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The Effects of Temperature on the Pathogenicity of Heat-Sensitive Mutants of the Entomopathogenic Fungus,Beauveria bassiana,toward the Migratory Grasshopper,Melanoplus sanguinipes
JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.
68, 160–165 (1996)
0074
The Effects of Temperature on the Pathogenicity of Heat-Sensitive Mutants of the Entomopathogenic Fungus, Beauveria bassiana, toward the Migratory Grasshopper, Melanoplus sanguinipes DWAYNE D. HEGEDUS1
GEORGE G. KHACHATOURIANS2
AND
Bioinsecticide Research Laboratory, Department of Applied Microbiology and Food Science, University of Saskatchewan, Canada, Saskatchewan S7N 5A8 Received March 10, 1995; accepted April 18, 1996
INTRODUCTION
Heat-sensitive (HS) mutants of Beauveria bassiana were shown to kill grasshoppers at the growthpermissive temperature (PT) of 20°C but not at 30°C or more, the nonpermissive temperatures (NPT). The migratory grasshopper, Melanoplus sanguinipes, exhibited near complete poikilothermy in the bioassay system employed for B. bassiana strains. Temperature shift, from 20 to 30°C, during the bioassays was used to halt the mutant cell development postinfection. The resultant mortality data were analyzed using median effect plots. Timing of the infection was correlated with the required time at PT for grasshopper death. An incubation period of 4.6 to 6.4 days, postinfection, at PT was needed in order for the values for onset of infection, mortality rates, and the LT50 for the three HS mutants to reach those insects infected with control strain, GK2016, at the PT. The time of PT exposure required to ultimately kill half of the test insect population, termed LE50, ranged from 3.9 to 5.1 days. A minimum time for the insect infection at PT, defined by the LE50 value, is required for infection to lead to mortality. This time, the initial 4.4 days of infection, may reflect certain critical event(s) after which, whether or not additional fungal growth at NPT occurs, grasshopper death is unavoidable. These observations should help the development of models for predicting the outcome of infection processes and elucidation of critical events. r 1996 Academic Press, Inc. KEY WORDS: heat-sensitive mutants; Beauveria bassiana; Melanoplus sanguinipes; fungal-pathogenesis models; median effect plots.
1 Present address: Department of Zoology, University of British Columbia, 6270 University Blvd., Vancouver, BC, Canada V6T 1Z4. 2 To whom correspondence should be addressed.
0022-2011/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Our present knowledge of fungal pathogenesis in insects indicates that infection occurs via a series of integrated, systematic interactions between the fungus and the insect (Khachatourians, 1991, 1996). These interactions are complex and dependent upon specific host–pathogen interactions (Charnley, 1989; Hajek and St. Leger, 1994). The temperature (Walstad et al., 1970) and relative humidity (Marcandier and Khachatourians, 1987) dependency of fungal growth and infection has been established and models able to predict disease outcome under varying environmental conditions have been developed (Carruthers et al., 1985). What are lacking are predictive models capable of integrating both pathogen and host factors in order to establish a correlation between fungal virulence factors, host responses, and environmental parameters. One possible method is to use heat-sensitive (HS) mutants that can infect insects only at a permissive temperature but not at a restrictive temperature (Hegedus and Khachatourians, 1994). Examination of the disease outcome in response to various times of exposure to the HS mutants at the permissive temperature (PT) can allow for the determination of events in the infection process, in terms of either time or physiology, that predict the outcome of the infection. In this report HS mutants were used to investigate the infection process of Beauveria bassiana in grasshoppers and to define such events. MATERIALS AND METHODS
Fungal strains. Beauveria bassiana (Balsamo) Vuillemin, GK2016, and the heat-sensitive mutants HS1, HS2, HS6, HS9, and HS11 derived from this strain (Hegedus and Khachatourians, 1994) were grown on YPG agar (0.2% yeast extract, 1% peptone, 2% glucose,
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TEMPERATURE EFFECTS ON PATHOGENICITY OF B. bassiana
1.5% agar) for 8–10 days at 20°C. Conidia were harvested and stored as described before (Hegedus et al., 1990). Grasshopper infection. Adult migratory grasshoppers of a nondiapause strain of Melanoplus sanguinipes were used for the bioassays. Prior to inoculation insects were anesthetized with a brief exposure to CO2 gas. Topical application of the insect’s thorax and abdomen was done by dipping for 3 sec in a suspension of 1 3 108 conidia/ml of distilled H2O; care was taken to prevent ingestion of the inoculum. Insects were individually housed in plastic cylindrical vials (4 cm wide 3 8 cm high) in an environmental incubator, set at either 20 or 32°C, 24-hr photoperiod, and a relative humidity of 20–35% (Marcandier and Khachatourians, 1987). The hypothesis that the infection process could be halted by adjusting the ambient temperature, obviating the use of antifungal antibiotics or sacrificing the insect, was tested via a ‘‘temperature shift’’ bioassay in which a fraction of the insects were transferred from the PT of 20°C to the NPT of 32°C at 1, 3, and 5 days postinoculation. Bioassays were conducted with 10 insects per treatment and each treatment was either duplicated or run a third time as indicated. Measurement of grasshopper internal body temperature. The grasshopper’s response to changes in ambient air temperature was determined with the aid of a temperature microprobe. The probe was constructed by fusing 0.003-inch copper and constantine electrodes with silver solder and inserting this into a 22-gauge syringe. The insect was punctured beneath the pronotum and the probe inserted as per Prange (1990). The syringe was removed and the probe fixed into