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Cytoplasmic-polyhedrosis virus and the development and fecundity of the pink bollworm
JOURNAL
OF INVERTEBRATE
PATHOLOGY
Cytoplasmic-Polyhedrosis Fecundity
15, 109-112
(1970)
Virus and the Development of the Pink Bollworml
H.R. BULLOCK,E. MARTINEZ, AND
C.
W.
STWRMER,
and
JR.~
Entomology Research Dieision, Agricultural Research Service, U.S. Department of Agriculture, Brownsville, Texas 78520 Received June 24, 1969 Larvae of the pink bollworm, Pectinophora gossypiella, treated with a cytoplasmicpolyhedrosis virus ( 100 viral inclusions/mm2 of diet surface) were 73.5% smaller than untreated larvae, pupation was reduced 50%, and pupal weights were reduced 20%. Only 22% of treated larvae produced adults, the longevity of diseased males and females was reduced 48 and 46%, respectively, and production of eggs was reduced 66% in diseased moths. However, hatch of eggs and mating were not affected by disease.
INTRODUCTION
A disease agent that was suspected to be in insect cultures at the Southwestern Cotton Insects Investigations laboratory in Brownsville, Texas, as early as 1963 was isolated from adult pink bollworms, Pectinophora gosstJpiell.u, and described as a cytoplasmic-polyhedrosis virus, Smithiavirus pectinophorae, by Ignoffo and Adams ( 1966). Ignoffo and Adams reported that when larvae fed on diets contaminated with the virus, pupal weights were significantly reduced, and pupation and adult emergence were retarded. They speculated that the disease may reduce longevity, fecundity, and viability of eggs. Subsequently, we isolated a similar, if not identical, virus( es) from cultures of the cabbage looper, Trichoplusia ni, the bollworm, Heliothis xea, the tobacco budworm, Heliothis virescens, the salt-marsh caterpillar, Estigmene acrea, and the beet armyworm, 1 In cooperation with the Texas Agricultural Experiment Station, Texas A&M University, College Station 77843. s Graduate student, Texas A&M University, under Cooperative Agreement NO. 12-14-100-9046
Spodoptera erigua ( Bullock, unpublished data). We therefore made a study to determine the effects of the virus on the development and fecundity of the pink bollworm. The results are reported here. MATERIALS AND METHODS The criteria used to evaluate the effect of virus on the development of the pink bollworms were: weight of lo-day-old larvae, weight of pupae, and percentages of pupation and emergence. The effects of the virus on fecundity were determined in a supplemental test from production of eggs, percentage of hatch, and percentage of mating. In all tests, we used cytoplasmic polyhedra isolated from larval HeZiothis nea prepared by washing the diseased midguts of larvae three times in a solution of 1-phenyl-2-thiourea ( 0.5 mg/ml of water), homogenizing the washed midgut in a Ten Broeck grinder, and filtering the homogenate through Kimwipes (TYPE 900-S )“. The 3 Use of trade name does endorsement of the product ment of Agriculture.
(33). 109
not necessarily by the U.S.
imply Depart-
110
BULLOCK ET AL.
