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Susceptibility of Heliothis armiger to a commercial nuclear polyhedrosis virus
JOURNAL
OF INVERTEBRATE
Susceptibility
PATHOLOGY
of /-/eliothis armiger to a Commercial Polyhedrosis Virus R. E.
Depurtment
46, 166-173 (1985)
TEAKLE,
of Primary
J. M. Industries.
JENSEN, Indooroopilly.
AND
Nuclear
J. E. GILES
Queensland,
Australia
4068
Received January 14, 1985; accepted March 26, 1985 The susceptibility of Heliothis armiger larvae of different ages to a commercial nuclear polyhedrosis virus (NPV), Elcar, was determined by bioassay. The median lethal dosage (LD,,) increased HO-fold during the first week of larval life at 25°C. i.e.. during development to early fourth instar, but daily feeding rate and thus potential virus acquisition also increased. A linear relationship was determined between log LD,, and larval length, indicating that larval length constitutes a useful index for estimating the susceptibility of larval populations. Median lethal times (LT,,s) were similar for larvae tested at ages of 0 to 7 days and ranged from 3.6 to 8.0 days at 30°C. The amount of virus produced in a single, infected neonate was equivalent to 1.4 x IO6 LD,,s for neonates, a 900.000-fold increase on the dose supplied. The data support the practice of directing the NPV against neonates. but, on the basis of larval susceptibility alone, the age of larvae at treatment may not always be critical. D 1982 Academic Press, Inc. KEY WORDS: Heliothis urmiger; Nuclear polyhedrosis virus: Elcar; Bioassays.
Williams and Payne, 1984). However, quantitative data on susceptibility of H. armiger to NPV were inadequate to allow larval mortality to be predicted over a range of doses and larval ages. In this study, the susceptibility of H. armiger larvae of different ages to Elcar was quantified by bioassay. In conjunction with this, the relative daily food consumption of larvae was measured, to provide a guide to optimal virus usage.
INTRODUCTION
A commercial formulation of nuclear polyhedrosis virus (NPV), Elcar, has been registered for use on cotton and sorghum in Queensland following laboratory and field assessment (Teakle, 1979; Teakle et al., 1983; Rogers et al., 1983). However, reliability in usage of Elcar will be achieved only with a better understanding of factors influencing efficacy, including those relating to larval susceptibility. This prompted us to study relationships between larval development and susceptibility to this virus formulation. Lepidoptera generally display a marked reduction in susceptibility to Baculovirus infections with increasing larval age. The magnitude of the reduction is dependent on the particular virus-host system (Boucias and Nordin, 1977; Payne et al., 1981). Studies have consistently shown a decrease in susceptibility to NPV with increasing larval age in Heliothis zea and H. virescens (Ignoffo, 1966; Allen and Ignoffo, 1969; Stacey et al., 1977) and in H. armiger from Africa (Daoust, 1974; Whitlock, 1977; Matter and Zohdy, 1981; Flattery, 1983;
MATERIALS
166 0022-201
l/85 $1.50
Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
Larvae were dosed by a modification of the method of Chauthani (1968), in which initial feeding on a synthetic diet was confined to an Elcar-contaminated area of surface by means of a wax overlay. Larvae. The laboratory culture of H. urmiger was derived from apparently healthy individuals from several collections from sorghum in southeast Queensland during 1978 and early 1979. Testing commenced during the second half of 1979. Larvae were reared in batches of 8 to 15 on an artificial diet (Shorey and Hale, 1965) to a predetermined age at 25°C. Larvae tested within 1 day of hatching were assigned an age of 0
Hdiothis
nrrnigcr
AND
day on the assumption that feeding had not commenced prior to testing. Larval weight and length were determined on the larvae assigned as untreated controls. Larval instars were determined on the basis of head capsule measurements (Twine, 1978a), assuming six instars. However, it was subsequently determined that the larvae in the laboratory culture completed development in five instars, and instars given are based on those of 40 larvae reared for similar periods under similar conditions. Virus. Elcar (batch 82561) was received from Sandoz Inc., Homestead, Florida, via Shell Chemical Australia Pty. Ltd., and was held at 7°C. Doses used were based on the results of preliminary bioassays. On the day of the test, 0.5 g of Elcar was suspended in deionized water at pH 7 in a volumetric flask. From this stock, a suspension equal to the highest concentration required was prepared in 0.05% Triton X-100 in 0.067 M phosphate buffer, pH 7.0. This was mixed for 5 min to break up clumps, and serial twofold dilutions were prepared in 0.067 M phosphate buffer. Bioassay. Sixty larvae for each of five dilutions of Elcar were used. Larvae were dosed in 28-ml clear-plastic cups containing artificial diet (formaldehyde omitted) (Ignoffo and Garcia, 1968) with a wax overlay. The diet was dispensed to a depth of 1 cm in the cups on the day before the test, allowed to set, and refrigerated at 7°C overnight. On the day of the bioassay, the diet surface was air-dried and covered with a 2to 3-mm layer of paraffin wax (mp, 56°C) dispensed at 170” to 200°C from a Pasteur pipette. Just before use, a 1.5-mm-diameter hole was drilled through the wax and approximately 1 mm into the diet. A l-p1 aliquot of Elcar suspension was introduced through the hole to the exposed diet using an Oxford Ultra-Micro sampler. An additional 60 control larvae received 1 ~1 of 0.05% Triton X-100 in phosphate buffer, pH 7.0. Cups were discarded if the wax became detached from the diet. Larvae were added and incubated at 29 ? 1°C in the dark. Larvae whose head cap-
COMMERCIAL
NPV
167
