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The recovery of virulent nuclear-polyhedrosis virus of the cabbage looper, Trichoplusia ni, from the feces of birds
JOURNAL
OF
INVERTEBRATE
PATHOLOGY
15,
1’73-176
(1970)
The Recovery of Virulent Nuclear-Polyhedrosis Virus of the Cabbage Looper, Trichoplusiu ni, from the Feces of Birds DONALD
Entomology
L. HOSTETI-ER
AND K. D. BIEVER
Research Division, Agricultural Research Service, U.S. Department Agriculture, Columbia, Mi.ssouri 65201 Received
June
of
18, 1969
English sparrows have been observed methodically searching cabbage plants for insects. Cabbage loopers, Trichoplusia ni, in the advanced stages of nuclear polyhedrosis, lose their natural defense mechanisms and become easy prey for birds. Examination of individual aqueous suspensions of bird feces collected from a cabbage field in St. Louis County, Missouri, in July 1968, revealed high numbers of polyhedra inclusion bodies in 9 of the 12 samples. Per OS inoculation of third instar T. ni larvae proved that all 12 suspensions contained virulent polyhedra (i.e., virus) capable of producing the polyhedrosis.
Steinhaus (1954) referring to various means of dissemination of insect pathogens, Several vegetable growers in the St. mentioned that carnivorous insects and inLouis, Missouri area have mentioned that sectivorous animals, such as birds, probably birds, especially the English sparrow, spread infectious agents after feeding on Passer domesticus, consume large numbers dead or dying insects. Bird (1955) reof insect larvae in their fields. The English ported that the stomach contents of two of sparrow, generally considered to be a four birds killed in a Scats pine plantation nuisance, is regarded to be beneficial by while feeding on virus-infected European these growers. pine sawfly larvae, Neodiprion sertifer, were An epizootic of nuclear polyhedrosis of highly infectious. He concluded that it the cabbage looper, Trichoplusiu ni, occurwas probable that viruses are transmitted red in a cabbage field in St. Louis County, through the droppings of birds and may be Missouri, in 1968. Small flocks of English distributed over long distances. Genung sparrows were observed methodically ( 1959) during a field experiment to detersearching the plants for insects prior, durmine the possibility of inducing the polying, and after the collapse of the looper hedrosis diseasein cabbage loopers prior to populations due to this virus. The activity its natural appearance, observed English of the sparrows was most noticeable during sparrows feeding on the loopers. Partial the early morning hours. They seemed to screening of test plants was required to be particularly attracted to cabbage loopers prevent feeding mortality from the sparshowing advanced signs of the virus disease. Larvae in this condition turn pale-yellow rows. The purpose of this investigation was to to white and move to the outer, more exdetermine if virulent polyhedrosis virus, posed portions of the plants, where they or polyhedra inclusion bodies (PIB), could become extremely susceptible to avian be recovered from field-collected bird feces. predation. 173
HOSTETTER ANn BIEVER
174
MATERIALS AND METHODS Twelve bird droppings were randomly collected from cabbage plants in a field in which the epizootic of nuclear polyhedrosis occurred. Each dropping was isolated in a sterile plastic tube and then stored at -8°C. One of the droppings was used to develop a procedure in a preliminary feeding test utilizing serial dilutions. One ml of distilled water was added to each 100 mg of feces. This suspension was examined microscopically, and counts of the polyhedra found were made using an A0 Spencer Bright Line, Improved Neubauer hemacytometer ( Table 1) . The polyhedral suspension was then frozen until it could be bioassayed. A preliminary feeding test was conducted which contained with the suspension 145,500,000 PIB/ml and with serial dilutions of 1O1, 102, 103, and lo4 to determine the virulence of the recovered polyhedra and to see if similar tests with the remaining feces would be justified. Five replications of 10 7’. ni larvae (3rd instar) were fed on approximately 100 g of a modified Vanderzant’s media poured into 6-0~ ice cream containers (waxed). TABLE
1
OF POLYHEDRA OF Trichoplusia ni Vmus THE FECES OF THE ENGLISH SPARROW AND THE CALCULATED DOSAGE APPLIED
COUNTS
FROM
TO THE TEST MEDIA
Sample 2 No. PIBa no. per 10 counts 1 4 2 600 183 3 4 298 29 5 669 6 7 3 24 8 48 9 10 218 7 11 291 12 0 Polyhedral
inclusion
No. PIB/ml 2.
x 106 3.0 x 10s 9.47 x 107 1.49 x 10s 1.45 x 10’ 3.34 x 10s 1.5 x 106 1.20 x 10’ 2.4 X 107
1.09 x 10s 3.5 X 106 1.45 x 10s bodies.