preparations were standardized by counting polyhedra with a hemocytometer (430 X magnification). The stock preparation was stored at 4°C. The test to determine the effects of virus on development was replicated six times with each replicate consisting of 600 firstinstar larvae from the laboratory colony, 300 fed on semisynthetic diet treated with the virus (Ouye, 1962; Ouye and Vanderzant, 1964) and 300 fed on untreated diet. The larvae were infested by placing them in individual cotton-plugged vials on diet contaminated with lo/u1 of viral suspension (100 viral inclusion bodies/mm’ of media surface). (The control diets were left untreated.) Insects were held at 27 2 10°C. Seven days later treated and untreated larvae were divided into two groups, and the number of living and dead larvae were recorded. One-half of the treated and onehalf of the untreated larvae were held to obtain data on pupation, weight of pupae, emergence of adults, number of eggs laid in 10 days, and hatch of eggs from five pairs of adults. Pupae were held at 27°C and weighed within 24 hr of pupation. Observations on pupation were made for 45 days because pupation was delayed in the treated larvae. Adults and eggs were held at room temperature (21 * 1.7”C and a relative humidity of 55-70%). However, few matings could be made with adults from treated larvae because so few survived treatment. Also, at 10 days after treatment, the other halves of treated and untreated larvae were weighed, and the midguts were diagnosed for frank polyhedrosis with a microscope. Usually, there were insufficient numbers of adults from treated larvae for comparison with adults from untreated larvae; therefore, an additional test was made with adults from larvae known to be diseased (The presence of disease was determined by sampling about 400 larvae, the feces
and/or vomit of which revealed inclusion bodies and thus infection with polyhedrosis virus.). As in the first tests, adults from diseased larvae were compared with healthy insects to determine comparative fecundity, longevity, and egg hatch. Subsequently, all adults in this test (five replicates of five pairs of adults) were examined for the presence of viral inclusion bodies, and the females were examined for spermatophores to determine the degree of mating. Progeny from diseased and healthy adults were fed on untreated diet and reared for subsequent diagnosis for viral infection. RESULTS AND DISCUSSION
The concentration of 100 virus inclusion bodies/mm2 of media produced 85% infection in first-instar pink bollworm larvae that survived for 10 days after feeding on treated diet. Infection ranged from 72.0 to 96.2%. The effects of the virus on weight, pupation, and emergence of pink bollworms are given in Table 1. Ten-day-old treated larvae were significantly smaller (73.5%) than untreated larvae (1% level of confidence); however, the weights of these larvae were less than if they had been reared at the normal rearing temperature of 29°C. (The insectary temperature was inadvertently held at 27°C during the first replicate, so larvae were held at this temperature in all subsequent tests to maintain uniformity. ) We did not observe any successive molts in infected larvae without increasesin body size as reported by Ignoffo and Adams (1966). The percentage of pupation in treated larvae was significantly less than in the untreated check (1% level) whether we counted the pupae that resulted from larvae living on day 7 of the test (42% reduction) or considered the rate of pupation of all larvae (51.4% reduction). Ignoffo and Adams (1966) reported
EFFECT
VIRUS
ON
Weight
(mg)
f
SD
Male
19.1
Untreated
6.8 -c 0.5
24.9 ?I 0.4
different different
20.8
-c 0.5
at 0.05 level at 0.01 level
LONGEVITY
in days
2 SD
Males
Diseased
13.9 k 3.2a
14.5 *
Healthy
26.9
27.0
at 0.05 level
All pupae
All larvae
57.0 2 6.6b
65.9 k 3.2”
22.2
98.2
96.3 + 1.4
66.7 t
t
TABLE 2 OF DLVEASED PINK BOLLWORM ( 5 REPLICATES )
1.2
k3.76 4.0
of confidence.
ADULTS
%
Females
5.9
% Emergence t SD from:
Table 2. The diseased adults of both sexes lived significantly fewer days ( 5% level), 48.3% and 46.3% less for males and females, respectively. Also diseased adult females laid 67% fewer eggs than healthy females. Thus, our data are in agreement with those of Neilson ( 1965) who reported that in four species of Lepidoptera, viral infection reduced the reproductive capacity of the hosts. He further reported that diseased adult fall cankerworms, AZsophikz pometaria, did not produce fertile eggs. In our tests, eggs from diseased pink bollworms did not have a reduced hatch. Jupin et al. (1968) have reported similar results with Drosophila infected with sigma virus: they found that sigma virus affected oogenesis in Drosophila but that the viability of laid eggs was not modified. Also, in our test, the incidence of disease in larvae hatched from eggs laid by diseased and healthy adults did not differ when they
Diagnosis
f
I? 4.8”
69.2 -r- 4.1
AND
of confidence. of confidence
AND FECTJND~Y Longevity
different
All larvae
Larvae living at 7 days
f- 0.9b 33.6
a retarded but not a reduced rate of pupation. Pupal weights of both males and females were significantly less in the treated than in the check (5% level for males and 1% level for females), and the male and female pupae from treated larvae weighed 17.3 and 23.3%, respectively, less than pupae from untreated larvae; these data are similar to those reported by Ignoffo and Adams ( 1966) for pupal weights (not differentiated by sex). Also, adult emergence was reduced in treated larvae whether the rate of emergence was based on number of pink bollworms reaching the pupal stage (31.6% less) or the number of larvae infested (66.6% less) compared with the untreated checks (1% level). Only 22% of treated larvae produced adults. Data on the results from the supplemental test of longevity and fecundity of diseased pink bollworms are presented in
0 Significantly
-C SD from:
Female
1.8 & 0.3b 17.2 & 0.5~
PUPATION,
% Pupation
Pupae
lo-day-old larvae
Treated ( 100 inclusions/mms)
Q Significantly b Significantly
111
BOLLWORM
TABLE 1 OF CYTOPLASMIC-POLYHEDROSIS VIRUS ON WEIGHT, EMERGENCE OF PINK BOLLWORM ( 6 Fhp~1cATEs )
EFFECT
Treatment
OF
3.1a
& 5.7
Eggs
hatched
Mated
No.
