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Pathogenicity of a granulosis virus of the Indian-meal moth
JOURNAL
OF
INVEltTEBRATE
PATHOLOGY
Yathogenicity
16,
339-:3~1
of a Granulosis
( 1970)
Virus
of the Indian-meal
Moth
D. K. HUNTER Stored-Product Agricultural
Imects Research Branch, Market Quality Research Dihiorl, USDA, Frecno, California 93727 Research Sewice, Receioed
February
3, 1970
A bioassay was conducted to determine the virulence of a gramdosis virus to firstinstar, Indian-meal moth larvae. The calculated LC,, was 18,000 capsules per gram of bran diet. Fiducial limits (0.05) were determined to be 20,900 and 15,900 capsules/g. Studies on time of death showed that a concentration of 8.6 x 104 capsules/g killed 51% of test larvae by 10 days after exposure to the virus. A subsequent specificity test showed that the virus failed to infect first-instar Epkestia elutella, Cadra cautella, and C. figulilella at a concentration of 8.6 x 10” caps&s/g.
INTRODUCTION
The Indian-meal moth, Plodiu interpunctella, is widely recognized as a serious pest of stored grain and other agricultural products. Arnott and Smith (1968) published a study on the ultrastructure and development of a granulosis virus in cells of this insect. They reported that the virus appeared to be mainly confined to the cytoplasm of the fat cells, although some evidence for its occurrence in the tracheae was also found. This report deals with such relationships as virulence, time of host death, and host-specificity as observed when larvae were perorally exposed to the pathogen. METHODS
AND RESULTS
Virus suspension for testing was prepared by comminuting infected late instar Plodia interpunctella larvae in a tissue grinder with distilled water. The crude suspension was twice centrifuged differentially at 4000 g for 5 min followed by 12,000 g for 1 hr at 10°C. The capsules were counted on a Petroff-Hausser bacteria counter with a phase-contrast microscope at 1250X . The stock inoculum was immediately diluted
and added to media in test containers as undesirable clumping of the capsulesbegins within a few hours. Test insects were obtained by placing newly emerged P. interpunctella adults in quart jars for mating and oviposition. Screen disks were substituted for the solid tops, and the jars were inverted and placed on bottom halves of disposable petri dishes. Eggs laid in the jars fell through the screens and were collected at 72-hr intervals. Scales and debris were removed with a fine stream of air. The eggs were surfaccsterilized in S% formaldehvde for 20 min, washed in distilled water, and then incubated at 27°C in plastic vials. After the eggs hatched, each of 25 firstinstar larvae were transferred to test chambers consisting of half-pint cartons supplied with 10 g of bran diet (Finney and Hrinkman, 1967)l into which 2 ml of the virlls suspension were mixed. Control cartons were treated with distilled water. Each test consisted of at least 2 trials. The treatments were replicated 3 times with 25 larvae per 1 A combination of sorbic hydroxybenzoate (4000 ppm) a fungistat.
acid \vas
+
methyl-p-
incoqxm~ttd as
340
IIUNTER
replicate. All mortalities were corrected by Abbott’s formula (Abbott, 1925). The concentration-mortality regression line was computed according to Finney (1964).
7-
6-
Concentration-Mortality The virus suspension was diluted with distifled water and mixed with diet in cartons to give concentrations of 8.6 X 104, 4.3 x 104, 2.15 x 104, 8.6 x 103, 4.3 x 103, and 2.15 X lo3 capsules/g. The larvae were introduced to the cartons and incubated at 27°C. Heavily infected individuals became noticeably white and sluggish and generally succumbed within 12 days. As the cadavers desiccated, they darkened and shriveled to about a third the size of healthy larvae. Mortalities recorded from the various concentrations were based on adult emergence and are given in Table 1. Figure 1 shows probit mortality plotted over log concentration. An equation describing the curve is given. The calculated LCBo was 18,000 capsules/g with the upper and lower fiducial limit (0.05) lying at 20,900 and 15,900 capsules/g, respectively. Time of Death Time of death was determined for the concentration of 8.6 X lo4 capsules/g at 27°C. The bran containing young larvae was poured from the treatment and control cartons on a sieve shaker having openings of 0.84 mm, spread evenly, and placed about 6 in beneath a 60-watt light bulb.
