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Comparative tests of various species of Bacillus of the “cereus Group” on larvae of Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae)
266
NOTES
examination of diseased fat-body tissues has revealed the presence of hexagonal particles, possibly icosahedra, approximately 60 rnp in diameter in the cytoplasm of the cells. The particles may be a noninclusion body type of insect virus and appear to be similar to those reported by Krieg and Huger (J. Znsect Pathol., 2, 274-288, 1960), by S’teinhausand Leutenegger (J. Insect Pathol., 5, 266-270, 1963), and by Leutenegger (Virology, 24, 200-204, 1964). No such particles were found in the healthy tissues. Huger (personal communication) has suggested that these particles may be parallels to the virus Moratorvirus lamellicornium Krieg and Huger in the fat cells of Melolontha in which there appears to be a breakdown of
Comparative of
Tests
the
JEAN
ADAMS
A.
WILCOX
Entomology Research Division Agricultural Research Service U. S. Department of Agriculture Beltsville, Maryland Accepted
Group” fumiferana
(Lepidoptera
R.
THEODORE
of Various
“cereus
Choristoneura
albuminoid sphereswith a transformation into virogenic stroma which disappear during the course of the disease (Figs. 2 and 3). The hexagonal shape of the pathogen is shown in Fig. 4. As diseased specimens become available, transmission and isolation and purification studies will be conducted.
April
12, 1965
Species
of
on Larvae
Bacillus of
(Clemens)
: Tortricidae)
’
Among the six species of entomopathogenous Bacillus of the so-called “cereus Group” (Heimpel and Angus, Bacterial. Rev., 24, 266-288, 1960), only two have been tested on the spruce budworm, Choristoneura jumijerana (Clemens): Bacillus thuringiensis var. thuringiensis Berliner (Angus et al., Can.
spraying of fir twigs with bacterial suspensions; (b) buccal administration of droplets of the Bacillus suspensionsto larvae; (c) immersion of larvae for 10 seconds in bacterial suspensions;(d) oral administration of Bacillus suspensionsto larvae reared on artificial diet (Berger, U. S. Dept. Agr. Agr. Res. Dept. Forestry, Forest Entomol. Pathol. Serv., Puhl. X3-84, 4 pp. (October 1963). The following speciesof spore- and crystalBranch, Bimonthly Progr. Rept., 17 (3) forming Bacillus were tested: B. thuringiensis 1961; Smirnoff, Can. Entomologist, 95, 127Berliner; B. thuringiensis 133, 1963) and Bacillus ceyeus Franklin and var. thuringiensis Franklin (Smirnoff, Can. Entomologist, 95, var. sotto Aoki and Chigasaki: B. cereus var. 127-133, 1963). Biological control studies of dendrolimus Talalaev; B. t huringiensis var. the spruce budworm warranted further tests alesti Toumanoff and Vago; B. entomocidus with species of Bacillus to establish their var. entomoridus Heimp and Angus; while comparative efficacy. This note reports on the B. cereus Franklin and Franklin (strain 44)” results obtained. 2 B. WY~US No. 44 was isolated by the author Four methods of infection were used: (a) from larvae of Trichiocamp~ts viminalis (Fallen) 1 Contribution No. 1204, Forest Entomology and Pathology Branch, Department of Forestry, Ottawa, Canada.
(Hymenoptera: Tenthredinidae), 1959. All varieties of Racillus were supplied by Dr. Angus, Insect Pathology Research Institute, Ste. Marie, Ontario, Canada.
other T. A. Sault
267
NOTES
PERCENTAGE
OF LARVAL
Varieties and
of Bacillus tested treatment used
B. thuringiensis Sprayed
MORTALITY
var.
foliage
Sprayed
var.
Buccal administration on foliage
Sprayed
var.
foliage
B. thuringiensis
var.
var.
