JOURNAL
OF INVERTEBRATE
PATHOLOGY
25, 343-348 (1975)
Bioassay of Nucleopolyhedrosis Larval lnstars of Malacosoma ALDO
Virus Against neustria
MAGNOLER
Stazione Sperimentale de1Sughero. 07029 Tempio Pausania, Sassari, Italy Received
June 27, 1974
The relative susceptibility of thirdand fourth-instar Malacosoma neustriu larvae to a nucleopolyhedrosis virus was studied by bioassay in the laboratory. Larval mortality increased with increased dosage, whereas the dosage-incubation relationship was reversed. Larval age inversely affected mortality and incubation. The computed LD,,‘s for third- and fourth-instar larvae were 1,405 and 12,320 polyhedral inclusion bodies (PIB’s)/larva. The median lethal doses calculated as number of PIB’s/mg body weight showed that third-instar larvae were only twice more susceptible to virus than fourths. The LT,, values for 3 x 105, 3 x IO*, and 3 x lo3 PIB’s/larva were 5.9,6.58, and 8.15 days, respectively, in third-instar assay; the corresponding figures for the two highest concentrations were 9.3 and 10.7 days in the older larvae. Lethally infected individuals died after one or, exceptionally, two molts. No correlation was found between pupal weight or adult emergence of survivors and the virus dose administered to the larvae.
The tent caterpillar, Malacosoma neustria. is one of the major forest pests in Sardinia. Population samples of this insect, collected at the end of an outbreak in 1964, were found to carry natural infection by a nucleopolyhedrosis virus (Sicker et al., 1965). During the same year, this virus caused considerable mortality among field populations. Studies were initiated to determine whether a nucleopolyhedrosis virus can be effectively used to control tent caterpillar infestations in cork oak stands. In a preliminary infectivity test, a virus isolate obtained from diseased individuals showed to be pathogenic for M. neustria larvae in the early stages of an outbreak. Since all caterpillars were not equally susceptible to virus infection, it was obvious to investigate the relationship between virus and host. Bioassay informations for other virusinfected Malacosoma specieswere reported (Bergold, 1953; Stairs, 1965). The present work attempts to quantify the virus dose required to attain different levels of mortality
for middle-aged larval stages of the tent caterpillar. MATERIALS
The nucleopolyhedrosis virus (NPV) used in this study was obtained from a field population during the 1968 infestation. Virusdiseased M. neustria larvae, collected in a cork forest, were macerated in water for several weeks and the resulting suspension was then strained through muslin and filtered. The filtrate was centrifuged at 655g for IO min once, and then at 4,095g for 20 min three times. The final pellet was resuspended in sterile distilled water and stored at 4°C. The number of polyhedra of this stock suspension was determined with a Thoma counting chamber. Based on 30 samples, the partially purified suspension contained an average of 1.79 + 0.16 x lo9 polyhedral inclusion bodies (PIB’s)/ml. For infection experiments, normally appearing M. neustria larvae were used. The insects were obtained from field-collected egg masses, previously sterilized with 10% formalin solution for 60 min. Larvae were grown on a semisynthetic
343 Copyright s 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
344
ALDO MAGNOLER
diet (Addy, 1969) and maintained at 24 + 0.5”C with 16-hr daylight throughout the experiment. Freshly molted third- and fourthinstar larvae, weighing 8.1 + 2.0 and 32.8 f 10.6 mg, respectively, were used in the bioassay. To estimate the pathogenicity of the virus, six decimal dilutions of the stock suspension were each tested against 25 larvae. With a microapplicator, 0.002 ml of each virus concentration were applied to the surface of a disk of diet, 1 mm thick x 2.5 mm in diameter, placed on the bottom of a small sterile vial. The vials were then infested with single larvae and plugged with Morton caps. Control larvae were fed sterile food. Those larvae which entirely consumed the treated disks within 36-48 hr only were included in the test, whereas all other larvae were discarded. Test larvae were transferred to individual sterile vials supplied with virus-free nutrient medium. Each treatment was replicated four times on third-instar larvae, and twice on fourth-instars. The tests were continued until the larvae had either died or pupated. The pupae were isolated in plastic cups for adult emergence and weighed to the nearest 0.1 mg 48 hr after pupation. Disease mortality was established by examining tissue smears under a phase contrast microscope.
