mats. Discharged conidia fell through the meshonto larvae. All conidial exposures were conducted at 20 °Cunder Xuorescent lighting.

After exposure, insects were placed individually in 59 mllidded portion cups (Fabri-Kal Corporation, Kalamazoo,MI) lined with moistened paper toweling. Cups were thenplaced in 14.1 l polyethylene storage containers (Rubber-maid Incorporated, Wooster, OH) that were also lined withmoistened paper toweling to maintain high RH and reducemoisture loss from the individual cups. Boxes were main-tained at 14 h light: 10 h dark and 20 °C except for tests oftemperature-related susceptibility, where larvae were main-tained at 14 h L: 10 h D and the designated temperature foreach treatment.

Larvae were maintained without food for 24 h to avoidpotential negative eVects of antimicrobials in the diet onconidial germination. After 24 h larvae were removed fromhumid storage containers, any dead insects were discardedand living larvae were transferred to lidded 29.6 ml plasticcups containing artiWcial diet and checked daily for mortal-ity for 14 days for all bioassays except those testing temper-ature-related susceptibility. For temperature bioassays,larvae were checked for 20 days to allow for potentialdelayed development of F. gastropachae at lower tempera-tures. Cadavers of any larvae that died were checked dailyfor 3 days to detect conidial production by F. gastropachae,and then dissected and examined under a compound

220 M.J. Filotas et al. / Biological Control 39 (2006) 218–224

microscope at 400£ to check for presence of internal rest-ing spores. Additionally, all larvae used in bioassays wereexamined microscopically to check for presence of sporesof the transovarially transmitted chronic microsporidianpathogen Nosema disstriae (Thom.) (Microsporidia: Nose-matidae), which is common in M. disstria populations anddiYcult to eliminate in laboratory colonies. Cadavers werechecked while looking for resting spores, and all other lar-vae not dying over the duration of bioassays were killed byfreezing, then dissected and examined at 400£ to check forpresence of microsporidian spores.

2.3. Concentration mortality bioassay

The general technique used to assess concentration-mor-tality relationships was similar to that described by Van-denberg and Soper (1979). Test insects were exposed tosporulating mycelia and the conidial concentration wascontrolled by varying the duration of exposure (5–45 min).To determine the actual concentrations received, two29.6 ml, Sigmacote-coated plastic cups containing ca 2 ml of1.5% water agar (WA) were placed on the rotating platformwith each set of experimental larvae. Concentration wasestimated as number of conidia/mm2 by counting Wve ran-dom microscope Welds per cup. Counts from the two cupswere averaged to obtain the Wnal estimate of concentrationfor each larval exposure. This yielded an array of concen-trations, ranging from 0.5 to 47.6 conidia/mm2. A total of 20concentrations were used. Because the supply of larvae waslimited throughout the experiment, only Wve to six larvaewere used per concentration. As a control, for each bioas-say 20 insects (in groups of Wve) were treated in an identicalmanner except they were not exposed to sporulating myce-lia. The entire experiment was conducted on three separatedates, resulting in a total of three replicate assays with ca.300 insects exposed to each isolate.

In all assays, only cadavers producing F. gastropachaeresting spores or conidia were considered to be infected.For both isolates, mortality of control larvae was low(<11%), and no control larvae died of F. gastropachaeinfections. Probit analysis was used to estimate slope andmedian lethal concentration (LC50 values) for each isolate(SAS Institute Inc, 2001). Infection of larvae by the micro-sporidian pathogen N. disstriae was also included as a pre-dictor variable in probit analyses. Regression analysis wasused to test the eVect of dosage and prevalence of N. disst-

riae (measured as arcsine-transformed percentage of larvaeinfected with microsporidian spores) on survival time (mea-sured as mean days to death per treated group) and arcsine-transformed percentage of cadavers producing conidia andresting spores (SAS Institute Inc, 2001).