sules were too large to fit into the hole soon widened the hole by chewing the wax to get to the diet. Mortality occurring after 2 days was recorded daily. Virus-killed larvae were soft and fragile and tended to flatten against the supporting substrate, usually the wax overlay or side of the container. Larvae with atypical symptoms were examined for virus polyhedra in the body contents by phase-contrast microscopy. When mortality and pupation were complete, LD,, values were computed by probit analysis (Finney, 1952). Computed median lethal doses (LD,,s) for larvae of the different ages are given in terms of polyhedra, assuming 4 x lo9 polyhedra/g Elcar. Compared with a standardized suspension of 4 x 10’ polyhedra, the potency of 1 g was 0.94 (0.69- 1.28) (95% confidence limits). Time-mortality response. Times to 50% mortality (LT,,s) were determined by the method of Gehan (1969), a method using life tables. Virus yield from infected neonate H. armiger. The virus contained in a virus-killed cadaver, previously dosed as a neonate with Elcar equivalent to 1.5 LD,,s, was quantified by bioassay, as viral activity may not be adequately represented by counts of polyhedra (Martignoni and Ignoffo, 1980). The larva, which died in the second instar, 5 days (at 30°C) after dosing, was carefully recovered from the wax overlay and suspended in 4 ml buffer, pH 7.0, with 0.05% Triton X-100. After mixing this stock suspension for 5 min. serial, threefold dilutions were used to dose neonate larvae using the bioassay method described above. From the computed LD,, value, the number of LD,,s for neonates in the virus-killed larva was calculated. Estimation of daily food consumption of H. armiger. Plastic cups (28 ml) half-filled with artificial diet and with a l-mm-thick wax overlay were prepared as for the bioassay. Larval access to the diet was provided via a l-mm-diameter hole drilled in the wax. After weighing, each cup received a neonate H. armiger larva and was incubated at 25°C. Daily food consumption was
168
TEAKLE,
JENSEN,
RESULTS Response
Data on length, weight, and dosage-mortality response for larval ages of 0 to 9 days are summarized in Table 1. Those for larvae of 0 day are from the combined results of three separate bioassays. Their high variability (heterogeneity factor, 3.61; df, 11) suggests that the method of bioassay has shortcomings for neonates. This could be attributed to low doses used, estimated to be as low as one polyhedron, and the small size of larvae and possibly extended period of viral ingestion. Only low mortality levels were recorded for larvae tested at ages of
Age-Related
Age
log LD,,
(days at 25°C)
Instar
L%
confidence limits; polyhedra)
(95%
Mean weight (mg IT SD)
0
I
1.7 + 0.2
0.1 c 0.20
1
I
3.0
t 0.5
0.3
2
I
3.4
+ 0.5
0.7 + 0.12
5.1
k 0.6
2.0 2 0.60
5.7 k 0.9
4.3 k 1.3
33‘ (23-45)
t 0.05
110 (87-
3 4
II
5
II, III (97.5, 2.5) II. III
6
1.6
13 -f 4.7
(Id. 90) III
13 k 1.4
29 + 5.6
7
IV
14 -t 1.5
38 2 16
8 9
IV IV, v (62.5, 37.5)
17 -c 2.7 22 e 2.5
68 k 26 142 f 36
a Based on 4 * y; mortality ’ From pooled d LD,, values
8.5
i
= 1.38 + 0.32 (age in days at 25°C) (r = 0.99).
I
DOSE-MORTALITY RESPONSES OF Heliothis armiger A COMMERCIAL NUCLEAR POLYHEDROS~S
Mean length (mm k SD)
Susceptibility
LD,, versus larval age. The relationship between log LD,, and larval age (0 to 7 days) could be described by the linear equation (Fig. 2)
TABLE SUMMARIZED
GILES
8 and 9 days, and the LD,, values, which were obtained by extrapolation, should be viewed with caution. Hence, the LD,, values for larvae of these ages are noted but generally not included in subsequent analysis of the results. Slopes for the dose-mortality relationships for larvae of various ages are shown in Figure 1. The dose-mortality responses of larvae of 0 to 7 days were consistent with a common slope of 1.69 (SE = 0.085). However, the dose-mortality responses of 8- and 9-day-old larvae had slopes that were significantly lower (P < 0.05) than those younger larvae. This indicates that the older larvae displayed greater variability in response to Elcar dose.
determined by removing, with a soft brush, the larva and larval faeces deposited on the wax overlay, and reweighing. The larval instar was also noted. This procedure was continued for 11 days, by which time the larvae were in the final instar. A final estimate was made at 25 days. Data were based on 25 to 45 larvae which developed normally. Ten similar cups without larvae were weighed as blanks.
Dosage-Mortality
AND
140) 140 (110-170) 350 (280-450) 800 (510-l 100) 2300 (1800-3000) 5100 (3700-7500) 3.4 x 1oa 4OOXlW
x IV/g product. (in probits); x, log dose (polyhedra). results of three bioassays. were obtained by extrapolation from low levels of mortality.
LARVAE VIRUS
DOSED WITH
ELCAR,
Dosage-mortality regression line Cy = bx t aJb
SE of B
Lb, (per mg body wt)
y = 1.76x
t 2.34
0.21
230
y = 2.19x
+ 1.28
0.30
170
y = 1.61~
+ 1.72
0.22
150
y = 1.93~
t 0.86
0.22
68
y = 1.66x
+ 0.78
0.21
82
y = 1.42.x + 0.87
0.22
64
y = 1.59.X t
0.34
0.22
80
y = 1.32x
t
0.11
0.23
130
Y = 0.49.x . y = 0.311
t
3.25
0.22
50,000
+ 3.29
0.23
3 x 106
Hdiofhis
W/X&~ AND COMMERCIAL
169
NPV
i!~g$y$y&c 10
102
103 104 105 Dose (polyhedral/larval1
106
107
106
109
FIG. 1. Dosage-mortality responses of Heliothis arlarvae aged 0 to 9 days to a commercial nuclear polyhedrosis virus. rniger
Providing temperatures remain within the “normal range” (Wigglesworth, 1965, p. 616), thermal summation can be used to estimate LD,, in terms of age at any range of temperatures. The transformation, age in days at 25°C =
lationship was found between LD,, and larval weight. Data from Table 1 indicate that the LD,, per unit of body weight declined to a minimum between larval ages of 2 and 7 days and then increased. LTSo versus larval age. LT,, values for larvae of ages 0 to 7 days varied from 3.6 to 8 days, and no consistent change in LT,, with larval age was apparent (Table 2). However, the LT,, declined with increasing dose for larvae of the same age. Insufficient data were available to compare LT,, values for larvae of particular ages in assays with the same final level of mortality. Recorded LT,, values overlapped with expected time to prepupation at dosing for
mean temperature - developmental 25 - developmental zero
zero
x age (days),
with a developmental zero of 10.6”C (Twine, 1978b), gives the expression log LD,,
= 1.38 + 0.32
mean temperature 14.4
LD,, versus larval length. A linear relationship determined between log LDso and larval length was defined by the equation (Fig. 3)
- 10.6
x
age (days).
larvae of 6 and 7 days, and also coincided with expected time to pupation for larvae aged 7 days. LT,, values were not obtained for larvae aged 8 and 9 days, as 50% mortality was not achieved in bioassays.
log LD,, = 1.35 + 0.17 (length in mm) (r = 0.98). 10,000 1 Lag mw= 1 35 + 0 17 length ,nm,
LD,, versus larval weight. No simple re-
1,000 3,000.
./
. loo-
*
/ 30.