RESULTS
AND DISCUSSION
Dose mortality data for both third- and fourth-instar M. neustriu larvae are summarized in Table 1. Mortality increased directly with dosage, but susceptibility to birus was inversely affected by the age of the larvae. Dosages of 3 x lo4 and 3 x lo5 PIB’s/larva only caused over 90% mortality when applied during the third- and fourthinstar, respectively. MicroscopicaI examination of dead larvae for polyhedra revealed that nonvirus deaths were less than 6% in any of the treatments. None of the control larvae died from virus disease. The pathogenicity of the NPV for thirdand fourth-instar larvae was compared by plotting the larval mortality as probits (Finney, 1964). The regression lines and the equations describing the curves for both larval stages are shown in Figure 1. The response lines were almost parallel, as it was confirmed by the similarity of the slopes (t (4) = 0.251, P > 0.05), and heterogeneity about each regression was nonsignificant (Table 2). The response line for younger larvae was the better fitting of the two, owing to the greater number of insects. However, it is suspected that the population quality (Laux, 1962; Franz and Laux, 1965) may have caused some deviation of larval
TABLE 1 Mortality of Third- and Fourth-Instar Malacosoma neustria Larvae Following Ingestion of Different Concentrations of Nuclear Polyhedra Third&star
Approx. No. PIB’s/larva
No. larvae in test
3x los 3x lo4 3 x lo3 3x lo2 3x 10 3 Control
63 67 69 66 66 68 68
assay0 Avg. percent larval mortality Unknown Polyhedrosis causes
‘All data based on four replicates. ‘All data based on two replicates.
100 95.5 62.3 19.7 4.5 0 0
0 4.5 4.3 4.5 1.5 5.9 1.5
Fourth-instar assay* Avg. percent larval mortality No. larvae in test
Polyhedrosis
Unknown causes
36 36 39 40 44 39 40
91.7 75.0 17.9 5.0 0 0 0
5.5 5.5 0 5.0 2.3 2.6 2.5
OF
r
responses from the straight line. Since variations of mortality responses were not excessive and the values for g were less than unity, the normal deviate for the 5% level of probability was used as multiplier of the standard error of Y to obtain the limits of probit mortality. These limits for the expected 10, 30, 50, 70, and 90% lethal doses were tabulated as PIB’s/larva or PIB’s/mg body weight (Table 3). A comparison based on the median lethal doses (LD5J showed that third-instar larvae were about nine times more susceptible to virus than fourthinstar larvae. However, only a twofold difference between the same two stages was obtained when the LD,, doses were calculated as number of inclusion bodies/mg of body weight. Further, the dosage-mortality lines indicated that 15 and 112 PIB’s/larva or 2 and 4 PIB’s/mg body weight were sufficient to induce lethal infections in a few third- and fourth-instar larvae, respectively. Using nuclear polyhedra against the forest tent caterpillar, Bergold (1953) estimated an oral LD,, of 5.5 x lo6 PIB’s/larva. This author, however, did not mention the specific larval stage used in his tests. Stairs (1965) showed that third-instar larvae of M. disstriu were 64 times more susceptible to a NPV than fourths. He pointed out that the
L3
l
a
n L4
y=O.3367+1.1400~
I
NPV
I:::. l
y~l.2919+l.17aox
1
345
BIOASSAY
Log
I
I
I
I
I
2
3
4
5
6
Oosagc,PIBs
/Larva
FIG. I. Dosage-mortality response following ingestion of nucleopolyhedrosis virus by thirdand fourthinstar Malacosoma neustria larvae.
TABLE 2 Analysis of Probit-Log Dose Assays for the Peroral Infection of Malacosoma neustria Larvae by a Nucleopolyhedrosis Virus
Slope (b) Standard error of the slope Heterogeneity about the regression: x2 (2) Goodness of fit (P) Value of g
Third-instar
Fourth-instar
1.1780 kO.0962
1.1400 io.1379
1.366 >0.50 0.0256
2.610 >0.20 0.0562
TABLE 3 Calculated Number of PIB’s/Larva and Number of PIB’s/mg Larval to Produce 10, 30, 50, 70, and 90% Mortality of Third- and Fourth-Instar Percent larval mortality
PIB’s/ larva
95% fiducial Lower
limits
10 30 50 70 90
115 504 1,405 3,917 17,208
67 339 916 2,614 9,932
10 30 50 IO 90
926 4,212 12,320 35,530 163,992
428 2,454 1,318 19,752 71,657
PIB’s/mg body weight
Upper Third-instar
Required neustria
Larvae
95% fiducial Lower
limits Upper
assay
196 149 2,024 5,868 29,815 Fourth-ins&x 2,000 1,435 20,513 63,916 375,300
Body Weight Malacosoma
14 62 114 485 2,132
8 42 121 324 1,231
24 93 251 727 3,694
28 130 376 1,084 5,003
13 15 225 603 2,186
61 227 628 1,950 11,449
assay
346
ALDO
MAGNOLER
LD,, for the larvae in the fourth-instar was about 2 x lo6 PIB’s/larva. Comparatively, smaller doses were needed to kill a high proportion of third- and fourth-instar M. neustria larvae. As a result, the NPV assayed may be considered highly pathogenic for the tent caterpillar in Sardinia. The time of mortality at different concentrations of the virus fed to third- and fourthinstar larvae was studied. Daily cumulative mortality data were plotted on log probability paper and straight lines were drawn through the plotted points (Fig. 2). Mortality of third-instars feeding on dosages 3 x lo5 and 3 x lo4 PIB’s/larva first occurred on the fifth day. Fourth-instar larvae treated with the same concentrations began to die after 6 and 7 days, respectively. An incubation time of 6 and 9 days was required for initial mortality, respectively, in thirdand fourth-instar larvae exposed to 3 x lo3 PIB’s/larva. The LT,, values, estimated according to Lichtfield (1949), were inversely related to the concentration of virus, irrespective of larval age (Table 4). In general,
i
L3
99 c
3Xld 3x18
Days FIG. 2. Time-mortality response of third- and fourth-instar Mulacosoma neustriu larvae to different concentrations of nucleopolyhedrosis virus.