position by allowing the small drop of hemolymph extruded upon puncture to coagulate around the probe. An identical probe was designed to record ambient air temperature. The electrodes were connected to a Model HH23 type J-K-T thermocouple data recorder (Omega Engineering Inc., Stamford, CT) capable of recording dual readings. The probes were found to be accurate to within 0.1°C when compared to a commercial digital thermometer. Examination of mutant morphology on infected insects. Grasshoppers that remained alive at the NPT 14 days after treatment with the HS mutants were examined for the presence of fungi on the cuticular surface. Fungi were removed from insect surfaces by washing with 0.5 ml of 0.02% Tween-20 in a 1.5-ml microcentrifuge tube for 30 sec and then pelleting the debris for 2 min prior to resuspension in distilled water. Description of fungal developmental stages was as per Bidochka et al. (1987) and HS mutant morphology as per Hegedus and Khachatourians (1994). Phase contrast and differential interference contrast microscopy was conducted using a Jeneval microscope (Carl Zeiss-
161
Jena, Germany) fitted with a Normarski prism attachment. Median effect plots. The relationship between the rate and levels of mortality (fraction, affected, fa; fraction unaffected, fu) with increased exposure to the NPT was examined using the Dose Response Analysis software program (Elsevier-Biosoft, Cambridge, UK) to conduct median effect plot analysis (Chou and Taladay, 1984). In this analysis the magnitude of the slope (m) of the line was used as an indicator of the rate at which the infection was progressing where larger m values corresponded to increasing rates of mortality. The point at which the regression line intercepts the Y axis was used as a measure of the time of infection onset or the point at which an exponential increase in mortality begins. Daily levels of insect mortality were corrected for control group mortality using the method of Abbott (1925). RESULTS AND DISCUSSION
Grasshopper Thermoregulation Grasshoppers are almost true poikilotherms in that body temperature closely resembles ambient air temperature provided that physical activity is restricted and that a source of radiant light energy is absent (Chappel and Whitman, 1990). Although our bioassay system satisfied these requirements this generalization was validated by constructing a temperature microprobe and measuring the insects’ response to changes in ambient air temperature. Examination of the thermoregulating properties of M. sanguinipes indicated that insect internal body temperature does not deviate by more than 1°C from that of the ambient temperature over the range of 12–54°C. At ambient temperatures of less than 15°C the insects increase their body temperature slightly, whereas at 17–20°C the insect and the ambient temperatures were equivalent and at temperatures higher than 20°C body temperature was slightly lower than ambient temperature. Only 8–10 min was required for the ambient air and insect body temperature to stabilize upon shifting from either 20–32°C or 32–20°C with the insect’s body temperature lagging behind. In accordance with these findings a temperature of 32°C was selected as the NPT in all bioassays since even a 1–2°C thermoregulation on behalf of the insect would leave the bioassay at a temperature that was sufficient to inhibit the growth of the HS mutant strains (Hegedus and Khachatourians, 1994). Grasshopper Bioassays The infectivity of the HS mutants on the migratory grasshopper, M. sanguinipes, at both the PT of 20°C and the NPT of 32°C was determined. As shown in Fig. 1 the wild-type strain, GK2016, was virulent at both
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type strain are similar at both temperatures. At the PT mutants HS2, HS6, and HS9 exhibited LT50 values ranging from 6.6 to 7.3 days, comparable to the value of 6.8 days observed with GK2016 (Table 1). Conversely, mutants HS1 and HS11, although pathogenic, exhibited LT50 values of 8.2 and 10.3 days, respectively. This may be indicative of an indirect effect caused by the HS mutation or a second mutation at a separate locus unrelated to heat-sensitivity that affects infectivity in these two strains. Microscopic observation of the mutant morphology on insect cuticle indicated that the HS phenotypes were similar to that observed previously in vitro (Hegedus and Khachatourians, 1994). All of the fungal inocula recovered contained ungerminated conidia with some showing structures such as swelling, germination, peg formation, and elongation of mycelia. The control strain,
FIG. 1. Daily mortality of grasshoppers topically inoculated with Beauveria bassiana GK2016 or heat-sensitive mutants and incubated at either 20°C (X) or 32°C (W). Results are the average of duplicate experiments with 10 insects per treatment.
temperatures, whereas the HS strains caused insect mortality at the PT but not at the NPT. The temperatures 20 and 32°C are approximately 5–6°C above and below the optimal growth temperature of 25–27°C for GK2016. The kinetics for the infection with the wildTABLE 1 Infectivity Parameters for Beauveria bassiana Heat-Sensitive Mutants on Grasshoppers Strain
Temperature (°C)
LT50 a (days)
m (SE) b
Intercept (SE)
r c,d
GK2016 GK2016 HS1 HS1 HS2 HS2 HS6 HS6 HS9 HS9 HS11 HS11
20 32 20 30 20 32 20 32 20 32 20 32
6.8 6.5 8.2 —e 6.6 — 7.3 16.5 6.8 43.3 10.3 —
4.34 (1.08) 3.73 (0.28) 10.56 (1.05) 0.92 (0.25) 4.26 (0.70) 0.65 (0.13) 8.81 (1.16) 2.85 (0.38) 6.24 (1.29) 0.89 (0.10) 8.80 (0.28) 0.55 (0.08)
23.62 (0.83) 23.04 (0.23) 29.68 (0.94) 21.81 (0.24) 23.49 (0.53) 21.34 (0.11) 27.60 (1.04) 23.47 (0.37) 25.20 (1.08) 21.46 (0.09) 28.92 (0.29) 21.30 (0.07)
0.87 0.98 0.99 0.79 0.93 0.89 0.97 0.93 0.91 0.93 0.99 0.90
a LT 50 is time required for 50% response as estimated from regression analysis. b SE is the estimated standard error as calculated by the computer program. c r is the linear regression coefficient. d Results shown are the average of duplicate experiments with 10 insects per treatment. e Values calculated were .90 days.
