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Analysis of Cellular Defense Reactions of the Migratory Grasshopper,Melanoplus sanguinipes,Infected with Heat-Sensitive Mutants ofBeauveria bassiana
JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.
68, 166–169 (1996)
0075
NOTE Analysis of Cellular Defense Reactions of the Migratory Grasshopper, Melanoplus sanguinipes, Infected with Heat-Sensitive Mutants of Beauveria bassiana Beauveria bassiana and other entomopathogenic fungi are being examined as potential biological insect control agents. Our present knowledge of fungal pathogenesis in insects indicates that it occurs via a series of integrated, systematic events progressing from spore attachment to germination, penetration, growth, and proliferation within the body of the host, interaction with insect defense mechanisms, and finally reemergence on the cadaver. The interactions that occur between the fungus and the insect are exceedingly complex and are dependent upon specific host–pathogen interactions (Hajek and St. Leger, 1994; Hegedus and Khachatourians, 1995; Khachatourians, 1996). Beauveria bassiana heat-sensitive (HS) mutants have been developed (Hegedus and Khachatourians, 1994) that do not infect Melanoplus sanguinipes at the nonpermissive temperature (NPT) of 32°C. Bioassays in which the infection was terminated by shifting the ambient air temperature indicated that events occurring within the first 4–5 days postinoculation were critical to the outcome of the infection (Hegedus and Khachatourians, 1996). Here we report on the interaction between several HS mutants and the insect’s cellular defense responses at the permissive temperature (PT) of 20°C and the NPT to further define the role of fungal morphology in the infection process. Beauveria bassiana strain GK2016 and the HS mutants HS2, HS6, HS9, and HS11 derived from this strain were grown in YPG broth (0.2% yeast extract, 1% peptone, 2% glucose) for 4–5 days at 20°C with shaking (150 rpm) and blastospores harvested as described previously (Hegedus et al., 1990). Adult migratory grasshoppers of a nondiapause strain of M. sanguinipes were used for the bioassays. Insects were anesthetized with CO2 gas and 3 µl of a suspension of 1 3 108 blastospores/ml of sterile dH2O was injected into the right hind coxa using a Hamilton syringe modified to prevent penetration beyond a depth of 2 mm. Insects were individually housed in plastic vials 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). Samples of hemolymph were obtained 1 or 14 days after treatment, as indicated, by cutting off the left rear appendage of live 0022-2011/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
insects and gently squeezing the insect to promote bleeding. The samples were mixed with an equal volume of anticoagulant buffer (0.098 M NaOH, 0.146 M NaCl, 0.17 M EDTA, and 0.041 M trisodium citrate, pH 4.5) and stored at 4°C. To assist the visualization of blastospores, and to determine the percentage which had germinated, blastospores were labeled with the fluorescent compound fluorescein isothiocyante (FITC; Sigma Chemical Co., St Louis, MO) according to Hung and Boucias (1992). There was no effect on the viability of the blastospores treated in this manner as 99% germinated in YPG broth (data not shown). Each treatment was replicated with seven insects from which duplicate 5-µl samples of the diluted hemolymph were placed on glass slides and covered with a coverslip and the entire sample scored. This method of inoculation and sampling results in approximately 50–200 blastospores per slide. All statistical analyses were done using the Statview Student version 1.0 program (Abacus Concepts Inc., Berkeley, CA). Mean percentage values were arc sin Œ p transformed and then subjected to the Student t test, analysis of variance, and multiple comparison using Fisher’s F test. Previously, examination of the cuticular surfaces of insects that had been topically treated with the HS mutants, followed by incubation at the NPT, revealed that mutant morphology closely resembled that observed in vitro under the same conditions (Hegedus and Khachatourians, 1994; Hegedus and Khachatourians, 1996). To determine if this was also occurring within the body cavity of the insect, as well as to examine the role of fungal morphology in the infection, hemolymph was examined from insects 14 days after injection and incubation at either the PT or NPT. At the PT abundant amounts of short hyphal bodies, blastospores, and budding mycelia were observed (Fig. 1). There were no visible differences between mutants HS2, HS6, and the wild-type strain, GK2016. Mutant HS9 produced long mycelia with an abundance of budding blastospores. At 32°C, GK2016 produced structures similar to that at 20°C. Conversely, mutant morphology was dramatically altered and resembled structures observed in vitro when the mutants were incubated at the NPT. Mutant HS6 produced highly elongated mycelia
166
167
NOTE
FIG. 1. Examination of hemolymph of grasshoppers injected with blastospores of Beauveria bassiana GK2016 or heat-sensitive mutants and incubated at either 20 or 32°C for 14 days. For GK2016 and HS9: A, B (20°C), C, D (32°C). HS2: A, B, C (20°C), D, E (32°C). HS6: A (20°C), B (32°C). Arrows indicate mycelim (m), budding mycelium (bm), blastospore (b), hemocyte (hc), spreading hemocyte (shc), germinated blastospore (gb), bacteria (ba), and blebbing of hemocyte (bl). 1 mm 5 1 µm.
