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Interinstar susceptibility of the fall webworm, Hyphantria cunea, to its nucleopolyhedrosis and granulosis viruses
JOURNAL
OF INVERTEBRATE
PATHOLOGY
30,68-75
(1977)
lnterinstar Susceptibility of the Fall Webworm, Hyphantria to Its Nucleopolyhedrosis and Granulosis Viruses’ D.G. Department
of Entomology,
BOUCIASANDG.
cunea,
L. NORDIN
University of Kentucky, Lexington,
Kentucky 40506
Received October 22, 1976 Comparative interinstar susceptibility of the fall webworm, Hyphantria cunea, to granulosis and nucleopolyhedrosis viruses was quantitated. As the larvae matured, susceptibility to either baculovirus preparation decreased. Mortality data converted to a dose per milligram body weight basis demonstrated that this decrease in susceptibility was only in part due to the difference in weight of the larvae tested. Lethal time data showed that incubation time for either baculovirus was dependent on the age of the larvae and the dose of virus assayed. A difference in interinstar susceptibility was observed between the granulosis virus and nucleopolyhedrosis virus.
INTRODUCTION
were equally susceptible to NPV isolates from three species of hemlock loopers. The fall webworm, Hy~hantria cunea, an occasional pest on fruit and shade trees, is susceptible to both a GV and an NPV. The ultrastructure and histopathology of both viruses have been previously described (Vasiljevic, 1962; Watanabe and Kobayashi, 1970; Nordin, 1971). Mortality studies concerning these viruses have been limited to the NPV. An early report by Szirmai (1957) demonstrated that early-instar f&l webworm larvae were more susceptible than the later-instar larvae. More recently, Nordin (1971) reported L&, values of 1.48 x 103 and 3.30 x 103 PIB/larvae for second- and third-instar fall webworm larvae, respectively. The purpose of this report is to compare the inter&star susceptibility of the fall webworm to its respective NPV and GV.
Quantitative determination of interinstar susceptibility of lepidopterous species to their respective baculoviruses has been mainly restricted to Subgroup A, the nucleopolyhedrosis viruses (NPV). In general, younger larvae are more susceptible to baculovirus infection than more mature larvae (Huger, 1963). This has been demonstrated for both a granulosis virus (GV) of Pseuduletiu ani~ancta (Tanada, 1956) and an NPV of ~e~~o?~~~zea (Ignoffo, 1966). The range of interinstar susceptibility appears to be dependent on the particular insect-virus system. Stairs (1965) demonstrated that first-instar Malacosoma disstria larvae were 1000 times more susceptible than third-&star larvae and 68,000 times more susceptible than fourth-instar larvae to an NPV. Likewise, Magnoler (1975) determined on a weight basis (m~lig~ms) that third-ins&u Malac~suma neustria larvae were twice as susceptible to an NPV as fourth-instar larvae. However, Cunningham (1970) reported that second-, third-, and fourth&tar Lambdina jiscella fiscella larvae
MATERIALS
AND METHODS
Rearing and Bioassay Procedure The culture of black-headed fall webworms used in these bioassays originated from larvae collected near Lexington, Kentucky, during the early summer of 1975. They were reared on a semisynthetic diet (Yearian et al., 1%6), following modifications described by Nordin (1971). The
f This paper (No. 76-11-169) was prepared in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director. 68 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-201 I
SUSCEPTIBILITY
OF HYPHANTRIA
stock culture went through four generations in the laboratory before the bioassays were initiated. Early second-, fourth-, and fifthinstar larvae, weighing 0.74 + 0.16, 4.12 -+ 0.51, and 14.70 + 3.32 mg, respectively, were used in these assays. The assay procedure used was similar to that of Magnoler (1975). Small disks of diet were cut, placed in sterile Petri dishes (35 x 10 mm), and surface contaminated with serial decimal dilutions of viral suspensions (1 pi/disk) using a microapplicator. Second-instar larvae were assayed in groups (five/disk). The amount of inoculum received by each of the five secondstar larvae was assumed to be 0.2 ~1. Fourth- and fifth-instar larvae were assayed individually (one/disk). Control larvae were placed on untreated diet disks. The treated and control larvae were placed in a 26°C incubator having a 12-hr photoperiod. After 48 hr, those larvae completely consuming the upper surface of the disk were individually transferred to uncontaminated diet creamers and returned to the 26°C incubator. Treatments consisting of 15 larvae/dilution were replicated three times for second-instar larvae and twice for fourth- and fifth-instar larvae. Mortality was monitored every 48 hr until pupation. Squashes prepared from all dead larvae were diagnosed using phase contrast optics. Only treated larvae displaying definitive nucleopolyhedrosis or granulosis were used in the calculations. Preparation and Quantitation Suspensions
of Viral
The NPV used in these assays was produced by injecting early sixth-instar larvae with infectious hemolymph previously extracted from heavily infected larvae. The injected larvae reared at 26°C succumbed to nucleopolyhedrosis within 8 days. These larvae were triturated in distilled water containing several crystals of phenylthiourea to prevent melanization and were filtered through four layers of cheescloth.