*
1oi
01234567 Age (days)
Age (days)
FIG. 2. Relationship between median lethal dosage (LD,,) and larval age for Heliothis armiger treated with a commercial nuclear polyhedrosis virus.
0
2
4
6 6 Length
10 12 14 (mm)
FIG. 3. Relationship between median lethal dosage (LD5,,) and larval length for Heliothis rrrmiger treated with a commercial nuclear polyhedrosis virus.
170
TEAKLE.
Larval age (days at 25°C) 0 1 2 3 4 5 6 7
Range of final mortality levels”
JENSEN,
Range of LT,,, value, (days at 30°C)”
96.6-73.2 96.4-66.7 83.7, 72.0 94.8-56.9 82.5. 53.6 84.5-66. I 78.2 74.4. 57.9
AND GII,ES
Mean period to prepupation at dosing (days at 30°C)”
3.6-4.6 4.4-s. I 4.8. 5.5 4. I-4.9 4.6, 5.7
-
4.7-5.8 6.1 4.8, 8.0
6.8 5.7 4.8 4.2 4.0
8 9
-
Mean period to pupation at dosing (day5 at 30°C)”
-
9.3 8.5 7.0 6.7 6.0
a Dashes indicate values were not obtained.
Virus Yield from Infected Neonate H. armiger The yield of virus from a larva infected shortly after hatching was determined by bioassay using neonate H. armiger as an indication of minimum virus production per larva following Elcar treatment. There were 1.4 x lo6 LD,+ for neonates in the virus-killed larva, a 900,000-fold increase on the dose applied. Using the LD,, values from Table 1, the comparable number of LD,,s for larvae from 0 to 7 days are given in Table 3. Relationship Between Larval Age and Diet Consumption The estimated daily consumption of artificial diet is given in Fig. 4 and is expressed by log (diet consumption in mg) -0.12 + 0.28 (age in days at 25°C) (7 = 0.99). Relationship Between LD,, and Diet Consumption A transformation relating LD,, to diet consumption was derived by substituting the age diet consumption expression into the equation for LD,, in terms of age. This gave
log LD,,
= 1.36 + 1.12 log (diet consumption in mg). DISCUSSION
The dosage-mortality response data for H. armiger and the commercial NPV, Elcar, indicate a progressive increase in the virus dose required to infect with increasing larval age, as was established by previous workers. The LD,, increased approximately 150-fold during the first week of larval life (at 2X), which involved development to the early fourth instar. Subsequently, resistance to infection developed more rapidly with increasing larval age, but accurate quantification was not possible owing to limitations on the maximum dose able to be administered. The dosage-mortality responses were parallel for larvae up to 7 days of age, but these differed from the slopes of 8- to 9-day-old larvae. Allen and Ignoffo (1969), working with H. zea, also found that the slopes of the dosage-mortality responses were similar for larvae of ages 3 to 7 days, and that these were different from the slope at the age of 8 days. From the age of 7 days, LT,, values started to overlap with the expected period to pupation. Thus changes associated with pupation could have inhibited viral development in a significant proportion of larvae at
armiger AND COMMERCIAL
Heliothis
TABLE YIELD
OF NPV
171
NPV
3
FROM A NEONATE Heliothis urmiger LARVA DOSED WITH ELCAR IN TERMS OF MEDIAN DOSAGES (LD,,s) FOR LARVAE OF DIFFERENT AGES
LETHAL
Larval age (days)
No. of LD,,s per NPV-infected neonate H. armiger ( x 10’)
0
1
2
3
4
5
6
I
1400
900
410
320
130
56
20
Y
this stage (Whitlock, 1977; Evans, 1981). We have recently obtained evidence (Teakle et al., unpubl.) which indicates that early inhibition of NPV infection in H. punctiger is gut-associated, as it was expressed when the virus was provided per OS, but not when it was injected intrahemocoelically. However, at 24 hr after molting to the final (fifth) instar, larvae had become much more resistant to injected virus. This could mean that the increase in the rate of resistance development in the fourth instar in H. armiger is due to the virus now having to contend with the gutassociated mechanism(s) plus another type of resistance mechanism. The linear relationship obtained between log LD,,, and larval length indicates that larval length constitutes a potentially useful and convenient parameter for determining the relative susceptibility of H. armiger
11 012
., 34 5 6 7 8 Larval Age (days)
910
FIG. 4. Relationship between daily food consumption by Heliothis armiger and larval age.
populations, provided larvae are no older than early fourth instar. Allen and Ignoffo (1969) reported a twofold increase in LD,, per milligram body weight between the larval ages of 3 and 8 days. Although a similar increase applied between 3- and 7-day-old larvae in the present study, the comparative significance is lost when it is noted that the LD,, per milligram body weight of 3-day-old larvae is also half of that for larvae aged 0 to 2 days, an age range not investigated by Allen and Jgnoffo (1969). Along with Payne et al. (1981), we know of no reason why the infectivity of a virus (which replicates in its host) should be related to the weight of the host. The rate of increase in LD,, was only slightly greater than the rate of increase in food consumption of larvae up to 7 days of age. In effect, this means that a constant dose rate should give a nearly constant mortality in larvae up to the early fourth instar, in situations where the food mass consumed is proportional to the contaminated surface area, as would be expected for foliage of flowers. Payne et al. (1981) reached a similar conclusion for a granulosis virus in Pieris rapae. This would also appear to explain why some species have failed to show an increase in tolerance to a Baculovirus with larval age, when the virus was presented as contaminated foliage after dipping in graded virus concentrations (e.g., Smith et al., 1956; Cunningham, 1970). This allowed increases in feeding rate (virus acquisition) to compensate for increasing tolerance to virus with larval age. While it is desirable that the NPV be
172
TEAKLE.
JENSEN.
directed against young larvae in order to minimize crop damage (Stacey et al., 1977) and to take advantage of their favorable feeding habits on cotton (Allen et al., 1966: Potter and Watson. 1983). precision in timing, for example, to coincide with larva1 hatch, may not be required to achieve a certain level of mortality of H. nr’miger at a particular application rate. For larvae up to the fourth instar, at least, the increase in tolerance to the NPV with age could be largely offset by the increase in the rate of virus acquisition. We have recently obtained data from sorghum sprayed with NPV which is consistent with this thesis (Teakle et al., 1985). ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Mrs. P. C. Greenway; and the advice of J. C. Mulder of Biometry Branch, and Dr. M. Bengston of this Branch and Dr. D. E. Pinnock. University of Adelaide, South Australia. This study was supervised by Dr. J. G. Atherton and Dr. G. H. S. Hooper of the University of Queensland. The Central Queensland Grain Sorghum Marketing Board kindly gave financial assistance.