TABLE 4 The LTso Values and 95% Fiducial Limits for Thiidand Fourth-Instar Malucosoma neustria Larvae Treated with Different Concentrations of a Nucleopolyhedrosis Virus 95% fiducial limits Approx. No. PIB’s/larva 3x lo5 3x lo4 3 x lo3 3x lo5 3x lo4
LT5o (days)
Lower (days)
Upper (days)
Third-ins& assay 5.90 5.71 6.10 6.58 6.81 6.36 8.15 7.72 8.61 Fourth-instar assay 9.30 8.85 9.77 11.49 10.70 9.96
Slope function 1.141 1.174 1.226 1.176 1.236
the time of mortality at a given dosage was longer in the older larvae. With a fourfold increase in body weight of the larvae the median lethal time increased 3.4 and 4.12 days, respectively, at dosages 3 x lo5 and 3 x lo4 PIB’s/larva. The increasing slope difference at the given decreasing concentrations provided evidence that the course of the disease was significantly altered by the age of the larvae at the lower dosage. With concentrations producing less than 50% mortality, the relation between larval age and period of lethal infection was reversed. A similar variation in incubation periods, observed in other virus-infected lepidoptera, was attributed to individual differences in resistance to infection (Morris, 1962). It should be noted that the interval between any two LT,, times was statistically significant. The time-mortality data for A4. neustria larvae indicated that the incubation period of the disease was shorter than that reported for the nucleopolyhedroses of other insects. In particular, it was shown that the LT,, for third-instar larvae of the gypsy moth exposed to dosages ranging from 2.5 x lo6 to 2.5 x lo3 PIB’s/larva varied between 8.1 and 13.1 days (Magnoler, 1974). Even first-instar larvae of the North American species of Malacosoma feeding on concentrations of lOa and lo5 PIB’s/ml, respectively, showed an LTso of 6.18 and 10.6 days (Stairs, 1964).
BIOASSAY
OF
the third-, fourth-, and fifth-instar averaged 77.4, 19.9, and 2.7% respectively, for the younger virus-treated larvae. When the virus was administered to the older larvae, 66.7 and 33.3% of the total mortality occurred in the fourth- and fifth-instar, respectively. Apparently, within the time required by the larvae to molt twice almost all individuals receiving a lethal dose of polyhedra succumbed to disease. An analogous lethal effect of nuclear polyhedra on larval instars was observed in other insect species (Ignoffo, 1966; Doane, 1967; Magnoler, 1974). The weights of pupae and adult emergence of survivors from virus-infected third- and fourth-instar larvae are given in Table 5. Apparently, pupal weights were not affected by the virus dose administered to the larvae, as it was confirmed by analyses of variance. No well-defined relationship existed between adult emergence and polyhedral dose among the survivors from various treatments. In fourth-instar assay only, the lowest values recorded at the two highest dosages were based on a few pupae and could not account for a drastic reduction in adult emergence. The overall adult emergence from pupae surviving virus application during the thirdand fourth-instar was 91.8%; adult emergence from the controls was 94.3%. Owing
Records on the stage at which larval mortality occurred showed that the age of the larvae at death was inversely related to the size of inoculum (Fig. 3). With the highest concentration of the virus about 97 and 85% of the larvae treated, respectively, in the third- and fourth-instar died before reaching the subsequent stage of development. The corresponding figures for third- and fourthinstar larvae ingesting a loo-fold lower viral dose were about 70 and 43% respectively. At dosage levels from 3 x lo5 to 3 x lo2 PIB’s/larva mortalities among third- and fourth-instars increased steadily in the subsequent stage of larval development. Based on the total number that died, mortality in
PlBr I Larva
FIG. 3. Percentage of Mulacosoma neusfriu larvae dead at various instars after ingestion of different concentrations of nuclear polyhedra by third(a) and fourth-instar (b) larvae.