CUNEA
TO VIRUSES
69
The polyhedra were purified by two cycles of differential centrifugation (5OOgfor 3 min, 20008 for 20 min). The NPV preparation was then layered on top of a 38% (w/w) sucrose solution and spun at 2000g for 1 hr. The polyhedra pelleted at the bottom but the contaminants remained in suspension. The polyhedra were resuspended in distilled water and sonicated for 5 min using a Sonic Dismembrator.2 The number of polyhedra in the stock suspension was determined to be 8.2 x IO8 PIB/ml using a Petroff-Hausser bacterial counting chamber. Serial decimal dilutions were prepared in distilled water and frozen until needed. The GV was propagated by feeding early fourth-instar fall webworm larvae diet contaminated with a heavy suspension of crude GV preparation. Infected larvae were homogenized in a tissue grinder with distilled water containing several crystals of phenylthiourea, and the homogenate was then filtered through four layers of cheesecloth. Two cycles of differential centrifugation (2000g for 5 min, 10,OOOg for 20 min) were followed by sucrose gradient centrifugation as outlined by Summers and Paschke (1970). Glass-distilled water was used instead of the 0.1 M phosphate buffer. The band containing the GV was removed, diluted IO-fold, and sonicated for 5 min. Two cycles of 10,OOOg centrifugation for 20 min removed the sucrose. The final pellet was resuspended in distilled water; decimal dilutions were prepared from this and frozen until needed. Quantitation of GV using the bacterial counting chamber was not satisfactory. The method of Williams and Backus (1949) was thus adopted to calculate the number of capsules per milliliter. Polystyrene latex particles3 having a diameter of 0.481 & 0.0061 pm at a concentration of 8.1 x lOlo particles/ml were used as a calibraESupplied by Artek Systems Inc., Farmingdale, New York. 3 Suppied by Ernest F. Fullman, Inc., Schenectady, New York.
70
BOUCIAS
AND NORDIN
I
I
I
I
I
I
I
1
2
3
4
5
6
7
Log
Dosage,
PI69
! Larva
FIG. 1. Mortality response of second-, fourth-, and fifth-instar larvae fed decimal serial dilutions of nucleopolyhedrosis virus. Shaded area indicates overlap between 95% confidence limits of the regression lines.
tion standard. Nine-tenths milliliter of this was added to 0.1 ml of the (10-l) dilution of the stock GV solution. The spectrophotometric reading of the (10p2) dilution gave a maximum absorbance value of 0.63 at 280 nm. The preparation was placed in a nebulizer-spraye? and sprayed onto Formvar-coated loo-mesh copper grids. Counting was done using a Philips 201 electron microscope operating at 80 kV. The ratio of capsules per particle provided an estimate of 5.20 + 1.29 x 1012 capsules/ ml as the concentration in the stock solution. RESULTS
AND DISCUSSION
Dose-mortality data for both the NPV and the GV assays are summarized in Table 1. Mortality from unknown causes was highest in the second-instar larval assays. These larvae were very small (0.74 mg/larva), and the unknown mortality in most cases might be attributed to the transfer procedure. An exception to this may be the highest concentration of NPV which caused 30% unknown mortality in both the second-instar and fourth-instar assays. Diagnosis of these dead larvae revealed a high concentration of rod-shaped
bacteria which were not detected in the original PIB suspension. Of all the control larvae only one second-instar larva died of viral disease. In all assays, larval mortality increased with increased dosage. Larval susceptibility to both viral preparations decreased with increased age. A dosage of 8.2 x 104 PIB/larvae caused 100% mortality for second instars, but a dosage of 4.1 x lo5 PIB/larva caused only 82% mortality for fourth-instar larvae and 56% mortality for fifth-instar larvae. Similarly, a dosage of 1.0 x 10’ capsules/larva caused 100% mortality for second-instar larvae whereas a dosage of 5.2 x lo7 capsules/larva caused 60% mortality for fourth-instar larvae and only 1% mortality for fifth-instar larvae. Probit analysis4 was used to calculate the dose-mortality regression lines in Figures 1 and 2 and the LDso data in Table 2. The values of the slopes of all the regression lines approximate unity (Fig. 1, 2). The wide confidence limits of these regression lines, owing in part to the limited number of larvae used in these bioassays, 4 SAS Computer University).
Program
(North
Carolina
State
1.
x 10'
x 103
x 102
x 10'
4.1
4.1
4.1
4.1
for
l
**
dead
larvae/(Totai
no.
larvae
dead
Corrected mortality Based on 3 replications Based on 2 replications
from
Control
0
28
28
30
- larvae
5.2
0
30
x 10'
4.1
5.2
0
x 107
4.1
5.2
7
x 103
4.1
unknown
x 104
x 10s
x 10s
x 10'
5.2
26
x 108
23
x 104
4.1
26
5.2
x 10:
4.1
Assay*** 5.2 x 103
x 104
x 10s
56
30
x 106
4.1
viral
Control
0
0
= No.
5.2
0
Fifth-Instar 96
5.2
x 106
x 107
4
28
.t
x 10s x 108
1
5.2
5.2
5.2
5.2
Assay***
27
Control
t
x 104
x 10s
x 106
x 10'
x 108
x 109
Control
1.0
1.0
1.0
1.0
1.0
1.0
Assay**
8
a2
100
Fourth-Instar
0
12
15
52
91
100
100
Second-lnstar
against
Capsules/ larvae
assayed
52
0
Control
viruses
Corrected* % mortality
granulosis
Larvae dead from unknown causes
and
16
23
21
0
5
7
23
39
45
28
from
a nucleopolyhedrosis
-NPV Larvae dead po lyhedrosis
data
29
30
20
31
29
x 10s
45
4.1
x 100
8.2
49
30
x 10'
8.2
4s
x 106
x 102
8.2
45
4.1
x 103
8.2
45
40
x 10'
8.2
40
Total no. larvae/dose
DOSe mortality
Control
x 10s
8.2
PIB's/larvae
Table
GV
webworm
causes)
0
4
3
19
64
100
0
0
7
17
60
92
100
29 3
52
85
100
100
100
Corrected' % mortalittC
larvae.
