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Susceptibility of Hyphantria cunea to a granulosis virus isolated from Diacrisia virginica
JOURNAL
OF INVERTEBRATE
Susceptibility
PATHOLOGY
30, 377-380
(1977)
of Hyphantria cunea to a Granulosis Isolated from Diacrisia virginical
Virus
D. G. BOUCIASAND G. L. NORDIN Department
of Entomology,
University
of Kentucky,
Lexington,
Kentucky
40506
Received February 7, 1977 The dosage-mortality response of Hyphantria cunea larvae to a granulosis virus isolated from virginica was studied. Serial decimal dilutions of the D. virginica granulosis virus were fed to early second-instar H. cunea larvae. the LD,, for this virus (7.9 x lo4 capsules/larva) was significantly greater than the LD,, calculated for the H. cunea granulosis virus (7.06 x lo4 capsules/larva) against the same instar of H. cunea. Time mortality studies demonstrated that the LT,, values for the D. virginica granulosis were four to six times greater than for comparable dosages of H. cunea granulosis virus. Based on the mortality response of assayed H. cunea, it is possible to distinguish between the D. virginica granulosis virus and the H. cunea granulosis virus. Diacrisia
of H. cunea. D. virginica larvae challenged with the H. cunea granulosis (HcGV), however, did not become infected. The purpose of this paper is to compare the mortality response of H. cunea larvae to DGV with mortality response data reported previously for HcGV (Boucias and Nordin, 1977).
INTRODUCTION
Granulosis viruses are considered to exhibit a high degree of host specificity. Ignoffo (1968) reported only six successful cross transmissions of granulosis viruses to alternate host species. The majority of these were restricted to species of the same genus or to species of the same family. More recently, Hunter and Hoffman (1972) demonstrated the susceptibility of Plodia interpunctella to a granulosis from Cadra cautella. Based on symptomology and mortality response, they were able to distinguish between P. interpunctella larvae infected with its own granulosis virus and those infected with C. cautella granulosis virus. Host-range studies in our laboratory have shown that the fall webworm, Hyphantria cunea, is susceptible to a granulosis virus isolated from the arctiid, Diacrisiu virginica. Cross transmission has been confirmed by infecting D. virginica larvae perorally with D. virginica granulosis virus (DGV) after having been passed through two generations
MATERIALS
r This paper (No. 77-7-13) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the director.
AND METHODS
The granulosis capsules used in these assays were purified from heavily infected D. virginica larvae as outlined by Summers and Paschke (1970). Decimal dilutions of the purified capsules were prepared in distilled water. Quantitation of capsules, using the technique of Williams and Backus (1949), provided an estimate of 11.2 x 10” +- 2.8 x 10” capsules/ml in the stock suspension. Serial concentrations of the granulosis capsules were applied to small disks of diet, 1 @disk, and were fed to early secondinstar H. cunea larvae, 5 larvae/disk. Each larva was assumed to receive 0.2 ~1 of inoculum. Treated and untreated larvae were placed in a 26°C incubator having a 12-hr photoperiod. Treatments consisting of 35 larvae/dilution were replicated twice.