whereas mutant HS9 germinated to produce short mycelia with some minor branching. No secondary blastospores or other hyphal bodies were observed. In addition, the hemolymph extracted from insects infected with the mutant strains at the NPT possessed many engorged hemocytes containing large numbers of vacuoles laden with bacteria (Fig. 1). Gram staining of the bacteria revealed gram-positive cocci in pairs as well as two types of gram-negative bacilli (data not shown). One form of bacillus was long, approximately 10–15 µm, present singly, while the other was shorter, about 3–5 µm, oblong and present in chains of two or three organisms. Blebbing of hemocytes was frequently observed, which was not so with GK2016-infected insects where the hemocytes appeared unaffected. The hemocytic defense response toward the HS mutants was determined by injecting insects with FITClabeled blatospores followed by incubation at the NPT. The percentage of blastospores germinating after 24 hr and the number of blastospores phagocytized by the hemocytes were determined. With insects treated with strain GK2016 or the mutants HS2, HS6, or HS9 the majority of the injected, labeled blastospores germinated (Table 1). Total germination with these strains
ranged from 74.3% for mutant HS6 to 94% for mutant HS2. The levels of germination and phagocytosis were not significantly different with mutants HS2 and HS9; however, the wild-type strain GK2016 and mutant HS6 exhibited lower levels of germinated, phagocytized blastospores. This difference resulted from a higher
TABLE 1 Phagocytosis and Germination of Beauveria bassiana Heat-Sensitive Mutant FITC-Labeled Blastospores Germinated a,b (% 6 SE)
Ungerminated (% 6 SE)
Strain
Phagocytosed
Free
Phagocytosed
Free
GK2016 HS2 HS6 HS9 HS11
55.5 (6.4)a,AC 80.8 (4.8)a,B 65.9 (5.0)a,AB 82.3 (5.6)a,B 24.5 (6.9)a,C
31.0 (5.9)b,A 13.2 (3.7)b,B 8.4 (4.7)b,B 7.4 (4.8)b,B 1.9 (2.3)b,B
10.7 (3.4)c,A 4.3 (1.6)bc,A 24.7 (4.1)c,B 10.1 (3.6)b,A 69.3 (7.6)c,C
2.7 (1.8)c,A 1.7 (1.0)bc,A 0.4 (0.4)b,A 0.0 (0.0)b,A 4.0 (3.4)b,A
a Seven insects per treatment. Samples were removed after 24 h and duplicate aliquots scored each containing 50–200 fungal cells. b Lower case letters indicate significant differences within rows and capital letters within columns. Total 100% within rows.
168
NOTE
percentage of unphagocytized, germinated blastospores for strain GK2016, while the overall germination of mutant HS6 was less than that of the other strains. Mutant HS11 exhibited significantly lower overall germination levels than any of the other strains. Only in insects treated with strain GK2016 were large numbers of free, germinated blastospores and free, newly formed hyphal bodies present. Interestingly, all of the strains were able to germinate within the hemocyte after phagocytosis to produce mycelia that protruded from the hemocytes. None of the mutant strains were able to proceed beyond this point to form secondary hyphal bodies. However, these newly formed mycelia, and the secondary hyphal bodies produced by strain GK2016, were only rarely found to be interacting with the circulating hemocytes. Defense responses other than phagocytosis, such as nodule formation and encapsulation, were also frequently observed but only with the original inoculum (Fig. 2). An extensive body of information concerned with various aspects of entomopathogenic fungal pathogenesis in insects has been generated. However, the chronology of events and the contribution of fungal physiology, hydrolytic enzymes, and toxins in bringing about insect mortality still remains ill-defined. In general, once inside the hemocoel the fungus proliferates rapidly and spreads throughout the body by means of the insect’s open circulatory system. Hyphal bodies fill the hemocoel at the time of insect death with tissues being invaded and degraded to varying degrees. Hyphae