RESPONSE OF FIRST-INSTAR Plodia
Y-5.135tl.231(X-4.147)
FIG. 1. Concentration-mortality instar Ho&a interpunctella granulosis virus.
The heat drove the live larvae out of the bran and forced them to drop through the mesh into the pan below, where they were counted. Table 2 shows the results of 2 trials over a 4-day span beginning 7 days after the first-instar larvae were introduced to the cartons. A standard treatment and the controls were allowed to incubate until the adults emerged in order to confirm the virulence of the inoculum. As shown, 51% of the test larvae succumbed to the virus by the 10th day after exposure. However, at this time, most of the surviving larvae in the treatments showed advanced symptoms of the disease,indicating that these individuals would have died within a few days.
TABLE 1 interpunctelh Adult
Capsules/g 8.6 x 104 4.3 x 104 2.15 X 104 8.6 x 103 4.3 x 103 2.15 x 103 Control
No. larvae/trial 75 75 75 75 75 75 75
Trial 15 18 36 42 34 51 57
1
curve of firstexposed to a
larvae
LARVAE TO GRANULOSIS
VIRUS
emergence Trial 7 17 19 25 30 46 55
2
Trial 2 2 11 25 32 46 54
3
Av.
% mortality 86 78 61 45 42 14
GRANULOSIS
TIME
OF DEATH
Days of exposure
No.
to vrirus
.--
7 8
9 10 25 ( Standard
)
OF
THE
INDIAS-MEAL
TABLE 2 OF FIRST-INSTAR Plodia interpunctella VIRUS AT 8.6 X 104 CAPSULES larvae/ trial ______--75 75 75 75 75
54 55 48
59
TR
CK
26
64
31
60
2; 31
3
55
As more than one species of lepidopterous pest may infest a stored product, it would naturallv be desirable if the same virus could be used to control the various species. A preliminary investigation of this possibility was made by attempting to infect three other pestiferous moths of the family Phycitidae. The species tested were: the tobacco moth, Ephestiu elutellu, the almond moth, Cadre cautelkz, and the raisin moth, C. figuliklla. P. interpunctella was used as a standard. The virus was mixed with diet to give a concentration of 8.6 X 10” capsules/g and first-instar larvae of all the species were introduced. The test larvae were incubated at 27°C and the resultant mortality was based on adult emergence. Table 3 shows the results of two trials. Only P. interpunctella experienced mortality due to virus; the other species were unaffected by this means of exposure. The
OF FOUR
tested
Plodia interpunctella Ephestia eluteEla Cadra cautella Cadra figulilella
EXPOSED
TR
75 75 75 75
Trial CK 72 61 52 66
% mortalit! 11.0
61
48 .38 38
71
r,‘1
51.5
39.5 40.0
2
96.0
response of these species to intrahemococlic injection of free virus was not investigatcxd. ACKKOWLEDGMEYIS The author wishes to thank T. II. laboratory for his excellent technical
ABBOTT, W. S. 1925. effectiveness of an tomol., 18, 265-267.
A method insecticide.
lIexe1 of this assistancc~.
of computing J. Econ. Ert-
ARNOTT, II. J., ANJ SMITH, K. hl. 1968. An ultrastructural study of the development of a granulosis virus in the cells of the moth, Plodia interpunctella ( Hbn). J. Ubrastrrrcf. Res., 21, 251-258. FINNEY, D. J. 1964. 318 pp. Cambridge New York. FIKXEY, ing
“Probit Univ.
G. L., AKD BRIWXAS, the navel orangeworm
J. Entomol.
SOL-. Amer..
TABLE 3 SPECIES OF PHYCITIDAE TO A GRANULOSIS FROM Plodia interpuncteb
No. larvae/ trial
TO G1~.4uu~os1s
-il
Adult --Species
LARVAE AT 27°C
A\
-_ CK
Specificity
RESPONSE
‘141
I\ZOTFI
VIRUS
cd.. and
I>. 1967. Rearin the laborator\~. 60, 1109-I 11 1.
