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Nuclear polyhedrosis virus production in Heliothis armigera infected at different larval ages
JOURNAL
OF INVERTEBRATE
PATHOLOGY
Nuclear Polyhedrosis
Virus Production in Heliothis at Different Larval Ages R.E.
Entomology
53, 21-24 (1989)
TEAKLE AND V.S.
armigera
Infected
BYRNE
Branch, Department of Primary Industries, Meiers Indooroopilly, Queensland, Australia 4068
Road,
Received December 8, 1986; accepted May 9, 1988 The quantity of virus produced per larva in Heliothis armigera larvae dosed at specified ages with a Heliothis nuclear polyhedrosis virus was determined in terms of both numbers of polyhedra and virus infectivity for H. arm&era, estimated by bioassay. The virus yield increased exponentially with age of larva at dosing in the age range 0 to 6 days, the overall increase being approximately lOO-fold. However, there was then a reduction in yield recorded for larvae dosed at 7 days. Additionally, the infectivity per polyhedron was significantly less than that of polyhedra from larvae dosed at lesser ages (P < 0.05). The ratio of virus produced to the corresponding dose to infect (LD,,) was at a maximum for larvae at hatching (0 day), further confirming the desirability of directing virus applications against newly hatched larvae. o 1989 Academic PWSS, IIIC.
INTRODUCTION A nuclear polyhedrosis virus (NPV) from H&this zea has been developed commercially as a specific Heliothis larvicide for use on field crops (Ignoffo and Couch, 1981). The virus formulation has been shown to be effective against other Heliothis species, including H. armigera which is a major pest species in Australia. An important feature of such a virus is its capacity to replicate in and spread through host populations. The efficiency of such spread depends on a number of factors, including the level of virus contamination in the environment of the susceptible larvae. Ignoffo (1966) estimated that at least 6 x IO9 virus polyhedra were produced per larva in late instars of H. zea, an amount of virus he defined as “one larval equivalent.” The average yield of virus per larva infected after 5 to 7 days at 30°C was 15 x IO9 polyhedra (Ignoffo, 1973). One NPVinfected H. armigera neonate at death (sec.-.nd instar) was estimated to contain 1.4 x IO6 LD,,‘s (4.5 x lo7 polyhedra), when assessed by bioassay using neonates (Teakle et al., 1985a). Evans et al. (1981) recorded a 170-fold increase in yield of polyhedra per larva from first to fifth instars of Mamestra bras-
sicae. The yield was directly correlated with larval weight at death, presumably reflecting a constant ratio of susceptible tissue to body weight. In Heliothis species, oviposition and subsequent larval development are often synchronized with flowering or panicle development of host plants. For example, oviposition on grain sorghum may be largely confined to a 5-day period between panicle emergence and commencement of flowering (Teakle et al., 1985b). Consequently, NPV applications, timed on the basis of panicle development, may be directed against predominantly newly hatched larvae. This results in the secondary release of virus from infected larvae into the environment of the Heliothis survivors. An understanding of the secondary cycles of virus production may be gained from a knowledge of the initial dose and susceptibility of the larvae treated, the amount of the virus released from infected larvae, and the susceptibility of surviving larvae at release of the virus. In the present study, the virus yield from larvae of H. armigera infected at different ages was determined both in terms of counts of virus polyhedra, and also in terms of virus activity, determined by bioassay. 21 0022-201 l/89 $1.50 Copyright All rights
Q 1989 by Academic Press, Inc of reproduction in any form resvved.
22
TEAKLE
AND
MATERIALS AND METHODS Infection of lurvue. Batches of larvae of H. armigera of specified ages (at 25°C) were dosed individually at 25°C with H. zea singly embedded nuclear polyhedrosis virus. Larval age at dosing was taken as the number of days after initial feeding. Thus neonates (
BYRNE
by Teakle et al. (1986). In this, initial feeding of the test neonates was confined to a small area of diet surface contaminated with a specified amount of virus. The number of LD,,‘s produced per larva could then be calculated from the expression: Number of polyhedra per larva LCss of virus from age sample x LCS,:LDS, RESULTS
ratio
AND DISCUSSION
Comparison of LC,, and LD,, Bioussuys Using H. urmigeru Neonates LCsO and LD,, ratios were determined on two occasions. Since the bioassay results were not significantly different (Z’ > 0.05) they were pooled and yielded the following results: LCso = 0.78 (0.70-0.86) polyhedra/mm* LD,, = 44.2 (38.0-51.7) ratio of LCsO to
(95% C.L.) diet surface; polyhedra; LD,, = 0.0176.