2.4. Temperature-related susceptibility

For each assay, fourth instar M. disstria larvae were sub-jected to conidial showers for ca. 10–15 min. The concentra-tion received per shower was determined by counting thenumber of conidia falling onto two 29.6 ml water agar cupsplaced adjacent to larvae on rotating platforms. Concentra-tions corresponded to 9.6§0.2 and 8.1§0.2 conidia/mm2

for ARSEF 5544 and 5545, respectively. Twenty-Wve larvaewere exposed at a time, then divided into groups of Wve andincubated at 10, 15, 20, 25, or 30 °C, respectively. Bioassaysconsisted of 6–8 conidial showers, corresponding to 30–40larvae per temperature treatment. As a control, 50 larvae(in two groups of 25) were placed on rotating platformswithout sporulating mycelia, with 10 larvae subsequentlyincubated at each of the Wve temperatures. This entireexperiment was conducted on three separate dates, for atotal of three replicate assays with ca. 75 larvae exposed toeach isolate/temperature combination.

Analysis of variance was used to determine the eVects oftemperature, isolate and prevalence of N. disstriae on arc-sine-transformed percentage mortality, survival time andarcsine-transformed percentage of cadavers producingconidia and resting spores. Means were compared usingTukey’s HSD test (SAS Institute Inc, 2001).

3. Results

3.1. Concentration mortality assay

LC50 values, averaged (§SE) across the three replicateassays were 0.5§0.4 and 36.5§ 16.9 conidia/mm2 forARSEF 5544 and 5545, respectively (Table 1). The two iso-lates diVered signiWcantly with respect to both LC50(tD3.17, dfD4, P < 0.04) and slope (tD 4.09, dfD 4,P < 0.02), with the New York isolate (ARSEF 5544) havingthe lesser LC50 and slope (Table 1). Due to the possibilitythat estimates of LC50, which are calculated from the ratioof two normally distributed values (slope and intercept),may not be normally distributed themselves, results from t-

Table 1Results of dose–response bioassays and prevalence of the chronic microsporidian pathogen, Nosema disstriae, for two isolates of Furia gastropachaeagainst fourth instar forest tent caterpillars

Means in each column followed by the same letter are not signiWcantly diVerent (P < 0.05, T test or ANOVA).a Three replicate assays per isolate, 20 doses per assay, 4–6 insects per concentration.b Analyses done using log10 conidia per square millimeter.

Isolate No. larvaea

Slope § SEb LC50 § SE (conidia/mm2)b

% N. disstriae infection § SE

Survival time §SE (days)

% Cadavers producing spores (§SE)

Conidia Resting spores Both

ARSEF 5544 302 0.5 § 0.2 a 0.5 § 0.4 a 4.2 § 1.7 a 5.2 § 0.1 a 19.6 + 3.3 a 54.4 + 4.4 a 25.4§ 4.3 aARSEF 5545 293 1.7 § 0.3 b 36.5 § 16.9 b 12.8 § 2.6 a 5.1 § 0.1 a 25.0 + 4.9 a 62.8 + 6.5 a 12.3§ 3.2 a

M.J. Filotas et al. / Biological Control 39 (2006) 218–224 221

tests on these data were conWrmed with nonparametricanalysis. This also indicated a signiWcant diVerence betweenisolates in LC50 (�

2D3.86, dfD1, P < 0.05, Kruskal–WallisTest).

Mean prevalence of N. disstriae among larvae used in allassays was relatively low (<13%), and did not diVer signiW-cantly between the two isolates (tD 1.45, dfD 4, P > 0.2,Table 1) or between control and treated larvae (tD0.10,dfD4, P > 0.9 for ARSEF 5544, tD 0.49, dfD 4, P > 0.6 forARSEF 5545, data not shown). In probit analyses, infectionof larvae by N. disstriae was not signiWcantly associatedwith infection by ARSEF 5544 (�2 < 1.5, dfD1, P > 0.2 forthe three replicate assays), or with infection by ARSEF5545 for two of the three replicate assays (�2 < 0.3, dfD1,P > 0.6). However, during the third assay with ARSEF5545, the presence of N. disstriae in larvae was signiWcantlynegatively associated with infection by F. gastropachae(�2D 7.47, dfD 1, P < 0.006;), and consequently N. disstriaewas included in estimates of LC50 and slope for this assayonly. For this assay, both LC50 and slope estimates weregreater when N. disstriae was included in the model(LC50D 70.2 conidia/mm2, slopeD2.2 when N. disstriae wasincluded versus LC50D11.2 conidia/mm2, slopeD 1.8 whenit was not).

