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Age-related susceptibility of Heliothis punctiger to a commercial formulation of nuclear polyhedrosis virus
JOURNAL
OF INVERTEBRATE
PATHOLOGY
47, 82-92 (1986)
Age-Related Susceptibility of Heliothis punctiger to a Commercial Formulation of Nuclear Polyhedrosis Virus R. E. TEAKLE, Entomology
Branch,
Department
J. M. JENSEN, of Primary
Industries,
AND J. E. GILES Indooroopilly,
Australia,
4068
Received October 24, 1984; accepted September 3, 1985 The susceptibility of Heliothis punctiger to specified doses of a commercial formulation of nuclear polyhedrosis virus (NPV), Elcar, is described in terms of larval age, length, and weight and time-mortality response. Our bioassays indicated that the susceptibility of newly molted thirdinstar larvae was independent of the temperature history and rates of larval development. Consequently, a generalized expression relating susceptibility to age, at a range of temperatures, was derived. In addition to a progressive decrease in susceptibility with increasing larval instar, fluctuations in susceptibility at various periods within instars were recorded. Both of these changes in susceptibility were apparently mediated by the gut, as no such changes were recorded when nonoccluded virions were injected into fourth-instar larvae of different ages and newly molted fifth-instar larvae. However, a large reduction in susceptibility to injected virus was recorded at 24 hr postmolt in the fifth (final) instar. This indicated that a different resistance mechanism, not mediated by the gut, was then operating. 0 1986 Academic press, IX. KEY WORDS: Heliothis punctiger; nuclear polyhedrosis virus; NPV, susceptibility to; biological control.
INTRODUCTION Heliothis
punctiger
with increasing age (Allen and Ignoffo, 1969; Teakle et al., 1985a), a phenomenon reported for other baculoviruses (Boucias et al., 1980; Evans, 1981; Payne et al., 1981). However, Keeley and Vinson (1975) showed that the susceptibility of H. zea to NPV diminished with injections of p-ecdysone. Therefore, hormonal changes at molting may influence susceptibility to NPV. Thus there is a need to define larval development in terms of both age (e.g., hours or days at a specified temperature) and instar and period postmolt. Few studies of susceptibility to baculoviruses within larval instars have been reported. Kobayashi et al. (1969) showed that a general decrease in susceptibility to NPV as silkworm (Bombyx mori) larvae aged was interrupted by increases in susceptibility at 24 or 48 hr postmolt in three instars and also at 72 or 96 hr postmolt (25°C) in the lifth instar. The change in susceptibility as larvae developed was less with virions extracted from polyhedra, injected intrahemocoelically (IH), than when intact polyhedra were administered per OS.
(syn. H. punctigera)
and Heliothis armiger, which is the subject of a separate study (Teakle et al., 1985a), are the major pest Heliothis spp. in Australia. H. punctiger attacks over a dozen horticultural and field crops including cotton and lucerne ( = alfalfa), for which pest management programs are being developed (Hearn et al., 1981; D. E. Pinnock, pers. commun.). More selective measures to control this pest are being sought, largely to conserve the considerable natural control which exists on many crops and to delay the development of resistance to broad-spectrum insecticides. An imported, commercially available nuclear polyhedrosis virus (NPV) specific for Heliothis spp. (Ignoffo, 1973) was found to be as potent for H. punctiger neonates as a local NPV isolate from H. punctiger (Teakle, 1979), but its virulence for later instar larvae is not known. The susceptibility to NPV of H. zea and H. armiger, assessed at daily intervals, showed a steady decline 82 0022-201 l/86 $1.50 Copyright 0 1986 by Academic Press, Inc. Au lights of reproduction in any form resewed.
Heliothis
puncriger
SUSCEPTIBILITY
They, therefore, postulated an ‘inhibitory mechanism associated with the gut. On the other hand, David (1978) reported that two strains of Pieris brassicae displayed differential susceptibility to a granulosis virus, regardless of whether the virus was administered per OS or IH. This suggests differences in a postgut defensive mechanism. Merritt (1977) recorded a decreasing susceptibility to per OS infection by an NPV in Spodoptera exigua as the larval age increased during the fourth instar, but the susceptibility of fourth- and fifth-instar larvae just after molting was similar. He also speculated that faster developing larvae might be more susceptible to the NPV than slower developing ones. No corresponding data are available for NPV in Heliothis spp. The potential for crop protection, safety, and facilities for production have already been established for the commercial NPV from H. zea but not for isolates from Heliothis in Australia. Host susceptibility constitutes one of the variables which, together with virus acquisition rate, virus stability, and dose achieved, needs to be defined to predict host mortality using the NPV (Pinneck and Brand, 1981; Payne, 1982). Therefore, in this study we have quantified changes in susceptibility to the commercial NPV associated with changes in larval age and size in H. punctiger.
MATERIALS
AND METHODS
Bioassay of Occluded NPV Larvae. The H. punctiger larvae were from an 8-year laboratory culture and normally completed their development in five instars at 30°C. To improve synchrony, larvae used were usually derived from eggs laid during an oviposition peak at dusk. Larvae were transferred at hatching to an artificial diet (Shorey and Hale, 1965) and kept individually at 30°C in “Costar” tissue culture trays sealed with 19.2-mm-diameter polypropylene balls (Neta Moulders, Mel-
TO
NPV
83
bourne, Victoria, Australia) (GrifIith et al., 1979). As larvae molted to the required instar (or hatched), sequential batches of 288 larvae (6 doses x 48 larvae) were assigned directly to contaminated diet for bioassay, or to fresh, uncontaminated diet in “Costar” trays for further incubation to the required age for bioassay. Analysis of variance of probit mortality was used to test for bias due to nonrandom assignment of larvae. The mean weight and length of larvae for each age were estimated from separate batches maintained under similar conditions, or, in some instances, from a sample of larvae actually used in bioassays. Each larva was weighed and length measured only once. Virus. A commercial NPV formulation, Elcar batch 82561, was obtained from Sandoz Inc., Homestead, Florida, and stored at 7°C. Its potency was determined as described by Teakle et al. (1983), except that the diet was dispensed into wells in “Costar” trays, with a diet surface area of 200 mm’ per well, and each well was contaminated with 30 ~1 of Elcar suspension. Relative to a semipurified suspension of 4 x lo9 polyhedra ex H. punctiger dosed with Elcar that had been standardized by hemocytometer count, the potency of 1 g Elcar was 0.94 (0.69- 1.28) (95% confidence limits). Bioassay. The method was based on one developed and used by I. P. Griffith (pers. commun.). Round- or flat-bottom microtiter trays or “Costar” trays, with holes of appropriate diameter drilled into the base of the wells, were placed on the surface of semimolten diet (Shorey and Hale, 1965. formaldehyde omitted; Ignoffo and Garcia, 1968) so that they were slightly embedded when the diet set. The diet surface which was exposed by the holes was then contaminated with the virus dose. The quantities of Elcar suspension, area of contaminated diet, and type of tray were selected to suit the larval instar. First-instar
84
TEAKLE,
JENSEN,
AND GILES