FIG. 2. Median effect plots of daily mortality of grasshoppers topically inoculated with Beauveria bassiana heat-sensitive mutants following temperature shift. Bioassays were incubated at 20°C for 1 (M), 3 (W) or 5 (Q) days followed by incubation at 32°C. Continual incubation at 20°C (N) and 32°C (X) are also shown. Results are the average of triplicate experiments with 10 insects per treatment.
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TEMPERATURE EFFECTS ON PATHOGENICITY OF B. bassiana
GK2016, and HS mutants at the permissive temperature did not show any unusual developmental blocks. The material from HS1-treated insects possessed lysed conidial cell walls similar to those observed in liquid culture after exposure to the NPT (Hegedus and Khachatourians, 1994). The conidia recovered from HS2-, HS9-, and HS11-infected grasshoppers were swollen. Short hyphal elements were observed but only rarely. Samples taken from HS6-treated insects exhibited long, thin hyphal elements similar to those found in vitro at the NPT. Thus, the temperature on the insect’s cuticular surface must approximate the ambient air temperature in order to allow expression of the HS mutations. Effect of Temperature Shift on Mortality The results of temperature shift bioassays conducted with strains HS2, HS6, and HS9 and subjected to median effect plot analysis are presented in Fig. 2. This analysis shows a progression of regression lines moving from the NPT to the PT values that was dependent upon the time of exposure to the PT (Table 2). In all cases, and more so for HS2 strain, the slope of the regression line (the indicator of mortality rate) for the single-day PT exposure closely paralleled that of the NPT exposure. A PT exposure of 3 days produced a noticeable increase in mortality rate, whereas 5 days of PT exposure produced rates that approached that of the constant PT incubation. In addition, the LT50 and
onset values approximated those of the constant PT incubation after only 5 days of PT exposure. The relationship between bioassay parameters during this period and those of the constant PT exposure were used to estimate the time of PT exposure required for comparable levels of control lethality, termed the LEc. In general, mutant HS2 would require the longest exposure time, 6.4 days, before reaching control levels whereas mutants HS6 and HS9 would require only 5.2 and 4.6 days, respectively. It is at this point when the bioassay parameters are indistinguishable from those observed with a normal infection. Plotting the final levels of mortality against the time of PT incubation created a curve able to predict the outcome of the disease (Fig. 3). Based upon this analysis the PT exposure time required to ultimately kill 50% of the population via an active infection or LE50 ranged from 3.9 days for HS6 to 5.1 days for HS2. An additional observation made from this series of experiments was that the outcome of the infection was related to the effect on mutant morphology after exposure to the NPT. The rates and final levels of mortality were greatest for HS6, intermediate for HS9, and least for HS2. The mutants’ ability to produce biomass and extend mycelia after a shift to the NPT, in decreasing order of ability, was HS6, HS9, and HS2 (Hegedus and Khachatourians, 1994). This is correlated with the degree of infectivity after the shift to the NPT which in terms of increasing capacity follows this same order.
TABLE 2 Infectivity Parameters for Beauveria bassiana Heat-Sensitive Mutants on Grasshoppers Following Temperature Shift
Strain
Treatment
LT50 a (days)
HS2 HS2 HS2 HS2 HS2 HS6 HS6 HS6 HS6 HS6 HS9 HS9 HS9 HS9 HS9 No treatment No treatment
20°C 32°C 20°C, 1 day 20°C, 3 day 20°C, 5 day 20°C 32°C 20°C, 1 day 20°C, 3 day 20°C, 5 day 20°C 32°C 20°C, 1 day 20°C, 3 day 20°C, 5 day 20°C 32°C
8.2 26.1 17.1 12.0 9.84 7.9 25.7 9.9 11.7 7.0 7.7 43.3 14.7 9.82 7.7 39.3 21.5
a
m (SE) b
Intercept (SE)
Final mortality (%) c
Abbott-corrected mortality (%) d
r e, f
5.02 (0.47) 1.00 (0.07) 0.97 (0.05) 2.22 (0.30) 3.61 (0.25) 7.53 (0.37) 1.39 (0.17) 2.23 (0.15) 1.88 (0.16) 6.92 (0.94) 5.92 (0.96) 0.89 (0.10) 1.86 (0.14) 2.93 (0.36) 6.71 (1.06) 1.17 (0.23) 1.68 (0.23)
24.60 (0.43) 21.42 (0.06) 21.20 (0.04) 22.40 (0.23) 23.58 (0.22) 26.74 (0.34) 21.96 (0.16) 22.22 (0.12) 22.01 (0.15) 25.86 (0.79) 25.27 (0.81) 21.46 (0.09) 22.17 (0.12) 22.90 (0.33) 25.94 (0.89) 21.86 (0.19) 22.24 (0.21)
83 33 43 36 60 97 25 48 49 90 100 22 40 50 67 20 27
79 8 25 16 47 96 1 31 32 87 100 0 21 34 57 — —
0.97 0.98 0.98 0.93 0.98 0.99 0.93 0.99 0.96 0.97 0.94 0.93 0.97 0.94 0.96 0.89 0.93
LT50 is time required for 50% response as estimated from regression analysis. SE is the estimated standard error as calculated by computer program. c Recorded 12 days postinoculation. d Adjusted for control mortality according to Abbott (1925). e r is the linear regression coefficient. f Results shown are the average of triplicate experiments with 10 insects per treatment. b
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FIG. 3. Final mortalities of grasshoppers exposed to the Beauveria bassiana heat-sensitive mutants HS2 (X), HS6 (T), or HS9 (N) for 12 days. Bioassays were incubated at the permissive temperature (PT) of 20°C for 1, 3, or 5 days followed by incubation at 32°C. Arrows indicate extrapolation of 50% lethal exposure time (LE50).