Mean survival time of larvae infected with both isolateswas 5.1–5.2 days (Table 1). Survival time was not signiW-cantly associated with dose for either isolate (F1,43D 3.5,P > 0.06 for ARSEF 5544, F1,47D 1.75, P > 0.1 for ARSEF5545) and did not diVer signiWcantly between the two iso-lates (F1,83D0.56, P > 0.4). The microsporidium had noeVect on survival time for either isolate (F1,43D 1.38, P > 0.2for ARSEF 5544; F1,47D 0.39, P > 0.5 for ARSEF 5545).

Resting spores and conidia, alone or in combination,were produced within sporulating cadavers by both isolates(Table 1). There was no signiWcant eVect of concentration(P > 0.4 for all isolates and spore types) or the microspori-dium (P > 0.09 for all isolates and spore types) on the typeof spore produced. Patterns of resting spore or conidia pro-duction did not diVer signiWcantly among isolates(F1,92D 2.52, P > 0.1; F1,92D 0.05, P > 0.8; and F1,92D2.42,P > 0.1 for cadavers producing conidia, resting spores andboth spore types, respectively), and for both isolates mostcadavers (>75%) produced resting spores, either alone or incombination with conidia (Table 1).

3.2. Temperature-related susceptibility

Infection of fourth instar forest tent caterpillars variedsigniWcantly with temperature for both isolates(F4,137D 20.17, P < 0.001 for ARSEF 5544; F4,137D2.85,P < 0.03 for ARSEF 5545). Percent infection by ARSEF5544 was maximized at lower temperatures (10–20 °C),while a signiWcant diVerence in the percentage of caterpil-lars infected by ARSEF 5545 was observed only between 15and 30 °C (Fig. 1A). Percent infection also diVered signiW-cantly between the two isolates (F1,277D5.92, P < 0.02) andthere was a signiWcant isolate by temperature interaction

(F4,277D 4.42, P < 0.002). While percent infection by ARSEF5544 was greater than 5545 at the two lowest temperatures(10 and 15 °C), at 25 °C signiWcantly more larvae wereinfected by the southern isolate, ARSEF 5545 (Fig. 1A).Control mortality in these assays was 620% at all tempera-tures except 30 °C, where it increased to 40%, probably dueto negative eVects of the high temperature on the quality ofthe artiWcial diet. For F. gastropachae-treated insects, mor-tality due to causes other than fungus (i.e., the diVerencebetween overall mortality and percent infection) was simi-lar to control mortality at each temperature.

Survival time of infected larvae also varied signiWcantlywith temperature (F4,64D 52.28, P < 0.0001 and F4,57D54.6,P < 0.001 for ARSEF 5544 and 5545, respectively), but notby isolate (F1,123D1.41, P > 0.2). For both isolates, lowestsurvival times were observed at 25 °C, whereas infected lar-vae survived almost 3 times as long at 10 °C (Fig. 1B).

The percentage of cadavers bearing resting spores and/or conidia was also aVected by temperature (for conidiaF4,82D9.79, P < 0.0001 and F4,75D3.45, P < 0.02 for ARSEF5544 and 5545, respectively; for resting spores F4,85D 11.07,

Fig. 1. EVect of temperature on (A) the percentage (§SE) of fourth instarforest tent caterpillars infected by two isolates of Furia gastropachaeconidia in the laboratory and (B) survival time (§SE) of infected larvae.For each isolate, bars labeled with the same letter are not signiWcantlydiVerent (P < 0.05, Tukey test). For each temperature, asterisks indicate asigniWcant diVerence between isolates.

