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Pathogenicity of a nuclear-polyhedrosis virus of the beet armyworm, Spodoptera exigua
JOURNAL
OF INVERTEBRATE
Pathogenicity
12, 83-85
PATHOLOGY
( 1968)
of a Nuclear-Polyhedrosis Spodoptera D. IS. HUNTERI.' Department
of Biological Rioerside, Received
Virus of the Beet Armyworm, exigua AND
Control, California December
I. M. HALL University 92502 15,
of
California,
1967
A bioassay of a nuclear-polyhedrosis virus was conducted with first- and fourthinstar larvae of the beet armyworm, Spodoptera exigua. Purified suspensions of polyhedra were layered on a diet at concentrations of 5, 15, 50, and I50 polyhedra/ mm?. Computed LDSO’S were 8.3 and 57.6 polyhedra/mm~ for the respective instars. ~~50’s ranged from 4.1 to 5.2 days for the first instar and 5.0 to 5.7 days for the fourth instar.
through organdy cloth and purified by differential centrifugation and fluorocarbon treatment ( Bergold, 1959). The suspension was buffered s at pH 7.0. Counting of the polyhedra was done on a Petroff-Hausser bacteria counter, and the stock suspension wds stored at -6°C. A semisynthetic artificial diet (Henneberry and Kishaba, 1966) was used to rear test larvae. Quantities (0.1 ml) of a polyhedral suspension were layered over the diet surfaces (Ignoffo, 1966) in l-02 clear plastic cups while distilled water at pH 7.0 was similarly layered for the controls. Uniformly aged larvae were placed individually in the cups and incubated at 27°C in a cabinet that provided a light cycle of 12 hr/day. The effects of hydrogen-ion concentration and formaldehyde (originally in the form of formaldehyde solution) on virulence were determined by methods similar to those reported by Ignoffo (1966) and Vail, Henneberry, and Kishaba (1968). In the bioassay, larval mortalities were recorded from day 4 until pupation, and determination was also made of adult emergence.
INTRODUCTION
Biological assays of insect viruses may be influenced by chemical conditions. Ignoffo (1966) reported that the activity of corn earworm virus material was best maintained at pH 7.0. Vail, Henneberry, and Kishaba (1968) demonstrated that virus on diet with formaldehyde was less pathogenic to cabbage-looper larvae. The effects of these chemical factors on virus-caused mortality in the beet armyworm, Sporloptera exigua, were investigated. A bioassay was then conducted wherein virus pathogenicity was not reduced by detrimental pH or formaldehyde. METHODS AND MATERIALS
Laboratory-reared larvae were infected per OS with field-collected virus material. Following death, the cadavers were temporarily stored at -6”C, and later thawed and blended with a fivefold amount of distilled water. The homogenate was strained 1 Extracted from a doctorate dissertation accepted by the Department of Biological Control, University of California, Riverside, July 24, 1967. 2 Present address: Stored-Product Insects Laboratory, U. S. Department of Agriculture, Fresno, California 93727.
:; Coleman Co., Maywood, 83
Buffer Ill.
Tablets,
Coleman
Instrument
84
HUNTER AND MALL RESULTS
AND
8-
DISCUSSION
Of the five PH values tested (3.0, 5.0, 7.0, 9.0, and ll.O), the highest larval mortality was obtained with polyhedral suspensions buffered at pH 7.0. Moreover, an eightfold increase in mortality occurred when larvae were exposed to polyhedra on a diet which did not contain formaldehyde. These results indicate that the influence of pH and of formaldehyde on mortality rates of this polyhedrosis are similar to those reported by earlier workers (Ignoffo, 1966; Vail, Henneberry, and Kishaba, 1968) with similar viruses. This would suggest that greater pathogenicity could be expected in a bioassay wherein these inhibiting factors were eliminated. In the bioassay with the beet armyworm, concentrations of 5, 15, 50, and 150 polyhedra/mms of diet surface were tested against first- and fourth-instar larvae. The results are shown in Table 1. Larval mortality increased with the dose of polyhedra administered. First-instar larvae were more susceptible to a given polyhedral concentration than the later-instar larvae. Heavily infected individuals were noticeably retarded and generally died before molting. Figure 1 shows mortality probits plotted over logarithmic dose. The curves were computed by probit analysis ( Finney, 1952). Equations describing the curves are given. The LD~*'S were co,mputed to be 8.3 and 57.6 polyhedra/mm’ of diet surface for the respective larval instars. FiTABLE RESPONSE
OF
FIRST- AND FOURTH-INSTAR VARIOUS
CONCENTRATIONS
7.
5
15 50 150
No. larvae per treatment
75 75 75 75
(X-1.17)
* Y=4.71+1.07
(X-1.50)
.
i 6 5 * 2 S %
1
4
*
/ .