0
Larvae dead from unknown causes
fall
30
5
ia
28
0
0
2
5
15
24
30
1
10
22
35
31
38
37
from
and fifth-instar
Larvae dead qranulosis
fourth-
29
29
30
28
29
30
27
28
29
30
26
26
30
40
40
45
45
35
40
40
Total no. larvae/dose
second-,
72
BOUCIAS
3
4
5 Log Dosage,
AND NORDIN
6
7
6
s
10
I Larva
Capsules
FIG. 2. Mortality response of second-, fourth-, and tifth-instar larvae fed decimal serial dilutions of granulosis virus. Shaded area indicates overlap between the 95% confidence limits of the regression lines.
also reflect the genetic variation in the laboratory population of the fall webworm. The dose-mortality regression lines do show a significant difference between the susceptibility of the second-instar larvae and the susceptibility of fourth- and fifthinstar larvae to either baculovirus preparation. Differences between fourthand fifth-instar larval susceptibility were not significant at the 95% confidence level as shown by the extensive overlap between the fiducial limits of their regression lines.
The relative susceptibility of the differentaged instars was determined by comparing their LD,, values (Table 2). It is important to note that within a single-age instar group an approximate lo-fold range was predicted between the upper and lower fiducial limits of their respective LD,, values. Second-instar larvae were the most susceptible instar to either the NPV or the GV. It took 100 times more capsules than polyhedra to cause the same mortality against second-instar larvae. The polyhedra
TABLE CALCULATED
NUMBER OF NUCLEOPOLYHEDROSIS TO KILL SO% OF THE TREATED
2 AND GRANULOSIS INCLUSIONS FALL WEBWORM LARVAE
Inclusions/larva
Inclusions/milligram
NPV Second instar Fourth instar Fifth instar GV Second instar Fourth instar Fifth instar
body weight
95% Confidence limits
95% Confidence limits Virus and larvae
REQUIRED
LDso value
Lower
Upper
LD5,, value
Lower
4.79 x 102 2.99 x 104 1.88 x 105
1.92 x 102 1.13 x 104 5.80 x 104
1.20 x 103 8.36 x 104 6.80 x 105
6.47 x 102 7.25 x 18 1.28 x 104
2.59 x 102 2.74 x 1W 3.53 x 103
1.62 x 103 2.03 x 104 4.62 x 1W
7.06 x lo4 2.74 x 10’ 1.87 x lo*
2.67 x 104 1.08 x 10’ 6.36 x 10’
1.79 x 105 7.31 x 10’ 6.25 x 108
9.54 x 104 6.65 x 106 1.27 x 10’
3.61 x lo4 2.62 x lo6 4.32 x 106
2.42 x lo5 1.77 x 10’ 4.24 x 10’
Upper
SUSCEPTIBILITY
HYPHANTRZA
OF
contain 25-30 viral bundles/PIB, each in turn containing 2-12 viral rods (Nordin, 1971). The granulosis capsule usually contains only 1 viral rod/capsule (Vasiljevic, 1962). Converting the number of capsules per larva and number of PlB per larva to number of viral rods per larva could potentially account for any numerical difference between the GV and NPV required to kill second-instar larvae. However, the interinstar susceptibility of fall webworm larvae demonstrates a distinct difference between the GV and NPV. Average second-instar larvae having an LD,, of 4.79 x lo2 PIB/larva were 62 and 392 times more susceptible than the average fourth- and fifth-instar larvae, respectively. After converting LD5,, values to a dose per milligram body weight basis, the susceptibility of second-instar larvae to nucleopolyhedrosis was 11 times greater than fourth-instar larvae and 19 times greater than fifth-instar larvae. Similar decreases in susceptibility to nucleopolyhedrosis with TABLE THE
age have been reported by other investigators (Stairs, 196.5; Doane, 1967; Magnoler, 1975). Challenged with GV, average secondinstar larvae were 388 times more susceptible than fourth-instar larvae and 2648 times more susceptible than fifth-instar larvae. On a dose per milligram body weight basis the second-instar larvae were 70 or 133 times more susceptible to granulosis than fourth- or fifth-instar larvae, respectively. Lethal time data for dosages of NPV and GV causing greater than 50% mortality are graphically depicted in Figures 3 and 4. Cumulative mortality was transformed to probits and graphed against log days. The amount and the time of initial mortality are dependent upon the age of the instar tested and the dosage used. Second-instar larvae fed 8.2 x lo5 PIB/larva or 5.2 x IO9 capsules/larva began to die after 4 days. Mortality of fourth- and fifth-instar larvae fed dosages of 4.1 x lo6 PIB/larva or 5.2 x log capsules/larva first occurred on 3
LT,, VALUES FOR THOSE DOSAGES OF NUCLEOPOLYHEDROSIS AND GRANULOSIS OR GREATER MORTALITY AGAINST SECOND-, FOURTH-, AND FIFTH-INSTAR FALL NPV
PIB/ larva
LTm (days)
Lower
95% Confidence limits Capsules/ larva
Upper Second-instar
x x x x
lo5 104 103 102
3.9 7.8 8.6 12.5
2.9 6.6 7.8 11.4
4.5 9.2 9.4 13.8
Fourth-instar 4.1 x 106 4.1 x 105 4.1 x 104
8.4 11.5 14.8
7.3 10.7 12.1
9.7 12.3 18.1 Fifth-instar
4.1 x 106 4.1 x 105
10.7 13.7
VIRUSES CAUSING 50% WEBWORM LARVAE GV
95% Confidence limits
8.2 8.2 8.2 8.2
73
CUNEA TO VIRUSES
10.1 11.6
11.4 16.2
LTm (days)
Lower
Upper
4.0 5.2 6.1 ,7.4 11.5
3.6 4.8 5.4 6.2 9.4
4.4 5.6 6.9 8.8 14.1
9.0 1’1.5 17.7
7.3 9.2 12.5
11.1 14.4 25.1
9.3 14.0
8.4 10.0
10.2 19.6
assay 1 1 1 1 1
x x x x x
109 108 10’ 106 105
assay 5.2 x 109 5.2 x 108 5.2 x 10’ assay 5.2 x IO9 5.2 x 108
BOUCIAS
74
1
I
I
III
2
4
6810
FIG. 3. Time-mortality polyhedrosis virus.