%
96
345
76.8
92
1037
79.3
112
BULLOCK ET AL.
fed on untreated diet, and there was no difference in mating between diseased and healthy adults. These data can be helpful in the construction of life tables to guide the management of insect cultures. Thus, one can predict the number of both adults and eggs that will be produced when the incidence of cytoplasmic disease is 85%. Changes in development and fecundity of the pink bollworm caused by cytoplasmic-polyhedrosis virus were therefore reflected by: (1) the weight of the larvae, (2) the rate of pupation and the weight of the pupae, (3) the rate of adult emergence and longevity, and (4) a reduction of about two-thirds in production of eggs. Mating and hatch were not affected by the virus in this experiment.
REFERENCES I~NOFFO, C. hl., AND ADAMS, J. R. 1966. A cytoplasmic-polyhedrosis virus, Smithiavirus pectinophorae, sp. n. of the pink bollworm, Pectinophora gossypiella ( Saunders ) . J. Invertebrate Pathol., 8, 59-66. JUPIN,
N., Action fertilite Pasteur,
PLUS, N., AND FLEUHIET, A. 1968. dune souche de virus sigma sur la des Drosophiles femelles. Ann. Inst.
114, 577-594.
NEILSON, M. M. 1965. Effects of a cytoplasmic polyhedrosis on adult Lepidoptera. 1. Invertebrate Pathol., 7, 306-314. OUYE,
M. T. 1962. Effects of antimicrobial agents on micro-organisms and pink bollworm development. J. &on. Entomol., 55, 854-857.
OUYE,
M. T., AND VANDERZANT, E. S. Vitamin requirements of the pink J. Econ. Entmol., 57, 427430.
1964. Bbollworm.
OF INVERTEBRATE
PATHOLOGY
Cytoplasmic-Polyhedrosis Fecundity
15, 109-112
(1970)
Virus and the Development of the Pink Bollworml
H.R. BULLOCK,E. MARTINEZ, AND
C.
W.
STWRMER,
and
JR.~
Entomology Research Dieision, Agricultural Research Service, U.S. Department of Agriculture, Brownsville, Texas 78520 Received June 24, 1969 Larvae of the pink bollworm, Pectinophora gossypiella, treated with a cytoplasmicpolyhedrosis virus ( 100 viral inclusions/mm2 of diet surface) were 73.5% smaller than untreated larvae, pupation was reduced 50%, and pupal weights were reduced 20%. Only 22% of treated larvae produced adults, the longevity of diseased males and females was reduced 48 and 46%, respectively, and production of eggs was reduced 66% in diseased moths. However, hatch of eggs and mating were not affected by disease.