4,
. /
33 0
Log
4
3
2
1 Dosage
FIG. 1. Dosage-mortality curves of first- and fourth-instar larvae of Spodopteru exigua exposed to nuclear-polyhedrosis virus.
ducial limits (95%) of the LDso'S were 7.7 to 8.4 polyhedra/mm? for the first instar and 42.3 to 86.3 polyhedra/mm2 for the fourth instar. LTSO).S for the dosestested are shown in Table 2. The data show that the time of mortality at a given dose was generally longer in the older larvae. Lack of distinct decreases in LQ"'s with increased dosage was probably due to the narrow dosage range tested. The percentage of adults that emerged in the treatments was always lower than in the controls, but the differences among the 1 LARVAE OF THE BEET OF NUCLEAR POLYHEDRA Percent
Dose of diet surface ( polyhedra/mm* )
. Y=5.39+1.73
1st instar
32 65 92 98
ARMYWORM
TO
mortality 4th
instar
15 24 43 71
PATHOGENICITY
TABLE L’l’,,‘s
(DAYS)
2
OF VIRUS-INFECTED
AT 27°C
IMW.M
Dose Larval
instar 1 4
treatments could not be correlated to polyhedral dose.
G. H. 1959. Purification of insect virus inclusion bodies with a fluorocarbon. J. Insect Path& 1, 9697. F’INNEY, D. J. 1952. “Probit Analysis, a Statistical Treatment of the Sigmoid Response Curve,” 2nd ed., 318 pp. Cambridge Universtiy Press, Cambridge. HENNEBERRY, T. J., AND KISHABA, A. N. 1966. Mass rearing cabbage loopers. In “Insect Colonization and Mass Rearing,” pp. 461477.
(polyhedra/mml)
5
15
50
150
4.1 5.5
5.2 5.7
4.2 5.1
5.1 5.0
C. M. 1966. Susceptibility instar of the bollworm, Heliothis tobacco budworm, Heliothis Heliothis nuclear-polyhedrosis vertebrate Pathol., 8, 531-536.
IGNOFFO,
REFERENCES BERGOLD,
85
OF vmus FClR BEET ARMYWORM
of the firstzea, and the virescens, to virus. J. In-
C. M., AND GARCIA, C. 1966. The relation of pH to the activity of inclusion bodies of the Heliothis nuclear-polyhedrosis. J. Invertebrate Pathol., 8, 226-227.
IGNOFFO,
VAIL,
T. S., KISHABA, A. N., K. Y. 1968. Sodium hypochlorite and formalin as antiviral agents against nuclear-polyhedrosis virus in larva of the cabbage looper. J. Invertebrate Pathol., 10, 84-93. P. V.,
AND
HENNEBERRY,
ARAKAWA,
OF INVERTEBRATE
Pathogenicity
12, 83-85
PATHOLOGY
( 1968)
of a Nuclear-Polyhedrosis Spodoptera D. IS. HUNTERI.' Department
of Biological Rioerside, Received
Virus of the Beet Armyworm, exigua AND
Control, California December
I. M. HALL University 92502 15,
of
California,
1967
A bioassay of a nuclear-polyhedrosis virus was conducted with first- and fourthinstar larvae of the beet armyworm, Spodoptera exigua. Purified suspensions of polyhedra were layered on a diet at concentrations of 5, 15, 50, and I50 polyhedra/ mm?. Computed LDSO’S were 8.3 and 57.6 polyhedra/mm~ for the respective instars. ~~50’s ranged from 4.1 to 5.2 days for the first instar and 5.0 to 5.7 days for the fourth instar.
through organdy cloth and purified by differential centrifugation and fluorocarbon treatment ( Bergold, 1959). The suspension was buffered s at pH 7.0. Counting of the polyhedra was done on a Petroff-Hausser bacteria counter, and the stock suspension wds stored at -6°C. A semisynthetic artificial diet (Henneberry and Kishaba, 1966) was used to rear test larvae. Quantities (0.1 ml) of a polyhedral suspension were layered over the diet surfaces (Ignoffo, 1966) in l-02 clear plastic cups while distilled water at pH 7.0 was similarly layered for the controls. Uniformly aged larvae were placed individually in the cups and incubated at 27°C in a cabinet that provided a light cycle of 12 hr/day. The effects of hydrogen-ion concentration and formaldehyde (originally in the form of formaldehyde solution) on virulence were determined by methods similar to those reported by Ignoffo (1966) and Vail, Henneberry, and Kishaba (1968). In the bioassay, larval mortalities were recorded from day 4 until pupation, and determination was also made of adult emergence.
INTRODUCTION
Biological assays of insect viruses may be influenced by chemical conditions. Ignoffo (1966) reported that the activity of corn earworm virus material was best maintained at pH 7.0. Vail, Henneberry, and Kishaba (1968) demonstrated that virus on diet with formaldehyde was less pathogenic to cabbage-looper larvae. The effects of these chemical factors on virus-caused mortality in the beet armyworm, Sporloptera exigua, were investigated. A bioassay was then conducted wherein virus pathogenicity was not reduced by detrimental pH or formaldehyde. METHODS AND MATERIALS
Laboratory-reared larvae were infected per OS with field-collected virus material. Following death, the cadavers were temporarily stored at -6”C, and later thawed and blended with a fivefold amount of distilled water. The homogenate was strained 1 Extracted from a doctorate dissertation accepted by the Department of Biological Control, University of California, Riverside, July 24, 1967. 2 Present address: Stored-Product Insects Laboratory, U. S. Department of Agriculture, Fresno, California 93727.