14
AND NORDIN
20
response of second-, fourth-, and fifth&star
21
4I
III 6810
larvae fed serial dilutions
of nucleo-
20'f'I
Dsys Fro. 4. Time-mortality virus.
response of second-, fourth-, and fifth-instar larvae fed serial dilutions of granulosis
SUSCEPTlBILITY
OF H~PHANTR~A
the sixth day. A decrease in viral concentration for any one assay caused either an increase in the time required for mortality or a decrease in the amount of initial mortality. The LT,, values calculated using the analysis of Litchfield (1949) are shown in Table 3. For any one assay, the LTJo values are inversely related to dosage. A significant difference existed between the LTSO values of second- and fourth-instar larvae when assayed with either virus preparation. Daoust (1974) and lgnoffo (1966) assaying an NPV against various Heliothis species showed a similar correlation between LTBO values and the age of the larvae tested. In conclusion, these data show that interinstar susceptibility of fall webworms to either granulosis or nucleopolyhedrosis is not entirely related to the weight of the larvae assayed. This can be seen by comparing the differences between the LDSO and LT,, values of second- and fourthinstar larvae to differences between the LDSo and LTSO values of second- and fifth-instar larvae. Fu~hermore, there is a distinct difference between the NPV and GV in terms of interinstar susceptibility. Based on the number of viral rods per larva, both viral preparations appear to be equally virulent to second-ins&u larvae. However, the susceptibility of the olderinstar larvae to granulosis is significantly less than their susceptibility to nucleopolyhedrosis. One may speculate that physiological changes during larval maturation have a greater effect on the virulence of the GV than on the virulence of the NPV. ACKNOWLEDGMENT We express appreciation to Miss Susan G. Burros for providing technical assistance during the course of this work.
REFERENCES CUNNINGHAM, J. C, 1970. Pathogenicity tests of nuclear polyh~rosis viruses infecting tlte eastern hemlock looper, Lambdina jisrellaria jiscelluria (Lepidoptera: Geometridae). Canad. Entomol., 102, 1534-1539.
CUNEA TO VIRUSES
75
DAOUST, R. A. 1974. Weight-related susceptibility of larvae of Heliothis armigera to a crude nuclear-polyhedrosis virus preparation. J. Znvertebr. Pathol.,
23,400~401.
DOANE, G. C. 1967. Bioassay of nuclear-polyhedrosis virus against larval instars of the gypsy moth. 1. Invertebr. Path&. , 9,376-386. HUGER, A. 1%3. Granulosis of insects. In “Insect Pathology, an Advanced Treatise” (E. A. Steinhaus, ed.), Vol 1, pp. 531-576. Academic Press, New York. IGNOFFO, C. M. 1966. Effects of age on mortality of Neliothis zea and Heliothis virescens larvae exposed to a nuclei-polyhedrosis virus. J. Znvertebr. Puthol., 8, 279-283. LJTCHFIELD, J. T. 1949. A method for rapid graphic solution of tome-~rcent curves. J. Pharma~ol. Exp. Ther., 97, 399-408. MAGNOLER, A. 1975. Bioassay of nucleopolyhedrosis virus against larval instars of Maiucosoma neustria. J. fnvertebr. Pathol., 25, 343-358. NORDIN, G. L. 1971. “Studies of a Nuclear Polyhedrosis Virus and Three Species of Microsporidia Pathogenic to the Fall Webworm Hyphantriu cunea (Drury).” Ph.D. Thesis, University of Illinois, Urbana, Ill. STAIRS, G. R. 1965. Quantitative differences in susceptibility to nuclear-poiyhedrosis virus among larval instars of the forest tent caterpillar, Maiatosoma disstria ~Hubner). J. fnvertebr. Puthol., 7,427-429.
SUMMERS, M. D., AND PASCHKE, J. D. 1970. Alkahliberated granulosis virus of Trichopiasia ni. I. Density gradient purification of virus components and some of their in vitro chemical and physical properties. 1. Invertebr. Pathol., 16, 227-240. SZIRMAI, J. 1957. Biologische Abwehr mitteb Virus zur Bekamp fung der Hyphantria cunea Drury. Acta Microbial. Hung., 4, 30-42. TANADA, Y. 1956. Some factors affecting the susceptibility of the ~myworm to virus infections. J. Econ. Entomol., 49,52-57. VASIWEVIC, L. 1962. Etude au microscope eiectronique du virus de la granulose de I’ecaille fileuse (Hyphantria cunea Drury). Colloq. Int. Insectes Paris, 383-391. WATANABE, H., AND KOBAYASHI, M. 1970. Histopathology of a granulosis in the larvae of the fall webworm, Hyphantria cunea. J. Invertebr. Pathol., X6,71-79.
WILLIAMS, R, C., AND BACKUS, R. C. 1949. Macromolecular weights determined by direct particle counting. I. The weight of the bushy stunt virus. J. Amer. Chem. Sot., 71,4052-4057. YEARIAN, W. C., GILBERT, K. L., AND WARREN, L. 0. 1966. Rearing of the fall webworm Hypha~tria c~~ea Dr. (Lepidoptera: Arctiidae) on a wheat germ medium. J. Kans. Entomol. Sot., 39, 49.5-499.