reforms an intact cell wall (Pendland and Boucias, 1993). The use of temperature-sensitive pathogens infective toward poikilothermic insects can complement histological examinations and provide a new avenue of research for the examination of disease processes (Hegedus and Khachatourians, 1996). Our results indicate that some critical events determine the minimum time required for the infectious processes to result in insect death. Bioassay of a collection of entomopathogenic fungi has shown LT50 values ranging from 4 to 20 days with M. sanguinipes (Khachatourians, 1992). It is reasonable to suggest that the time required for the irreversibility of infection process may vary among hosts and pathogens. Use of HS mutants may set the lower time limit for critical events and determine whether or not an isolate’s virulence enhanced by passage through a host (Hayden et al., 1992) can change specific disease milestones. The development of other temperature-sensitive mutants affecting specific virulence determinants, such as hydrolytic enzyme activity or toxin production, could be used to define the molecular nature and role of these components as critical events at specific stages of the infection process. ACKNOWLEDGMENTS
Grasshoppers inoculated with mutant HS6 conidia, after 5 days of PT incubation, showed rates and final levels of mortality that closely resembled those of the constant PT incubation. This mutant is deficient in septa formation at the NPT; however, it continues to grow, forming long uncompartmentalized mycelia that eventually fracture. Exposure of mutant HS2 to the NPT results in an abrupt cessation of biomass accumulation and inhibition of further apical extension of the mycelia. In liquid culture, mutant HS9 exhibits many of the same physiological responses as that of HS2; however, apical extension and biomass accumulation are not inhibited as abruptly. The model developed using the HS mutants/grasshopper system indicated that a critical event leading to a lethal event, described as the LE50, occurs at approximately 3.9 to 5.1 days postinoculation. This event is the point beyond which the majority of the population will succumb to the infecting agent. Drawing comparisons to other fungal–insect interactions, the point established here would allow for conidial germination and cuticular penetration to occur in approximately 2 days, with 2–3 days remaining for invasion and degradation of tissues in the body cavity. In Lepidoptera it was during this later 2- to 3-day period that B. bassiana was shown to proliferate within the body cavity by means of pseudo-protoplasts refractive to phagocytosis by hemocytes. After this time phagocytic cells are rendered incompetent, possibly by fungal toxins, and the fungus
D.D.H. acknowledges support in the form of graduate scholarships from the University of Saskatchewan, the Natural Sciences and Engineering Research Council of Canada, and the Pest Management Alternatives Office. This work was supported by the Saskatchewan Department of Agriculture and Food through the Agriculture Development Strategic Research Programs Fund. We also thank J. Falkowsky for technical assistance.
REFERENCES Abbott, W. S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267. Bidochka, M. J., Pfeifer, T. A., and Khachatourians, G. G. 1987. Development of the entomopathogenic fungus Beauveria bassiana in liquid cultures. Mycopathologia 99, 77–83. Carruthers, R. I., Feng, Z., Robson, D. S., and Roberts, D. W. 1985. In vivo temperature-dependent development of Beauveria bassiana (Deuteromycotina:Hyphomycetes) mycosis of the European corn borer, Ostrinia nubilalis (Lepidoptera:Pyralidae). J. Invertebr. Pathol. 46, 305–311. Chappell, M. A., and Whitman, D. W. 1990. Grasshopper thermoregulation. In ‘‘Biology of Grasshoppers’’ (R. F. Chapman and A. Joern, Eds.), pp. 143–172. Wiley, New York. Charnley, A. K. 1989. Mechanisms of fungal pathogenesis in insects. In ‘‘Biotechnology of Fungi for Improving Plant Growth’’ (J. M. Whipps and R. D. Lumsden, Eds.), pp. 85–125. Cambridge University Press, Cambridge. Chou, T-C., and Taladay, P. 1984. Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 22, 27–55. Hajek, A. E., and St. Leger, R. J. 1994. Interactions between fungal pathogens and insect hosts. Annu. Rev. Entomol. 39, 293–322. Hayden, T. P., Bidochka, M. J., and Khachatourians, G. G. 1992. Entomopathogenicity of several fungi toward the English grain