REFERENCES ALLEN. G. E.. GREGORY. B. G.. AND BKAZZEL. J. R. 1966. Integration of the Heliothis nuclear polyhedrosis virus into a biological control program for cotton. J. Econ. Entomol.. 59, 1333-1336. ALLEN, G. E.. AND IGNOFFO, C. M. 1969. The nucleopolyhedrosis virus of Heliothis: Quantitative in vivo estimates of virulence. J. fni,errebr. Patl70/., 13, 378-381. BOIJCIAS, D. G., AND NORDIN. G. L. 1977. Interinstar susceptibility of the fall webworm, Hyphantria cctnea. to its nucleopolyhedrosis and granulosis viruses. J. Invertebr. Puthol., 30, 68-75. CHAUTHANI. A. R. 1968. Bioassay technique for insect viruses. J. Inlaertehr. Puthol.. 11, 242-245. CUNNINGHAM, J. C. 1970. Pathogenicity tests of nuclear polyhedrosis viruses infecting the eastern hemlock looper, Lambdinu jkelluriu fiscellnrict (Lepidoptera: Geometridae) Cunad. Enrorno/., 102, 1534- 1539. DAOUST, R. A. 1974. Weight-related susceptibility of larvae of Heliothis ~rmigevu to a crude nuclearpolyhedrosis virus preparation. J. Inverrebr. Purhol..
23, 400-401.
EVANS. H. F. 1981. Quantitative assessment of the relationships between dosage and response of the nu-
AND GILES clear polyhedrosis virus of M~lr)r~~.s/,.trl~~.o.s.~i~.t/c. J. Inr’er-tebr. Pa/ho/.. 37, 101 - 109. FINNI:).. E. J. 19.52. “Probit Analysis.” 2nd ed. Cambridge Univ. Press, London/New York. t;LATTERY. K. E. 1983. Bioassav of a uurified nuclear polyhedrosis virus against Heliotfris rrrrui,vo-rr. Ann. Appl. Biol., 102, 301-304. G~HAN. E. A. 1969. Estimating survival functions from the life table. J. Ckronic Dis.. 21, 629-644. IGNOFFO. C. M. 1966. Effects of age on mortality of Heliothis ;YN and Heliothis t9irescen.s larvae exposed to a nuclear-polyhedrosis virus. J. fnt,errebr. Puthol.,
8, 279-282.
C. M.. AND GARCIA. C. 1968. Formalin inactivation of nuclear-polyhedrosis virus. /. fnverrehr. Parhol.. 10, 430-437. MAKTIGNONI. M. E.. AND IGNOFFO. C. M. 1980. Biological activity of Bacl&)rirr!.r preparations: In View assay. 117 “Proceedings, Conference of Project V. Microbiological Control of Insect Pests. of the US/ USSR Joint Working Group on the Production of Substances by Microbiological Means. 2nd: 1980 Clearwater Beach. Florida, USA.” pp. 138- 153. MATTER. M. M.. ANT) ZOHDY. N. Z. M. 1981. Biotic efficiency of Bucillus thuringiensis Berl. and a nuclear-polyhedrosis virus on larvae of the American bollworm. Heliothis urI)zi,yeru Hbn. (Lepid., Noctuidael. Z. Ange\t,. Ent.. 92. 336-343. PAYNE, C. C.. TATCHELL. G. M., AND WILLIAMS, C. F. 1981. The comparative susceptibilities of Pieris hrassicue and P. txpae to a granulosis virus from P. hrussicue. J. Invertehr. Pathol.. 38, ?73280. POTTER. M. F.. AND WATSON, T. F. 1983. Timing of nuclear polyhedrosis virus-bait spray combinations for control of egg and larval stages of tobacco budworm (Lepidoptera: Noctuidae). J. Econ. Entornol.. 76, 446-448. ROGERS. D. J.. TEAKLE. R. E.. AND BRIEK. H. B. 1983. Evaluation of Heiiothis nuclear polyhedrosis virus for control of Heliorhi., trrmiger on navy beans in Queensland. Gen. Appl. Enromol., 15, 31-34. SHORE’~‘.H. H.. AND HALE. R. L. 1965. Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. J. Econ. Entomol.. 58, 522-524. SMITH. 0. J.. HUGHES, K. M.. DUNN. P. H., AND HALL, I. M. 1956. A granulosis virus disease of the western grape leaf skeletonizer and its transmission. Cunud. Entornol. 88, 507-515. STACEY. A. L.. YOUNG. S. Y.. AND YEARIAN. W. C. 1977. Effect of larval age and mortality level on damage to cotton by Heliothis zeu infected with IGNOFFO.
Bucnlo~Gws
heliorhis.
J. Econ.
Enromol.,
70, 383-
386. TEAKLE. R. E. 1979. Relative pathogenicity of nuclear polyhedrosis viruses from Heliofhis pnnctigera and Heliothi.5 zeu for larvae of Heliothis urmigern and Heliothis puncligeru. J. Inverrebr. Puthol.. 34, 23l237.
He[iothis ~rnzip~r AND COMMERCIAL TEAKLE. R. E.. PAGE, F. D.. SABINE, B. N. E., AND GILES. J. E. 1983. Evaluation of Heliothis nuclear polyhedrosis virus for control of Heliothis armiger on sorghum in Queensland. Australia. Gen. Appl. Entomol.. 15, 11-18. TEAKLE, R. E., JENSEN, J. E., AND MULDER. J. C. 1985. Susceptibility of He/i&is armiger (Lepidoptera:Noctuidae) on sorghum to a nuclear polyhedrosis virus. J. Econ. Entomol., In Press. TWINE. P. H. 1978a. Variation in the number of larval instars of Heliothis urmigera (Hiibner) (Lepidoptera: Noctuidae). J. Aust. Entomol. Sot., 17, 289292.
NPV
173
TWINE, P. H. 1978b. Effect of temperature on the development of larvae and pupae of the corn earworm, Heliothis ctrmigera (Hiibner) (Lepidoptera: Noctuidae). Queerzsl. J. Agric. Anim. Sri., 35, 23-28. WHITLOCK. V. H. 1977. Effect of larval maturation on mortality induced by nuclear polyhedrosis and granulosis virus infections of Heliothis armigeru. J. Invertebr. Pathol., 30, 80-86. WIGGLESWORTH, V. B. 1965. “The Principles of Insect Physiology,” 6th ed. Methuen, London. WILLIAMS,?. F., AND PAYNE, C. C. 1984. The susceptibility of Heliorhis armigeru larvae to three nuclear polyhedrosis viruses. Ann. Appl. Bid.. 104, 4OS412.