Amounts of NPV per Larva in H. urmigera at Death after Dosing at Specified Larval Ages The amounts of virus per dosed larva at death, estimated in terms of numbers of polyhedra and numbers of LD,,‘s for neonate H. urmigeru, are given in Figure 1. The number of LD,,‘s recorded per larva increased exponentially with larval age at dosing to a maximum at 6 days. The increase from 0 to 6 days was about loo-fold and could be expressed by the equation: log LDSO’s per larva = 7.30 + 0.25 [larval age (25°C) at dosing] (R2 = 0.99). (1) The number of LD,,-,‘s of virus recorded from larvae infected at 7 days was less than that for larvae dosed at 5 and 6 days, and only slightly higher than that for larvae dosed at 4 days. Furthermore, the LCs, obtained for the virus from larvae dosed at 7 days was higher than those for virus from
NPV
PRODUCTION
IN INFECTED
‘3
H. armigera
significantly less (P < 0.05) than that of polyhedra from larvae of lesser ages. The ratio of virus yield after infection to the dose required to infect (LD,,) at different larval ages (or “productivity ratio” of Evans et al. (1981), i.e., virus produced divided by LD,) would give an indication of the potential virus increase resulting from an infection. The rate of increase in LD,, with larval age can be expressed by the equation: log LD,,
AGE
OF LARVAE
(DAYS
AT 253
FIG. 1. Production of nuclear polyhedrosis virus in Heliothis arm&era dosed at different larval ages: W, number of polyhedra produced per larva when larvae were infected at age indicated; 0, number of LD,, values of virus produced per larva (determined by bioassay using H. armigera neonates).
larvae dosed at lesser ages (Table 1). Since the LCso values were based on doses determined by polyhedral count, the relative size should reflect the virus activity per polyhedron. This indicated that the activity per polyhedron from larvae dosed at 7 days was TABLE BIOASSAYS
OF NUCLEAR POLYHEDROSIS PREVIOUSLY DOSED
= 1.38 f 0.32 (age in days at 25°C) (R2 = 0.98) (2)
(Teakle et al., 1985a). A transformation lating virus yield to LD,, was derived substituting the age-LD,, expression into the equation for virus yield in terms age (1). This gave:
reby (2) of
log LD,,‘s (for neonates) per larva = 6.22 + 0.78 (log LD5, in polyhedra) (3) This indicates a maximum yield of virus relative to dose to infect at age 0. This ratio then declined slowly with larval age at infection to 6 days. To optimize virus applications with respect to potential secondary virus infection, applications should there1
VIRUS HARVESTED FROM LARVAE OF Heliothis WITH THE VIRUS AT DIFFERENT AGES
armigera
LGO Age of larvae at dosing (days at 25°C)
(95% confidence limits) (polyhedra/mm’ diet surface)” 0.78 (0.70-4l.86)” 0.99 (0.46-2.35) 0.54 (0.46-0.63) 1.95 (1.54-2.40) 1.55 (1.31-1.86) 0.77 (0.67-0.88) 1.51 (1.21-1.90) 3.19 (2.55-3.91)
Regression equation: Y=bx+c log dose/probit mortality Y= Y= Y= Y = Y= Y= Y= Y=
2.49x + 1.33x + 2.62x + 2.52.x + 2.16x + 3.01x + 1.81x + 2.69x +
5.27’ 5.0 5.7 4.3 4.6 5.3 4.7 3.6
n Doses based on hemocytometer counts of polyhedra. Diet-surface-contamination neonates as test insects. ’ Computed from pooled data of two bioassays.
armigera
SEofh 0.15 0.21 0.25 0.31 0.19 0.27 0.16 0.36 method used with H.