:; Coleman Co., Maywood, 83
Buffer Ill.
Tablets,
Coleman
Instrument
84
HUNTER AND MALL RESULTS
AND
8-
DISCUSSION
Of the five PH values tested (3.0, 5.0, 7.0, 9.0, and ll.O), the highest larval mortality was obtained with polyhedral suspensions buffered at pH 7.0. Moreover, an eightfold increase in mortality occurred when larvae were exposed to polyhedra on a diet which did not contain formaldehyde. These results indicate that the influence of pH and of formaldehyde on mortality rates of this polyhedrosis are similar to those reported by earlier workers (Ignoffo, 1966; Vail, Henneberry, and Kishaba, 1968) with similar viruses. This would suggest that greater pathogenicity could be expected in a bioassay wherein these inhibiting factors were eliminated. In the bioassay with the beet armyworm, concentrations of 5, 15, 50, and 150 polyhedra/mms of diet surface were tested against first- and fourth-instar larvae. The results are shown in Table 1. Larval mortality increased with the dose of polyhedra administered. First-instar larvae were more susceptible to a given polyhedral concentration than the later-instar larvae. Heavily infected individuals were noticeably retarded and generally died before molting. Figure 1 shows mortality probits plotted over logarithmic dose. The curves were computed by probit analysis ( Finney, 1952). Equations describing the curves are given. The LD~*'S were co,mputed to be 8.3 and 57.6 polyhedra/mm’ of diet surface for the respective larval instars. FiTABLE RESPONSE
OF
FIRST- AND FOURTH-INSTAR VARIOUS
CONCENTRATIONS
7.
5
15 50 150
No. larvae per treatment
75 75 75 75
(X-1.17)
* Y=4.71+1.07
(X-1.50)
.
i 6 5 * 2 S %
1
4
*
/ .
4,
. /
33 0
Log
4
3
2
1 Dosage
FIG. 1. Dosage-mortality curves of first- and fourth-instar larvae of Spodopteru exigua exposed to nuclear-polyhedrosis virus.
ducial limits (95%) of the LDso'S were 7.7 to 8.4 polyhedra/mm? for the first instar and 42.3 to 86.3 polyhedra/mm2 for the fourth instar. LTSO).S for the dosestested are shown in Table 2. The data show that the time of mortality at a given dose was generally longer in the older larvae. Lack of distinct decreases in LQ"'s with increased dosage was probably due to the narrow dosage range tested. The percentage of adults that emerged in the treatments was always lower than in the controls, but the differences among the 1 LARVAE OF THE BEET OF NUCLEAR POLYHEDRA Percent
Dose of diet surface ( polyhedra/mm* )
. Y=5.39+1.73
1st instar
32 65 92 98
ARMYWORM
TO
mortality 4th
instar
15 24 43 71
PATHOGENICITY
TABLE L’l’,,‘s
(DAYS)
2
OF VIRUS-INFECTED
AT 27°C
IMW.M
Dose Larval
instar 1 4
treatments could not be correlated to polyhedral dose.
G. H. 1959. Purification of insect virus inclusion bodies with a fluorocarbon. J. Insect Path& 1, 9697. F’INNEY, D. J. 1952. “Probit Analysis, a Statistical Treatment of the Sigmoid Response Curve,” 2nd ed., 318 pp. Cambridge Universtiy Press, Cambridge. HENNEBERRY, T. J., AND KISHABA, A. N. 1966. Mass rearing cabbage loopers. In “Insect Colonization and Mass Rearing,” pp. 461477.
(polyhedra/mml)
5
15
50
150
4.1 5.5
5.2 5.7
4.2 5.1
5.1 5.0
C. M. 1966. Susceptibility instar of the bollworm, Heliothis tobacco budworm, Heliothis Heliothis nuclear-polyhedrosis vertebrate Pathol., 8, 531-536.
IGNOFFO,
REFERENCES BERGOLD,
85
OF vmus FClR BEET ARMYWORM
of the firstzea, and the virescens, to virus. J. In-
C. M., AND GARCIA, C. 1966. The relation of pH to the activity of inclusion bodies of the Heliothis nuclear-polyhedrosis. J. Invertebrate Pathol., 8, 226-227.
IGNOFFO,
VAIL,
T. S., KISHABA, A. N., K. Y. 1968. Sodium hypochlorite and formalin as antiviral agents against nuclear-polyhedrosis virus in larva of the cabbage looper. J. Invertebrate Pathol., 10, 84-93. P. V.,
AND
HENNEBERRY,
ARAKAWA,