OF INVERTEBRATE
Susceptibility
PATHOLOGY
of /-/eliothis armiger to a Commercial Polyhedrosis Virus R. E.
Depurtment
46, 166-173 (1985)
TEAKLE,
of Primary
J. M. Industries.
JENSEN, Indooroopilly.
AND
Nuclear
J. E. GILES
Queensland,
Australia
4068
Received January 14, 1985; accepted March 26, 1985 The susceptibility of Heliothis armiger larvae of different ages to a commercial nuclear polyhedrosis virus (NPV), Elcar, was determined by bioassay. The median lethal dosage (LD,,) increased HO-fold during the first week of larval life at 25°C. i.e.. during development to early fourth instar, but daily feeding rate and thus potential virus acquisition also increased. A linear relationship was determined between log LD,, and larval length, indicating that larval length constitutes a useful index for estimating the susceptibility of larval populations. Median lethal times (LT,,s) were similar for larvae tested at ages of 0 to 7 days and ranged from 3.6 to 8.0 days at 30°C. The amount of virus produced in a single, infected neonate was equivalent to 1.4 x IO6 LD,,s for neonates, a 900.000-fold increase on the dose supplied. The data support the practice of directing the NPV against neonates. but, on the basis of larval susceptibility alone, the age of larvae at treatment may not always be critical. D 1982 Academic Press, Inc. KEY WORDS: Heliothis urmiger; Nuclear polyhedrosis virus: Elcar; Bioassays.
Williams and Payne, 1984). However, quantitative data on susceptibility of H. armiger to NPV were inadequate to allow larval mortality to be predicted over a range of doses and larval ages. In this study, the susceptibility of H. armiger larvae of different ages to Elcar was quantified by bioassay. In conjunction with this, the relative daily food consumption of larvae was measured, to provide a guide to optimal virus usage.
INTRODUCTION
A commercial formulation of nuclear polyhedrosis virus (NPV), Elcar, has been registered for use on cotton and sorghum in Queensland following laboratory and field assessment (Teakle, 1979; Teakle et al., 1983; Rogers et al., 1983). However, reliability in usage of Elcar will be achieved only with a better understanding of factors influencing efficacy, including those relating to larval susceptibility. This prompted us to study relationships between larval development and susceptibility to this virus formulation. Lepidoptera generally display a marked reduction in susceptibility to Baculovirus infections with increasing larval age. The magnitude of the reduction is dependent on the particular virus-host system (Boucias and Nordin, 1977; Payne et al., 1981). Studies have consistently shown a decrease in susceptibility to NPV with increasing larval age in Heliothis zea and H. virescens (Ignoffo, 1966; Allen and Ignoffo, 1969; Stacey et al., 1977) and in H. armiger from Africa (Daoust, 1974; Whitlock, 1977; Matter and Zohdy, 1981; Flattery, 1983;
MATERIALS
166 0022-201
l/85 $1.50
Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
Larvae were dosed by a modification of the method of Chauthani (1968), in which initial feeding on a synthetic diet was confined to an Elcar-contaminated area of surface by means of a wax overlay. Larvae. The laboratory culture of H. urmiger was derived from apparently healthy individuals from several collections from sorghum in southeast Queensland during 1978 and early 1979. Testing commenced during the second half of 1979. Larvae were reared in batches of 8 to 15 on an artificial diet (Shorey and Hale, 1965) to a predetermined age at 25°C. Larvae tested within 1 day of hatching were assigned an age of 0
Hdiothis
nrrnigcr
AND
day on the assumption that feeding had not commenced prior to testing. Larval weight and length were determined on the larvae assigned as untreated controls. Larval instars were determined on the basis of head capsule measurements (Twine, 1978a), assuming six instars. However, it was subsequently determined that the larvae in the laboratory culture completed development in five instars, and instars given are based on those of 40 larvae reared for similar periods under similar conditions. Virus. Elcar (batch 82561) was received from Sandoz Inc., Homestead, Florida, via Shell Chemical Australia Pty. Ltd., and was held at 7°C. Doses used were based on the results of preliminary bioassays. On the day of the test, 0.5 g of Elcar was suspended in deionized water at pH 7 in a volumetric flask. From this stock, a suspension equal to the highest concentration required was prepared in 0.05% Triton X-100 in 0.067 M phosphate buffer, pH 7.0. This was mixed for 5 min to break up clumps, and serial twofold dilutions were prepared in 0.067 M phosphate buffer. Bioassay. Sixty larvae for each of five dilutions of Elcar were used. Larvae were dosed in 28-ml clear-plastic cups containing artificial diet (formaldehyde omitted) (Ignoffo and Garcia, 1968) with a wax overlay. The diet was dispensed to a depth of 1 cm in the cups on the day before the test, allowed to set, and refrigerated at 7°C overnight. On the day of the bioassay, the diet surface was air-dried and covered with a 2to 3-mm layer of paraffin wax (mp, 56°C) dispensed at 170” to 200°C from a Pasteur pipette. Just before use, a 1.5-mm-diameter hole was drilled through the wax and approximately 1 mm into the diet. A l-p1 aliquot of Elcar suspension was introduced through the hole to the exposed diet using an Oxford Ultra-Micro sampler. An additional 60 control larvae received 1 ~1 of 0.05% Triton X-100 in phosphate buffer, pH 7.0. Cups were discarded if the wax became detached from the diet. Larvae were added and incubated at 29 ? 1°C in the dark. Larvae whose head cap-
COMMERCIAL
NPV
167