24
TEAKLE
AND
fore be timed to coincide with larval hatch. This would also give the greatest opportunity for virus released from infected larvae to be encountered by uninfected larvae before developmental resistance has reached a high level. The enormous amounts of infective virus produced by even very young larvae would explain how epizootics can develop in dense field or laboratory populations from small foci of virus contamination. ACKNOWLEDGMENTS Miss C. Howitt of the Biometry Branch gave statistical advice, and Dr. M. Bengston of the Entomology Branch kindly critically reviewed the manuscript. Drs. J. G. Atherton and G. H. S. Hooper of the University of Queensland supervised the study.
REFERENCES H. F., LOMER, C. J., AND KELLY, D. C. (1981). Growth of nuclear polyhedrosis virus in larvae of the cabbage moth, Mamestra brassicae L. Arch. Virol., 70, 207-214.
EVANS,
BYRNE
C. M. (1966). Standardization containing insect viruses. J. Invertebr.
IGNOFFO,
of products Pathol.,
8,
547-548. IGNOFFO,
C. M. (1973). Development of a viral insecticide: Concept to commercialization. Exp. Parasi-
tel., 33, 389-406. IGNOFFO, C. M., AND COUCH, T. cleopolyhedrosis virus of Heliothis
L. (1981). The nuspecies as a microbial insecticide. In “Microbial Control of Pests and Plant Diseases 1970-1980” (H. D. Burges, Ed.), pp. 329-362. Academic Press, London/New York. IGNOFFO, C. M., AND SHAPIRO, M. (1978). Characteristics of baculovirus preparations processed from living and dead larvae. J. Econ. Entomol., 71, 186188. TEAKLE, R. E., JENSEN, J. M., AND GILES, J. E. (1985a). Susceptibility of Heliothis armiger to a commercial nuclear polyhedrosis virus. .I. Znvertebr. Pathol., 46, 166-173. TEAKLE, R. E., JENSEN, J. M., AND GILES, J. E. (1986). Age-related suscentibilitv of Heliothis punctiger to a commercial formulation of nuclear polyhedrosis virus. J. Znvertebr. Pathol., 47,82-92. TEAKLE, R. E., JENSEN, J. M., AND MULDER, J. C. (1985b). Susceptibility of Heliothis armiger (Lepidoptera: Noctuidae) on sorghum to a nuclear polyhedrosis virus. J. Econ. Entomol., 78, 1373-1378.
OF INVERTEBRATE
PATHOLOGY
Nuclear Polyhedrosis
Virus Production in Heliothis at Different Larval Ages R.E.
Entomology
53, 21-24 (1989)
TEAKLE AND V.S.
armigera
Infected
BYRNE
Branch, Department of Primary Industries, Meiers Indooroopilly, Queensland, Australia 4068
Road,
Received December 8, 1986; accepted May 9, 1988 The quantity of virus produced per larva in Heliothis armigera larvae dosed at specified ages with a Heliothis nuclear polyhedrosis virus was determined in terms of both numbers of polyhedra and virus infectivity for H. arm&era, estimated by bioassay. The virus yield increased exponentially with age of larva at dosing in the age range 0 to 6 days, the overall increase being approximately lOO-fold. However, there was then a reduction in yield recorded for larvae dosed at 7 days. Additionally, the infectivity per polyhedron was significantly less than that of polyhedra from larvae dosed at lesser ages (P < 0.05). The ratio of virus produced to the corresponding dose to infect (LD,,) was at a maximum for larvae at hatching (0 day), further confirming the desirability of directing virus applications against newly hatched larvae. o 1989 Academic PWSS, IIIC.