OF INVERTEBRATE
PATHOLOGY
30,68-75
(1977)
lnterinstar Susceptibility of the Fall Webworm, Hyphantria to Its Nucleopolyhedrosis and Granulosis Viruses’ D.G. Department
of Entomology,
BOUCIASANDG.
cunea,
L. NORDIN
University of Kentucky, Lexington,
Kentucky 40506
Received October 22, 1976 Comparative interinstar susceptibility of the fall webworm, Hyphantria cunea, to granulosis and nucleopolyhedrosis viruses was quantitated. As the larvae matured, susceptibility to either baculovirus preparation decreased. Mortality data converted to a dose per milligram body weight basis demonstrated that this decrease in susceptibility was only in part due to the difference in weight of the larvae tested. Lethal time data showed that incubation time for either baculovirus was dependent on the age of the larvae and the dose of virus assayed. A difference in interinstar susceptibility was observed between the granulosis virus and nucleopolyhedrosis virus.
INTRODUCTION
were equally susceptible to NPV isolates from three species of hemlock loopers. The fall webworm, Hy~hantria cunea, an occasional pest on fruit and shade trees, is susceptible to both a GV and an NPV. The ultrastructure and histopathology of both viruses have been previously described (Vasiljevic, 1962; Watanabe and Kobayashi, 1970; Nordin, 1971). Mortality studies concerning these viruses have been limited to the NPV. An early report by Szirmai (1957) demonstrated that early-instar f&l webworm larvae were more susceptible than the later-instar larvae. More recently, Nordin (1971) reported L&, values of 1.48 x 103 and 3.30 x 103 PIB/larvae for second- and third-instar fall webworm larvae, respectively. The purpose of this report is to compare the inter&star susceptibility of the fall webworm to its respective NPV and GV.
Quantitative determination of interinstar susceptibility of lepidopterous species to their respective baculoviruses has been mainly restricted to Subgroup A, the nucleopolyhedrosis viruses (NPV). In general, younger larvae are more susceptible to baculovirus infection than more mature larvae (Huger, 1963). This has been demonstrated for both a granulosis virus (GV) of Pseuduletiu ani~ancta (Tanada, 1956) and an NPV of ~e~~o?~~~zea (Ignoffo, 1966). The range of interinstar susceptibility appears to be dependent on the particular insect-virus system. Stairs (1965) demonstrated that first-instar Malacosoma disstria larvae were 1000 times more susceptible than third-&star larvae and 68,000 times more susceptible than fourth-instar larvae to an NPV. Likewise, Magnoler (1975) determined on a weight basis (m~lig~ms) that third-ins&u Malac~suma neustria larvae were twice as susceptible to an NPV as fourth-instar larvae. However, Cunningham (1970) reported that second-, third-, and fourth&tar Lambdina jiscella fiscella larvae
MATERIALS
AND METHODS
Rearing and Bioassay Procedure The culture of black-headed fall webworms used in these bioassays originated from larvae collected near Lexington, Kentucky, during the early summer of 1975. They were reared on a semisynthetic diet (Yearian et al., 1%6), following modifications described by Nordin (1971). The
f This paper (No. 76-11-169) was prepared in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Director. 68 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-201 I
SUSCEPTIBILITY
OF HYPHANTRIA
stock culture went through four generations in the laboratory before the bioassays were initiated. Early second-, fourth-, and fifthinstar larvae, weighing 0.74 + 0.16, 4.12 -+ 0.51, and 14.70 + 3.32 mg, respectively, were used in these assays. The assay procedure used was similar to that of Magnoler (1975). Small disks of diet were cut, placed in sterile Petri dishes (35 x 10 mm), and surface contaminated with serial decimal dilutions of viral suspensions (1 pi/disk) using a microapplicator. Second-instar larvae were assayed in groups (five/disk). The amount of inoculum received by each of the five secondstar larvae was assumed to be 0.2 ~1. Fourth- and fifth-instar larvae were assayed individually (one/disk). Control larvae were placed on untreated diet disks. The treated and control larvae were placed in a 26°C incubator having a 12-hr photoperiod. After 48 hr, those larvae completely consuming the upper surface of the disk were individually transferred to uncontaminated diet creamers and returned to the 26°C incubator. Treatments consisting of 15 larvae/dilution were replicated three times for second-instar larvae and twice for fourth- and fifth-instar larvae. Mortality was monitored every 48 hr until pupation. Squashes prepared from all dead larvae were diagnosed using phase contrast optics. Only treated larvae displaying definitive nucleopolyhedrosis or granulosis were used in the calculations. Preparation and Quantitation Suspensions
of Viral
The NPV used in these assays was produced by injecting early sixth-instar larvae with infectious hemolymph previously extracted from heavily infected larvae. The injected larvae reared at 26°C succumbed to nucleopolyhedrosis within 8 days. These larvae were triturated in distilled water containing several crystals of phenylthiourea to prevent melanization and were filtered through four layers of cheescloth.