larvae received 0.33 ~1 of virus suspension (times to 50% mortality) were estimated by via l-mm-diameter holes, whereas second- the method of Gehan (1969), a method instar larvae received 1 ~1 of virus suspen- using life tables. sion via 2-mm-diameter holes. Later instars received 1 ~1 of virus suspension on diet Bioassay of Nonoccluded Virions exposed by 3-mm-diameter holes (Fig. 1). Larvae. Fourth- and fifth-instar larvae The stock suspension of the virus was reared to predetermined ages at 30°C were prepared in water, pK 7, containing 0.01% used. They were immobilized by cooling Triton X-100 mixed in an MSE homogejust before use. nizer for 5 min on the day of the test. Serial, Virus. Virions from larval hemolymph twofold dilutions were prepared in 0.067 M were used. Six-day-old (25°C) H. punctiger phosphate buffer, pH 7, containing 0.01% arvae were dosed with Elcar at 6 X lo3 Triton X-100 and 5% green food dye (Vin- polyhedra/mm2 diet surface, and bled after tage Products, Alderley, Queensland, Aus- 4 days at 30°C. The hemolymph was mixed tralia) added to trace the virus. The doses with 4 vol of cold (5°C) tissue culture meof 1 ~1 were delivered by means of an Ox- dium (MM medium) (Mitsuhashi and Marford Ultra-Micro Sampler, whereas the amorosch, 1964) to make handling easier. doses of 0.33 ~1 were delivered via a 0.5 ~1 After centrifugation at 4300g for 10 min, the Drummond Microcap capillary reduced to supernatant was passed through a Millipore two-thirds of its original length. The capil- filter, pore size 0.45 pm, and stored in OSlary was cemented to an Oxford “slimline” ml volumes in liquid N (Vaughn, 1972). Sampler tip (Cat. No. 910), which was Doses were expressed as dilutions of this taped to clear tubing of the same external stock virus suspension, which were made diameter. The tubing had a mouth-piece in cold MM medium. with cotton wool plug at the other end so Bioassay. Intrahemocoelic injections of that the virus dose could be expelled by 1 ~1 of virus dilutions were made using a “Burkard” microapplicator with an blowing (Fig. 1). The test insects were added and confined “Agla” all-glass syringe and a 30-gauge in the wells of microtiter trays by g-mm- needle. Usually 48, but sometimes 24, diameter steel balls (Griffith and Smith, larvae for each of five virus dilutions were 1977), or in wells of “Costar” trays by 19.2- used. Control larvae received 1 ~1 of MM mm-diameter polypropylene balls (Griffith medium. et al., 1979). Batches of 48 larvae (of apAfter injection, the larvae were mainpropriate age) per dose were used for each tained on artificial diet in “Costar” trays at of five twofold dilutions of Elcar. For con- 30°C. Mortality was recorded daily until all trols, a further batch of 48 larvae was the surviving larvae had pupated. treated with Elcar at the highest concentration used, after it had been boiled for 5 min RESULTS to inactivate the virus. Larvae which conDosage-Mortality Responses;. LDjO vs. sumed the dose within the allotted time (24 Larval Age hr or less), as indicated by the consumption of the green dye on the diet surface, were Table 1 records the dosage-mortality transferred to uncontaminated diet in data for H. punctiger larvae at defined “Costar” trays and further incubated at stages of development, and combines the 30°C to death or pupation. Mortality was results of up to five bioassays using larvae age. The slopes of the recorded daily and dosage-mortality re- of a particular sponses were computed by probit analysis dosage-mortality responses from larval (Finney, 1952). hatch to newly molted fourth instar were Time-mortality response. LT,, values typically low (Meynell and Meynell, 1965),
Heliofhis
punctiger
SUSCEPTIBILITY
TO NPV
FIG. 1. Microtiter trays and “Costar” tray with base of the wells drilled to expose diet to be contaminated with the virus for bioassay. The virus was applied using an Oxford Ultra-Micro Sampler (left) or a Sampler tip containing a Drummond Microcap capillary (inset) from which the liquid could be expelled by blowing. ./ m.s--.II.*
85
86
TEAKLE,
JENSEN, TABLE
SUMMARIZED
1
DOSAGE-MORTALITY RESPONSES OF Heliothis NUCLEAR POLYHEDROSIS VIRUS
Larval age Hr at Instar + 30°C hr at 30°C
Length 2 SD (mm)
Weight k SD (md
0 1st +o 6 +6 12 +12 24 +24 30 +30
1.5 1.7 1.9 2.6 3.0
-r- 0.13 k 0.16 2 0.10 2 0.26 2 0.33
0.06 0.09 0.12 0.38 0.47
42 2nd +0 48 +6 54 +12 66 +24
2.8 3.5 4.1 5.2
2 -I2 +-
74 3rd +0 86 +12 98 +24
5.5 I!Z 0.55 3.2 + 0.55 7.5 * 0.89 8.3 f 1.64 9.6 +- 1.02 17.2 + 3.71
AND GILES
punctiger LARVAE DOSED WITH A COMMERCIAL FORMULATION, ELCAR
LD,, (95% confidence limits (polyhedra)P
Dosage-mortality response (Y = bx + a)b
SE of b
~JWw body wt
r 0.01 2 0.01 It 0.02 zk 0.07 k 0.13
34 (28-42)c 76 (60- 102)‘ 51 (42-61)c 46 (30-62) 73 (58-89)
Y Y Y Y Y
= = = = =
1.55x + 1.37~ + 1.73x + 1.63~ + 2.2.5.x +
2.63 2.42 2.04 2.28 0.81
0.17 0.17 0.17 0.25 0.28
543 888 413 120 154
0.31 0.51 k 0.08 0.29 0.86 2 0.13 0.43 1.4 + 0.18 0.46 3.1 f 0.67
76 (66-86)d 80 (67-95)c 103 (80-133)‘+ 120 (94-147)C
Y Y Y Y
= = = =
1.80.x + 190x + 1.71.X + 1.43x +
1.90 1.37 1.57 2.02
0.29 0.19 0.20 0.16
15.5 93 74 38
Y = 1.49x + 1.26 Y = 1.08x + 0.42 Y = 1.75x - 0.30
0.09 0.12 0.13
98 252 64
115 4th +0 9.4 2 0.93 20.1 * 3.40 5,500 (4,000-7,000)’ 127 + 12 12.1 2 0.99 48.0 +- 7.76 24,000 (12,000-241,OOO)f 139 +24 16.6 t 1.73 87.1 2 12.70 6,000 (4,000-9,000)
Y = 1.08.x + 0.96 0.15 Y = 0.70.X + 1.93 0.22 Y = 1.17~ + 0.59 0.23
272 492 69
159 5th +0
Y = 0.63x + 0.99
16.4 + 1.41 86.4 2 9.21
320 (280-360) 2,100 (1,700-2,600)d 1,100 (950-1,300)d
2,100,000’
0.33
24,400
a Assuming 4 x IO9 polyhedra/g Elcar. b Y = mortality in probits, x = log dose (polyhedra). c Two bioassays combined. d Three bioassays combined. e Five bioassays combined. f LD, value obtained by extrapolation.
ranging from 1.08 to 2.25, and indicated a wide range of tolerances to the NPV within the populations. However, the similarity of most of the slopes indicated similar changes in response to increasing virus dose at most of the larval ages. The median lethal dosages (LD,,s) showed a general increase with larval instar (Table 1). Newly hatched larvae showed a 2-fold increase in LD,, in the first 6 hr, and there was a 160-fold increase from larval hatch to newly molted fourth instar. The individual LD,,, values are shown in Figure 2, common plots being used for LDS, values obtained with larvae from the same batch. Within an instar, larvae were most susceptible to the NPV (had lowest LD,,) at hatching or molting. Little overall decline in susceptibility occurred during the
first instar. No consistent change with larval age was recorded within the second instar, whereas within the third instar there was a trend for the general decline in susceptibility to be interrupted by an increase in susceptibility between 12 and 24 hr postmolt. Similar increases in susceptibility were recorded from 6 or 12 hr posthatch in the first instar or 12 hr postmolt in the fourth instar. As only low mortality was achieved with fourth-instar + 12-hr and fifth-instar + 0-hr larvae, the LD,, values were obtained by extrapolation in the probit analysis, and were not included in further analysis. Similarly, a low LD,, of 18.4 polyhedra obtained for newly molted second-instar larvae in one batch was inconsistent with other data and excluded from further analysis, although larvae from
Heliofhis
punctiger
SUSCEPTIBILITY
TO NPV
87
I i I I I I . I
IV ;
$9, ! I’. I 1. ? . ,, \ ! @
12
24 159
LARVAL
AGE
(HOURS
AT
3O’C)
FIG. 2. Relationships between median lethal dosage (LD,,) and larval age for Heliothis puncriger dosed with a commercial formulation of a nuclear polyhedrosis virus. Circled values were excluded from curve fitting.
the same batch tested normally later at different stages. The trend in the relationship between the LD,, and the larval age could be described by the quadratic equation log LD,,
= 1.68 - 0.0026 (age30) + 0.00019 (age30)2
where age30 is in hours at 30°C (R2 = 89%).
A generalized equation relating the susceptibility of the larvae to the NPV to their age at a range of temperatures can be derived, provided temperature remains within the normal range (Wigglesworth, 1965). This is obtained by substituting in the quadratic equation the transformation: age30 = age x (m.t. - d.z.)/(30 - d.z.)