0

10

20

30

40

50

60

70

10 15 20 25 30Temperature (ºC)

noitcefnI %

ARSEF 5544 (NY)ARSEF 5545 (MD)

a

a

a

b

b b

ababa

ab

**

*

0

2

4

6

8

10

12

14

10 15 20 25 30

Temperature (ºC)

)syad( emi

T lavivruS

ARSEF 5544 (NY)

ARSEF 5545 (MD)

a

ccd

d

cc

bb

a

d

A

B

222 M.J. Filotas et al. / Biological Control 39 (2006) 218–224

P < 0.0001 and F4,75D 3.32, P < 0.02 for ARSEF 5544 and5545, respectively). The percentage of cadavers bearingboth spore types was always low and not associated withtemperature (P > 0.08 for both isolates). For ARSEF 5544,resting spore production was greatest and production ofconidia was lowest at the two lowest temperatures (10 and15 °C) (Fig. 2A). Spore production by ARSEF 5545 diVeredonly between 15 and 25 °C, with more cadavers bearingresting spores and fewer cadavers bearing conidia at 15 °C(Fig. 2B). Spore production did not diVer signiWcantlybetween the two isolates (P > 0.12 for all spore productionpatterns). There was no signiWcant eVect of conidial con-centration or prevalence of the microsporidian pathogen N.disstriae on types of spore produced (P > 0.07 for all tests).

4. Discussion

Standard protocols for dose–response bioassays havebeen notoriously diYcult to apply to Entomophthoralesdue to problems associated with manipulation of their frag-ile conidia (Papierok and Hajek, 1997). The “spore showertechnique”, in which test insects are exposed to a series ofconidial showers with duration varied to generate diVerent

Fig. 2. EVect of temperature on the percentage of fourth instar forest tentcaterpillar cadavers bearing diVerent types of Furia gastropachae sporesfor (A) ARSEF 5544 and (B) ARSEF 5545. For each isolate and sporetype, bars labeled with the same letter are not signiWcantly diVerent(P < 0.05, Tukey test).

0

10

20

30

40

50

60

70

80

90

100

10 15 20 25 30

Temperature (ºC)

gnicudor

p srevadac

%eahcaportsag .

Fserops

conidia onlyresting spores onlyboth spore types

a

b b

a

a

a

a

a

aaa

c

bcb

a

0

10

20

30

40

50

60

70

80

90

100

10 15 20 25 30

Temperature (ºC)

gnicudor

p srevadac

%eahcaportsag .

Fserops

conidia onlyresting spores onlyboth spore types

aba

ab

b

ab

a

abab

ab

b

aa

a

A

B

concentrations, was Wrst described by Wilding (1976), andhas since been applied successfully to a number of entom-ophthoralean pathogens (e.g., Vandenberg and Soper, 1979;Pell et al., 1993; Feng et al., 1998; Xu and Feng, 2000). Inour study, we modiWed this technique slightly by placinginsects on a motorized platform, which was constantlyrotating below the sporulating cultures. We found that thisallowed a more even distribution of conidia, particularlywhen insects are exposed to multiple mycelial mats (whichmay be sporulating at diVerent rates), and minimizedmechanical disruption of fragile mycelia. Slope and LC50estimates (Table 1) resulting from this technique werewithin the range reported for other Entomophthorales(Vandenberg and Soper, 1979; Papierok, 1982; Pell et al.,1993; Xu and Feng, 2000).

The average survival time of ca. 5 days for M. disstrialarvae dying of F. gastropachae also fell within the range of4.5–7 days reported for other species of Entomophthoralesattacking Lepidoptera (Vandenberg and Soper, 1979;Shimazu and Soper, 1986). As a potential natural controlagent of M. disstria, these survival times are promising,since they are more than 4 days faster than the 9.6§ 1.2days estimated for M. disstria nucleopolyhedrovirus(MdNPV), the other major M. disstria pathogen (Eblingand Kaupp, 1999).

The production of resting spores in a majority (>70%) ofcadavers infected with both isolates is consistent with previ-ous results for this host/pathogen system (Filotas et al.,2003; Filotas and Hajek, 2004). Existing evidence suggeststhat in the Weld F. gastropachae does not encounter hostinsects until late in the life cycle due to the tendency of earlyinstar forest tent caterpillars to remain in their natal tree(Carruthers and Soper, 1987). There is consequently limitedopportunity for the fungus to initiate cycles of secondaryinfection (by conidia) throughout the season; thus, wehypothesize that F. gastropachae invests heavily in restingspore production to ensure its long term survival.