PIB/mm2 106.0 15,860.O 5006.0 7876.0 766.0 17,656.0 79.0 634.0 1268.0 5762.0 185.0 7962.0
The media surfaces were treated with 0.3 ml of the original suspension and serial dilutions. Each suspension was pipetted onto the surface of the diet and spread evenly with a sterile glass rod. The surface area of the treated diet was calculated, and the number of PIB/mm” was computed (Table 1). The cups were covered with a sterile glass petri-dish cover to enable clear observations of larvae without disturbance. Two hundred and fifty larvae, in lots of 10, were fed on media treated with 0.3 ml distilled water as a control, The cups were incubated at 22°C under constant illumination. The larvae were allowed to feed until they died of the polyhedrosis virus or until they pupated. Observations for mortality were made daily, and dead learvae were removed on the day of death. Tests were terminated 12 days after the initial exposure. The percentage mortality for each suspension was corrected utilizing Abbott’s formula ( 1925). This testing procedure, without serial dilutions, was utilized for the remaining 11 samples; 50 larvae were set up as controls. RESULTS AND DISCUSSION
The preliminary feeding test using serial dilutions (sample 12) demonstrated that the PIB (virus particles) were virulent at all dilution levels tested (Fig. 1). The percent mortality for the original, lOi, and lo2 suspensions was 100%; for the 103, 89.7; and 104, 56.6%. Dilutions greater than lo” indicated that the mortality rate is directly proportional to the amount of virulent virus in the suspension. Total mortality in the controls for sample 12 and the 4 serial dilutions was 9%, and for samples 1 through 5, 6% mortality occurred. Mortality in the controls for samples 6 through 11 was high due to viral contamination and was not plotted on Fig. 3. Suspensions 1, 7, and 11 showed the lowest initial and total mortality (Figs. 2
RECOVERY
‘DAY
0
2
1
3
OF
4
100 90 % Q 80 9 a 70 9 ;
60
2
50
2
40
1 -I
---.-
1.
Percent
SW. x2
Trichophsia
FROM
BIRD
5 DAYS
FIG.
VIRUS
FECES
6
- - - POST
ni mortality
7
8
9
10
11
EXPOSURE
for
sample
12 and serial
dilutions.
#l
‘21 r’l 30 !z $ 20 6 a
10 0 1
2
3
4
5 DAYS
FIG.
2.
Percent
6
- - - POST
T. ni mortality,
and 3), and contained signifkantly smaller amounts of virus (Table 1). Since individual larvae were not isolated in the test containers secondary infection probably occurred due to direct contact with infected larvae in the same unit and not as a result of ingestion of the originally contaminated media. A more precise time of mortality would have been expected had there been
I
R
9
10
11
EXPOSURE
samples
1 through
5.
a higher dosage supplied in these tests. Some of the droppings collected may have come from sparrows that had not recently eaten an infected larva or had previously eliminated the majority of ingested polyhedra prior to sampling. The probability of birds ingesting infected, rather than noninfected, T. ni Iarvae is greater due to the change in color of
176
HOSTE’ITER
---..-_ --
1
AND
r //’,’,‘i II [ /I / I/ / 1”
#6 #7 it8 ii9 YlO
I
I
2
3
FIG.
BIEVER
3.
4
Percent
,
5 6 1 DAYS - - - POST EXPOSURE
T. ni mortality,
infected larvae, involving a loss of camouflage, as well as the tendency of the infected larvae to move to the higher, more exposed, portions of the plants. It is quite conceivable that natural epizootics of nuclear polyhedrosis in T. ni populations could be disseminated by sparrows over long distances. This study demonstrates that the cabbageIooper nucIear-polyhedrosis virus survives the digestive processes of the sparrow in sufhcient quantities to readily disseminate the disease. Dissemination of this virus through bird feces is a method of insect
,/--- .’ /......’ / _._.*........ .A.
r
8
samples
9
6 through
10
,
11
12
11.
disease transmission that should be considered when studying epizootics in insect populations. REFERENCES ABBOTT,
w.
1925. Method of insecticide.
S.
effectiveness
for computing the J. Econ. Entcnnol.,
18, 265-267. Bmn,
F.
T.
1955.
Can. Entomologist
Virus
diseases
of
sawflies.
87, 124127.
W. G. 1959. Observations on and preliminary experiments with a polyhedrosis virus for control of cabbage looper, Trichoplusia ni (Hbn). Florida Entomologist 42, 99-104. STEINFLUS, E. A. 1954. The effects of disease on insect populations. HiZgardia 23, 245.