TEMPERATURE EFFECTS ON PATHOGENICITY OF B. bassiana aphid (Homoptera: Acrididae) and enhancement of virulence with host passage of Paecilomyces farinosus. J. Econ. Entomol. 85, 58–64. Hegedus, D. D., Bidochka, M. J., and Khachatourians, G. G. 1990. Beauveria bassiana submerged conidia production in a defined medium containing chitin, two hexosamines or glucose. Appl. Microbiol. Biotechnol. 33, 641–647. Hegedus, D. D., and Khachatourians, G. G. 1994. Isolation and characterization of conditional lethal mutants of Beauveria bassiana. Can. J. Microbiol. 40, 766–776. Hegedus, D. D., and Khachatourians, G. G. 1995. The impact of biotechnology on hyphomycetous fungal insect biocontrol agents. Biotechnol. Adv. 13, 455–490. Hegedus, D. D., and Khachatourians, G. G. 1996. Analysis of cellular defense reactions of the migratory grasshopper, Melanoplus sanguinipes, infected with heat-sensitive mutants of Beauveria bassiana. J. Invertebr. Pathol. 68, 166–169. Khachatourians, G. G. 1991. Physiology and genetics of entomopathogenic fungi. In ‘‘Handbook of Applied Mycology,’’ Vol. II, ‘‘Humans, Animals and Insects’’ (D. K. Arora, L. Ajello, and K. G. Mukerji, Eds.), pp. 613–661. Marcel Dekker, New York. Khachatourians, G. G. 1992. Virulence of five Beauveria strains,
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Paecilomyces farinosus, and Verticillium lecanii against the migratory grasshopper, Melanoplus sanguinipes. J. Invertebr. Pathol. 59, 212–214. Khachatourians, G. G. 1996. The relationship between biochemistry and molecular biology of entomopathogenic fungi and insect diseases. In ‘‘The Mycota,’’ Vol. VI, ‘‘Human and Animal Relationships’’ (D. H. Howard and J. D. Miller, Eds.), pp. 331–363. SpringerVerlag, Berlin. Marcandier, S., and Khachatourians, G. G. 1987. Susceptibility of the migratory grasshopper, Melanoplus sanguinipes (Fab.) (Orthoptera: Acrididae), to Beauveria bassiana (Bals.) Vuillemin (Hyphomycete): Influence of relative humidity. Can. Entomol. 119, 893–900. Pendland, J. C., and Boucias, D. G. 1993. Evasion of host defense by in vivo-produced protoplast-like cells of the insect mycopathogen Beauveria bassiana. J. Bacteriol. 175, 5962–5969. Prange, H. D. 1990. Temperature regulation by respiratory evaporation in grasshoppers. J. Exp. Biol. 154, 463–474. Walstad, J. D., Anderson, R. F., and Stambaugh, W. J. 1970. Effects of environmental conditions on two species of muscardine fungi (Beauveria bassiana and Metarrhizium anisopliae). J. Invertebr. Pathol. 16, 221–226.
68, 160–165 (1996)
0074
The Effects of Temperature on the Pathogenicity of Heat-Sensitive Mutants of the Entomopathogenic Fungus, Beauveria bassiana, toward the Migratory Grasshopper, Melanoplus sanguinipes DWAYNE D. HEGEDUS1
GEORGE G. KHACHATOURIANS2
AND
Bioinsecticide Research Laboratory, Department of Applied Microbiology and Food Science, University of Saskatchewan, Canada, Saskatchewan S7N 5A8 Received March 10, 1995; accepted April 18, 1996
INTRODUCTION
Heat-sensitive (HS) mutants of Beauveria bassiana were shown to kill grasshoppers at the growthpermissive temperature (PT) of 20°C but not at 30°C or more, the nonpermissive temperatures (NPT). The migratory grasshopper, Melanoplus sanguinipes, exhibited near complete poikilothermy in the bioassay system employed for B. bassiana strains. Temperature shift, from 20 to 30°C, during the bioassays was used to halt the mutant cell development postinfection. The resultant mortality data were analyzed using median effect plots. Timing of the infection was correlated with the required time at PT for grasshopper death. An incubation period of 4.6 to 6.4 days, postinfection, at PT was needed in order for the values for onset of infection, mortality rates, and the LT50 for the three HS mutants to reach those insects infected with control strain, GK2016, at the PT. The time of PT exposure required to ultimately kill half of the test insect population, termed LE50, ranged from 3.9 to 5.1 days. A minimum time for the insect infection at PT, defined by the LE50 value, is required for infection to lead to mortality. This time, the initial 4.4 days of infection, may reflect certain critical event(s) after which, whether or not additional fungal growth at NPT occurs, grasshopper death is unavoidable. These observations should help the development of models for predicting the outcome of infection processes and elucidation of critical events. r 1996 Academic Press, Inc. KEY WORDS: heat-sensitive mutants; Beauveria bassiana; Melanoplus sanguinipes; fungal-pathogenesis models; median effect plots.
1 Present address: Department of Zoology, University of British Columbia, 6270 University Blvd., Vancouver, BC, Canada V6T 1Z4. 2 To whom correspondence should be addressed.