Pupal Weight and Adult Emergence Malacosoma neustria Larvae Treated
Approx. No. PIB’s/larva
TABLE 5 of Survivors from Third- and Fourth-Instar with Various Concentrations of Polyhedra Mean pupal weight (mg)
No. pupae Males
347
NPV
Females
Males Third-instar
3 x lo3 3x lo2 3x 10 3 Control
14 23 30 26 29
9 21 32 38 38
3 x lo5 3 x lo4 3 x lo3 3 x lo2 3x 10 3 Control
0 4 18 15 20 14 16
1 3 14 21 23 2.4 23
Percent adult emergence
Females
Males
Females
669.9 664.2 662.4 660.9 612.9
85.1 91.3 93.3 92.3 93.1
88.9 92.6 96.9 89.5 91.4
594.0 651.1 611.4 663.9 652.3 658.9 619.3
0 15 .o 94.4 93.3 90.0 85.1 93.1
0 66.1 92.9 90.5 91.3 95.8 91.3
assay 285.5 268.6 269.0 264.5 261.0
Fourth-instar
assay 0 292.0 265.3 263.1 251.3 262.6 264.8
348
ALDO
MAGNOLER
to the small size of samples, no definite conclusion can be drawn on the effects of the virus on postlarval stages. However, the results on M. neustriu agree in general with those reported for other virus-infected lepidoptera (Ignoffo, 1964, 1965; Vail and Hall, 1969; Magnoler, 1974). It should be noted that no virus dead pupae were found in any of the treatments or among the controls. From the present study it may be concluded that the Sardinian strain of M. neustria is highly susceptible to the NPV. Larval mortality varied directly with dosage. However, a twofold resistance to virus infection was developed by the larvae with a fourfold increase in body weight. The dosage-incubation relationship was reversed. At a given dosage of virus inoculum, the period of lethal infection was generally longer in the older larvae. The larvae surviving a second molt after virus ingestion developed and emerged normally comparable to the controls. REFERENCES
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Pathol..
9,376-386.
FINNEY, D. J. 1964. “Probit Analysis,” Second ed. Cambridge University Press, London/New York. FRANZ, J. M. AND LAUX, W. 1965. Individual differences in Malacosoma neustria (L.). Proc. 12th Intern.
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IGNOFFO, C. M. 1966. Susceptibility of the first-instar of the bollworm, Heliothis zea, and the tobacco budworm, Heliothis virescens, to Heliothis nuclearpolyhedrosis virus. J. Znvertebr. Pathol.. 8,531-536. LAUX, W. 1962. Individuelle Unterschiede in Verhalten und Leistung des Ringelspinners, Malacosoma neustria (L.). 2. Angew. Zool., 49,465-524. LICHTFIELD, J. T. 1949. A method for rapid graphic solution of time-per cent effect curves. J. Pharmacol. Exp. Therap.,
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MAGNOLER, A. 1974. Bioassay of a nucleopolyhedrosis virus of the gypsy moth, Porthetria dispar. J. Invertebr.
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W., MAGNOLER, A., AND HUGER, A. 1965. ijber ein verziigertes Absterben von viruskranken Raupen des Ringelspinners, Malacosoma neustriu nach Behandlung mit einem Eacillus-thur(L.), ingiensis-PrPparat. Z. Pfkrankh. Pflschutz., 72,599605. STAIRS, G. R. 1964. Infection of Malacosoma dissrria Httbner with nuclear-polyhedrosis virus from other species of Malacosoma (Lepidoptera, Lasiocampidae). J. Insect Pathol.. 6, 164-169. STAIRS, G. R. 1965. Quantitative differences in susceptibility to nuclear-polyhedrosis virus among larval instars of the forest tent caterpillar, Malacosoma disstria (Hitbner). J. Invertebr. Pathol., 7,427-429. VAIL, P. V., AND HALL, I. M. 1969. Susceptibility of the pupa of the cabbage looper, Trichoplusia ni, to nucleopolyhedrosis virus. J. Invertebr. Puthol.. 14, 227236. SICKER,