50
96
4
SO
70
100
5
20
50
100
30.7
53.5
96.0
4 6.5
5
15
85
11
30
50
80
8
20
30
40
9
20
50
100
5
18.8
36.2
14
0
8;
10 5
76.2
9
50
100
70
80 100
5 -
20
SO
28.5
42.5
91.3
20
30
70
12
30
60
100
3
50
0
80
6
30
90
100
3
32.5
45.0
87.5
35
45
75
1.1
30
60
80
8
SO
80
90
6
20
70
33.7
63.7
100 -_ 86.2
5 8
6.7
6
to larvae
larvae
Oral administration on artificial diet
20 60
entomocidus:
foliage
Buccal administration on foliage
Average
20
to larvae
.i\verage
Immersed
13
44
to larvae
larvae
Oral administration on artificial diet
Sprayed
88
33
alesti:
foliage
Buccal administration on foliage
B. entomocidus
120
to larvae
Average
Immersed
48
Sumber of days for total mortality
to larvae
larvae
Oral administration on artificial diet
Sprayed
mortality hours
.-~DMINISTERED
dendrolimzrs:
Buccal administration on foliage Immersed
24
VARIOUSLY
to larvae
Average B. ceretu
Percent after
Bacillus
to larvae
larvae
Oral administration on artificial diet
OF
sotto:
foliage
Immersed
SPECIES
to larvae
Average B. thuriagiensis
1
to larvae
larvae
Oral administration on artificial diet
TABLE BY DIFFERENT
thuringiensis:
Buccal administration on foliage Immersed
CAUSED
to larvae
268
NOTES TABLE
Varieties of Bacillus tested and treatment used
1 (Continued) Percent after 24
mortality hours
Number of days for total mortality
48
120
26
0
53
14
60
70 70
70
7
30
80
9
60
70
80
7 -
44.0
52.5
70.7
9.2
33
49
82
9
30
60
80
5
50
70
100
5
30
80
100
35.7
64.7
B. cereus: Sprayed
foliage
Buccal administration on foliage Immersed
to larvae
larvae
Oral administration on artificial diet
to larvae
Average Mechanical Sprayed
mixture: foliage
Buccal administration on foliage Immersed
larvae
Oral administration on artificial diet Average
to larvae
to larvae
was the only acrystalliferous sporeforming BacilZus tested. All cultures were grown on Difco nutrient agar and incubated at 28’C for 10 days, i.e. until lysis was completed. The bacilli were collected and kept in peptonized water (0.25 g peptone/lOO ml of distilled water) at a concentration of one part of Bacillus to 10 parts of peptonized water. The concentrations of bacterial suspensions were standardized by transparency and the number of spores was estimated (2 X log) by counting with the aid of a microscope. In addition to the six pure cultures, two mixtures were also tested: (a) a mechanical mixture of suspensions from cultures of the six species grown separately; (b) a mixture of the six species grown together. In the feeding tests, 1700 fourth-instar larvae of C. jumijerana were used. These larvae were obtained from the Insect Pathology Research Institute at Sault Ste. Marie, Ontario, and were free of the native microsporidia Per&a fumiferanae Thomson. The larvae were reared individually in wellaerated glass vials, at 21-22’C and 7070 relative humidity. Dead larvae were collected
90.5
7 .s
daily and microscopically analyzed; only those containing bacterial cells in the hemolymph were considered as having died from Bacillus infection. The results of the tests with the six pure cultures and the mechanical mixture are presented in Table 1. The data obtained with the six cultures grown together were not significant and have been deleted from the table. From the tests reported in this paper I have drawn the following conclusions: 1. The larvae of C. jumijerana are susceptible to the five spore- and crystal-forming Bacillus and also to the sporeforming B. cereus. The microorganisms were pathogenic to C. jumijerana and caused mortality only when bacterial cells were present in the hemolymph. 2. The period of mortality due to B. thuvingiensis and B. cereus was twice as long (13-14 days) in this case as in previous tests with larvae infected by the native microsporidia (Smirnoff, Can. Entomologist, 95, 127-133, 1963). This suggests that the microsporidia act as a stressor for the Bacillus. 3. The following order of efficacy of the
269
NOTES
TABLE AVERAGE
PERCENT
MORTALITY
CAUSED
BY EACH
2 OF THE
FOUR
Percent after Methods
of
treatment
Sprayed
foliage
Buccal
administration
on
on
to
larvae
administration artificial
to diet
hours
of total
davs for mortality
120
20.7
22.8
70.3
13.6
27.5
53.7
80.7
6.5
40.0
52.5
77.5
7.5
26.2
62.5
91.2
5.1
of
of a grant-in-aid Council
to of
A. J. Musgrave Canada
method, for each of the six pure cultures, and the two mixtures of cultures depended not only on the species tested, but also on the method of treatment (Table 2). The difference in percentage and in rate of mortality between buccal administration and spraying of foliage is as yet unknown but could possibly be due to bactericidal and bacteriostatic effect of the fir foliage on the Bacillus.