0022-2011/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
Our present knowledge of fungal pathogenesis in insects indicates that infection occurs via a series of integrated, systematic interactions between the fungus and the insect (Khachatourians, 1991, 1996). These interactions are complex and dependent upon specific host–pathogen interactions (Charnley, 1989; Hajek and St. Leger, 1994). The temperature (Walstad et al., 1970) and relative humidity (Marcandier and Khachatourians, 1987) dependency of fungal growth and infection has been established and models able to predict disease outcome under varying environmental conditions have been developed (Carruthers et al., 1985). What are lacking are predictive models capable of integrating both pathogen and host factors in order to establish a correlation between fungal virulence factors, host responses, and environmental parameters. One possible method is to use heat-sensitive (HS) mutants that can infect insects only at a permissive temperature but not at a restrictive temperature (Hegedus and Khachatourians, 1994). Examination of the disease outcome in response to various times of exposure to the HS mutants at the permissive temperature (PT) can allow for the determination of events in the infection process, in terms of either time or physiology, that predict the outcome of the infection. In this report HS mutants were used to investigate the infection process of Beauveria bassiana in grasshoppers and to define such events. MATERIALS AND METHODS
Fungal strains. Beauveria bassiana (Balsamo) Vuillemin, GK2016, and the heat-sensitive mutants HS1, HS2, HS6, HS9, and HS11 derived from this strain (Hegedus and Khachatourians, 1994) were grown on YPG agar (0.2% yeast extract, 1% peptone, 2% glucose,
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TEMPERATURE EFFECTS ON PATHOGENICITY OF B. bassiana
1.5% agar) for 8–10 days at 20°C. Conidia were harvested and stored as described before (Hegedus et al., 1990). Grasshopper infection. Adult migratory grasshoppers of a nondiapause strain of Melanoplus sanguinipes were used for the bioassays. Prior to inoculation insects were anesthetized with a brief exposure to CO2 gas. Topical application of the insect’s thorax and abdomen was done by dipping for 3 sec in a suspension of 1 3 108 conidia/ml of distilled H2O; care was taken to prevent ingestion of the inoculum. Insects were individually housed in plastic cylindrical vials (4 cm wide 3 8 cm high) in an environmental incubator, set at either 20 or 32°C, 24-hr photoperiod, and a relative humidity of 20–35% (Marcandier and Khachatourians, 1987). The hypothesis that the infection process could be halted by adjusting the ambient temperature, obviating the use of antifungal antibiotics or sacrificing the insect, was tested via a ‘‘temperature shift’’ bioassay in which a fraction of the insects were transferred from the PT of 20°C to the NPT of 32°C at 1, 3, and 5 days postinoculation. Bioassays were conducted with 10 insects per treatment and each treatment was either duplicated or run a third time as indicated. Measurement of grasshopper internal body temperature. The grasshopper’s response to changes in ambient air temperature was determined with the aid of a temperature microprobe. The probe was constructed by fusing 0.003-inch copper and constantine electrodes with silver solder and inserting this into a 22-gauge syringe. The insect was punctured beneath the pronotum and the probe inserted as per Prange (1990). The syringe was removed and the probe fixed into position by allowing the small drop of hemolymph extruded upon puncture to coagulate around the probe. An identical probe was designed to record ambient air temperature. The electrodes were connected to a Model HH23 type J-K-T thermocouple data recorder (Omega Engineering Inc., Stamford, CT) capable of recording dual readings. The probes were found to be accurate to within 0.1°C when compared to a commercial digital thermometer. Examination of mutant morphology on infected insects. Grasshoppers that remained alive at the NPT 14 days after treatment with the HS mutants were examined for the presence of fungi on the cuticular surface. Fungi were removed from insect surfaces by washing with 0.5 ml of 0.02% Tween-20 in a 1.5-ml microcentrifuge tube for 30 sec and then pelleting the debris for 2 min prior to resuspension in distilled water. Description of fungal developmental stages was as per Bidochka et al. (1987) and HS mutant morphology as per Hegedus and Khachatourians (1994). Phase contrast and differential interference contrast microscopy was conducted using a Jeneval microscope (Carl Zeiss-
161
Jena, Germany) fitted with a Normarski prism attachment. Median effect plots. The relationship between the rate and levels of mortality (fraction, affected, fa; fraction unaffected, fu) with increased exposure to the NPT was examined using the Dose Response Analysis software program (Elsevier-Biosoft, Cambridge, UK) to conduct median effect plot analysis (Chou and Taladay, 1984). In this analysis the magnitude of the slope (m) of the line was used as an indicator of the rate at which the infection was progressing where larger m values corresponded to increasing rates of mortality. The point at which the regression line intercepts the Y axis was used as a measure of the time of infection onset or the point at which an exponential increase in mortality begins. Daily levels of insect mortality were corrected for control group mortality using the method of Abbott (1925). RESULTS AND DISCUSSION
Grasshopper Thermoregulation Grasshoppers are almost true poikilotherms in that body temperature closely resembles ambient air temperature provided that physical activity is restricted and that a source of radiant light energy is absent (Chappel and Whitman, 1990). Although our bioassay system satisfied these requirements this generalization was validated by constructing a temperature microprobe and measuring the insects’ response to changes in ambient air temperature. Examination of the thermoregulating properties of M. sanguinipes indicated that insect internal body temperature does not deviate by more than 1°C from that of the ambient temperature over the range of 12–54°C. At ambient temperatures of less than 15°C the insects increase their body temperature slightly, whereas at 17–20°C the insect and the ambient temperatures were equivalent and at temperatures higher than 20°C body temperature was slightly lower than ambient temperature. Only 8–10 min was required for the ambient air and insect body temperature to stabilize upon shifting from either 20–32°C or 32–20°C with the insect’s body temperature lagging behind. In accordance with these findings a temperature of 32°C was selected as the NPT in all bioassays since even a 1–2°C thermoregulation on behalf of the insect would leave the bioassay at a temperature that was sufficient to inhibit the growth of the HS mutant strains (Hegedus and Khachatourians, 1994). Grasshopper Bioassays The infectivity of the HS mutants on the migratory grasshopper, M. sanguinipes, at both the PT of 20°C and the NPT of 32°C was determined. As shown in Fig. 1 the wild-type strain, GK2016, was virulent at both
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type strain are similar at both temperatures. At the PT mutants HS2, HS6, and HS9 exhibited LT50 values ranging from 6.6 to 7.3 days, comparable to the value of 6.8 days observed with GK2016 (Table 1). Conversely, mutants HS1 and HS11, although pathogenic, exhibited LT50 values of 8.2 and 10.3 days, respectively. This may be indicative of an indirect effect caused by the HS mutation or a second mutation at a separate locus unrelated to heat-sensitivity that affects infectivity in these two strains. Microscopic observation of the mutant morphology on insect cuticle indicated that the HS phenotypes were similar to that observed previously in vitro (Hegedus and Khachatourians, 1994). All of the fungal inocula recovered contained ungerminated conidia with some showing structures such as swelling, germination, peg formation, and elongation of mycelia. The control strain,
FIG. 1. Daily mortality of grasshoppers topically inoculated with Beauveria bassiana GK2016 or heat-sensitive mutants and incubated at either 20°C (X) or 32°C (W). Results are the average of duplicate experiments with 10 insects per treatment.