sules were too large to fit into the hole soon widened the hole by chewing the wax to get to the diet. Mortality occurring after 2 days was recorded daily. Virus-killed larvae were soft and fragile and tended to flatten against the supporting substrate, usually the wax overlay or side of the container. Larvae with atypical symptoms were examined for virus polyhedra in the body contents by phase-contrast microscopy. When mortality and pupation were complete, LD,, values were computed by probit analysis (Finney, 1952). Computed median lethal doses (LD,,s) for larvae of the different ages are given in terms of polyhedra, assuming 4 x lo9 polyhedra/g Elcar. Compared with a standardized suspension of 4 x 10’ polyhedra, the potency of 1 g was 0.94 (0.69- 1.28) (95% confidence limits). Time-mortality response. Times to 50% mortality (LT,,s) were determined by the method of Gehan (1969), a method using life tables. Virus yield from infected neonate H. armiger. The virus contained in a virus-killed cadaver, previously dosed as a neonate with Elcar equivalent to 1.5 LD,,s, was quantified by bioassay, as viral activity may not be adequately represented by counts of polyhedra (Martignoni and Ignoffo, 1980). The larva, which died in the second instar, 5 days (at 30°C) after dosing, was carefully recovered from the wax overlay and suspended in 4 ml buffer, pH 7.0, with 0.05% Triton X-100. After mixing this stock suspension for 5 min. serial, threefold dilutions were used to dose neonate larvae using the bioassay method described above. From the computed LD,, value, the number of LD,,s for neonates in the virus-killed larva was calculated. Estimation of daily food consumption of H. armiger. Plastic cups (28 ml) half-filled with artificial diet and with a l-mm-thick wax overlay were prepared as for the bioassay. Larval access to the diet was provided via a l-mm-diameter hole drilled in the wax. After weighing, each cup received a neonate H. armiger larva and was incubated at 25°C. Daily food consumption was
168
TEAKLE,
JENSEN,
RESULTS Response
Data on length, weight, and dosage-mortality response for larval ages of 0 to 9 days are summarized in Table 1. Those for larvae of 0 day are from the combined results of three separate bioassays. Their high variability (heterogeneity factor, 3.61; df, 11) suggests that the method of bioassay has shortcomings for neonates. This could be attributed to low doses used, estimated to be as low as one polyhedron, and the small size of larvae and possibly extended period of viral ingestion. Only low mortality levels were recorded for larvae tested at ages of
Age-Related
Age
log LD,,
(days at 25°C)
Instar
L%
confidence limits; polyhedra)
(95%
Mean weight (mg IT SD)
0
I
1.7 + 0.2
0.1 c 0.20
1
I
3.0
t 0.5
0.3
2
I
3.4
+ 0.5
0.7 + 0.12
5.1
k 0.6
2.0 2 0.60
5.7 k 0.9
4.3 k 1.3
33‘ (23-45)
t 0.05
110 (87-
3 4
II
5
II, III (97.5, 2.5) II. III
6
1.6
13 -f 4.7
(Id. 90) III
13 k 1.4
29 + 5.6
7
IV
14 -t 1.5
38 2 16
8 9
IV IV, v (62.5, 37.5)
17 -c 2.7 22 e 2.5
68 k 26 142 f 36
a Based on 4 * y; mortality ’ From pooled d LD,, values
8.5
i
= 1.38 + 0.32 (age in days at 25°C) (r = 0.99).
I
DOSE-MORTALITY RESPONSES OF Heliothis armiger A COMMERCIAL NUCLEAR POLYHEDROS~S
Mean length (mm k SD)
Susceptibility
LD,, versus larval age. The relationship between log LD,, and larval age (0 to 7 days) could be described by the linear equation (Fig. 2)
TABLE SUMMARIZED
GILES
8 and 9 days, and the LD,, values, which were obtained by extrapolation, should be viewed with caution. Hence, the LD,, values for larvae of these ages are noted but generally not included in subsequent analysis of the results. Slopes for the dose-mortality relationships for larvae of various ages are shown in Figure 1. The dose-mortality responses of larvae of 0 to 7 days were consistent with a common slope of 1.69 (SE = 0.085). However, the dose-mortality responses of 8- and 9-day-old larvae had slopes that were significantly lower (P < 0.05) than those younger larvae. This indicates that the older larvae displayed greater variability in response to Elcar dose.
determined by removing, with a soft brush, the larva and larval faeces deposited on the wax overlay, and reweighing. The larval instar was also noted. This procedure was continued for 11 days, by which time the larvae were in the final instar. A final estimate was made at 25 days. Data were based on 25 to 45 larvae which developed normally. Ten similar cups without larvae were weighed as blanks.
Dosage-Mortality
AND
140) 140 (110-170) 350 (280-450) 800 (510-l 100) 2300 (1800-3000) 5100 (3700-7500) 3.4 x 1oa 4OOXlW
x IV/g product. (in probits); x, log dose (polyhedra). results of three bioassays. were obtained by extrapolation from low levels of mortality.
LARVAE VIRUS
DOSED WITH
ELCAR,
Dosage-mortality regression line Cy = bx t aJb
SE of B
Lb, (per mg body wt)
y = 1.76x
t 2.34
0.21
230
y = 2.19x
+ 1.28
0.30
170
y = 1.61~
+ 1.72
0.22
150
y = 1.93~
t 0.86
0.22
68
y = 1.66x
+ 0.78
0.21
82
y = 1.42.x + 0.87
0.22
64
y = 1.59.X t
0.34
0.22
80
y = 1.32x
t
0.11
0.23
130
Y = 0.49.x . y = 0.311
t
3.25
0.22
50,000
+ 3.29
0.23
3 x 106
Hdiofhis
W/X&~ AND COMMERCIAL
169
NPV
i!~g$y$y&c 10
102
103 104 105 Dose (polyhedral/larval1
106
107
106
109
FIG. 1. Dosage-mortality responses of Heliothis arlarvae aged 0 to 9 days to a commercial nuclear polyhedrosis virus. rniger
Providing temperatures remain within the “normal range” (Wigglesworth, 1965, p. 616), thermal summation can be used to estimate LD,, in terms of age at any range of temperatures. The transformation, age in days at 25°C =
lationship was found between LD,, and larval weight. Data from Table 1 indicate that the LD,, per unit of body weight declined to a minimum between larval ages of 2 and 7 days and then increased. LTSo versus larval age. LT,, values for larvae of ages 0 to 7 days varied from 3.6 to 8 days, and no consistent change in LT,, with larval age was apparent (Table 2). However, the LT,, declined with increasing dose for larvae of the same age. Insufficient data were available to compare LT,, values for larvae of particular ages in assays with the same final level of mortality. Recorded LT,, values overlapped with expected time to prepupation at dosing for
mean temperature - developmental 25 - developmental zero
zero
x age (days),
with a developmental zero of 10.6”C (Twine, 1978b), gives the expression log LD,,
= 1.38 + 0.32
mean temperature 14.4
LD,, versus larval length. A linear relationship determined between log LDso and larval length was defined by the equation (Fig. 3)
- 10.6
x
age (days).
larvae of 6 and 7 days, and also coincided with expected time to pupation for larvae aged 7 days. LT,, values were not obtained for larvae aged 8 and 9 days, as 50% mortality was not achieved in bioassays.
log LD,, = 1.35 + 0.17 (length in mm) (r = 0.98). 10,000 1 Lag mw= 1 35 + 0 17 length ,nm,
LD,, versus larval weight. No simple re-
1,000 3,000.
./
. loo-
*
/ 30.
*
1oi
01234567 Age (days)
Age (days)
FIG. 2. Relationship between median lethal dosage (LD,,) and larval age for Heliothis armiger treated with a commercial nuclear polyhedrosis virus.