CUNEA
TO VIRUSES
69
The polyhedra were purified by two cycles of differential centrifugation (5OOgfor 3 min, 20008 for 20 min). The NPV preparation was then layered on top of a 38% (w/w) sucrose solution and spun at 2000g for 1 hr. The polyhedra pelleted at the bottom but the contaminants remained in suspension. The polyhedra were resuspended in distilled water and sonicated for 5 min using a Sonic Dismembrator.2 The number of polyhedra in the stock suspension was determined to be 8.2 x IO8 PIB/ml using a Petroff-Hausser bacterial counting chamber. Serial decimal dilutions were prepared in distilled water and frozen until needed. The GV was propagated by feeding early fourth-instar fall webworm larvae diet contaminated with a heavy suspension of crude GV preparation. Infected larvae were homogenized in a tissue grinder with distilled water containing several crystals of phenylthiourea, and the homogenate was then filtered through four layers of cheesecloth. Two cycles of differential centrifugation (2000g for 5 min, 10,OOOg for 20 min) were followed by sucrose gradient centrifugation as outlined by Summers and Paschke (1970). Glass-distilled water was used instead of the 0.1 M phosphate buffer. The band containing the GV was removed, diluted IO-fold, and sonicated for 5 min. Two cycles of 10,OOOg centrifugation for 20 min removed the sucrose. The final pellet was resuspended in distilled water; decimal dilutions were prepared from this and frozen until needed. Quantitation of GV using the bacterial counting chamber was not satisfactory. The method of Williams and Backus (1949) was thus adopted to calculate the number of capsules per milliliter. Polystyrene latex particles3 having a diameter of 0.481 & 0.0061 pm at a concentration of 8.1 x lOlo particles/ml were used as a calibraESupplied by Artek Systems Inc., Farmingdale, New York. 3 Suppied by Ernest F. Fullman, Inc., Schenectady, New York.
70
BOUCIAS
AND NORDIN
I
I
I
I
I
I
I
1
2
3
4
5
6
7
Log
Dosage,
PI69
! Larva
FIG. 1. Mortality response of second-, fourth-, and fifth-instar larvae fed decimal serial dilutions of nucleopolyhedrosis virus. Shaded area indicates overlap between 95% confidence limits of the regression lines.
tion standard. Nine-tenths milliliter of this was added to 0.1 ml of the (10-l) dilution of the stock GV solution. The spectrophotometric reading of the (10p2) dilution gave a maximum absorbance value of 0.63 at 280 nm. The preparation was placed in a nebulizer-spraye? and sprayed onto Formvar-coated loo-mesh copper grids. Counting was done using a Philips 201 electron microscope operating at 80 kV. The ratio of capsules per particle provided an estimate of 5.20 + 1.29 x 1012 capsules/ ml as the concentration in the stock solution. RESULTS
AND DISCUSSION
Dose-mortality data for both the NPV and the GV assays are summarized in Table 1. Mortality from unknown causes was highest in the second-instar larval assays. These larvae were very small (0.74 mg/larva), and the unknown mortality in most cases might be attributed to the transfer procedure. An exception to this may be the highest concentration of NPV which caused 30% unknown mortality in both the second-instar and fourth-instar assays. Diagnosis of these dead larvae revealed a high concentration of rod-shaped
bacteria which were not detected in the original PIB suspension. Of all the control larvae only one second-instar larva died of viral disease. In all assays, larval mortality increased with increased dosage. Larval susceptibility to both viral preparations decreased with increased age. A dosage of 8.2 x 104 PIB/larvae caused 100% mortality for second instars, but a dosage of 4.1 x lo5 PIB/larva caused only 82% mortality for fourth-instar larvae and 56% mortality for fifth-instar larvae. Similarly, a dosage of 1.0 x 10’ capsules/larva caused 100% mortality for second-instar larvae whereas a dosage of 5.2 x lo7 capsules/larva caused 60% mortality for fourth-instar larvae and only 1% mortality for fifth-instar larvae. Probit analysis4 was used to calculate the dose-mortality regression lines in Figures 1 and 2 and the LDso data in Table 2. The values of the slopes of all the regression lines approximate unity (Fig. 1, 2). The wide confidence limits of these regression lines, owing in part to the limited number of larvae used in these bioassays, 4 SAS Computer University).
Program
(North
Carolina
State
1.
x 10'
x 103
x 102
x 10'
4.1
4.1
4.1
4.1
for
l
**
dead
larvae/(Totai
no.
larvae
dead
Corrected mortality Based on 3 replications Based on 2 replications
from
Control
0
28
28
30
- larvae
5.2
0
30
x 10'
4.1
5.2
0
x 107
4.1
5.2
7
x 103
4.1
unknown
x 104
x 10s
x 10s
x 10'
5.2
26
x 108
23
x 104
4.1
26
5.2
x 10:
4.1
Assay*** 5.2 x 103
x 104
x 10s
56
30
x 106
4.1
viral
Control
0
0
= No.
5.2
0
Fifth-Instar 96
5.2
x 106
x 107
4
28
.t
x 10s x 108
1
5.2
5.2
5.2
5.2
Assay***
27
Control
t
x 104
x 10s
x 106
x 10'
x 108
x 109
Control
1.0
1.0
1.0
1.0
1.0
1.0
Assay**
8
a2
100
Fourth-Instar
0
12
15
52
91
100
100
Second-lnstar
against
Capsules/ larvae
assayed
52
0
Control
viruses
Corrected* % mortality
granulosis
Larvae dead from unknown causes
and
16
23
21
0
5
7
23
39
45
28
from
a nucleopolyhedrosis
-NPV Larvae dead po lyhedrosis
data
29
30
20
31
29
x 10s
45
4.1
x 100
8.2
49
30
x 10'
8.2
4s
x 106
x 102
8.2
45
4.1
x 103
8.2
45
40
x 10'
8.2
40
Total no. larvae/dose
DOSe mortality
Control
x 10s
8.2
PIB's/larvae
Table
GV
webworm
causes)
0
4
3
19
64
100
0
0
7
17
60
92
100
29 3
52
85
100
100
100
Corrected' % mortalittC
larvae.