eventually emerge from the cadaver producing aerial spores capable of initiating the cycle once again (Zacharuk, 1981). Entomopathogenic fungi are capable of producing various antibiotic-like substances concomitantly with invasion of the body cavity that may allow the fungus to suppress bacterial competition for host nutrients (Gillespie and Claydon, 1989). Bacteria were not observed in the hemolymph of insects infected with the wild-type strain, GK2016, but were present in the mutant-infected samples in which the growth of the fungus was inhibited by the NPT. Even though the mutants germinated and protruded from the phagocytic cell, suppression of further growth of the fungus by the restrictive temperature allowed opportunistic bacteria to monopolize the environment. It has also been postulated that fungal surface carbohydrates play a major role in phagocyte recognition of fungal elements (Pendland and Boucias, 1986). In vivo-produced hyphal bodies of B. bassiana, which are not recognized by phagocytic cells, were osmotically sensitive and lacked a properly formed cell wall. The cell wall was reformed only 48–60 hr postinfection which also corresponded with suppression of hemocyte phagocytic activity, possibly by fungal metabolites (Pendland and Boucias, 1993). These same phenomenon may be occurring when B. bassiana infects M. sanguinipes since hemolymph extracted from strain GK2016-infected grasshoppers possessed hemocytes that appeared unaltered but did not phagocytize the newly formed fungal elements. The hemocytes themselves were competent in dealing
FIG. 2. Phagocytosis and germination of FITC-labeled blastospores from Beauveria bassiana GK2016 or heat-sensitive mutants after injection into grasshoppers and incubation at 32°C for 24 hr. Composite I is GK2016: A, 1 hr; B, C, and D, 24 hr. Composite II is temperature-sensitive mutants: A, B (HS2), C, D (HS6), E (HS9), F, G (HS11). Arrows indicate labeled blastospores (lb), hemocyte (hc), newly formed blastospore (b), mycelia (m), nodule (n), spreading hemocyte (shc), and capsule formation (cp). 1 mm 5 5 µm.
169
NOTE
with foreign agents since hemolymph extracted from mutant-infected insects possessed many engorged hemocytes containing large numbers of vacuoles laden with bacteria. However, the reduced efficiency with which hemocytes phagocytized in vitro produced GK2016 FITC-labeled blastospores versus the HS mutants at the NPT may indicate that suppression of phagocytic activity is also occurring during the active infection. The approach of using HS mutants of mycopathogens to examine pathogenesis in poikilothermic organisms may prove useful in determining the role of specific components or responses in the infection process. Traditionally, loss-of-function mutants were useful in identifying factors required for infection. Design of temperature-sensitive mutants affecting these same functions may make it possible to determine the role of these factors at specific stages in the infection and thus decipher the intricate nature of specific host–pathogen interactions. KEY WORDS: Beauveria bassiana; heat-sensitive mutants; infection, Melanoplus sanguinipes; defense response. 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 Gillespie, A. T., and Claydon, N. 1989. The use of entomogenous fungi for pest control and the role of toxins in pathogenesis. Pestic. Sci. 27, 203–215. Hajek, A. E., and St. Leger, R. J. 1994. Interactions between fungal pathogens and insect hosts. Annu. Rev. Entomol. 39, 293–322. 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. The effects of temperature on the pathogenicity of heat-sensitive mutants of the entomopathogenic fungus Beauveria bassiana: Toward the migratory grasshopper, Melanoplus sanguinipes. J. Invertebr. Pathol. 68, 160–165. Hung, S., and Boucias, D. G. 1992. Influence of Beauveria bassiana on the cellular defense response of the beet armyworm, Spodoptera exigua. J. Invertebr. Pathol. 60, 152–158. Khachatourians, G. G. 1996. The relationship between biochemistry and molecular biology of entomopathogenic fungi and insect diseases. In ‘‘The Mycota,’’ Vol. VI, ‘‘Animal and Human 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, 901–907. Pendland, J. C., and Boucias, D. G. 1986. Lectin binding characteristics of several entomogenous hyphomycetes: Possible relationship to insect hemagglutinins. Mycologia 78, 818–824. 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. Zacharuk, R. Y. 1981. Fungal disease of terrestrial insects. In ‘‘Pathogenesis of Invertebrate Microbial Diseases’’ (E. W. Davidson, Ed.), pp. 367–402. Allanheld, Osmun, Totowa, NJ.
DWAYNE D. HEGEDUS1 GEORGE G. KHACHATOURIANS2 Bioinsecticide Research Laboratory Department of Applied Microbiology and Food Science University of Saskatchewan Saskatchewan, Canada, S7N 0W0 Received March 10, 1995; accepted March 15, 1996 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.