377 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-2011
378
BOUCIAS
AND NORDIN
After 48 hr, those larvae consuming the upper surface of the disk were transferred to uncontaminated diet creamers (one larva/ creamer) and were returned to 26°C. Mortality was monitored every 48 hr until pupation. Fat tissue removed from the dead larvae was examined using phase-contrast optics. Only larvae showing definitive signs of granulosis were used in the calculations. RESULTS
sules/larva; lower limit: 3.0 x lo5 capsules/ larva). This value is significantly greater than the LD,, calculated for HcGV which is 7.06 x lo4 capsules/larva (upper limit: 1.79 x lo5 capsules/larva; lower limit: 2.67 x lo4 capsules/larva). Cross-infectivity tests by Hunter and Hoffman (1972) have shown a similar difference in the susceptibility of Plodia interpunctella infected with its own granulosis virus, compared with a granulosis virus isolate from Cadra cautella. Cumulative time-mortality data for dosages of DGV causing over 50% mortality are graphically depicted in Figure 2. In all cases the test larvae responded in a heterogenous fashion. The DGV is able to kill a small portion of the treated population within 15 days. The majority of treated larvae, however, did not begin to succumb until after 30 days postinoculation. LTSo values, calculated using the method of Litchfield (1949), are shown in Table 2. The incubation time required to kill 50% of the treated DGV was much greater than the values reported for HcGV-treated larvae (Boucias and Nordin, 1977). A dosage of 2.2 x lo* DGV capsules/larva, causing 100% mortality, had an LT,, value of 3.5 days. A dosage of 1.0 x IO7 HcGV capsules/larva, also causing 100% mortality, had an LT,, value of 6.1 days. The maximum calculated LT,, value for the DGV, 43 days, was achieved with a dosage of
AND DISCUSSION
Dosage-mortality
data for second-instar infected with DGV are summarized in Table 1. The number of larvae dying from granulosis increased with increased dosage. A small percentage of the tested larvae died from unknown causes. This mortality can be attributed either to the handling procedure or to the individual rearing of these normally gregarious larvae. None of the control larvae died from granulosis. Probit analysis2 was used to calculate the dosage-mortality regression line (Fig. 1). The slope of this regression line was 0.74 as opposed to a slope value of 1 .Ol previously reported for second-instar larvae fed the HcGV isolate (Boucias and Nordin, 1977). The calculated LD,, for DGV was 7.9 x lo5 capsules/larva (upper limit: 2.0 x lo6 capH. cunea larvae
2 SAS Computer University.
Program,
North
Carolina
State TABLE
1
DOSAGE-MORTALITY RESPONSE FORA GRANULOSISVIRUS FROM DIACRISIAVIRGINICA AGAINST SECOND-INSTAR HYPHANTRIACUNEA LARVAE
No. of capsules/larvae 2.2 2.2
x 108 x 10’
2.2 2.2 2.2
x 106
x 105 x 104
Control a Corrected mortality from unknown causes.
Total No. of larvae/dose 50 60 60 60 60 55
ASSAYED
No. of larvae dead from granulosis
No. of larvae dead from unknown causes
44 48
6 4
100
34
4
15 11
0
61 25 20 0
0
4 5
Corrected mortality” (%) 86
= No. of larvae dead from granulosis per total No. of larvae - No. of larvae dead
379
AND DGV FROM D~A~R~~IA
H~~~A~~RlA
TABLE 7-
2
THE
LTSo VALUES FOR THOSE DOSAGES OF DIACRISIA GRANULOSIS VIRUS CAUSING OVER 50% MORTALITY AGAINST SECOND-INSTAR HYPHANTR~A CUNEA LARVAE
Y=iZO + 74x
6-
95%
No. of capsules/larvae I 4
I 5
I 6
I 7
I 8
2.2 x 108 2.2 x 107 2.2 x 106
I 9
FOG. 1. Hyphanrria
Dosage-mo~~ity response of second-instar cunea larvae fed serial decimal dilutions of granulosis virus from Diacrisia virginica. Curved lines are the 95% confidence limits on the calculated dose-mortality line.
2.2 x 10” capsules/larva (61% mortality), as compared with the m~imum c~~uIated LT,, value for H&V, 11.5 days, which was achieved with a dosage of 1.0 x 105 capsules/larva (52% mortality). The reason for the prolonged incubation of DGV in H. cunea is not known. Infected larvae at 10 days postinoculation demonstrated typical symptoms of granulosis. As infection progressed, the integument lost all normal pigment, becoming white on both its dorsal and ventral surfaces,
Confidence limits
LT, (days)
Lower
Upper
35 39 43
33 29 26
37 52 71
The intact fat body, as observed with phase-contrast optics, contained huge numbers of capsules. The majority of larvae continued to develop until the prepupal or “wandering stage,” at which time they succumbed. In conclusion, it is possible to differentiate the DGV from the HcGV based on the dosage-mortality response of H. cunea larvae. Comparisons between the LDSO values have shown that DGV is less virulent to H. cunea larvae than is HcGV. Timemortality data also demonstrate that the LTS, values for dosages of DGV are four to six times greater than for compa~ble dosages of HcGV. Further studies are underway to determine what mechanisms
Days
FIG. 2. Time-mortality response of second-instar Hjphantriu cunea larvae fed serial decimal dilutions of granulosis virus from Diacrisia virginica. Dashed lines indicate the extrapolation to 84% mortality required for the LT,, calculation.