88
TEAKLE,
JENSEN,
where m.t. = mean temperature (“C) and d.z. = developmental zero (“C). This is derived in the assumption that the temperature history of the H. punctiger does not influence the susceptibility of the NPV. Effect of Temperature History Susceptibility to NPV
on
No significant difference in susceptibility to the NPV between temperature groups reared at 20” and 30°C respectively, was found. The respective LD,,s (95% confidence limits) were 208 (129-292) and 236 (164-322) and relative susceptibility of larvae at 20°C versus those at 30°C was 1.07 (0.68-1.17). The respective times from hatch to third instar at 20” and 30°C indicated a developmental zero of 13.7”C by thermal summation. However, times to pupation at a range of temperatures, 16” to 3 l”C, indicated a developmental zero of 14.85”C for the laboratory culture by extrapolation from simple linear regression (unpubl.). Similar analysis of data on times to pupation from Cullen (1969) indicated a developmental zero of 12.4”C for field-collected H. punctiger in South Australia. Influence of Rate of Larval Development on Susceptibility to NPV As randomization of larvae was not practicable, it was necessary to determine whether the method of assigning larvae to
SUSCEPTIBILITY
Route of infection Per osa Injectio& Batch I Batch II
(LD,,)
OF Heliothis POLYHEDROSIS
AND GILES
age treatments introduced bias. Four sequential batches of newly molted, third-instar H. punctiger larvae, tested by bioassay, gave the following LD,,s (95% confidence limits): 293 (216-385), 404 (314-523), 286 (203-388), and 310 (200448). The estimated larval ages for the respective batches overlapped, owing to the time involved in transferring the newly hatched larvae to diet initially. Analysis of variance of probit mortalities for batch variation in H. punctiger indicated that they were not significantly different in susceptibility to the NPV (P > 0.05). From this, it was concluded that the method of assigning larvae to age treatments did not bias the results. LD,, for Nonoccluded Larval Age
Virus vs
Little change in susceptibility to nonoceluded virus administered by intrahemocoelic injection was recorded as larvae increased in age from early fourth to early fifth instar (Table 2). This contrasted with the results obtained when larvae of similar ages were dosed with Elcar per OS, as these showed fluctuating susceptibility within the fourth instar, and a large overall decrease in susceptibility (Table 2). This suggests that such changes were mediated by the gut. However, when testing of susceptibility to nonoccluded virus was extended into the
TABLE 2 punctiger IN FOURTH AND FIFTH INSTARS TO INFECTION VIRUS BY PER OS AND INTRAHEMOCOELIC ROUTES
BY A NUCLEAR
Larval age 4th + 0 hr 5.5 x 103 4.9 x (Y = 1.57.X + 4.3 x (Y = 1.61x +
10-4 10.20) 10-4 10.42)
4th + 12 hr 23.6 x 103b 3.1 x 10-h (Y = 1.28x + 9.49)
4th + 24 hr 6.0 x lo3
5th + 0 hr 2110 x 10rb
4.6 x lO-4 (Y = 1.04x + 8.47)
0 LD,, expressed as number of virus polyhedra, assuming 4 x lo9 per g Elcar. b Obtained by extrapolation. c LD, expressed as ml diluted infectious hemolymph. d Regression equation in brackets, Y = mortality in probits, x = log dose (polyhedra).
3.1 x 10-J (Y = 0.81x + 7.84)
Heliofhis
puncriger
SUSCEPTIBILITY
fifth (final) instar, a high level of resistance was recorded at 24 hr postmolt (Table 3), indicating the development of a different form of resistance, not mediated by the gut. From the dosage-mortality response of newly molted fifth-instar larvae dosed by IH injection, the reduction in mortality from 81.8 to 5% in the 24 hr after molting is equivalent to a 1380-fold increase in tolerance to the virus. LD,,
vs. Larval
TO NPV
x9
4 Log LD50 = 1.19 + 0.234 length (mm) .
i
Size
LD,, vs. larval length. A linear relationship between log LD,, and larval length was described by
log LD,, (polyhedra) = 1.19 + 0.234 length (mm) CR2 = 88%)
(Fig. 3). Larval length constitutes a convenient index from which the susceptibility, or tolerance, to the NPV of H. punctiger can be determined, although the elasticity of larvae could provide a source of error. LD,, vs. larval weight. No simple relationship was apparent between LD,, and larval weight or LD,, per milligram body weight. It is apparent from Table 1 that larvae of similar weight but different instar can differ markedly in their susceptibility to the NPV. Even within instars, the LD,, per milligram body weight differed by factors of 7.4, 4.1, and 3.9 for first to third instars, respectively. Table 1 shows that LD,, per milligram body weight takes a maximum value between 0 and 24 hr within each instar, except for the second instar in which LD,, per milligram body weight deTABLE
3
MORTALITY (%) OF FIFTH-INSTAR Heliorhis punctiger OF DIFFERENT AGES AFTER INJECTIONWITH NONOCCLUDEDNUCLEARPOLYHEDROSISVIRLJS Larval age (hr) Dose”
0
24
48
72
96
4 x 10-4 32 x 1O-4
58.3 81.8
0 5
0 0
0 0
0 0
‘I ml of diluted infectious hemolymph.
(prepupa)
2
4 LARVAL
6 LENGTH
6
10
knm)
FIG. 3. Relationship between median lethal dosage (LD,,,) and larval length for Heliothis puncriger dosed with a commercial formulation of a nuclear polyhedrosis virus.
creased continuously and 24 hr. Time-Mortality
with age between
0
Response
The values of median lethal time (LT,,) for larvae to fourth instar (not given) ranged from 3.1 to 6.2 days, but there was no consistent change m LT,, with larval age. However, larvae of similar age declined in LT,, with increasing Elcar dose. The period to pupation and LT,, coincided at approximately 24 hr postmolt in the fourth instar. Thus changes associated with pupation may have interfered with viral infection contracted at this age. The degree of interference would then increase as the larval age at exposure to the virus further approached pupation (Evans, 1981). DISCUSSION
The decrease in susceptibility to the NPV recorded between instars and the fluctuations in susceptibility within instars were
90
TEAKLE,
JENSEN,
recorded only when the virus was dosed per OS. It was observed that third-instar larvae at 12 hr postmolt showed a higher rate of food passage than at 0 and 24 hr postmolt (Teakle, unpubl.), possibly reducing the opportunity for virion release and attachment in the midgut, reflected in the increased LD,,. Inter- and intrainstar changes were not found when the gut was bypassed by IH injection of the virus. This would suggest that hormonal changes at molting did not influence susceptibility to the virus, unless such hormonal involvement was confined to the midgut. The evidence supports the concept of a gut-associated inhibitory mechanism, as postulated by Kobayashi et al, (1969). Tinsley (1975) also hypothesized that the defensive mechanisms were directed toward the restriction or “containment” of baculovirus particles to the midgut tissue, and prevention of entry into the hemocoel. A major reduction in susceptibility to IHadministered virus was recorded in larvae 24 hr after they had molted to the final instar. The additive effects of this plus changes associated with the midgut are presumably responsible for the large increase in LD,, for virus administered per OS between late fourth instar and newly molted fifth instar. The larvae which succumbed were apparently those in which the virus gained access to the hemocoel before the onset of this “new” resistance mechanism. The timing of this mechanism suggests that changes associated with pupation (Vail and Gough, 1970) might be implicated. This additional resistance mechanism would confer considerable survival advantage, as it occurs when larvae approach their maximum feeding rate, and hence maximum potential rate of virus acquisition. In addition, the level of environmental contamination during a developing epizootic could be high, owing to release of virus from early infected larvae. Burges (1971) and Briese (1981) concluded that there was no substantial evidence for the development of resistance to viruses in the
AND GILES
field. Therefore, survival during epizootics may depend more on chance avoidance of lethal doses in the early, susceptible stages, rather than on the possession of special genes for resistance. The considerable variation in the LD,, values sometimes recorded for insect batches of the same rated age may reflect rapid, temporary changes in susceptibility, and this should be taken into account when intraspecific and interspecific comparisons of the susceptibility of larvae to NPV isolates are made, as was done by Ignoffo et al. (1983). Therefore, only consistent and relatively large differences should be considered important. It also underscores the need for precision in the age-selection of larvae for bioassays, when the larvae acquire a defined dose. On the other hand, where larvae feed without restriction on a contaminated food substrate, differences in rates of feeding (and therefore virus acquisition) may largely offset differences in agerelated susceptibility. In this event, the susceptibility can remain effectively unchanged over a range of larval ages. We have recently obtained data from H. armiger in the laboratory (Teakle et al., 1985a) and on sorghum in the field (Teakle et al., 1985b) which suggests that feeding rate increased at a rate which compensated for increases in tolerance to the virus for at least the first three instars. This would apply only to the consumption of very small or lamina-type structures where the surface area, and thus dose, is directly related to the mass consumed.