temperatures, whereas the HS strains caused insect mortality at the PT but not at the NPT. The temperatures 20 and 32°C are approximately 5–6°C above and below the optimal growth temperature of 25–27°C for GK2016. The kinetics for the infection with the wildTABLE 1 Infectivity Parameters for Beauveria bassiana Heat-Sensitive Mutants on Grasshoppers Strain
Temperature (°C)
LT50 a (days)
m (SE) b
Intercept (SE)
r c,d
GK2016 GK2016 HS1 HS1 HS2 HS2 HS6 HS6 HS9 HS9 HS11 HS11
20 32 20 30 20 32 20 32 20 32 20 32
6.8 6.5 8.2 —e 6.6 — 7.3 16.5 6.8 43.3 10.3 —
4.34 (1.08) 3.73 (0.28) 10.56 (1.05) 0.92 (0.25) 4.26 (0.70) 0.65 (0.13) 8.81 (1.16) 2.85 (0.38) 6.24 (1.29) 0.89 (0.10) 8.80 (0.28) 0.55 (0.08)
23.62 (0.83) 23.04 (0.23) 29.68 (0.94) 21.81 (0.24) 23.49 (0.53) 21.34 (0.11) 27.60 (1.04) 23.47 (0.37) 25.20 (1.08) 21.46 (0.09) 28.92 (0.29) 21.30 (0.07)
0.87 0.98 0.99 0.79 0.93 0.89 0.97 0.93 0.91 0.93 0.99 0.90
a LT 50 is time required for 50% response as estimated from regression analysis. b SE is the estimated standard error as calculated by the computer program. c r is the linear regression coefficient. d Results shown are the average of duplicate experiments with 10 insects per treatment. e Values calculated were .90 days.
FIG. 2. Median effect plots of daily mortality of grasshoppers topically inoculated with Beauveria bassiana heat-sensitive mutants following temperature shift. Bioassays were incubated at 20°C for 1 (M), 3 (W) or 5 (Q) days followed by incubation at 32°C. Continual incubation at 20°C (N) and 32°C (X) are also shown. Results are the average of triplicate experiments with 10 insects per treatment.
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GK2016, and HS mutants at the permissive temperature did not show any unusual developmental blocks. The material from HS1-treated insects possessed lysed conidial cell walls similar to those observed in liquid culture after exposure to the NPT (Hegedus and Khachatourians, 1994). The conidia recovered from HS2-, HS9-, and HS11-infected grasshoppers were swollen. Short hyphal elements were observed but only rarely. Samples taken from HS6-treated insects exhibited long, thin hyphal elements similar to those found in vitro at the NPT. Thus, the temperature on the insect’s cuticular surface must approximate the ambient air temperature in order to allow expression of the HS mutations. Effect of Temperature Shift on Mortality The results of temperature shift bioassays conducted with strains HS2, HS6, and HS9 and subjected to median effect plot analysis are presented in Fig. 2. This analysis shows a progression of regression lines moving from the NPT to the PT values that was dependent upon the time of exposure to the PT (Table 2). In all cases, and more so for HS2 strain, the slope of the regression line (the indicator of mortality rate) for the single-day PT exposure closely paralleled that of the NPT exposure. A PT exposure of 3 days produced a noticeable increase in mortality rate, whereas 5 days of PT exposure produced rates that approached that of the constant PT incubation. In addition, the LT50 and
onset values approximated those of the constant PT incubation after only 5 days of PT exposure. The relationship between bioassay parameters during this period and those of the constant PT exposure were used to estimate the time of PT exposure required for comparable levels of control lethality, termed the LEc. In general, mutant HS2 would require the longest exposure time, 6.4 days, before reaching control levels whereas mutants HS6 and HS9 would require only 5.2 and 4.6 days, respectively. It is at this point when the bioassay parameters are indistinguishable from those observed with a normal infection. Plotting the final levels of mortality against the time of PT incubation created a curve able to predict the outcome of the disease (Fig. 3). Based upon this analysis the PT exposure time required to ultimately kill 50% of the population via an active infection or LE50 ranged from 3.9 days for HS6 to 5.1 days for HS2. An additional observation made from this series of experiments was that the outcome of the infection was related to the effect on mutant morphology after exposure to the NPT. The rates and final levels of mortality were greatest for HS6, intermediate for HS9, and least for HS2. The mutants’ ability to produce biomass and extend mycelia after a shift to the NPT, in decreasing order of ability, was HS6, HS9, and HS2 (Hegedus and Khachatourians, 1994). This is correlated with the degree of infectivity after the shift to the NPT which in terms of increasing capacity follows this same order.