68, 166–169 (1996)
0075
NOTE Analysis of Cellular Defense Reactions of the Migratory Grasshopper, Melanoplus sanguinipes, Infected with Heat-Sensitive Mutants of Beauveria bassiana Beauveria bassiana and other entomopathogenic fungi are being examined as potential biological insect control agents. Our present knowledge of fungal pathogenesis in insects indicates that it occurs via a series of integrated, systematic events progressing from spore attachment to germination, penetration, growth, and proliferation within the body of the host, interaction with insect defense mechanisms, and finally reemergence on the cadaver. The interactions that occur between the fungus and the insect are exceedingly complex and are dependent upon specific host–pathogen interactions (Hajek and St. Leger, 1994; Hegedus and Khachatourians, 1995; Khachatourians, 1996). Beauveria bassiana heat-sensitive (HS) mutants have been developed (Hegedus and Khachatourians, 1994) that do not infect Melanoplus sanguinipes at the nonpermissive temperature (NPT) of 32°C. Bioassays in which the infection was terminated by shifting the ambient air temperature indicated that events occurring within the first 4–5 days postinoculation were critical to the outcome of the infection (Hegedus and Khachatourians, 1996). Here we report on the interaction between several HS mutants and the insect’s cellular defense responses at the permissive temperature (PT) of 20°C and the NPT to further define the role of fungal morphology in the infection process. Beauveria bassiana strain GK2016 and the HS mutants HS2, HS6, HS9, and HS11 derived from this strain were grown in YPG broth (0.2% yeast extract, 1% peptone, 2% glucose) for 4–5 days at 20°C with shaking (150 rpm) and blastospores harvested as described previously (Hegedus et al., 1990). Adult migratory grasshoppers of a nondiapause strain of M. sanguinipes were used for the bioassays. Insects were anesthetized with CO2 gas and 3 µl of a suspension of 1 3 108 blastospores/ml of sterile dH2O was injected into the right hind coxa using a Hamilton syringe modified to prevent penetration beyond a depth of 2 mm. Insects were individually housed in plastic vials 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). Samples of hemolymph were obtained 1 or 14 days after treatment, as indicated, by cutting off the left rear appendage of live 0022-2011/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
insects and gently squeezing the insect to promote bleeding. The samples were mixed with an equal volume of anticoagulant buffer (0.098 M NaOH, 0.146 M NaCl, 0.17 M EDTA, and 0.041 M trisodium citrate, pH 4.5) and stored at 4°C. To assist the visualization of blastospores, and to determine the percentage which had germinated, blastospores were labeled with the fluorescent compound fluorescein isothiocyante (FITC; Sigma Chemical Co., St Louis, MO) according to Hung and Boucias (1992). There was no effect on the viability of the blastospores treated in this manner as 99% germinated in YPG broth (data not shown). Each treatment was replicated with seven insects from which duplicate 5-µl samples of the diluted hemolymph were placed on glass slides and covered with a coverslip and the entire sample scored. This method of inoculation and sampling results in approximately 50–200 blastospores per slide. All statistical analyses were done using the Statview Student version 1.0 program (Abacus Concepts Inc., Berkeley, CA). Mean percentage values were arc sin Œ p transformed and then subjected to the Student t test, analysis of variance, and multiple comparison using Fisher’s F test. Previously, examination of the cuticular surfaces of insects that had been topically treated with the HS mutants, followed by incubation at the NPT, revealed that mutant morphology closely resembled that observed in vitro under the same conditions (Hegedus and Khachatourians, 1994; Hegedus and Khachatourians, 1996). To determine if this was also occurring within the body cavity of the insect, as well as to examine the role of fungal morphology in the infection, hemolymph was examined from insects 14 days after injection and incubation at either the PT or NPT. At the PT abundant amounts of short hyphal bodies, blastospores, and budding mycelia were observed (Fig. 1). There were no visible differences between mutants HS2, HS6, and the wild-type strain, GK2016. Mutant HS9 produced long mycelia with an abundance of budding blastospores. At 32°C, GK2016 produced structures similar to that at 20°C. Conversely, mutant morphology was dramatically altered and resembled structures observed in vitro when the mutants were incubated at the NPT. Mutant HS6 produced highly elongated mycelia
166
167
NOTE
FIG. 1. Examination of hemolymph of grasshoppers injected with blastospores of Beauveria bassiana GK2016 or heat-sensitive mutants and incubated at either 20 or 32°C for 14 days. For GK2016 and HS9: A, B (20°C), C, D (32°C). HS2: A, B, C (20°C), D, E (32°C). HS6: A (20°C), B (32°C). Arrows indicate mycelim (m), budding mycelium (bm), blastospore (b), hemocyte (hc), spreading hemocyte (shc), germinated blastospore (gb), bacteria (ba), and blebbing of hemocyte (bl). 1 mm 5 1 µm.