ACKNOWLEDGMENTS
Dr. D. E. Pinnock, University of Adelaide, Dr. I. P. Griffith, Victorian College of Pharmacy, Parkville, Victoria, and Dr. M. Bengston and Mr. J. C. Mulder of this Department provided valuable advice. This study was also supervised by Dr. J. G. Atherton and Dr. G. H. S. Hooper of the University of Queensland. Financial support was provided by the Central Queensland Grain Sorghum Marketing Board. This assistance is gratefully acknowledged.
Heliothis
punctiger
SUSCEPTIBILITY
REFERENCES ALLEN, G. E., AND IGNOFFO, C. M. 1969. The nucleopolyhedrosis virus of Heiiothis: Quantitative in vivo estimates of virulence. J. Znvertebr. Pathol., 13, 378-381. BOLJCIAS, D. G., JOHNSON. D. W., AND ALLEN, G. E. 1980. Effects of host age, virus dosage. and temperature on the infectivity of a nucleopolyhedrosis virus against velvetbean caterpillar, Anficarsia gemmatulis, larvae. Environ. Entomol., 9, 59-61. BRIESE. D. T. 1981. Resistance of insect species to microbial pathogens. In “Pathogenesis of Invertebrate Microbial Diseases” (E. W. Davidson, ed.). pp. 51 l-545. Allenheld. Osmun, Totowa. NJ. BURGES. H. D. 1971. Possibilities of pest resistance to microbial control agents. In “Microbial Control of Insects and Mites” (H. D. Burges and N. W. Hussey eds.). pp. 445-457. Academic Press, London/New York. CULLEN. J. M. 1969. “The Reproduction and Survival of ffeliothis punctigera Wallengren in South Australia.” Ph.D. thesis, University of Adelaide, South Australia. DAVID. W. A. L. 1978. The granulosis virus of Pieris hrassicae (L.) and its relationship with its host. Adv. I’irus Res., 22, 112-161. EVANS. H. F. 1981. Quantitative assessment of the relationships between dosage and response of the nuclear polyhedrosis virus of Mamestra brassicue. J. Znvertebr. Pathol.. 31, 101-109. F~NNE’I’, E. J. 1952. “Probit Analysis.” 2nd ed. Cambridge Univ. Press. London/New York. GEHAN, E. A. 1969. Estimating survival functions from the life table. .Z. Chron. Dis.. 21, 629-644. GRIFFITH, I. P., AND SMITH, A. M. 1977. A convenient method for rearing lepidopteran larvae in isolation. J. Aust.
Enfomol.
Sot..
16, 366.
GRIFFITH, 1. P., SMtrH. A. M., AND WILLIAMSON, W. E. P. 1979. Raising potato moth larvae (Phthorimaea opercu/el/u (Zeller): Lepidoptera: Gelechiidae) in isolation. J. Aust. Entomol. Sot., 18, 348. HEARN, A. B.. IVES. P. M.. ROOM, P. M., THOMPSON, N. J.. AND WILSON, L. T. 1981. Computer-based cotton pest management in Australia. Field Crops Rrs.. 4, 321-332. IGNOFFO, C. M. 1973. Development of a viral insecticide: Concept to commercialization. Exp. Parasitol..
33, 380-406.
IGNOFFO, C. M., AND GARCIA, C. 1968. Formalin inactivation of nuclear-polyhedrosis virus. J. Znverrebr.
Pathol..
10, 430-432.
IGNOFFO, C. M.. MCINTOSH, A. M., AND GARCIA. C. 1983. Susceptibility of larvae of Heliothis zea. H. virescens, and H. armigera (Lep.: Noctuidae) to 3 Baculoviruses. Entomophaga, 28, 1-8.
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KEELEY, L. L., AND VINSON, S. B. 1975. B-Ecdysone effects on the development of nucleopolyhedrosis in Heliothis spp., J. Znvertebr. Pathol., 26, 121-123. KOBAYASHI, M.. YAMAGUCHI, S.. AND YOKOYAMA, Y. 1969. Influence of the larval development on the susceptibility of the silkworm, Bombyx mori L.. to the nuclear-polyhedrosis virus. J. Sericcrlt. Sci. Japan, 38, 481-487. MERRITT, D. L. 1977. “Factors Influencing the Susceptibility of the Beet Armyworm, Spodopteru e.i-. igua (Hubnet?. to a Nuclear Polyhedrosis Virus.’ Ph.D. thesis, University of California. Berkeley. MEYNELL. G. G., AND MEYNELL, E. 1965. “Theory and Practice in Experimental Bacteriology.” Cambridge Univ. Press. Cambridge. J., AND MARAMOROSCH, K. 1964. Leafhopper tissue culture: Embryonic, nymphal and imaginal tissues from aseptic insects. Contrih
MITSUHASHI.
Boyce
Thomp.son
Zmt.,
22, 435-460.
PAYNE. C. C. 1982. Insect viruses as control agents. Parasitology,
84, 35-77.
PAYNE, C. C., TATCHELL, G. M., AND WILLIAMS, C. F. 1981. The comparative susceptibilities of Pieris brussicae and P. rapae to a granulosis virus from P. hrcwic,ae. J. Zn\,ertebr. Pathol.. 38, 273280.
PINNOCK, D. E.. AND BRAND, R. J. 1981. A quantitative approach to the ecology of the use of pathogens for insect control. In “Microbial Control of Pest\ and Plant Diseases 1970-1980” (H. D. Burges, ed.). pp. 654-665. Academic Press. London/New York. SHOREY. 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, 572-524. TEAKLE, R. E. 1979. Relative pathogenicity of nuclear polyhedrosis viruses from Heliothis punctigera and Heliothis zecr for larvae of Heliothis armigera and Heliothis 237.
ptmctigero.
J. Invertebr.
Pathol..
34, 23 I --
TEAKLE. R. E.. JENSEN.J. M.. AND GILES. J. E. 1985a. Susceptibility of Heliothis armiger to a commercial nuclear polyhedrosis virus. J. Invertebr. Pathol. 46. 166-173. TEAKLE, R. E.. JENSEN, J. M.. AND MULDEK, J. C‘. 1985b. Susceptibility of Heliothis curniger (Lepidoptera: Noctuidae) on sorghum to nuclear polyhedrosis virus. J. &on. Entomol. 78, in press. TEAKLE. R. E., PAGE. F. D., SAHINE. B. N. E.. ANI) GILES, J. E. 1983. Evaluation of Heliothis nuclear polyhedrosis virus for control of Heliothk rrrmiyer
92
TEAKLE,
JENSEN,
on sorghum in Queensland, Australia. Gen. Appl. Entomol., 15, 11-18. TINSLEY, T. W. 1975. Factors affecting virus infection of insect gut tissue. In “Invertebrate ImmunityMechanisms of Invertebrate Vector-Parasite Reiations” (K. Maramorosch and R. E. Shope, eds.), pp. 55-63. Academic Press, New York/London. VAIL, P. V., AND GOUGH, D. 1970. Susceptibility of the
AND GILES
pupal stage of the cabbage looper, Trichoplusia ni, to nuclear-polyhedrosis virus. J. Invertebr. Pathol., 15, 211-218. VAUGHN, J. L. 1972. Long-term storage of hemolymph from insects infected with nuclear polyhedrosis virus. J. Znvertebr. Pathol., 20, 367-368. WIGGLESWORTH, V. B. 1965. “The Principles of Insect Physiology,” 6th ed. Methuen, London.