380
BOUCIAS
may be causing differences in virulence between the two viral isolates. REFERENCES BOUCIAS, D. G., AND NORDIN, G. L. 1977. Interinstar susceptibility of the fall webworm, Hyphantria cunea (Drury) to its nucleopolyhedrosis and granulosis viruses. J. Invertebr. Pathol. 30, 68-75. HUNTER, D. K., AND HOFFMAN, D. F. 1972. Cross infection of a granulosis virus of Cadra cautella with observations on its ultrastructure in infected cells of Plodia interpunctella. .I. Invertebr. Pathol., 20, 4- 10.
AND
NORDIN IGNOFFO,
C.
M.
1968.
Specificity
of insect
viruses.
Bull. Entomol. Sot. Amer., 14, 255-276. LITCHFIELD, .I. T. 1949. solution of time-percent
A method curves.
for
rapid
graphic
J. Pharmacol. Exp.
Ther., 97, 399-408. SUMMERS, M. D., AND PASHKE, J. D. 1970. Alkaliliberated granulosis virus of Trichoplusia ni I. Density gradient purification of virus components and some of their in vitro chemical and physical properties. J. Invertebr. Pathol., 16, 227-240. WILLIAMS, R. C., AND BACKUS, R. C. 1949. Macromolecular weights determined by direct particle counting. I. The weight of the bushy stunt virus.
J. Amer. Chem. Sot.,
71, 40.52-4057.
OF INVERTEBRATE
Susceptibility
PATHOLOGY
30, 377-380
(1977)
of Hyphantria cunea to a Granulosis Isolated from Diacrisia virginical
Virus
D. G. BOUCIASAND G. L. NORDIN Department
of Entomology,
University
of Kentucky,
Lexington,
Kentucky
40506
Received February 7, 1977 The dosage-mortality response of Hyphantria cunea larvae to a granulosis virus isolated from virginica was studied. Serial decimal dilutions of the D. virginica granulosis virus were fed to early second-instar H. cunea larvae. the LD,, for this virus (7.9 x lo4 capsules/larva) was significantly greater than the LD,, calculated for the H. cunea granulosis virus (7.06 x lo4 capsules/larva) against the same instar of H. cunea. Time mortality studies demonstrated that the LT,, values for the D. virginica granulosis were four to six times greater than for comparable dosages of H. cunea granulosis virus. Based on the mortality response of assayed H. cunea, it is possible to distinguish between the D. virginica granulosis virus and the H. cunea granulosis virus. Diacrisia
of H. cunea. D. virginica larvae challenged with the H. cunea granulosis (HcGV), however, did not become infected. The purpose of this paper is to compare the mortality response of H. cunea larvae to DGV with mortality response data reported previously for HcGV (Boucias and Nordin, 1977).
INTRODUCTION
Granulosis viruses are considered to exhibit a high degree of host specificity. Ignoffo (1968) reported only six successful cross transmissions of granulosis viruses to alternate host species. The majority of these were restricted to species of the same genus or to species of the same family. More recently, Hunter and Hoffman (1972) demonstrated the susceptibility of Plodia interpunctella to a granulosis from Cadra cautella. Based on symptomology and mortality response, they were able to distinguish between P. interpunctella larvae infected with its own granulosis virus and those infected with C. cautella granulosis virus. Host-range studies in our laboratory have shown that the fall webworm, Hyphantria cunea, is susceptible to a granulosis virus isolated from the arctiid, Diacrisiu virginica. Cross transmission has been confirmed by infecting D. virginica larvae perorally with D. virginica granulosis virus (DGV) after having been passed through two generations
MATERIALS
r This paper (No. 77-7-13) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the director.