GENUNG,
OF
INVERTEBRATE
PATHOLOGY
15,
1’73-176
(1970)
The Recovery of Virulent Nuclear-Polyhedrosis Virus of the Cabbage Looper, Trichoplusiu ni, from the Feces of Birds DONALD
Entomology
L. HOSTETI-ER
AND K. D. BIEVER
Research Division, Agricultural Research Service, U.S. Department Agriculture, Columbia, Mi.ssouri 65201 Received
June
of
18, 1969
English sparrows have been observed methodically searching cabbage plants for insects. Cabbage loopers, Trichoplusia ni, in the advanced stages of nuclear polyhedrosis, lose their natural defense mechanisms and become easy prey for birds. Examination of individual aqueous suspensions of bird feces collected from a cabbage field in St. Louis County, Missouri, in July 1968, revealed high numbers of polyhedra inclusion bodies in 9 of the 12 samples. Per OS inoculation of third instar T. ni larvae proved that all 12 suspensions contained virulent polyhedra (i.e., virus) capable of producing the polyhedrosis.
Steinhaus (1954) referring to various means of dissemination of insect pathogens, Several vegetable growers in the St. mentioned that carnivorous insects and inLouis, Missouri area have mentioned that sectivorous animals, such as birds, probably birds, especially the English sparrow, spread infectious agents after feeding on Passer domesticus, consume large numbers dead or dying insects. Bird (1955) reof insect larvae in their fields. The English ported that the stomach contents of two of sparrow, generally considered to be a four birds killed in a Scats pine plantation nuisance, is regarded to be beneficial by while feeding on virus-infected European these growers. pine sawfly larvae, Neodiprion sertifer, were An epizootic of nuclear polyhedrosis of highly infectious. He concluded that it the cabbage looper, Trichoplusiu ni, occurwas probable that viruses are transmitted red in a cabbage field in St. Louis County, through the droppings of birds and may be Missouri, in 1968. Small flocks of English distributed over long distances. Genung sparrows were observed methodically ( 1959) during a field experiment to detersearching the plants for insects prior, durmine the possibility of inducing the polying, and after the collapse of the looper hedrosis diseasein cabbage loopers prior to populations due to this virus. The activity its natural appearance, observed English of the sparrows was most noticeable during sparrows feeding on the loopers. Partial the early morning hours. They seemed to screening of test plants was required to be particularly attracted to cabbage loopers prevent feeding mortality from the sparshowing advanced signs of the virus disease. Larvae in this condition turn pale-yellow rows. The purpose of this investigation was to to white and move to the outer, more exdetermine if virulent polyhedrosis virus, posed portions of the plants, where they or polyhedra inclusion bodies (PIB), could become extremely susceptible to avian be recovered from field-collected bird feces. predation. 173
HOSTETTER ANn BIEVER
174
MATERIALS AND METHODS Twelve bird droppings were randomly collected from cabbage plants in a field in which the epizootic of nuclear polyhedrosis occurred. Each dropping was isolated in a sterile plastic tube and then stored at -8°C. One of the droppings was used to develop a procedure in a preliminary feeding test utilizing serial dilutions. One ml of distilled water was added to each 100 mg of feces. This suspension was examined microscopically, and counts of the polyhedra found were made using an A0 Spencer Bright Line, Improved Neubauer hemacytometer ( Table 1) . The polyhedral suspension was then frozen until it could be bioassayed. A preliminary feeding test was conducted which contained with the suspension 145,500,000 PIB/ml and with serial dilutions of 1O1, 102, 103, and lo4 to determine the virulence of the recovered polyhedra and to see if similar tests with the remaining feces would be justified. Five replications of 10 7’. ni larvae (3rd instar) were fed on approximately 100 g of a modified Vanderzant’s media poured into 6-0~ ice cream containers (waxed). TABLE
1
OF POLYHEDRA OF Trichoplusia ni Vmus THE FECES OF THE ENGLISH SPARROW AND THE CALCULATED DOSAGE APPLIED
COUNTS
FROM
TO THE TEST MEDIA
Sample 2 No. PIBa no. per 10 counts 1 4 2 600 183 3 4 298 29 5 669 6 7 3 24 8 48 9 10 218 7 11 291 12 0 Polyhedral
inclusion
No. PIB/ml 2.
x 106 3.0 x 10s 9.47 x 107 1.49 x 10s 1.45 x 10’ 3.34 x 10s 1.5 x 106 1.20 x 10’ 2.4 X 107
1.09 x 10s 3.5 X 106 1.45 x 10s bodies.