OF INVERTEBRATE
PATHOLOGY
47, 82-92 (1986)
Age-Related Susceptibility of Heliothis punctiger to a Commercial Formulation of Nuclear Polyhedrosis Virus R. E. TEAKLE, Entomology
Branch,
Department
J. M. JENSEN, of Primary
Industries,
AND J. E. GILES Indooroopilly,
Australia,
4068
Received October 24, 1984; accepted September 3, 1985 The susceptibility of Heliothis punctiger to specified doses of a commercial formulation of nuclear polyhedrosis virus (NPV), Elcar, is described in terms of larval age, length, and weight and time-mortality response. Our bioassays indicated that the susceptibility of newly molted thirdinstar larvae was independent of the temperature history and rates of larval development. Consequently, a generalized expression relating susceptibility to age, at a range of temperatures, was derived. In addition to a progressive decrease in susceptibility with increasing larval instar, fluctuations in susceptibility at various periods within instars were recorded. Both of these changes in susceptibility were apparently mediated by the gut, as no such changes were recorded when nonoccluded virions were injected into fourth-instar larvae of different ages and newly molted fifth-instar larvae. However, a large reduction in susceptibility to injected virus was recorded at 24 hr postmolt in the fifth (final) instar. This indicated that a different resistance mechanism, not mediated by the gut, was then operating. 0 1986 Academic press, IX. KEY WORDS: Heliothis punctiger; nuclear polyhedrosis virus; NPV, susceptibility to; biological control.
INTRODUCTION Heliothis
punctiger
with increasing age (Allen and Ignoffo, 1969; Teakle et al., 1985a), a phenomenon reported for other baculoviruses (Boucias et al., 1980; Evans, 1981; Payne et al., 1981). However, Keeley and Vinson (1975) showed that the susceptibility of H. zea to NPV diminished with injections of p-ecdysone. Therefore, hormonal changes at molting may influence susceptibility to NPV. Thus there is a need to define larval development in terms of both age (e.g., hours or days at a specified temperature) and instar and period postmolt. Few studies of susceptibility to baculoviruses within larval instars have been reported. Kobayashi et al. (1969) showed that a general decrease in susceptibility to NPV as silkworm (Bombyx mori) larvae aged was interrupted by increases in susceptibility at 24 or 48 hr postmolt in three instars and also at 72 or 96 hr postmolt (25°C) in the lifth instar. The change in susceptibility as larvae developed was less with virions extracted from polyhedra, injected intrahemocoelically (IH), than when intact polyhedra were administered per OS.
(syn. H. punctigera)
and Heliothis armiger, which is the subject of a separate study (Teakle et al., 1985a), are the major pest Heliothis spp. in Australia. H. punctiger attacks over a dozen horticultural and field crops including cotton and lucerne ( = alfalfa), for which pest management programs are being developed (Hearn et al., 1981; D. E. Pinnock, pers. commun.). More selective measures to control this pest are being sought, largely to conserve the considerable natural control which exists on many crops and to delay the development of resistance to broad-spectrum insecticides. An imported, commercially available nuclear polyhedrosis virus (NPV) specific for Heliothis spp. (Ignoffo, 1973) was found to be as potent for H. punctiger neonates as a local NPV isolate from H. punctiger (Teakle, 1979), but its virulence for later instar larvae is not known. The susceptibility to NPV of H. zea and H. armiger, assessed at daily intervals, showed a steady decline 82 0022-201 l/86 $1.50 Copyright 0 1986 by Academic Press, Inc. Au lights of reproduction in any form resewed.
Heliothis
puncriger
SUSCEPTIBILITY
They, therefore, postulated an ‘inhibitory mechanism associated with the gut. On the other hand, David (1978) reported that two strains of Pieris brassicae displayed differential susceptibility to a granulosis virus, regardless of whether the virus was administered per OS or IH. This suggests differences in a postgut defensive mechanism. Merritt (1977) recorded a decreasing susceptibility to per OS infection by an NPV in Spodoptera exigua as the larval age increased during the fourth instar, but the susceptibility of fourth- and fifth-instar larvae just after molting was similar. He also speculated that faster developing larvae might be more susceptible to the NPV than slower developing ones. No corresponding data are available for NPV in Heliothis spp. The potential for crop protection, safety, and facilities for production have already been established for the commercial NPV from H. zea but not for isolates from Heliothis in Australia. Host susceptibility constitutes one of the variables which, together with virus acquisition rate, virus stability, and dose achieved, needs to be defined to predict host mortality using the NPV (Pinneck and Brand, 1981; Payne, 1982). Therefore, in this study we have quantified changes in susceptibility to the commercial NPV associated with changes in larval age and size in H. punctiger.
MATERIALS
AND METHODS
Bioassay of Occluded NPV Larvae. The H. punctiger larvae were from an 8-year laboratory culture and normally completed their development in five instars at 30°C. To improve synchrony, larvae used were usually derived from eggs laid during an oviposition peak at dusk. Larvae were transferred at hatching to an artificial diet (Shorey and Hale, 1965) and kept individually at 30°C in “Costar” tissue culture trays sealed with 19.2-mm-diameter polypropylene balls (Neta Moulders, Mel-
TO
NPV
83
bourne, Victoria, Australia) (GrifIith et al., 1979). As larvae molted to the required instar (or hatched), sequential batches of 288 larvae (6 doses x 48 larvae) were assigned directly to contaminated diet for bioassay, or to fresh, uncontaminated diet in “Costar” trays for further incubation to the required age for bioassay. Analysis of variance of probit mortality was used to test for bias due to nonrandom assignment of larvae. The mean weight and length of larvae for each age were estimated from separate batches maintained under similar conditions, or, in some instances, from a sample of larvae actually used in bioassays. Each larva was weighed and length measured only once. Virus. A commercial NPV formulation, Elcar batch 82561, was obtained from Sandoz Inc., Homestead, Florida, and stored at 7°C. Its potency was determined as described by Teakle et al. (1983), except that the diet was dispensed into wells in “Costar” trays, with a diet surface area of 200 mm’ per well, and each well was contaminated with 30 ~1 of Elcar suspension. Relative to a semipurified suspension of 4 x lo9 polyhedra ex H. punctiger dosed with Elcar that had been standardized by hemocytometer count, the potency of 1 g Elcar was 0.94 (0.69- 1.28) (95% confidence limits). Bioassay. The method was based on one developed and used by I. P. Griffith (pers. commun.). Round- or flat-bottom microtiter trays or “Costar” trays, with holes of appropriate diameter drilled into the base of the wells, were placed on the surface of semimolten diet (Shorey and Hale, 1965. formaldehyde omitted; Ignoffo and Garcia, 1968) so that they were slightly embedded when the diet set. The diet surface which was exposed by the holes was then contaminated with the virus dose. The quantities of Elcar suspension, area of contaminated diet, and type of tray were selected to suit the larval instar. First-instar
84
TEAKLE,
JENSEN,
AND GILES