INTRODUCTION A nuclear polyhedrosis virus (NPV) from H&this zea has been developed commercially as a specific Heliothis larvicide for use on field crops (Ignoffo and Couch, 1981). The virus formulation has been shown to be effective against other Heliothis species, including H. armigera which is a major pest species in Australia. An important feature of such a virus is its capacity to replicate in and spread through host populations. The efficiency of such spread depends on a number of factors, including the level of virus contamination in the environment of the susceptible larvae. Ignoffo (1966) estimated that at least 6 x IO9 virus polyhedra were produced per larva in late instars of H. zea, an amount of virus he defined as “one larval equivalent.” The average yield of virus per larva infected after 5 to 7 days at 30°C was 15 x IO9 polyhedra (Ignoffo, 1973). One NPVinfected H. armigera neonate at death (sec.-.nd instar) was estimated to contain 1.4 x IO6 LD,,‘s (4.5 x lo7 polyhedra), when assessed by bioassay using neonates (Teakle et al., 1985a). Evans et al. (1981) recorded a 170-fold increase in yield of polyhedra per larva from first to fifth instars of Mamestra bras-
sicae. The yield was directly correlated with larval weight at death, presumably reflecting a constant ratio of susceptible tissue to body weight. In Heliothis species, oviposition and subsequent larval development are often synchronized with flowering or panicle development of host plants. For example, oviposition on grain sorghum may be largely confined to a 5-day period between panicle emergence and commencement of flowering (Teakle et al., 1985b). Consequently, NPV applications, timed on the basis of panicle development, may be directed against predominantly newly hatched larvae. This results in the secondary release of virus from infected larvae into the environment of the Heliothis survivors. An understanding of the secondary cycles of virus production may be gained from a knowledge of the initial dose and susceptibility of the larvae treated, the amount of the virus released from infected larvae, and the susceptibility of surviving larvae at release of the virus. In the present study, the virus yield from larvae of H. armigera infected at different ages was determined both in terms of counts of virus polyhedra, and also in terms of virus activity, determined by bioassay. 21 0022-201 l/89 $1.50 Copyright All rights
Q 1989 by Academic Press, Inc of reproduction in any form resvved.
22
TEAKLE
AND
MATERIALS AND METHODS Infection of lurvue. Batches of larvae of H. armigera of specified ages (at 25°C) were dosed individually at 25°C with H. zea singly embedded nuclear polyhedrosis virus. Larval age at dosing was taken as the number of days after initial feeding. Thus neonates (
BYRNE
by Teakle et al. (1986). In this, initial feeding of the test neonates was confined to a small area of diet surface contaminated with a specified amount of virus. The number of LD,,‘s produced per larva could then be calculated from the expression: Number of polyhedra per larva LCss of virus from age sample x LCS,:LDS, RESULTS
ratio
AND DISCUSSION
Comparison of LC,, and LD,, Bioussuys Using H. urmigeru Neonates LCsO and LD,, ratios were determined on two occasions. Since the bioassay results were not significantly different (Z’ > 0.05) they were pooled and yielded the following results: LCso = 0.78 (0.70-0.86) polyhedra/mm* LD,, = 44.2 (38.0-51.7) ratio of LCsO to
(95% C.L.) diet surface; polyhedra; LD,, = 0.0176.
Amounts of NPV per Larva in H. urmigera at Death after Dosing at Specified Larval Ages The amounts of virus per dosed larva at death, estimated in terms of numbers of polyhedra and numbers of LD,,‘s for neonate H. urmigeru, are given in Figure 1. The number of LD,,‘s recorded per larva increased exponentially with larval age at dosing to a maximum at 6 days. The increase from 0 to 6 days was about loo-fold and could be expressed by the equation: log LDSO’s per larva = 7.30 + 0.25 [larval age (25°C) at dosing] (R2 = 0.99). (1) The number of LD,,-,‘s of virus recorded from larvae infected at 7 days was less than that for larvae dosed at 5 and 6 days, and only slightly higher than that for larvae dosed at 4 days. Furthermore, the LCs, obtained for the virus from larvae dosed at 7 days was higher than those for virus from
NPV
PRODUCTION
IN INFECTED
‘3
H. armigera
significantly less (P < 0.05) than that of polyhedra from larvae of lesser ages. The ratio of virus yield after infection to the dose required to infect (LD,,) at different larval ages (or “productivity ratio” of Evans et al. (1981), i.e., virus produced divided by LD,) would give an indication of the potential virus increase resulting from an infection. The rate of increase in LD,, with larval age can be expressed by the equation: log LD,,
AGE
OF LARVAE
(DAYS
AT 253
FIG. 1. Production of nuclear polyhedrosis virus in Heliothis arm&era dosed at different larval ages: W, number of polyhedra produced per larva when larvae were infected at age indicated; 0, number of LD,, values of virus produced per larva (determined by bioassay using H. armigera neonates).