PIB/mm2 106.0 15,860.O 5006.0 7876.0 766.0 17,656.0 79.0 634.0 1268.0 5762.0 185.0 7962.0
The media surfaces were treated with 0.3 ml of the original suspension and serial dilutions. Each suspension was pipetted onto the surface of the diet and spread evenly with a sterile glass rod. The surface area of the treated diet was calculated, and the number of PIB/mm” was computed (Table 1). The cups were covered with a sterile glass petri-dish cover to enable clear observations of larvae without disturbance. Two hundred and fifty larvae, in lots of 10, were fed on media treated with 0.3 ml distilled water as a control, The cups were incubated at 22°C under constant illumination. The larvae were allowed to feed until they died of the polyhedrosis virus or until they pupated. Observations for mortality were made daily, and dead learvae were removed on the day of death. Tests were terminated 12 days after the initial exposure. The percentage mortality for each suspension was corrected utilizing Abbott’s formula ( 1925). This testing procedure, without serial dilutions, was utilized for the remaining 11 samples; 50 larvae were set up as controls. RESULTS AND DISCUSSION
The preliminary feeding test using serial dilutions (sample 12) demonstrated that the PIB (virus particles) were virulent at all dilution levels tested (Fig. 1). The percent mortality for the original, lOi, and lo2 suspensions was 100%; for the 103, 89.7; and 104, 56.6%. Dilutions greater than lo” indicated that the mortality rate is directly proportional to the amount of virulent virus in the suspension. Total mortality in the controls for sample 12 and the 4 serial dilutions was 9%, and for samples 1 through 5, 6% mortality occurred. Mortality in the controls for samples 6 through 11 was high due to viral contamination and was not plotted on Fig. 3. Suspensions 1, 7, and 11 showed the lowest initial and total mortality (Figs. 2
RECOVERY
‘DAY
0
2
1
3
OF
4
100 90 % Q 80 9 a 70 9 ;
60
2
50
2
40
1 -I
---.-
1.
Percent
SW. x2
Trichophsia
FROM
BIRD
5 DAYS
FIG.
VIRUS
FECES
6
- - - POST
ni mortality
7
8
9
10
11
EXPOSURE
for
sample
12 and serial
dilutions.
#l
‘21 r’l 30 !z $ 20 6 a
10 0 1
2
3
4
5 DAYS
FIG.
2.
Percent
6
- - - POST
T. ni mortality,
and 3), and contained signifkantly smaller amounts of virus (Table 1). Since individual larvae were not isolated in the test containers secondary infection probably occurred due to direct contact with infected larvae in the same unit and not as a result of ingestion of the originally contaminated media. A more precise time of mortality would have been expected had there been
I
R
9
10
11
EXPOSURE
samples
1 through
5.
a higher dosage supplied in these tests. Some of the droppings collected may have come from sparrows that had not recently eaten an infected larva or had previously eliminated the majority of ingested polyhedra prior to sampling. The probability of birds ingesting infected, rather than noninfected, T. ni Iarvae is greater due to the change in color of
176
HOSTE’ITER
---..-_ --
1
AND
r //’,’,‘i II [ /I / I/ / 1”
#6 #7 it8 ii9 YlO
I
I
2
3
FIG.
BIEVER
3.
4
Percent
,
5 6 1 DAYS - - - POST EXPOSURE
T. ni mortality,
infected larvae, involving a loss of camouflage, as well as the tendency of the infected larvae to move to the higher, more exposed, portions of the plants. It is quite conceivable that natural epizootics of nuclear polyhedrosis in T. ni populations could be disseminated by sparrows over long distances. This study demonstrates that the cabbageIooper nucIear-polyhedrosis virus survives the digestive processes of the sparrow in sufhcient quantities to readily disseminate the disease. Dissemination of this virus through bird feces is a method of insect
,/--- .’ /......’ / _._.*........ .A.
r
8
samples
9
6 through
10
,
11
12
11.
disease transmission that should be considered when studying epizootics in insect populations. REFERENCES ABBOTT,
w.
1925. Method of insecticide.
S.
effectiveness
for computing the J. Econ. Entcnnol.,
18, 265-267. Bmn,
F.
T.
1955.
Can. Entomologist
Virus
diseases
of
sawflies.
87, 124127.
W. G. 1959. Observations on and preliminary experiments with a polyhedrosis virus for control of cabbage looper, Trichoplusia ni (Hbn). Florida Entomologist 42, 99-104. STEINFLUS, E. A. 1954. The effects of disease on insect populations. HiZgardia 23, 245.
GENUNG,