larvae received 0.33 ~1 of virus suspension (times to 50% mortality) were estimated by via l-mm-diameter holes, whereas second- the method of Gehan (1969), a method instar larvae received 1 ~1 of virus suspen- using life tables. sion via 2-mm-diameter holes. Later instars received 1 ~1 of virus suspension on diet Bioassay of Nonoccluded Virions exposed by 3-mm-diameter holes (Fig. 1). Larvae. Fourth- and fifth-instar larvae The stock suspension of the virus was reared to predetermined ages at 30°C were prepared in water, pK 7, containing 0.01% used. They were immobilized by cooling Triton X-100 mixed in an MSE homogejust before use. nizer for 5 min on the day of the test. Serial, Virus. Virions from larval hemolymph twofold dilutions were prepared in 0.067 M were used. Six-day-old (25°C) H. punctiger phosphate buffer, pH 7, containing 0.01% arvae were dosed with Elcar at 6 X lo3 Triton X-100 and 5% green food dye (Vin- polyhedra/mm2 diet surface, and bled after tage Products, Alderley, Queensland, Aus- 4 days at 30°C. The hemolymph was mixed tralia) added to trace the virus. The doses with 4 vol of cold (5°C) tissue culture meof 1 ~1 were delivered by means of an Ox- dium (MM medium) (Mitsuhashi and Marford Ultra-Micro Sampler, whereas the amorosch, 1964) to make handling easier. doses of 0.33 ~1 were delivered via a 0.5 ~1 After centrifugation at 4300g for 10 min, the Drummond Microcap capillary reduced to supernatant was passed through a Millipore two-thirds of its original length. The capil- filter, pore size 0.45 pm, and stored in OSlary was cemented to an Oxford “slimline” ml volumes in liquid N (Vaughn, 1972). Sampler tip (Cat. No. 910), which was Doses were expressed as dilutions of this taped to clear tubing of the same external stock virus suspension, which were made diameter. The tubing had a mouth-piece in cold MM medium. with cotton wool plug at the other end so Bioassay. Intrahemocoelic injections of that the virus dose could be expelled by 1 ~1 of virus dilutions were made using a “Burkard” microapplicator with an blowing (Fig. 1). The test insects were added and confined “Agla” all-glass syringe and a 30-gauge in the wells of microtiter trays by g-mm- needle. Usually 48, but sometimes 24, diameter steel balls (Griffith and Smith, larvae for each of five virus dilutions were 1977), or in wells of “Costar” trays by 19.2- used. Control larvae received 1 ~1 of MM mm-diameter polypropylene balls (Griffith medium. et al., 1979). Batches of 48 larvae (of apAfter injection, the larvae were mainpropriate age) per dose were used for each tained on artificial diet in “Costar” trays at of five twofold dilutions of Elcar. For con- 30°C. Mortality was recorded daily until all trols, a further batch of 48 larvae was the surviving larvae had pupated. treated with Elcar at the highest concentration used, after it had been boiled for 5 min RESULTS to inactivate the virus. Larvae which conDosage-Mortality Responses;. LDjO vs. sumed the dose within the allotted time (24 Larval Age hr or less), as indicated by the consumption of the green dye on the diet surface, were Table 1 records the dosage-mortality transferred to uncontaminated diet in data for H. punctiger larvae at defined “Costar” trays and further incubated at stages of development, and combines the 30°C to death or pupation. Mortality was results of up to five bioassays using larvae age. The slopes of the recorded daily and dosage-mortality re- of a particular sponses were computed by probit analysis dosage-mortality responses from larval (Finney, 1952). hatch to newly molted fourth instar were Time-mortality response. LT,, values typically low (Meynell and Meynell, 1965),
Heliofhis
punctiger
SUSCEPTIBILITY
TO NPV
FIG. 1. Microtiter trays and “Costar” tray with base of the wells drilled to expose diet to be contaminated with the virus for bioassay. The virus was applied using an Oxford Ultra-Micro Sampler (left) or a Sampler tip containing a Drummond Microcap capillary (inset) from which the liquid could be expelled by blowing. ./ m.s--.II.*
85
86
TEAKLE,
JENSEN, TABLE
SUMMARIZED
1
DOSAGE-MORTALITY RESPONSES OF Heliothis NUCLEAR POLYHEDROSIS VIRUS
Larval age Hr at Instar + 30°C hr at 30°C
Length 2 SD (mm)
Weight k SD (md
0 1st +o 6 +6 12 +12 24 +24 30 +30
1.5 1.7 1.9 2.6 3.0
-r- 0.13 k 0.16 2 0.10 2 0.26 2 0.33
0.06 0.09 0.12 0.38 0.47
42 2nd +0 48 +6 54 +12 66 +24
2.8 3.5 4.1 5.2
2 -I2 +-
74 3rd +0 86 +12 98 +24
5.5 I!Z 0.55 3.2 + 0.55 7.5 * 0.89 8.3 f 1.64 9.6 +- 1.02 17.2 + 3.71
AND GILES
punctiger LARVAE DOSED WITH A COMMERCIAL FORMULATION, ELCAR
LD,, (95% confidence limits (polyhedra)P
Dosage-mortality response (Y = bx + a)b
SE of b
~JWw body wt
r 0.01 2 0.01 It 0.02 zk 0.07 k 0.13
34 (28-42)c 76 (60- 102)‘ 51 (42-61)c 46 (30-62) 73 (58-89)
Y Y Y Y Y
= = = = =
1.55x + 1.37~ + 1.73x + 1.63~ + 2.2.5.x +
2.63 2.42 2.04 2.28 0.81
0.17 0.17 0.17 0.25 0.28
543 888 413 120 154
0.31 0.51 k 0.08 0.29 0.86 2 0.13 0.43 1.4 + 0.18 0.46 3.1 f 0.67
76 (66-86)d 80 (67-95)c 103 (80-133)‘+ 120 (94-147)C
Y Y Y Y
= = = =
1.80.x + 190x + 1.71.X + 1.43x +
1.90 1.37 1.57 2.02
0.29 0.19 0.20 0.16
15.5 93 74 38
Y = 1.49x + 1.26 Y = 1.08x + 0.42 Y = 1.75x - 0.30
0.09 0.12 0.13
98 252 64
115 4th +0 9.4 2 0.93 20.1 * 3.40 5,500 (4,000-7,000)’ 127 + 12 12.1 2 0.99 48.0 +- 7.76 24,000 (12,000-241,OOO)f 139 +24 16.6 t 1.73 87.1 2 12.70 6,000 (4,000-9,000)
Y = 1.08.x + 0.96 0.15 Y = 0.70.X + 1.93 0.22 Y = 1.17~ + 0.59 0.23
272 492 69
159 5th +0
Y = 0.63x + 0.99
16.4 + 1.41 86.4 2 9.21
320 (280-360) 2,100 (1,700-2,600)d 1,100 (950-1,300)d
2,100,000’
0.33
24,400
a Assuming 4 x IO9 polyhedra/g Elcar. b Y = mortality in probits, x = log dose (polyhedra). c Two bioassays combined. d Three bioassays combined. e Five bioassays combined. f LD, value obtained by extrapolation.
ranging from 1.08 to 2.25, and indicated a wide range of tolerances to the NPV within the populations. However, the similarity of most of the slopes indicated similar changes in response to increasing virus dose at most of the larval ages. The median lethal dosages (LD,,s) showed a general increase with larval instar (Table 1). Newly hatched larvae showed a 2-fold increase in LD,, in the first 6 hr, and there was a 160-fold increase from larval hatch to newly molted fourth instar. The individual LD,,, values are shown in Figure 2, common plots being used for LDS, values obtained with larvae from the same batch. Within an instar, larvae were most susceptible to the NPV (had lowest LD,,) at hatching or molting. Little overall decline in susceptibility occurred during the
first instar. No consistent change with larval age was recorded within the second instar, whereas within the third instar there was a trend for the general decline in susceptibility to be interrupted by an increase in susceptibility between 12 and 24 hr postmolt. Similar increases in susceptibility were recorded from 6 or 12 hr posthatch in the first instar or 12 hr postmolt in the fourth instar. As only low mortality was achieved with fourth-instar + 12-hr and fifth-instar + 0-hr larvae, the LD,, values were obtained by extrapolation in the probit analysis, and were not included in further analysis. Similarly, a low LD,, of 18.4 polyhedra obtained for newly molted second-instar larvae in one batch was inconsistent with other data and excluded from further analysis, although larvae from
Heliofhis
punctiger
SUSCEPTIBILITY
TO NPV
87
I i I I I I . I
IV ;
$9, ! I’. I 1. ? . ,, \ ! @
12
24 159
LARVAL
AGE
(HOURS
AT
3O’C)
FIG. 2. Relationships between median lethal dosage (LD,,) and larval age for Heliothis puncriger dosed with a commercial formulation of a nuclear polyhedrosis virus. Circled values were excluded from curve fitting.
the same batch tested normally later at different stages. The trend in the relationship between the LD,, and the larval age could be described by the quadratic equation log LD,,
= 1.68 - 0.0026 (age30) + 0.00019 (age30)2
where age30 is in hours at 30°C (R2 = 89%).
A generalized equation relating the susceptibility of the larvae to the NPV to their age at a range of temperatures can be derived, provided temperature remains within the normal range (Wigglesworth, 1965). This is obtained by substituting in the quadratic equation the transformation: age30 = age x (m.t. - d.z.)/(30 - d.z.)