W. A.
SMIRNOFF
Forest Research Laboratory, Sillery, Quebec, Canada. Accepted
Nuclear
March
29, 1965
Equivalents
in
Microorganisms in Sitophilus granarius (Linnaeus) ’
The mycetomal microorganisms of the granary weevil, Sitophilus granarius (Linnaeus), supposedly play a useful but not always essentialpart in that insect’s nutrition (Musgrave, Can. Entomologist, 96, 377-389, 1964; Schneider, Z. Morphol. Oekol. Tiere, 44, 555-625, 1956). Kolb (2. Morphol. Oekol. Tiere, 48, 1-71, 1959) using Giemsaand other stains was able
acknowledged.
.
48
Evidence
Mycetomal
Research
.4verage number
mortality
larvae
Histochemical
1 Receipt
USED
24
Bacillus was determined. The percentage given is the average mortality obtained after 120 hours for the four methods of treatment described: (a) B. thuringiensis, 96.0%; (b) B. dendrolimus, 91.3% ; (c) B. alesti, 87.5%; (d) B. entomocidus, 86.2%; (e) B. sotto, 76.2r0; (f) B. cereus, 70.770. The mechanical mixture of the six species gave 90.5% mortality, while the six speciesgrown together gave 41.270 mortality. In the control rearings the larval mortality never passed 5-77oafter 120 hours. The reasons for the low percentage obtained with the mixed insemination are the object of a separate study. 4. The average percent mortality for each
National
OF TREATMENT
larvae
foliage
Immersed Oral
used
METHODS
is
gratefully
by
to identify DNA in the symbiotes of the ant, Camponotus, and the fruit fly, Ceratitis, but was unable to identify it in those of Sitophi1US.
Recently, after a number of preliminary trials, successin detecting DNA in the microorganisms of the mycetome in Sitophilus granarius has been achieved in this laboratory by the following technique. Mycetomes of larvae and mesentera of adults were prepared as smears on glass slides. Following fixation for 3 minutes in the
NOTES
examination of diseased fat-body tissues has revealed the presence of hexagonal particles, possibly icosahedra, approximately 60 rnp in diameter in the cytoplasm of the cells. The particles may be a noninclusion body type of insect virus and appear to be similar to those reported by Krieg and Huger (J. Znsect Pathol., 2, 274-288, 1960), by S’teinhausand Leutenegger (J. Insect Pathol., 5, 266-270, 1963), and by Leutenegger (Virology, 24, 200-204, 1964). No such particles were found in the healthy tissues. Huger (personal communication) has suggested that these particles may be parallels to the virus Moratorvirus lamellicornium Krieg and Huger in the fat cells of Melolontha in which there appears to be a breakdown of
Comparative of
Tests
the
JEAN
ADAMS
A.
WILCOX
Entomology Research Division Agricultural Research Service U. S. Department of Agriculture Beltsville, Maryland Accepted
Group” fumiferana
(Lepidoptera
R.
THEODORE
of Various
“cereus
Choristoneura
albuminoid sphereswith a transformation into virogenic stroma which disappear during the course of the disease (Figs. 2 and 3). The hexagonal shape of the pathogen is shown in Fig. 4. As diseased specimens become available, transmission and isolation and purification studies will be conducted.
April
12, 1965
Species
of
on Larvae
Bacillus of
(Clemens)
: Tortricidae)
’
Among the six species of entomopathogenous Bacillus of the so-called “cereus Group” (Heimpel and Angus, Bacterial. Rev., 24, 266-288, 1960), only two have been tested on the spruce budworm, Choristoneura jumijerana (Clemens): Bacillus thuringiensis var. thuringiensis Berliner (Angus et al., Can.