AND METHODS
The granulosis capsules used in these assays were purified from heavily infected D. virginica larvae as outlined by Summers and Paschke (1970). Decimal dilutions of the purified capsules were prepared in distilled water. Quantitation of capsules, using the technique of Williams and Backus (1949), provided an estimate of 11.2 x 10” +- 2.8 x 10” capsules/ml in the stock suspension. Serial concentrations of the granulosis capsules were applied to small disks of diet, 1 @disk, and were fed to early secondinstar H. cunea larvae, 5 larvae/disk. Each larva was assumed to receive 0.2 ~1 of inoculum. Treated and untreated larvae were placed in a 26°C incubator having a 12-hr photoperiod. Treatments consisting of 35 larvae/dilution were replicated twice.
377 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-2011
378
BOUCIAS
AND NORDIN
After 48 hr, those larvae consuming the upper surface of the disk were transferred to uncontaminated diet creamers (one larva/ creamer) and were returned to 26°C. Mortality was monitored every 48 hr until pupation. Fat tissue removed from the dead larvae was examined using phase-contrast optics. Only larvae showing definitive signs of granulosis were used in the calculations. RESULTS
sules/larva; lower limit: 3.0 x lo5 capsules/ larva). This value is significantly greater than the LD,, calculated for HcGV which is 7.06 x lo4 capsules/larva (upper limit: 1.79 x lo5 capsules/larva; lower limit: 2.67 x lo4 capsules/larva). Cross-infectivity tests by Hunter and Hoffman (1972) have shown a similar difference in the susceptibility of Plodia interpunctella infected with its own granulosis virus, compared with a granulosis virus isolate from Cadra cautella. Cumulative time-mortality data for dosages of DGV causing over 50% mortality are graphically depicted in Figure 2. In all cases the test larvae responded in a heterogenous fashion. The DGV is able to kill a small portion of the treated population within 15 days. The majority of treated larvae, however, did not begin to succumb until after 30 days postinoculation. LTSo values, calculated using the method of Litchfield (1949), are shown in Table 2. The incubation time required to kill 50% of the treated DGV was much greater than the values reported for HcGV-treated larvae (Boucias and Nordin, 1977). A dosage of 2.2 x lo* DGV capsules/larva, causing 100% mortality, had an LT,, value of 3.5 days. A dosage of 1.0 x IO7 HcGV capsules/larva, also causing 100% mortality, had an LT,, value of 6.1 days. The maximum calculated LT,, value for the DGV, 43 days, was achieved with a dosage of
AND DISCUSSION
Dosage-mortality
data for second-instar infected with DGV are summarized in Table 1. The number of larvae dying from granulosis increased with increased dosage. A small percentage of the tested larvae died from unknown causes. This mortality can be attributed either to the handling procedure or to the individual rearing of these normally gregarious larvae. None of the control larvae died from granulosis. Probit analysis2 was used to calculate the dosage-mortality regression line (Fig. 1). The slope of this regression line was 0.74 as opposed to a slope value of 1 .Ol previously reported for second-instar larvae fed the HcGV isolate (Boucias and Nordin, 1977). The calculated LD,, for DGV was 7.9 x lo5 capsules/larva (upper limit: 2.0 x lo6 capH. cunea larvae
2 SAS Computer University.
Program,
North
Carolina
State TABLE
1
DOSAGE-MORTALITY RESPONSE FORA GRANULOSISVIRUS FROM DIACRISIAVIRGINICA AGAINST SECOND-INSTAR HYPHANTRIACUNEA LARVAE
No. of capsules/larvae 2.2 2.2
x 108 x 10’
2.2 2.2 2.2
x 106
x 105 x 104
Control a Corrected mortality from unknown causes.