88
TEAKLE,
JENSEN,
where m.t. = mean temperature (“C) and d.z. = developmental zero (“C). This is derived in the assumption that the temperature history of the H. punctiger does not influence the susceptibility of the NPV. Effect of Temperature History Susceptibility to NPV
on
No significant difference in susceptibility to the NPV between temperature groups reared at 20” and 30°C respectively, was found. The respective LD,,s (95% confidence limits) were 208 (129-292) and 236 (164-322) and relative susceptibility of larvae at 20°C versus those at 30°C was 1.07 (0.68-1.17). The respective times from hatch to third instar at 20” and 30°C indicated a developmental zero of 13.7”C by thermal summation. However, times to pupation at a range of temperatures, 16” to 3 l”C, indicated a developmental zero of 14.85”C for the laboratory culture by extrapolation from simple linear regression (unpubl.). Similar analysis of data on times to pupation from Cullen (1969) indicated a developmental zero of 12.4”C for field-collected H. punctiger in South Australia. Influence of Rate of Larval Development on Susceptibility to NPV As randomization of larvae was not practicable, it was necessary to determine whether the method of assigning larvae to
SUSCEPTIBILITY
Route of infection Per osa Injectio& Batch I Batch II
(LD,,)
OF Heliothis POLYHEDROSIS
AND GILES
age treatments introduced bias. Four sequential batches of newly molted, third-instar H. punctiger larvae, tested by bioassay, gave the following LD,,s (95% confidence limits): 293 (216-385), 404 (314-523), 286 (203-388), and 310 (200448). The estimated larval ages for the respective batches overlapped, owing to the time involved in transferring the newly hatched larvae to diet initially. Analysis of variance of probit mortalities for batch variation in H. punctiger indicated that they were not significantly different in susceptibility to the NPV (P > 0.05). From this, it was concluded that the method of assigning larvae to age treatments did not bias the results. LD,, for Nonoccluded Larval Age
Virus vs
Little change in susceptibility to nonoceluded virus administered by intrahemocoelic injection was recorded as larvae increased in age from early fourth to early fifth instar (Table 2). This contrasted with the results obtained when larvae of similar ages were dosed with Elcar per OS, as these showed fluctuating susceptibility within the fourth instar, and a large overall decrease in susceptibility (Table 2). This suggests that such changes were mediated by the gut. However, when testing of susceptibility to nonoccluded virus was extended into the
TABLE 2 punctiger IN FOURTH AND FIFTH INSTARS TO INFECTION VIRUS BY PER OS AND INTRAHEMOCOELIC ROUTES
BY A NUCLEAR
Larval age 4th + 0 hr 5.5 x 103 4.9 x (Y = 1.57.X + 4.3 x (Y = 1.61x +
10-4 10.20) 10-4 10.42)
4th + 12 hr 23.6 x 103b 3.1 x 10-h (Y = 1.28x + 9.49)
4th + 24 hr 6.0 x lo3
5th + 0 hr 2110 x 10rb
4.6 x lO-4 (Y = 1.04x + 8.47)
0 LD,, expressed as number of virus polyhedra, assuming 4 x lo9 per g Elcar. b Obtained by extrapolation. c LD, expressed as ml diluted infectious hemolymph. d Regression equation in brackets, Y = mortality in probits, x = log dose (polyhedra).
3.1 x 10-J (Y = 0.81x + 7.84)
Heliofhis
puncriger
SUSCEPTIBILITY
fifth (final) instar, a high level of resistance was recorded at 24 hr postmolt (Table 3), indicating the development of a different form of resistance, not mediated by the gut. From the dosage-mortality response of newly molted fifth-instar larvae dosed by IH injection, the reduction in mortality from 81.8 to 5% in the 24 hr after molting is equivalent to a 1380-fold increase in tolerance to the virus. LD,,
vs. Larval
TO NPV
x9
4 Log LD50 = 1.19 + 0.234 length (mm) .
i
Size
LD,, vs. larval length. A linear relationship between log LD,, and larval length was described by
log LD,, (polyhedra) = 1.19 + 0.234 length (mm) CR2 = 88%)
(Fig. 3). Larval length constitutes a convenient index from which the susceptibility, or tolerance, to the NPV of H. punctiger can be determined, although the elasticity of larvae could provide a source of error. LD,, vs. larval weight. No simple relationship was apparent between LD,, and larval weight or LD,, per milligram body weight. It is apparent from Table 1 that larvae of similar weight but different instar can differ markedly in their susceptibility to the NPV. Even within instars, the LD,, per milligram body weight differed by factors of 7.4, 4.1, and 3.9 for first to third instars, respectively. Table 1 shows that LD,, per milligram body weight takes a maximum value between 0 and 24 hr within each instar, except for the second instar in which LD,, per milligram body weight deTABLE
3
MORTALITY (%) OF FIFTH-INSTAR Heliorhis punctiger OF DIFFERENT AGES AFTER INJECTIONWITH NONOCCLUDEDNUCLEARPOLYHEDROSISVIRLJS Larval age (hr) Dose”
0
24
48
72
96
4 x 10-4 32 x 1O-4
58.3 81.8
0 5
0 0
0 0
0 0
‘I ml of diluted infectious hemolymph.
(prepupa)
2
4 LARVAL
6 LENGTH
6
10
knm)
FIG. 3. Relationship between median lethal dosage (LD,,,) and larval length for Heliothis puncriger dosed with a commercial formulation of a nuclear polyhedrosis virus.
creased continuously and 24 hr. Time-Mortality
with age between
0
Response
The values of median lethal time (LT,,) for larvae to fourth instar (not given) ranged from 3.1 to 6.2 days, but there was no consistent change m LT,, with larval age. However, larvae of similar age declined in LT,, with increasing Elcar dose. The period to pupation and LT,, coincided at approximately 24 hr postmolt in the fourth instar. Thus changes associated with pupation may have interfered with viral infection contracted at this age. The degree of interference would then increase as the larval age at exposure to the virus further approached pupation (Evans, 1981). DISCUSSION
The decrease in susceptibility to the NPV recorded between instars and the fluctuations in susceptibility within instars were
90
TEAKLE,
JENSEN,
recorded only when the virus was dosed per OS. It was observed that third-instar larvae at 12 hr postmolt showed a higher rate of food passage than at 0 and 24 hr postmolt (Teakle, unpubl.), possibly reducing the opportunity for virion release and attachment in the midgut, reflected in the increased LD,,. Inter- and intrainstar changes were not found when the gut was bypassed by IH injection of the virus. This would suggest that hormonal changes at molting did not influence susceptibility to the virus, unless such hormonal involvement was confined to the midgut. The evidence supports the concept of a gut-associated inhibitory mechanism, as postulated by Kobayashi et al, (1969). Tinsley (1975) also hypothesized that the defensive mechanisms were directed toward the restriction or “containment” of baculovirus particles to the midgut tissue, and prevention of entry into the hemocoel. A major reduction in susceptibility to IHadministered virus was recorded in larvae 24 hr after they had molted to the final instar. The additive effects of this plus changes associated with the midgut are presumably responsible for the large increase in LD,, for virus administered per OS between late fourth instar and newly molted fifth instar. The larvae which succumbed were apparently those in which the virus gained access to the hemocoel before the onset of this “new” resistance mechanism. The timing of this mechanism suggests that changes associated with pupation (Vail and Gough, 1970) might be implicated. This additional resistance mechanism would confer considerable survival advantage, as it occurs when larvae approach their maximum feeding rate, and hence maximum potential rate of virus acquisition. In addition, the level of environmental contamination during a developing epizootic could be high, owing to release of virus from early infected larvae. Burges (1971) and Briese (1981) concluded that there was no substantial evidence for the development of resistance to viruses in the
AND GILES
field. Therefore, survival during epizootics may depend more on chance avoidance of lethal doses in the early, susceptible stages, rather than on the possession of special genes for resistance. The considerable variation in the LD,, values sometimes recorded for insect batches of the same rated age may reflect rapid, temporary changes in susceptibility, and this should be taken into account when intraspecific and interspecific comparisons of the susceptibility of larvae to NPV isolates are made, as was done by Ignoffo et al. (1983). Therefore, only consistent and relatively large differences should be considered important. It also underscores the need for precision in the age-selection of larvae for bioassays, when the larvae acquire a defined dose. On the other hand, where larvae feed without restriction on a contaminated food substrate, differences in rates of feeding (and therefore virus acquisition) may largely offset differences in agerelated susceptibility. In this event, the susceptibility can remain effectively unchanged over a range of larval ages. We have recently obtained data from H. armiger in the laboratory (Teakle et al., 1985a) and on sorghum in the field (Teakle et al., 1985b) which suggests that feeding rate increased at a rate which compensated for increases in tolerance to the virus for at least the first three instars. This would apply only to the consumption of very small or lamina-type structures where the surface area, and thus dose, is directly related to the mass consumed.