spraying of fir twigs with bacterial suspensions; (b) buccal administration of droplets of the Bacillus suspensionsto larvae; (c) immersion of larvae for 10 seconds in bacterial suspensions;(d) oral administration of Bacillus suspensionsto larvae reared on artificial diet (Berger, U. S. Dept. Agr. Agr. Res. Dept. Forestry, Forest Entomol. Pathol. Serv., Puhl. X3-84, 4 pp. (October 1963). The following speciesof spore- and crystalBranch, Bimonthly Progr. Rept., 17 (3) forming Bacillus were tested: B. thuringiensis 1961; Smirnoff, Can. Entomologist, 95, 127Berliner; B. thuringiensis 133, 1963) and Bacillus ceyeus Franklin and var. thuringiensis Franklin (Smirnoff, Can. Entomologist, 95, var. sotto Aoki and Chigasaki: B. cereus var. 127-133, 1963). Biological control studies of dendrolimus Talalaev; B. t huringiensis var. the spruce budworm warranted further tests alesti Toumanoff and Vago; B. entomocidus with species of Bacillus to establish their var. entomoridus Heimp and Angus; while comparative efficacy. This note reports on the B. cereus Franklin and Franklin (strain 44)” results obtained. 2 B. WY~US No. 44 was isolated by the author Four methods of infection were used: (a) from larvae of Trichiocamp~ts viminalis (Fallen) 1 Contribution No. 1204, Forest Entomology and Pathology Branch, Department of Forestry, Ottawa, Canada.
(Hymenoptera: Tenthredinidae), 1959. All varieties of Racillus were supplied by Dr. Angus, Insect Pathology Research Institute, Ste. Marie, Ontario, Canada.
other T. A. Sault
267
NOTES
PERCENTAGE
OF LARVAL
Varieties and
of Bacillus tested treatment used
B. thuringiensis Sprayed
MORTALITY
var.
foliage
Sprayed
var.
Buccal administration on foliage
Sprayed
var.
foliage
B. thuringiensis
var.
var.
50
96
4
SO
70
100
5
20
50
100
30.7
53.5
96.0
4 6.5
5
15
85
11
30
50
80
8
20
30
40
9
20
50
100
5
18.8
36.2
14
0
8;
10 5
76.2
9
50
100
70
80 100
5 -
20
SO
28.5
42.5
91.3
20
30
70
12
30
60
100
3
50
0
80
6
30
90
100
3
32.5
45.0
87.5
35
45
75
1.1
30
60
80
8
SO
80
90
6
20
70
33.7
63.7
100 -_ 86.2
5 8
6.7
6
to larvae
larvae
Oral administration on artificial diet
20 60
entomocidus:
foliage
Buccal administration on foliage
Average
20
to larvae
.i\verage
Immersed
13
44
to larvae
larvae
Oral administration on artificial diet
Sprayed
88
33
alesti:
foliage
Buccal administration on foliage
B. entomocidus
120
to larvae
Average
Immersed
48
Sumber of days for total mortality
to larvae
larvae
Oral administration on artificial diet
Sprayed
mortality hours
.-~DMINISTERED
dendrolimzrs:
Buccal administration on foliage Immersed
24
VARIOUSLY
to larvae
Average B. ceretu
Percent after
Bacillus
to larvae
larvae
Oral administration on artificial diet
OF
sotto:
foliage
Immersed
SPECIES
to larvae
Average B. thuriagiensis
1
to larvae
larvae
Oral administration on artificial diet
TABLE BY DIFFERENT
thuringiensis:
Buccal administration on foliage Immersed
CAUSED
to larvae
268
NOTES TABLE
Varieties of Bacillus tested and treatment used
1 (Continued) Percent after 24
mortality hours
Number of days for total mortality
48
120
26
0
53
14
60
70 70
70
7
30
80
9
60
70
80
7 -
44.0
52.5
70.7
9.2
33
49
82
9
30
60
80
5
50
70
100
5
30
80
100
35.7
64.7
B. cereus: Sprayed
foliage
Buccal administration on foliage Immersed
to larvae
larvae
Oral administration on artificial diet
to larvae
Average Mechanical Sprayed
mixture: foliage
Buccal administration on foliage Immersed
larvae
Oral administration on artificial diet Average
to larvae
to larvae
was the only acrystalliferous sporeforming BacilZus tested. All cultures were grown on Difco nutrient agar and incubated at 28’C for 10 days, i.e. until lysis was completed. The bacilli were collected and kept in peptonized water (0.25 g peptone/lOO ml of distilled water) at a concentration of one part of Bacillus to 10 parts of peptonized water. The concentrations of bacterial suspensions were standardized by transparency and the number of spores was estimated (2 X log) by counting with the aid of a microscope. In addition to the six pure cultures, two mixtures were also tested: (a) a mechanical mixture of suspensions from cultures of the six species grown separately; (b) a mixture of the six species grown together. In the feeding tests, 1700 fourth-instar larvae of C. jumijerana were used. These larvae were obtained from the Insect Pathology Research Institute at Sault Ste. Marie, Ontario, and were free of the native microsporidia Per&a fumiferanae Thomson. The larvae were reared individually in wellaerated glass vials, at 21-22’C and 7070 relative humidity. Dead larvae were collected