TABLE 2 Infectivity Parameters for Beauveria bassiana Heat-Sensitive Mutants on Grasshoppers Following Temperature Shift
Strain
Treatment
LT50 a (days)
HS2 HS2 HS2 HS2 HS2 HS6 HS6 HS6 HS6 HS6 HS9 HS9 HS9 HS9 HS9 No treatment No treatment
20°C 32°C 20°C, 1 day 20°C, 3 day 20°C, 5 day 20°C 32°C 20°C, 1 day 20°C, 3 day 20°C, 5 day 20°C 32°C 20°C, 1 day 20°C, 3 day 20°C, 5 day 20°C 32°C
8.2 26.1 17.1 12.0 9.84 7.9 25.7 9.9 11.7 7.0 7.7 43.3 14.7 9.82 7.7 39.3 21.5
a
m (SE) b
Intercept (SE)
Final mortality (%) c
Abbott-corrected mortality (%) d
r e, f
5.02 (0.47) 1.00 (0.07) 0.97 (0.05) 2.22 (0.30) 3.61 (0.25) 7.53 (0.37) 1.39 (0.17) 2.23 (0.15) 1.88 (0.16) 6.92 (0.94) 5.92 (0.96) 0.89 (0.10) 1.86 (0.14) 2.93 (0.36) 6.71 (1.06) 1.17 (0.23) 1.68 (0.23)
24.60 (0.43) 21.42 (0.06) 21.20 (0.04) 22.40 (0.23) 23.58 (0.22) 26.74 (0.34) 21.96 (0.16) 22.22 (0.12) 22.01 (0.15) 25.86 (0.79) 25.27 (0.81) 21.46 (0.09) 22.17 (0.12) 22.90 (0.33) 25.94 (0.89) 21.86 (0.19) 22.24 (0.21)
83 33 43 36 60 97 25 48 49 90 100 22 40 50 67 20 27
79 8 25 16 47 96 1 31 32 87 100 0 21 34 57 — —
0.97 0.98 0.98 0.93 0.98 0.99 0.93 0.99 0.96 0.97 0.94 0.93 0.97 0.94 0.96 0.89 0.93
LT50 is time required for 50% response as estimated from regression analysis. SE is the estimated standard error as calculated by computer program. c Recorded 12 days postinoculation. d Adjusted for control mortality according to Abbott (1925). e r is the linear regression coefficient. f Results shown are the average of triplicate experiments with 10 insects per treatment. b
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FIG. 3. Final mortalities of grasshoppers exposed to the Beauveria bassiana heat-sensitive mutants HS2 (X), HS6 (T), or HS9 (N) for 12 days. Bioassays were incubated at the permissive temperature (PT) of 20°C for 1, 3, or 5 days followed by incubation at 32°C. Arrows indicate extrapolation of 50% lethal exposure time (LE50).
reforms an intact cell wall (Pendland and Boucias, 1993). The use of temperature-sensitive pathogens infective toward poikilothermic insects can complement histological examinations and provide a new avenue of research for the examination of disease processes (Hegedus and Khachatourians, 1996). Our results indicate that some critical events determine the minimum time required for the infectious processes to result in insect death. Bioassay of a collection of entomopathogenic fungi has shown LT50 values ranging from 4 to 20 days with M. sanguinipes (Khachatourians, 1992). It is reasonable to suggest that the time required for the irreversibility of infection process may vary among hosts and pathogens. Use of HS mutants may set the lower time limit for critical events and determine whether or not an isolate’s virulence enhanced by passage through a host (Hayden et al., 1992) can change specific disease milestones. The development of other temperature-sensitive mutants affecting specific virulence determinants, such as hydrolytic enzyme activity or toxin production, could be used to define the molecular nature and role of these components as critical events at specific stages of the infection process. ACKNOWLEDGMENTS
Grasshoppers inoculated with mutant HS6 conidia, after 5 days of PT incubation, showed rates and final levels of mortality that closely resembled those of the constant PT incubation. This mutant is deficient in septa formation at the NPT; however, it continues to grow, forming long uncompartmentalized mycelia that eventually fracture. Exposure of mutant HS2 to the NPT results in an abrupt cessation of biomass accumulation and inhibition of further apical extension of the mycelia. In liquid culture, mutant HS9 exhibits many of the same physiological responses as that of HS2; however, apical extension and biomass accumulation are not inhibited as abruptly. The model developed using the HS mutants/grasshopper system indicated that a critical event leading to a lethal event, described as the LE50, occurs at approximately 3.9 to 5.1 days postinoculation. This event is the point beyond which the majority of the population will succumb to the infecting agent. Drawing comparisons to other fungal–insect interactions, the point established here would allow for conidial germination and cuticular penetration to occur in approximately 2 days, with 2–3 days remaining for invasion and degradation of tissues in the body cavity. In Lepidoptera it was during this later 2- to 3-day period that B. bassiana was shown to proliferate within the body cavity by means of pseudo-protoplasts refractive to phagocytosis by hemocytes. After this time phagocytic cells are rendered incompetent, possibly by fungal toxins, and the fungus
D.D.H. acknowledges support in the form of graduate scholarships from the University of Saskatchewan, the Natural Sciences and Engineering Research Council of Canada, and the Pest Management Alternatives Office. This work was supported by the Saskatchewan Department of Agriculture and Food through the Agriculture Development Strategic Research Programs Fund. We also thank J. Falkowsky for technical assistance.
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