0
2
4
6 6 Length
10 12 14 (mm)
FIG. 3. Relationship between median lethal dosage (LD5,,) and larval length for Heliothis rrrmiger treated with a commercial nuclear polyhedrosis virus.
170
TEAKLE.
Larval age (days at 25°C) 0 1 2 3 4 5 6 7
Range of final mortality levels”
JENSEN,
Range of LT,,, value, (days at 30°C)”
96.6-73.2 96.4-66.7 83.7, 72.0 94.8-56.9 82.5. 53.6 84.5-66. I 78.2 74.4. 57.9
AND GII,ES
Mean period to prepupation at dosing (days at 30°C)”
3.6-4.6 4.4-s. I 4.8. 5.5 4. I-4.9 4.6, 5.7
-
4.7-5.8 6.1 4.8, 8.0
6.8 5.7 4.8 4.2 4.0
8 9
-
Mean period to pupation at dosing (day5 at 30°C)”
-
9.3 8.5 7.0 6.7 6.0
a Dashes indicate values were not obtained.
Virus Yield from Infected Neonate H. armiger The yield of virus from a larva infected shortly after hatching was determined by bioassay using neonate H. armiger as an indication of minimum virus production per larva following Elcar treatment. There were 1.4 x lo6 LD,+ for neonates in the virus-killed larva, a 900,000-fold increase on the dose applied. Using the LD,, values from Table 1, the comparable number of LD,,s for larvae from 0 to 7 days are given in Table 3. Relationship Between Larval Age and Diet Consumption The estimated daily consumption of artificial diet is given in Fig. 4 and is expressed by log (diet consumption in mg) -0.12 + 0.28 (age in days at 25°C) (7 = 0.99). Relationship Between LD,, and Diet Consumption A transformation relating LD,, to diet consumption was derived by substituting the age diet consumption expression into the equation for LD,, in terms of age. This gave
log LD,,
= 1.36 + 1.12 log (diet consumption in mg). DISCUSSION
The dosage-mortality response data for H. armiger and the commercial NPV, Elcar, indicate a progressive increase in the virus dose required to infect with increasing larval age, as was established by previous workers. The LD,, increased approximately 150-fold during the first week of larval life (at 2X), which involved development to the early fourth instar. Subsequently, resistance to infection developed more rapidly with increasing larval age, but accurate quantification was not possible owing to limitations on the maximum dose able to be administered. The dosage-mortality responses were parallel for larvae up to 7 days of age, but these differed from the slopes of 8- to 9-day-old larvae. Allen and Ignoffo (1969), working with H. zea, also found that the slopes of the dosage-mortality responses were similar for larvae of ages 3 to 7 days, and that these were different from the slope at the age of 8 days. From the age of 7 days, LT,, values started to overlap with the expected period to pupation. Thus changes associated with pupation could have inhibited viral development in a significant proportion of larvae at
armiger AND COMMERCIAL
Heliothis
TABLE YIELD
OF NPV
171
NPV
3
FROM A NEONATE Heliothis urmiger LARVA DOSED WITH ELCAR IN TERMS OF MEDIAN DOSAGES (LD,,s) FOR LARVAE OF DIFFERENT AGES
LETHAL
Larval age (days)
No. of LD,,s per NPV-infected neonate H. armiger ( x 10’)
0
1
2
3
4
5
6
I
1400
900
410
320
130
56
20
Y
this stage (Whitlock, 1977; Evans, 1981). We have recently obtained evidence (Teakle et al., unpubl.) which indicates that early inhibition of NPV infection in H. punctiger is gut-associated, as it was expressed when the virus was provided per OS, but not when it was injected intrahemocoelically. However, at 24 hr after molting to the final (fifth) instar, larvae had become much more resistant to injected virus. This could mean that the increase in the rate of resistance development in the fourth instar in H. armiger is due to the virus now having to contend with the gutassociated mechanism(s) plus another type of resistance mechanism. The linear relationship obtained between log LD,,, and larval length indicates that larval length constitutes a potentially useful and convenient parameter for determining the relative susceptibility of H. armiger
11 012
., 34 5 6 7 8 Larval Age (days)
910
FIG. 4. Relationship between daily food consumption by Heliothis armiger and larval age.
populations, provided larvae are no older than early fourth instar. Allen and Ignoffo (1969) reported a twofold increase in LD,, per milligram body weight between the larval ages of 3 and 8 days. Although a similar increase applied between 3- and 7-day-old larvae in the present study, the comparative significance is lost when it is noted that the LD,, per milligram body weight of 3-day-old larvae is also half of that for larvae aged 0 to 2 days, an age range not investigated by Allen and Jgnoffo (1969). Along with Payne et al. (1981), we know of no reason why the infectivity of a virus (which replicates in its host) should be related to the weight of the host. The rate of increase in LD,, was only slightly greater than the rate of increase in food consumption of larvae up to 7 days of age. In effect, this means that a constant dose rate should give a nearly constant mortality in larvae up to the early fourth instar, in situations where the food mass consumed is proportional to the contaminated surface area, as would be expected for foliage of flowers. Payne et al. (1981) reached a similar conclusion for a granulosis virus in Pieris rapae. This would also appear to explain why some species have failed to show an increase in tolerance to a Baculovirus with larval age, when the virus was presented as contaminated foliage after dipping in graded virus concentrations (e.g., Smith et al., 1956; Cunningham, 1970). This allowed increases in feeding rate (virus acquisition) to compensate for increasing tolerance to virus with larval age. While it is desirable that the NPV be
172
TEAKLE.
JENSEN.
directed against young larvae in order to minimize crop damage (Stacey et al., 1977) and to take advantage of their favorable feeding habits on cotton (Allen et al., 1966: Potter and Watson. 1983). precision in timing, for example, to coincide with larva1 hatch, may not be required to achieve a certain level of mortality of H. nr’miger at a particular application rate. For larvae up to the fourth instar, at least, the increase in tolerance to the NPV with age could be largely offset by the increase in the rate of virus acquisition. We have recently obtained data from sorghum sprayed with NPV which is consistent with this thesis (Teakle et al., 1985). ACKNOWLEDGMENTS We gratefully acknowledge the technical assistance of Mrs. P. C. Greenway; and the advice of J. C. Mulder of Biometry Branch, and Dr. M. Bengston of this Branch and Dr. D. E. Pinnock. University of Adelaide, South Australia. This study was supervised by Dr. J. G. Atherton and Dr. G. H. S. Hooper of the University of Queensland. The Central Queensland Grain Sorghum Marketing Board kindly gave financial assistance.