ACKNOWLEDGMENTS
Dr. D. E. Pinnock, University of Adelaide, Dr. I. P. Griffith, Victorian College of Pharmacy, Parkville, Victoria, and Dr. M. Bengston and Mr. J. C. Mulder of this Department provided valuable advice. This study was also supervised by Dr. J. G. Atherton and Dr. G. H. S. Hooper of the University of Queensland. Financial support was provided by the Central Queensland Grain Sorghum Marketing Board. This assistance is gratefully acknowledged.
Heliothis
punctiger
SUSCEPTIBILITY
REFERENCES ALLEN, G. E., AND IGNOFFO, C. M. 1969. The nucleopolyhedrosis virus of Heiiothis: Quantitative in vivo estimates of virulence. J. Znvertebr. Pathol., 13, 378-381. BOLJCIAS, D. G., JOHNSON. D. W., AND ALLEN, G. E. 1980. Effects of host age, virus dosage. and temperature on the infectivity of a nucleopolyhedrosis virus against velvetbean caterpillar, Anficarsia gemmatulis, larvae. Environ. Entomol., 9, 59-61. BRIESE. D. T. 1981. Resistance of insect species to microbial pathogens. In “Pathogenesis of Invertebrate Microbial Diseases” (E. W. Davidson, ed.). pp. 51 l-545. Allenheld. Osmun, Totowa. NJ. BURGES. H. D. 1971. Possibilities of pest resistance to microbial control agents. In “Microbial Control of Insects and Mites” (H. D. Burges and N. W. Hussey eds.). pp. 445-457. Academic Press, London/New York. CULLEN. J. M. 1969. “The Reproduction and Survival of ffeliothis punctigera Wallengren in South Australia.” Ph.D. thesis, University of Adelaide, South Australia. DAVID. W. A. L. 1978. The granulosis virus of Pieris hrassicae (L.) and its relationship with its host. Adv. I’irus Res., 22, 112-161. EVANS. H. F. 1981. Quantitative assessment of the relationships between dosage and response of the nuclear polyhedrosis virus of Mamestra brassicue. J. Znvertebr. Pathol.. 31, 101-109. F~NNE’I’, E. J. 1952. “Probit Analysis.” 2nd ed. Cambridge Univ. Press. London/New York. GEHAN, E. A. 1969. Estimating survival functions from the life table. .Z. Chron. Dis.. 21, 629-644. GRIFFITH, I. P., AND SMITH, A. M. 1977. A convenient method for rearing lepidopteran larvae in isolation. J. Aust.
Enfomol.
Sot..
16, 366.
GRIFFITH, 1. P., SMtrH. A. M., AND WILLIAMSON, W. E. P. 1979. Raising potato moth larvae (Phthorimaea opercu/el/u (Zeller): Lepidoptera: Gelechiidae) in isolation. J. Aust. Entomol. Sot., 18, 348. HEARN, A. B.. IVES. P. M.. ROOM, P. M., THOMPSON, N. J.. AND WILSON, L. T. 1981. Computer-based cotton pest management in Australia. Field Crops Rrs.. 4, 321-332. IGNOFFO, C. M. 1973. Development of a viral insecticide: Concept to commercialization. Exp. Parasitol..
33, 380-406.
IGNOFFO, C. M., AND GARCIA, C. 1968. Formalin inactivation of nuclear-polyhedrosis virus. J. Znverrebr.
Pathol..
10, 430-432.
IGNOFFO, C. M.. MCINTOSH, A. M., AND GARCIA. C. 1983. Susceptibility of larvae of Heliothis zea. H. virescens, and H. armigera (Lep.: Noctuidae) to 3 Baculoviruses. Entomophaga, 28, 1-8.
TO
91
NPV
KEELEY, L. L., AND VINSON, S. B. 1975. B-Ecdysone effects on the development of nucleopolyhedrosis in Heliothis spp., J. Znvertebr. Pathol., 26, 121-123. KOBAYASHI, M.. YAMAGUCHI, S.. AND YOKOYAMA, Y. 1969. Influence of the larval development on the susceptibility of the silkworm, Bombyx mori L.. to the nuclear-polyhedrosis virus. J. Sericcrlt. Sci. Japan, 38, 481-487. MERRITT, D. L. 1977. “Factors Influencing the Susceptibility of the Beet Armyworm, Spodopteru e.i-. igua (Hubnet?. to a Nuclear Polyhedrosis Virus.’ Ph.D. thesis, University of California. Berkeley. MEYNELL. G. G., AND MEYNELL, E. 1965. “Theory and Practice in Experimental Bacteriology.” Cambridge Univ. Press. Cambridge. J., AND MARAMOROSCH, K. 1964. Leafhopper tissue culture: Embryonic, nymphal and imaginal tissues from aseptic insects. Contrih
MITSUHASHI.
Boyce
Thomp.son
Zmt.,
22, 435-460.
PAYNE. C. C. 1982. Insect viruses as control agents. Parasitology,
84, 35-77.
PAYNE, C. C., TATCHELL, G. M., AND WILLIAMS, C. F. 1981. The comparative susceptibilities of Pieris brussicae and P. rapae to a granulosis virus from P. hrcwic,ae. J. Zn\,ertebr. Pathol.. 38, 273280.
PINNOCK, D. E.. AND BRAND, R. J. 1981. A quantitative approach to the ecology of the use of pathogens for insect control. In “Microbial Control of Pest\ and Plant Diseases 1970-1980” (H. D. Burges, ed.). pp. 654-665. Academic Press. London/New York. SHOREY. 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, 572-524. TEAKLE, R. E. 1979. Relative pathogenicity of nuclear polyhedrosis viruses from Heliothis punctigera and Heliothis zecr for larvae of Heliothis armigera and Heliothis 237.
ptmctigero.
J. Invertebr.
Pathol..
34, 23 I --
TEAKLE. R. E.. JENSEN.J. M.. AND GILES. J. E. 1985a. Susceptibility of Heliothis armiger to a commercial nuclear polyhedrosis virus. J. Invertebr. Pathol. 46. 166-173. TEAKLE, R. E.. JENSEN, J. M.. AND MULDEK, J. C‘. 1985b. Susceptibility of Heliothis curniger (Lepidoptera: Noctuidae) on sorghum to nuclear polyhedrosis virus. J. &on. Entomol. 78, in press. TEAKLE. R. E., PAGE. F. D., SAHINE. B. N. E.. ANI) GILES, J. E. 1983. Evaluation of Heliothis nuclear polyhedrosis virus for control of Heliothk rrrmiyer
92
TEAKLE,
JENSEN,
on sorghum in Queensland, Australia. Gen. Appl. Entomol., 15, 11-18. TINSLEY, T. W. 1975. Factors affecting virus infection of insect gut tissue. In “Invertebrate ImmunityMechanisms of Invertebrate Vector-Parasite Reiations” (K. Maramorosch and R. E. Shope, eds.), pp. 55-63. Academic Press, New York/London. VAIL, P. V., AND GOUGH, D. 1970. Susceptibility of the
AND GILES
pupal stage of the cabbage looper, Trichoplusia ni, to nuclear-polyhedrosis virus. J. Invertebr. Pathol., 15, 211-218. VAUGHN, J. L. 1972. Long-term storage of hemolymph from insects infected with nuclear polyhedrosis virus. J. Znvertebr. Pathol., 20, 367-368. WIGGLESWORTH, V. B. 1965. “The Principles of Insect Physiology,” 6th ed. Methuen, London.