ISOLATEI)
emergence
1
Trial TR -____ 0 60 55 61
Analysis,“ 2nd Press. London
2
CK
TR
74 55 53 59
0 68 59 71
Average % ruortalit~ 100 0 0 0
--
OF
INVEltTEBRATE
PATHOLOGY
Yathogenicity
16,
339-:3~1
of a Granulosis
( 1970)
Virus
of the Indian-meal
Moth
D. K. HUNTER Stored-Product Agricultural
Imects Research Branch, Market Quality Research Dihiorl, USDA, Frecno, California 93727 Research Sewice, Receioed
February
3, 1970
A bioassay was conducted to determine the virulence of a gramdosis virus to firstinstar, Indian-meal moth larvae. The calculated LC,, was 18,000 capsules per gram of bran diet. Fiducial limits (0.05) were determined to be 20,900 and 15,900 capsules/g. Studies on time of death showed that a concentration of 8.6 x 104 capsules/g killed 51% of test larvae by 10 days after exposure to the virus. A subsequent specificity test showed that the virus failed to infect first-instar Epkestia elutella, Cadra cautella, and C. figulilella at a concentration of 8.6 x 10” caps&s/g.
INTRODUCTION
The Indian-meal moth, Plodiu interpunctella, is widely recognized as a serious pest of stored grain and other agricultural products. Arnott and Smith (1968) published a study on the ultrastructure and development of a granulosis virus in cells of this insect. They reported that the virus appeared to be mainly confined to the cytoplasm of the fat cells, although some evidence for its occurrence in the tracheae was also found. This report deals with such relationships as virulence, time of host death, and host-specificity as observed when larvae were perorally exposed to the pathogen. METHODS
AND RESULTS
Virus suspension for testing was prepared by comminuting infected late instar Plodia interpunctella larvae in a tissue grinder with distilled water. The crude suspension was twice centrifuged differentially at 4000 g for 5 min followed by 12,000 g for 1 hr at 10°C. The capsules were counted on a Petroff-Hausser bacteria counter with a phase-contrast microscope at 1250X . The stock inoculum was immediately diluted
and added to media in test containers as undesirable clumping of the capsulesbegins within a few hours. Test insects were obtained by placing newly emerged P. interpunctella adults in quart jars for mating and oviposition. Screen disks were substituted for the solid tops, and the jars were inverted and placed on bottom halves of disposable petri dishes. Eggs laid in the jars fell through the screens and were collected at 72-hr intervals. Scales and debris were removed with a fine stream of air. The eggs were surfaccsterilized in S% formaldehvde for 20 min, washed in distilled water, and then incubated at 27°C in plastic vials. After the eggs hatched, each of 25 firstinstar larvae were transferred to test chambers consisting of half-pint cartons supplied with 10 g of bran diet (Finney and Hrinkman, 1967)l into which 2 ml of the virlls suspension were mixed. Control cartons were treated with distilled water. Each test consisted of at least 2 trials. The treatments were replicated 3 times with 25 larvae per 1 A combination of sorbic hydroxybenzoate (4000 ppm) a fungistat.
acid \vas
+
methyl-p-
incoqxm~ttd as
340
IIUNTER
replicate. All mortalities were corrected by Abbott’s formula (Abbott, 1925). The concentration-mortality regression line was computed according to Finney (1964).
7-
6-
Concentration-Mortality The virus suspension was diluted with distifled water and mixed with diet in cartons to give concentrations of 8.6 X 104, 4.3 x 104, 2.15 x 104, 8.6 x 103, 4.3 x 103, and 2.15 X lo3 capsules/g. The larvae were introduced to the cartons and incubated at 27°C. Heavily infected individuals became noticeably white and sluggish and generally succumbed within 12 days. As the cadavers desiccated, they darkened and shriveled to about a third the size of healthy larvae. Mortalities recorded from the various concentrations were based on adult emergence and are given in Table 1. Figure 1 shows probit mortality plotted over log concentration. An equation describing the curve is given. The calculated LCBo was 18,000 capsules/g with the upper and lower fiducial limit (0.05) lying at 20,900 and 15,900 capsules/g, respectively. Time of Death Time of death was determined for the concentration of 8.6 X lo4 capsules/g at 27°C. The bran containing young larvae was poured from the treatment and control cartons on a sieve shaker having openings of 0.84 mm, spread evenly, and placed about 6 in beneath a 60-watt light bulb.