0
Larvae dead from unknown causes
fall
30
5
ia
28
0
0
2
5
15
24
30
1
10
22
35
31
38
37
from
and fifth-instar
Larvae dead qranulosis
fourth-
29
29
30
28
29
30
27
28
29
30
26
26
30
40
40
45
45
35
40
40
Total no. larvae/dose
second-,
72
BOUCIAS
3
4
5 Log Dosage,
AND NORDIN
6
7
6
s
10
I Larva
Capsules
FIG. 2. Mortality response of second-, fourth-, and tifth-instar larvae fed decimal serial dilutions of granulosis virus. Shaded area indicates overlap between the 95% confidence limits of the regression lines.
also reflect the genetic variation in the laboratory population of the fall webworm. The dose-mortality regression lines do show a significant difference between the susceptibility of the second-instar larvae and the susceptibility of fourth- and fifthinstar larvae to either baculovirus preparation. Differences between fourthand fifth-instar larval susceptibility were not significant at the 95% confidence level as shown by the extensive overlap between the fiducial limits of their regression lines.
The relative susceptibility of the differentaged instars was determined by comparing their LD,, values (Table 2). It is important to note that within a single-age instar group an approximate lo-fold range was predicted between the upper and lower fiducial limits of their respective LD,, values. Second-instar larvae were the most susceptible instar to either the NPV or the GV. It took 100 times more capsules than polyhedra to cause the same mortality against second-instar larvae. The polyhedra
TABLE CALCULATED
NUMBER OF NUCLEOPOLYHEDROSIS TO KILL SO% OF THE TREATED
2 AND GRANULOSIS INCLUSIONS FALL WEBWORM LARVAE
Inclusions/larva
Inclusions/milligram
NPV Second instar Fourth instar Fifth instar GV Second instar Fourth instar Fifth instar
body weight
95% Confidence limits
95% Confidence limits Virus and larvae
REQUIRED
LDso value
Lower
Upper
LD5,, value
Lower
4.79 x 102 2.99 x 104 1.88 x 105
1.92 x 102 1.13 x 104 5.80 x 104
1.20 x 103 8.36 x 104 6.80 x 105
6.47 x 102 7.25 x 18 1.28 x 104
2.59 x 102 2.74 x 1W 3.53 x 103
1.62 x 103 2.03 x 104 4.62 x 1W
7.06 x lo4 2.74 x 10’ 1.87 x lo*
2.67 x 104 1.08 x 10’ 6.36 x 10’
1.79 x 105 7.31 x 10’ 6.25 x 108
9.54 x 104 6.65 x 106 1.27 x 10’
3.61 x lo4 2.62 x lo6 4.32 x 106
2.42 x lo5 1.77 x 10’ 4.24 x 10’
Upper
SUSCEPTIBILITY
HYPHANTRZA
OF
contain 25-30 viral bundles/PIB, each in turn containing 2-12 viral rods (Nordin, 1971). The granulosis capsule usually contains only 1 viral rod/capsule (Vasiljevic, 1962). Converting the number of capsules per larva and number of PlB per larva to number of viral rods per larva could potentially account for any numerical difference between the GV and NPV required to kill second-instar larvae. However, the interinstar susceptibility of fall webworm larvae demonstrates a distinct difference between the GV and NPV. Average second-instar larvae having an LD,, of 4.79 x lo2 PIB/larva were 62 and 392 times more susceptible than the average fourth- and fifth-instar larvae, respectively. After converting LD5,, values to a dose per milligram body weight basis, the susceptibility of second-instar larvae to nucleopolyhedrosis was 11 times greater than fourth-instar larvae and 19 times greater than fifth-instar larvae. Similar decreases in susceptibility to nucleopolyhedrosis with TABLE THE
age have been reported by other investigators (Stairs, 196.5; Doane, 1967; Magnoler, 1975). Challenged with GV, average secondinstar larvae were 388 times more susceptible than fourth-instar larvae and 2648 times more susceptible than fifth-instar larvae. On a dose per milligram body weight basis the second-instar larvae were 70 or 133 times more susceptible to granulosis than fourth- or fifth-instar larvae, respectively. Lethal time data for dosages of NPV and GV causing greater than 50% mortality are graphically depicted in Figures 3 and 4. Cumulative mortality was transformed to probits and graphed against log days. The amount and the time of initial mortality are dependent upon the age of the instar tested and the dosage used. Second-instar larvae fed 8.2 x lo5 PIB/larva or 5.2 x IO9 capsules/larva began to die after 4 days. Mortality of fourth- and fifth-instar larvae fed dosages of 4.1 x lo6 PIB/larva or 5.2 x log capsules/larva first occurred on 3
LT,, VALUES FOR THOSE DOSAGES OF NUCLEOPOLYHEDROSIS AND GRANULOSIS OR GREATER MORTALITY AGAINST SECOND-, FOURTH-, AND FIFTH-INSTAR FALL NPV
PIB/ larva
LTm (days)
Lower
95% Confidence limits Capsules/ larva
Upper Second-instar
x x x x
lo5 104 103 102
3.9 7.8 8.6 12.5
2.9 6.6 7.8 11.4
4.5 9.2 9.4 13.8
Fourth-instar 4.1 x 106 4.1 x 105 4.1 x 104
8.4 11.5 14.8
7.3 10.7 12.1
9.7 12.3 18.1 Fifth-instar
4.1 x 106 4.1 x 105
10.7 13.7
VIRUSES CAUSING 50% WEBWORM LARVAE GV
95% Confidence limits
8.2 8.2 8.2 8.2
73
CUNEA TO VIRUSES
10.1 11.6
11.4 16.2
LTm (days)
Lower
Upper
4.0 5.2 6.1 ,7.4 11.5
3.6 4.8 5.4 6.2 9.4
4.4 5.6 6.9 8.8 14.1
9.0 1’1.5 17.7
7.3 9.2 12.5
11.1 14.4 25.1
9.3 14.0
8.4 10.0
10.2 19.6
assay 1 1 1 1 1
x x x x x
109 108 10’ 106 105
assay 5.2 x 109 5.2 x 108 5.2 x 10’ assay 5.2 x IO9 5.2 x 108
BOUCIAS
74
1
I
I
III
2
4
6810
FIG. 3. Time-mortality polyhedrosis virus.