REFERENCES ALLEN. G. E.. GREGORY. B. G.. AND BKAZZEL. J. R. 1966. Integration of the Heliothis nuclear polyhedrosis virus into a biological control program for cotton. J. Econ. Entomol.. 59, 1333-1336. ALLEN, G. E.. AND IGNOFFO, C. M. 1969. The nucleopolyhedrosis virus of Heliothis: Quantitative in vivo estimates of virulence. J. fni,errebr. Patl70/., 13, 378-381. BOIJCIAS, D. G., AND NORDIN. G. L. 1977. Interinstar susceptibility of the fall webworm, Hyphantria cctnea. to its nucleopolyhedrosis and granulosis viruses. J. Invertebr. Puthol., 30, 68-75. CHAUTHANI. A. R. 1968. Bioassay technique for insect viruses. J. Inlaertehr. Puthol.. 11, 242-245. CUNNINGHAM, J. C. 1970. Pathogenicity tests of nuclear polyhedrosis viruses infecting the eastern hemlock looper, Lambdinu jkelluriu fiscellnrict (Lepidoptera: Geometridae) Cunad. Enrorno/., 102, 1534- 1539. DAOUST, R. A. 1974. Weight-related susceptibility of larvae of Heliothis ~rmigevu to a crude nuclearpolyhedrosis virus preparation. J. Inverrebr. Purhol..
23, 400-401.
EVANS. H. F. 1981. Quantitative assessment of the relationships between dosage and response of the nu-
AND GILES clear polyhedrosis virus of M~lr)r~~.s/,.trl~~.o.s.~i~.t/c. J. Inr’er-tebr. Pa/ho/.. 37, 101 - 109. FINNI:).. E. J. 19.52. “Probit Analysis.” 2nd ed. Cambridge Univ. Press, London/New York. t;LATTERY. K. E. 1983. Bioassav of a uurified nuclear polyhedrosis virus against Heliotfris rrrrui,vo-rr. Ann. Appl. Biol., 102, 301-304. G~HAN. E. A. 1969. Estimating survival functions from the life table. J. Ckronic Dis.. 21, 629-644. IGNOFFO. C. M. 1966. Effects of age on mortality of Heliothis ;YN and Heliothis t9irescen.s larvae exposed to a nuclear-polyhedrosis virus. J. fnt,errebr. Puthol.,
8, 279-282.
C. M.. AND GARCIA. C. 1968. Formalin inactivation of nuclear-polyhedrosis virus. /. fnverrehr. Parhol.. 10, 430-437. MAKTIGNONI. M. E.. AND IGNOFFO. C. M. 1980. Biological activity of Bacl&)rirr!.r preparations: In View assay. 117 “Proceedings, Conference of Project V. Microbiological Control of Insect Pests. of the US/ USSR Joint Working Group on the Production of Substances by Microbiological Means. 2nd: 1980 Clearwater Beach. Florida, USA.” pp. 138- 153. MATTER. M. M.. ANT) ZOHDY. N. Z. M. 1981. Biotic efficiency of Bucillus thuringiensis Berl. and a nuclear-polyhedrosis virus on larvae of the American bollworm. Heliothis urI)zi,yeru Hbn. (Lepid., Noctuidael. Z. Ange\t,. Ent.. 92. 336-343. PAYNE, C. C.. TATCHELL. G. M., AND WILLIAMS, C. F. 1981. The comparative susceptibilities of Pieris hrassicue and P. txpae to a granulosis virus from P. hrussicue. J. Invertehr. Pathol.. 38, ?73280. POTTER. M. F.. AND WATSON, T. F. 1983. Timing of nuclear polyhedrosis virus-bait spray combinations for control of egg and larval stages of tobacco budworm (Lepidoptera: Noctuidae). J. Econ. Entornol.. 76, 446-448. ROGERS. D. J.. TEAKLE. R. E.. AND BRIEK. H. B. 1983. Evaluation of Heiiothis nuclear polyhedrosis virus for control of Heliorhi., trrmiger on navy beans in Queensland. Gen. Appl. Enromol., 15, 31-34. SHORE’~‘.H. H.. AND HALE. R. L. 1965. Mass-rearing of the larvae of nine noctuid species on a simple artificial medium. J. Econ. Entomol.. 58, 522-524. SMITH. 0. J.. HUGHES, K. M.. DUNN. P. H., AND HALL, I. M. 1956. A granulosis virus disease of the western grape leaf skeletonizer and its transmission. Cunud. Entornol. 88, 507-515. STACEY. A. L.. YOUNG. S. Y.. AND YEARIAN. W. C. 1977. Effect of larval age and mortality level on damage to cotton by Heliothis zeu infected with IGNOFFO.
Bucnlo~Gws
heliorhis.
J. Econ.
Enromol.,
70, 383-
386. TEAKLE. R. E. 1979. Relative pathogenicity of nuclear polyhedrosis viruses from Heliofhis pnnctigera and Heliothi.5 zeu for larvae of Heliothis urmigern and Heliothis puncligeru. J. Inverrebr. Puthol.. 34, 23l237.
He[iothis ~rnzip~r AND COMMERCIAL TEAKLE. R. E.. PAGE, F. D.. SABINE, B. N. E., AND GILES. J. E. 1983. Evaluation of Heliothis nuclear polyhedrosis virus for control of Heliothis armiger on sorghum in Queensland. Australia. Gen. Appl. Entomol.. 15, 11-18. TEAKLE, R. E., JENSEN, J. E., AND MULDER. J. C. 1985. Susceptibility of He/i&is armiger (Lepidoptera:Noctuidae) on sorghum to a nuclear polyhedrosis virus. J. Econ. Entomol., In Press. TWINE. P. H. 1978a. Variation in the number of larval instars of Heliothis urmigera (Hiibner) (Lepidoptera: Noctuidae). J. Aust. Entomol. Sot., 17, 289292.
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TWINE, P. H. 1978b. Effect of temperature on the development of larvae and pupae of the corn earworm, Heliothis ctrmigera (Hiibner) (Lepidoptera: Noctuidae). Queerzsl. J. Agric. Anim. Sri., 35, 23-28. WHITLOCK. V. H. 1977. Effect of larval maturation on mortality induced by nuclear polyhedrosis and granulosis virus infections of Heliothis armigeru. J. Invertebr. Pathol., 30, 80-86. WIGGLESWORTH, V. B. 1965. “The Principles of Insect Physiology,” 6th ed. Methuen, London. WILLIAMS,?. F., AND PAYNE, C. C. 1984. The susceptibility of Heliorhis armigeru larvae to three nuclear polyhedrosis viruses. Ann. Appl. Bid.. 104, 4OS412.