RESPONSE OF FIRST-INSTAR Plodia
Y-5.135tl.231(X-4.147)
FIG. 1. Concentration-mortality instar Ho&a interpunctella granulosis virus.
The heat drove the live larvae out of the bran and forced them to drop through the mesh into the pan below, where they were counted. Table 2 shows the results of 2 trials over a 4-day span beginning 7 days after the first-instar larvae were introduced to the cartons. A standard treatment and the controls were allowed to incubate until the adults emerged in order to confirm the virulence of the inoculum. As shown, 51% of the test larvae succumbed to the virus by the 10th day after exposure. However, at this time, most of the surviving larvae in the treatments showed advanced symptoms of the disease,indicating that these individuals would have died within a few days.
TABLE 1 interpunctelh Adult
Capsules/g 8.6 x 104 4.3 x 104 2.15 X 104 8.6 x 103 4.3 x 103 2.15 x 103 Control
No. larvae/trial 75 75 75 75 75 75 75
Trial 15 18 36 42 34 51 57
1
curve of firstexposed to a
larvae
LARVAE TO GRANULOSIS
VIRUS
emergence Trial 7 17 19 25 30 46 55
2
Trial 2 2 11 25 32 46 54
3
Av.
% mortality 86 78 61 45 42 14
GRANULOSIS
TIME
OF DEATH
Days of exposure
No.
to vrirus
.--
7 8
9 10 25 ( Standard
)
OF
THE
INDIAS-MEAL
TABLE 2 OF FIRST-INSTAR Plodia interpunctella VIRUS AT 8.6 X 104 CAPSULES larvae/ trial ______--75 75 75 75 75
54 55 48
59
TR
CK
26
64
31
60
2; 31
3
55
As more than one species of lepidopterous pest may infest a stored product, it would naturallv be desirable if the same virus could be used to control the various species. A preliminary investigation of this possibility was made by attempting to infect three other pestiferous moths of the family Phycitidae. The species tested were: the tobacco moth, Ephestiu elutellu, the almond moth, Cadre cautelkz, and the raisin moth, C. figuliklla. P. interpunctella was used as a standard. The virus was mixed with diet to give a concentration of 8.6 X 10” capsules/g and first-instar larvae of all the species were introduced. The test larvae were incubated at 27°C and the resultant mortality was based on adult emergence. Table 3 shows the results of two trials. Only P. interpunctella experienced mortality due to virus; the other species were unaffected by this means of exposure. The
OF FOUR
tested
Plodia interpunctella Ephestia eluteEla Cadra cautella Cadra figulilella
EXPOSED
TR
75 75 75 75
Trial CK 72 61 52 66
% mortalit! 11.0
61
48 .38 38
71
r,‘1
51.5
39.5 40.0
2
96.0
response of these species to intrahemococlic injection of free virus was not investigatcxd. ACKKOWLEDGMEYIS The author wishes to thank T. II. laboratory for his excellent technical
ABBOTT, W. S. 1925. effectiveness of an tomol., 18, 265-267.
A method insecticide.
lIexe1 of this assistancc~.
of computing J. Econ. Ert-
ARNOTT, II. J., ANJ SMITH, K. hl. 1968. An ultrastructural study of the development of a granulosis virus in the cells of the moth, Plodia interpunctella ( Hbn). J. Ubrastrrrcf. Res., 21, 251-258. FINNEY, D. J. 1964. 318 pp. Cambridge New York. FIKXEY, ing
“Probit Univ.
G. L., AKD BRIWXAS, the navel orangeworm
J. Entomol.
SOL-. Amer..
TABLE 3 SPECIES OF PHYCITIDAE TO A GRANULOSIS FROM Plodia interpuncteb
No. larvae/ trial
TO G1~.4uu~os1s
-il
Adult --Species
LARVAE AT 27°C
A\
-_ CK
Specificity
RESPONSE
‘141
I\ZOTFI
VIRUS
cd.. and
I>. 1967. Rearin the laborator\~. 60, 1109-I 11 1.
ISOLATEI)
emergence
1
Trial TR -____ 0 60 55 61
Analysis,“ 2nd Press. London
2
CK
TR
74 55 53 59
0 68 59 71
Average % ruortalit~ 100 0 0 0
--