larvae dosed at lesser ages (Table 1). Since the LCso values were based on doses determined by polyhedral count, the relative size should reflect the virus activity per polyhedron. This indicated that the activity per polyhedron from larvae dosed at 7 days was TABLE BIOASSAYS
OF NUCLEAR POLYHEDROSIS PREVIOUSLY DOSED
= 1.38 f 0.32 (age in days at 25°C) (R2 = 0.98) (2)
(Teakle et al., 1985a). A transformation lating virus yield to LD,, was derived substituting the age-LD,, expression into the equation for virus yield in terms age (1). This gave:
reby (2) of
log LD,,‘s (for neonates) per larva = 6.22 + 0.78 (log LD5, in polyhedra) (3) This indicates a maximum yield of virus relative to dose to infect at age 0. This ratio then declined slowly with larval age at infection to 6 days. To optimize virus applications with respect to potential secondary virus infection, applications should there1
VIRUS HARVESTED FROM LARVAE OF Heliothis WITH THE VIRUS AT DIFFERENT AGES
armigera
LGO Age of larvae at dosing (days at 25°C)
(95% confidence limits) (polyhedra/mm’ diet surface)” 0.78 (0.70-4l.86)” 0.99 (0.46-2.35) 0.54 (0.46-0.63) 1.95 (1.54-2.40) 1.55 (1.31-1.86) 0.77 (0.67-0.88) 1.51 (1.21-1.90) 3.19 (2.55-3.91)
Regression equation: Y=bx+c log dose/probit mortality Y= Y= Y= Y = Y= Y= Y= Y=
2.49x + 1.33x + 2.62x + 2.52.x + 2.16x + 3.01x + 1.81x + 2.69x +
5.27’ 5.0 5.7 4.3 4.6 5.3 4.7 3.6
n Doses based on hemocytometer counts of polyhedra. Diet-surface-contamination neonates as test insects. ’ Computed from pooled data of two bioassays.
armigera
SEofh 0.15 0.21 0.25 0.31 0.19 0.27 0.16 0.36 method used with H.
24
TEAKLE
AND
fore be timed to coincide with larval hatch. This would also give the greatest opportunity for virus released from infected larvae to be encountered by uninfected larvae before developmental resistance has reached a high level. The enormous amounts of infective virus produced by even very young larvae would explain how epizootics can develop in dense field or laboratory populations from small foci of virus contamination. ACKNOWLEDGMENTS Miss C. Howitt of the Biometry Branch gave statistical advice, and Dr. M. Bengston of the Entomology Branch kindly critically reviewed the manuscript. Drs. J. G. Atherton and G. H. S. Hooper of the University of Queensland supervised the study.
REFERENCES H. F., LOMER, C. J., AND KELLY, D. C. (1981). Growth of nuclear polyhedrosis virus in larvae of the cabbage moth, Mamestra brassicae L. Arch. Virol., 70, 207-214.
EVANS,
BYRNE
C. M. (1966). Standardization containing insect viruses. J. Invertebr.
IGNOFFO,
of products Pathol.,
8,
547-548. IGNOFFO,
C. M. (1973). Development of a viral insecticide: Concept to commercialization. Exp. Parasi-
tel., 33, 389-406. IGNOFFO, C. M., AND COUCH, T. cleopolyhedrosis virus of Heliothis
L. (1981). The nuspecies as a microbial insecticide. In “Microbial Control of Pests and Plant Diseases 1970-1980” (H. D. Burges, Ed.), pp. 329-362. Academic Press, London/New York. IGNOFFO, C. M., AND SHAPIRO, M. (1978). Characteristics of baculovirus preparations processed from living and dead larvae. J. Econ. Entomol., 71, 186188. TEAKLE, R. E., JENSEN, J. M., AND GILES, J. E. (1985a). Susceptibility of Heliothis armiger to a commercial nuclear polyhedrosis virus. .I. Znvertebr. Pathol., 46, 166-173. TEAKLE, R. E., JENSEN, J. M., AND GILES, J. E. (1986). Age-related suscentibilitv of Heliothis punctiger to a commercial formulation of nuclear polyhedrosis virus. J. Znvertebr. Pathol., 47,82-92. TEAKLE, R. E., JENSEN, J. M., AND MULDER, J. C. (1985b). Susceptibility of Heliothis armiger (Lepidoptera: Noctuidae) on sorghum to a nuclear polyhedrosis virus. J. Econ. Entomol., 78, 1373-1378.