Total No. of larvae/dose 50 60 60 60 60 55
ASSAYED
No. of larvae dead from granulosis
No. of larvae dead from unknown causes
44 48
6 4
100
34
4
15 11
0
61 25 20 0
0
4 5
Corrected mortality” (%) 86
= No. of larvae dead from granulosis per total No. of larvae - No. of larvae dead
379
AND DGV FROM D~A~R~~IA
H~~~A~~RlA
TABLE 7-
2
THE
LTSo VALUES FOR THOSE DOSAGES OF DIACRISIA GRANULOSIS VIRUS CAUSING OVER 50% MORTALITY AGAINST SECOND-INSTAR HYPHANTR~A CUNEA LARVAE
Y=iZO + 74x
6-
95%
No. of capsules/larvae I 4
I 5
I 6
I 7
I 8
2.2 x 108 2.2 x 107 2.2 x 106
I 9
FOG. 1. Hyphanrria
Dosage-mo~~ity response of second-instar cunea larvae fed serial decimal dilutions of granulosis virus from Diacrisia virginica. Curved lines are the 95% confidence limits on the calculated dose-mortality line.
2.2 x 10” capsules/larva (61% mortality), as compared with the m~imum c~~uIated LT,, value for H&V, 11.5 days, which was achieved with a dosage of 1.0 x 105 capsules/larva (52% mortality). The reason for the prolonged incubation of DGV in H. cunea is not known. Infected larvae at 10 days postinoculation demonstrated typical symptoms of granulosis. As infection progressed, the integument lost all normal pigment, becoming white on both its dorsal and ventral surfaces,
Confidence limits
LT, (days)
Lower
Upper
35 39 43
33 29 26
37 52 71
The intact fat body, as observed with phase-contrast optics, contained huge numbers of capsules. The majority of larvae continued to develop until the prepupal or “wandering stage,” at which time they succumbed. In conclusion, it is possible to differentiate the DGV from the HcGV based on the dosage-mortality response of H. cunea larvae. Comparisons between the LDSO values have shown that DGV is less virulent to H. cunea larvae than is HcGV. Timemortality data also demonstrate that the LTS, values for dosages of DGV are four to six times greater than for compa~ble dosages of HcGV. Further studies are underway to determine what mechanisms
Days
FIG. 2. Time-mortality response of second-instar Hjphantriu cunea larvae fed serial decimal dilutions of granulosis virus from Diacrisia virginica. Dashed lines indicate the extrapolation to 84% mortality required for the LT,, calculation.
380
BOUCIAS
may be causing differences in virulence between the two viral isolates. REFERENCES BOUCIAS, D. G., AND NORDIN, G. L. 1977. Interinstar susceptibility of the fall webworm, Hyphantria cunea (Drury) to its nucleopolyhedrosis and granulosis viruses. J. Invertebr. Pathol. 30, 68-75. HUNTER, D. K., AND HOFFMAN, D. F. 1972. Cross infection of a granulosis virus of Cadra cautella with observations on its ultrastructure in infected cells of Plodia interpunctella. .I. Invertebr. Pathol., 20, 4- 10.
AND
NORDIN IGNOFFO,
C.
M.
1968.
Specificity
of insect
viruses.
Bull. Entomol. Sot. Amer., 14, 255-276. LITCHFIELD, .I. T. 1949. solution of time-percent
A method curves.
for
rapid
graphic
J. Pharmacol. Exp.
Ther., 97, 399-408. SUMMERS, M. D., AND PASHKE, J. D. 1970. Alkaliliberated granulosis virus of Trichoplusia ni I. Density gradient purification of virus components and some of their in vitro chemical and physical properties. J. Invertebr. Pathol., 16, 227-240. WILLIAMS, R. C., AND BACKUS, R. C. 1949. Macromolecular weights determined by direct particle counting. I. The weight of the bushy stunt virus.
J. Amer. Chem. Sot.,
71, 40.52-4057.