90.5
7 .s
daily and microscopically analyzed; only those containing bacterial cells in the hemolymph were considered as having died from Bacillus infection. The results of the tests with the six pure cultures and the mechanical mixture are presented in Table 1. The data obtained with the six cultures grown together were not significant and have been deleted from the table. From the tests reported in this paper I have drawn the following conclusions: 1. The larvae of C. jumijerana are susceptible to the five spore- and crystal-forming Bacillus and also to the sporeforming B. cereus. The microorganisms were pathogenic to C. jumijerana and caused mortality only when bacterial cells were present in the hemolymph. 2. The period of mortality due to B. thuvingiensis and B. cereus was twice as long (13-14 days) in this case as in previous tests with larvae infected by the native microsporidia (Smirnoff, Can. Entomologist, 95, 127-133, 1963). This suggests that the microsporidia act as a stressor for the Bacillus. 3. The following order of efficacy of the
269
NOTES
TABLE AVERAGE
PERCENT
MORTALITY
CAUSED
BY EACH
2 OF THE
FOUR
Percent after Methods
of
treatment
Sprayed
foliage
Buccal
administration
on
on
to
larvae
administration artificial
to diet
hours
of total
davs for mortality
120
20.7
22.8
70.3
13.6
27.5
53.7
80.7
6.5
40.0
52.5
77.5
7.5
26.2
62.5
91.2
5.1
of
of a grant-in-aid Council
to of
A. J. Musgrave Canada
method, for each of the six pure cultures, and the two mixtures of cultures depended not only on the species tested, but also on the method of treatment (Table 2). The difference in percentage and in rate of mortality between buccal administration and spraying of foliage is as yet unknown but could possibly be due to bactericidal and bacteriostatic effect of the fir foliage on the Bacillus.
W. A.
SMIRNOFF
Forest Research Laboratory, Sillery, Quebec, Canada. Accepted
Nuclear
March
29, 1965
Equivalents
in
Microorganisms in Sitophilus granarius (Linnaeus) ’
The mycetomal microorganisms of the granary weevil, Sitophilus granarius (Linnaeus), supposedly play a useful but not always essentialpart in that insect’s nutrition (Musgrave, Can. Entomologist, 96, 377-389, 1964; Schneider, Z. Morphol. Oekol. Tiere, 44, 555-625, 1956). Kolb (2. Morphol. Oekol. Tiere, 48, 1-71, 1959) using Giemsaand other stains was able
acknowledged.
.
48
Evidence
Mycetomal
Research
.4verage number
mortality
larvae
Histochemical
1 Receipt
USED
24
Bacillus was determined. The percentage given is the average mortality obtained after 120 hours for the four methods of treatment described: (a) B. thuringiensis, 96.0%; (b) B. dendrolimus, 91.3% ; (c) B. alesti, 87.5%; (d) B. entomocidus, 86.2%; (e) B. sotto, 76.2r0; (f) B. cereus, 70.770. The mechanical mixture of the six species gave 90.5% mortality, while the six speciesgrown together gave 41.270 mortality. In the control rearings the larval mortality never passed 5-77oafter 120 hours. The reasons for the low percentage obtained with the mixed insemination are the object of a separate study. 4. The average percent mortality for each
National
OF TREATMENT
larvae
foliage
Immersed Oral
used
METHODS
is
gratefully
by
to identify DNA in the symbiotes of the ant, Camponotus, and the fruit fly, Ceratitis, but was unable to identify it in those of Sitophi1US.
Recently, after a number of preliminary trials, successin detecting DNA in the microorganisms of the mycetome in Sitophilus granarius has been achieved in this laboratory by the following technique. Mycetomes of larvae and mesentera of adults were prepared as smears on glass slides. Following fixation for 3 minutes in the