14
AND NORDIN
20
response of second-, fourth-, and fifth&star
21
4I
III 6810
larvae fed serial dilutions
of nucleo-
20'f'I
Dsys Fro. 4. Time-mortality virus.
response of second-, fourth-, and fifth-instar larvae fed serial dilutions of granulosis
SUSCEPTlBILITY
OF H~PHANTR~A
the sixth day. A decrease in viral concentration for any one assay caused either an increase in the time required for mortality or a decrease in the amount of initial mortality. The LT,, values calculated using the analysis of Litchfield (1949) are shown in Table 3. For any one assay, the LTJo values are inversely related to dosage. A significant difference existed between the LTSO values of second- and fourth-instar larvae when assayed with either virus preparation. Daoust (1974) and lgnoffo (1966) assaying an NPV against various Heliothis species showed a similar correlation between LTBO values and the age of the larvae tested. In conclusion, these data show that interinstar susceptibility of fall webworms to either granulosis or nucleopolyhedrosis is not entirely related to the weight of the larvae assayed. This can be seen by comparing the differences between the LDSO and LT,, values of second- and fourthinstar larvae to differences between the LDSo and LTSO values of second- and fifth-instar larvae. Fu~hermore, there is a distinct difference between the NPV and GV in terms of interinstar susceptibility. Based on the number of viral rods per larva, both viral preparations appear to be equally virulent to second-ins&u larvae. However, the susceptibility of the olderinstar larvae to granulosis is significantly less than their susceptibility to nucleopolyhedrosis. One may speculate that physiological changes during larval maturation have a greater effect on the virulence of the GV than on the virulence of the NPV. ACKNOWLEDGMENT We express appreciation to Miss Susan G. Burros for providing technical assistance during the course of this work.
REFERENCES CUNNINGHAM, J. C, 1970. Pathogenicity tests of nuclear polyh~rosis viruses infecting tlte eastern hemlock looper, Lambdina jisrellaria jiscelluria (Lepidoptera: Geometridae). Canad. Entomol., 102, 1534-1539.
CUNEA TO VIRUSES
75
DAOUST, R. A. 1974. Weight-related susceptibility of larvae of Heliothis armigera to a crude nuclear-polyhedrosis virus preparation. J. Znvertebr. Pathol.,
23,400~401.
DOANE, G. C. 1967. Bioassay of nuclear-polyhedrosis virus against larval instars of the gypsy moth. 1. Invertebr. Path&. , 9,376-386. HUGER, A. 1%3. Granulosis of insects. In “Insect Pathology, an Advanced Treatise” (E. A. Steinhaus, ed.), Vol 1, pp. 531-576. Academic Press, New York. IGNOFFO, C. M. 1966. Effects of age on mortality of Neliothis zea and Heliothis virescens larvae exposed to a nuclei-polyhedrosis virus. J. Znvertebr. Puthol., 8, 279-283. LJTCHFIELD, J. T. 1949. A method for rapid graphic solution of tome-~rcent curves. J. Pharma~ol. Exp. Ther., 97, 399-408. MAGNOLER, A. 1975. Bioassay of nucleopolyhedrosis virus against larval instars of Maiucosoma neustria. J. fnvertebr. Pathol., 25, 343-358. NORDIN, G. L. 1971. “Studies of a Nuclear Polyhedrosis Virus and Three Species of Microsporidia Pathogenic to the Fall Webworm Hyphantriu cunea (Drury).” Ph.D. Thesis, University of Illinois, Urbana, Ill. STAIRS, G. R. 1965. Quantitative differences in susceptibility to nuclear-poiyhedrosis virus among larval instars of the forest tent caterpillar, Maiatosoma disstria ~Hubner). J. fnvertebr. Puthol., 7,427-429.
SUMMERS, M. D., AND PASCHKE, J. D. 1970. Alkahliberated granulosis virus of Trichopiasia ni. I. Density gradient purification of virus components and some of their in vitro chemical and physical properties. 1. Invertebr. Pathol., 16, 227-240. SZIRMAI, J. 1957. Biologische Abwehr mitteb Virus zur Bekamp fung der Hyphantria cunea Drury. Acta Microbial. Hung., 4, 30-42. TANADA, Y. 1956. Some factors affecting the susceptibility of the ~myworm to virus infections. J. Econ. Entomol., 49,52-57. VASIWEVIC, L. 1962. Etude au microscope eiectronique du virus de la granulose de I’ecaille fileuse (Hyphantria cunea Drury). Colloq. Int. Insectes Paris, 383-391. WATANABE, H., AND KOBAYASHI, M. 1970. Histopathology of a granulosis in the larvae of the fall webworm, Hyphantria cunea. J. Invertebr. Pathol., X6,71-79.
WILLIAMS, R, C., AND BACKUS, R. C. 1949. Macromolecular weights determined by direct particle counting. I. The weight of the bushy stunt virus. J. Amer. Chem. Sot., 71,4052-4057. YEARIAN, W. C., GILBERT, K. L., AND WARREN, L. 0. 1966. Rearing of the fall webworm Hypha~tria c~~ea Dr. (Lepidoptera: Arctiidae) on a wheat germ medium. J. Kans. Entomol. Sot., 39, 49.5-499.