whereas mutant HS9 germinated to produce short mycelia with some minor branching. No secondary blastospores or other hyphal bodies were observed. In addition, the hemolymph extracted from insects infected with the mutant strains at the NPT possessed many engorged hemocytes containing large numbers of vacuoles laden with bacteria (Fig. 1). Gram staining of the bacteria revealed gram-positive cocci in pairs as well as two types of gram-negative bacilli (data not shown). One form of bacillus was long, approximately 10–15 µm, present singly, while the other was shorter, about 3–5 µm, oblong and present in chains of two or three organisms. Blebbing of hemocytes was frequently observed, which was not so with GK2016-infected insects where the hemocytes appeared unaffected. The hemocytic defense response toward the HS mutants was determined by injecting insects with FITClabeled blatospores followed by incubation at the NPT. The percentage of blastospores germinating after 24 hr and the number of blastospores phagocytized by the hemocytes were determined. With insects treated with strain GK2016 or the mutants HS2, HS6, or HS9 the majority of the injected, labeled blastospores germinated (Table 1). Total germination with these strains
ranged from 74.3% for mutant HS6 to 94% for mutant HS2. The levels of germination and phagocytosis were not significantly different with mutants HS2 and HS9; however, the wild-type strain GK2016 and mutant HS6 exhibited lower levels of germinated, phagocytized blastospores. This difference resulted from a higher
TABLE 1 Phagocytosis and Germination of Beauveria bassiana Heat-Sensitive Mutant FITC-Labeled Blastospores Germinated a,b (% 6 SE)
Ungerminated (% 6 SE)
Strain
Phagocytosed
Free
Phagocytosed
Free
GK2016 HS2 HS6 HS9 HS11
55.5 (6.4)a,AC 80.8 (4.8)a,B 65.9 (5.0)a,AB 82.3 (5.6)a,B 24.5 (6.9)a,C
31.0 (5.9)b,A 13.2 (3.7)b,B 8.4 (4.7)b,B 7.4 (4.8)b,B 1.9 (2.3)b,B
10.7 (3.4)c,A 4.3 (1.6)bc,A 24.7 (4.1)c,B 10.1 (3.6)b,A 69.3 (7.6)c,C
2.7 (1.8)c,A 1.7 (1.0)bc,A 0.4 (0.4)b,A 0.0 (0.0)b,A 4.0 (3.4)b,A
a Seven insects per treatment. Samples were removed after 24 h and duplicate aliquots scored each containing 50–200 fungal cells. b Lower case letters indicate significant differences within rows and capital letters within columns. Total 100% within rows.
168
NOTE
percentage of unphagocytized, germinated blastospores for strain GK2016, while the overall germination of mutant HS6 was less than that of the other strains. Mutant HS11 exhibited significantly lower overall germination levels than any of the other strains. Only in insects treated with strain GK2016 were large numbers of free, germinated blastospores and free, newly formed hyphal bodies present. Interestingly, all of the strains were able to germinate within the hemocyte after phagocytosis to produce mycelia that protruded from the hemocytes. None of the mutant strains were able to proceed beyond this point to form secondary hyphal bodies. However, these newly formed mycelia, and the secondary hyphal bodies produced by strain GK2016, were only rarely found to be interacting with the circulating hemocytes. Defense responses other than phagocytosis, such as nodule formation and encapsulation, were also frequently observed but only with the original inoculum (Fig. 2). An extensive body of information concerned with various aspects of entomopathogenic fungal pathogenesis in insects has been generated. However, the chronology of events and the contribution of fungal physiology, hydrolytic enzymes, and toxins in bringing about insect mortality still remains ill-defined. In general, once inside the hemocoel the fungus proliferates rapidly and spreads throughout the body by means of the insect’s open circulatory system. Hyphal bodies fill the hemocoel at the time of insect death with tissues being invaded and degraded to varying degrees. Hyphae