Furia gastropachae conidia caused mycoses from 10 to30 °C, similar to the range of activity reported for restingspores of this species (Perry and Fleming, 1989; Filotasand Hajek, 2004). However, percent infection by the NewYork isolate was maximized at temperatures 620 °C,while that of the more southern Maryland isolate wassimilar across temperatures, with a signiWcant diVerenceobserved only between 15 and 30 °C. Additionally, whileinfection by the NY isolate was greater than that of theMD isolate at lower temperatures (10 and 15 °C), at 25 °Clevels of infection by this southern isolate were actuallygreater than that of the northern isolate. Average annualtemperatures in the area of origin are 8.7 and 14.0 °C,respectively, for the NY and MD isolates, while tempera-tures occurring during the spring period when host insectsare active range from 7–24 °C for New York and 13–29 °Cfor Maryland (Northeast Regional Climate Center, Cor-nell). Thus, while one cannot make deWnitive conclusionsbecause only one isolate was tested from each area, it ispossible that these results may be due to adaptation to the

M.J. Filotas et al. / Biological Control 39 (2006) 218–224 223

diVerent prevailing temperatures occurring in the placesof origin of these geographically separated isolates.Regardless, for both isolates maximal percent infectionwas observed within the range of temperatures typicallyobserved during the early spring period in which the hostis active. Adaptation to temperatures prevailing when pri-mary hosts are likely to be active has been shown for sev-eral other species of Entomophthorales (Milner andLutton, 1983; Shimazu and Soper, 1986; Hajek et al.,1990; Tillotson et al., 1990).

Temperature also inXuenced the type of spore pro-duced within cadavers for both isolates, with the propor-tion of resting spores decreasing and the proportion ofconidia increasing as temperature increased (Fig. 2). Pro-duction of resting spores, the overwintering stage, byEntomophthorales tends to be correlated with tempera-tures occurring when host populations begin to decline(Glare et al., 1989; Hajek and Shimazu, 1996; Thomsenet al., 2001). Forest tent caterpillars hatch in late Apriland pupate in late June (Fitzgerald, 1995). Thus, declinesin host abundance are associated with increasing tempera-tures, making the observed results somewhat surprising.However, in this host/pathogen system, F. gastropachaedoes not encounter M. disstria larvae until late in the lifecycle, when later instars leave their natal trees to feedindependently (Carruthers and Soper, 1987). Thus, for F.gastropachae, the increased abundance of cadavers pro-ducing conidia at higher temperatures may therefore beassociated with the onset of wandering instars and henceincreased opportunity for within season infection of newhosts by conidia.

These results conXict with those of a previous study inwhich we exposed forest tent caterpillars to F. gastropachaeresting spores in soil collected from the same location as theNY isolate (Filotas and Hajek, 2004). In that study, weobserved a signiWcant decrease in production of conidia by M.disstria cadavers as temperature increased from 15 to 23°C,while resting spore production was unaVected by temperature(Filotas and Hajek, 2004). The conXicting results between ourpresent study and previous Wndings may be attributable to thefact that mycoses initiated by the two diVerent types of sporesmay respond to temperature diVerently.

Previous investigations have shown that F. gastropachaecan be important in the natural control of outbreak popu-lations of M. disstria. Our results indicate that F. gastrop-achae can be highly virulent, although there is variabilitybetween the two isolates. Although activity was optimizedat cooler temperatures, this fungal pathogen caused mortal-ity across all temperatures tested. The data suggest that F.gastropachae could be a promising agent for use in foresttent caterpillar population suppression, especially if meansfor mass production could be developed.

Acknowledgments

We thank D. Richter and students for supplying M. dis-stria egg masses, B. McCron of the Great Lakes Forestry

Center, Natural Resources Canada for providing the recipefor the modiWed Addy diet, S. Wraight, and F. Vermeylenfor advice on statistical analyses, L. Bauer and L. Solter foradvice on microsporidia, C. Castillo Davis for initial designand F. Labonte for construction of “caterpillar-go-rounds”used for conidial showers and M. Wheeler, N. Bertoia, M.Bertoia, J. McNeil, and S. Bretschger for technical assis-tance. J. Nyrop provided helpful comments on earlier ver-sions of this manuscript. This work was supported in partby a McIntire-Stennis grant to A.E.H. and a Natural Sci-ence and Engineering Research Council of Canada gradu-ate fellowship to M.J.F.

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