eventually emerge from the cadaver producing aerial spores capable of initiating the cycle once again (Zacharuk, 1981). Entomopathogenic fungi are capable of producing various antibiotic-like substances concomitantly with invasion of the body cavity that may allow the fungus to suppress bacterial competition for host nutrients (Gillespie and Claydon, 1989). Bacteria were not observed in the hemolymph of insects infected with the wild-type strain, GK2016, but were present in the mutant-infected samples in which the growth of the fungus was inhibited by the NPT. Even though the mutants germinated and protruded from the phagocytic cell, suppression of further growth of the fungus by the restrictive temperature allowed opportunistic bacteria to monopolize the environment. It has also been postulated that fungal surface carbohydrates play a major role in phagocyte recognition of fungal elements (Pendland and Boucias, 1986). In vivo-produced hyphal bodies of B. bassiana, which are not recognized by phagocytic cells, were osmotically sensitive and lacked a properly formed cell wall. The cell wall was reformed only 48–60 hr postinfection which also corresponded with suppression of hemocyte phagocytic activity, possibly by fungal metabolites (Pendland and Boucias, 1993). These same phenomenon may be occurring when B. bassiana infects M. sanguinipes since hemolymph extracted from strain GK2016-infected grasshoppers possessed hemocytes that appeared unaltered but did not phagocytize the newly formed fungal elements. The hemocytes themselves were competent in dealing
FIG. 2. Phagocytosis and germination of FITC-labeled blastospores from Beauveria bassiana GK2016 or heat-sensitive mutants after injection into grasshoppers and incubation at 32°C for 24 hr. Composite I is GK2016: A, 1 hr; B, C, and D, 24 hr. Composite II is temperature-sensitive mutants: A, B (HS2), C, D (HS6), E (HS9), F, G (HS11). Arrows indicate labeled blastospores (lb), hemocyte (hc), newly formed blastospore (b), mycelia (m), nodule (n), spreading hemocyte (shc), and capsule formation (cp). 1 mm 5 5 µm.
169
NOTE
with foreign agents since hemolymph extracted from mutant-infected insects possessed many engorged hemocytes containing large numbers of vacuoles laden with bacteria. However, the reduced efficiency with which hemocytes phagocytized in vitro produced GK2016 FITC-labeled blastospores versus the HS mutants at the NPT may indicate that suppression of phagocytic activity is also occurring during the active infection. The approach of using HS mutants of mycopathogens to examine pathogenesis in poikilothermic organisms may prove useful in determining the role of specific components or responses in the infection process. Traditionally, loss-of-function mutants were useful in identifying factors required for infection. Design of temperature-sensitive mutants affecting these same functions may make it possible to determine the role of these factors at specific stages in the infection and thus decipher the intricate nature of specific host–pathogen interactions. KEY WORDS: Beauveria bassiana; heat-sensitive mutants; infection, Melanoplus sanguinipes; defense response. 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 Gillespie, A. T., and Claydon, N. 1989. The use of entomogenous fungi for pest control and the role of toxins in pathogenesis. Pestic. Sci. 27, 203–215. Hajek, A. E., and St. Leger, R. J. 1994. Interactions between fungal pathogens and insect hosts. Annu. Rev. Entomol. 39, 293–322. 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. The effects of temperature on the pathogenicity of heat-sensitive mutants of the entomopathogenic fungus Beauveria bassiana: Toward the migratory grasshopper, Melanoplus sanguinipes. J. Invertebr. Pathol. 68, 160–165. Hung, S., and Boucias, D. G. 1992. Influence of Beauveria bassiana on the cellular defense response of the beet armyworm, Spodoptera exigua. J. Invertebr. Pathol. 60, 152–158. Khachatourians, G. G. 1996. The relationship between biochemistry and molecular biology of entomopathogenic fungi and insect diseases. In ‘‘The Mycota,’’ Vol. VI, ‘‘Animal and Human 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, 901–907. Pendland, J. C., and Boucias, D. G. 1986. Lectin binding characteristics of several entomogenous hyphomycetes: Possible relationship to insect hemagglutinins. Mycologia 78, 818–824. 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. Zacharuk, R. Y. 1981. Fungal disease of terrestrial insects. In ‘‘Pathogenesis of Invertebrate Microbial Diseases’’ (E. W. Davidson, Ed.), pp. 367–402. Allanheld, Osmun, Totowa, NJ.
DWAYNE D. HEGEDUS1 GEORGE G. KHACHATOURIANS2 Bioinsecticide Research Laboratory Department of Applied Microbiology and Food Science University of Saskatchewan Saskatchewan, Canada, S7N 0W0 Received March 10, 1995; accepted March 15, 1996 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.