INTRODUCTION
A disease agent that was suspected to be in insect cultures at the Southwestern Cotton Insects Investigations laboratory in Brownsville, Texas, as early as 1963 was isolated from adult pink bollworms, Pectinophora gosstJpiell.u, and described as a cytoplasmic-polyhedrosis virus, Smithiavirus pectinophorae, by Ignoffo and Adams ( 1966). Ignoffo and Adams reported that when larvae fed on diets contaminated with the virus, pupal weights were significantly reduced, and pupation and adult emergence were retarded. They speculated that the disease may reduce longevity, fecundity, and viability of eggs. Subsequently, we isolated a similar, if not identical, virus( es) from cultures of the cabbage looper, Trichoplusia ni, the bollworm, Heliothis xea, the tobacco budworm, Heliothis virescens, the salt-marsh caterpillar, Estigmene acrea, and the beet armyworm, 1 In cooperation with the Texas Agricultural Experiment Station, Texas A&M University, College Station 77843. s Graduate student, Texas A&M University, under Cooperative Agreement NO. 12-14-100-9046
Spodoptera erigua ( Bullock, unpublished data). We therefore made a study to determine the effects of the virus on the development and fecundity of the pink bollworm. The results are reported here. MATERIALS AND METHODS The criteria used to evaluate the effect of virus on the development of the pink bollworms were: weight of lo-day-old larvae, weight of pupae, and percentages of pupation and emergence. The effects of the virus on fecundity were determined in a supplemental test from production of eggs, percentage of hatch, and percentage of mating. In all tests, we used cytoplasmic polyhedra isolated from larval HeZiothis nea prepared by washing the diseased midguts of larvae three times in a solution of 1-phenyl-2-thiourea ( 0.5 mg/ml of water), homogenizing the washed midgut in a Ten Broeck grinder, and filtering the homogenate through Kimwipes (TYPE 900-S )“. The 3 Use of trade name does endorsement of the product ment of Agriculture.
(33). 109
not necessarily by the U.S.
imply Depart-
110
BULLOCK ET AL.
preparations were standardized by counting polyhedra with a hemocytometer (430 X magnification). The stock preparation was stored at 4°C. The test to determine the effects of virus on development was replicated six times with each replicate consisting of 600 firstinstar larvae from the laboratory colony, 300 fed on semisynthetic diet treated with the virus (Ouye, 1962; Ouye and Vanderzant, 1964) and 300 fed on untreated diet. The larvae were infested by placing them in individual cotton-plugged vials on diet contaminated with lo/u1 of viral suspension (100 viral inclusion bodies/mm’ of media surface). (The control diets were left untreated.) Insects were held at 27 2 10°C. Seven days later treated and untreated larvae were divided into two groups, and the number of living and dead larvae were recorded. One-half of the treated and onehalf of the untreated larvae were held to obtain data on pupation, weight of pupae, emergence of adults, number of eggs laid in 10 days, and hatch of eggs from five pairs of adults. Pupae were held at 27°C and weighed within 24 hr of pupation. Observations on pupation were made for 45 days because pupation was delayed in the treated larvae. Adults and eggs were held at room temperature (21 * 1.7”C and a relative humidity of 55-70%). However, few matings could be made with adults from treated larvae because so few survived treatment. Also, at 10 days after treatment, the other halves of treated and untreated larvae were weighed, and the midguts were diagnosed for frank polyhedrosis with a microscope. Usually, there were insufficient numbers of adults from treated larvae for comparison with adults from untreated larvae; therefore, an additional test was made with adults from larvae known to be diseased (The presence of disease was determined by sampling about 400 larvae, the feces
and/or vomit of which revealed inclusion bodies and thus infection with polyhedrosis virus.). As in the first tests, adults from diseased larvae were compared with healthy insects to determine comparative fecundity, longevity, and egg hatch. Subsequently, all adults in this test (five replicates of five pairs of adults) were examined for the presence of viral inclusion bodies, and the females were examined for spermatophores to determine the degree of mating. Progeny from diseased and healthy adults were fed on untreated diet and reared for subsequent diagnosis for viral infection. RESULTS AND DISCUSSION
The concentration of 100 virus inclusion bodies/mm2 of media produced 85% infection in first-instar pink bollworm larvae that survived for 10 days after feeding on treated diet. Infection ranged from 72.0 to 96.2%. The effects of the virus on weight, pupation, and emergence of pink bollworms are given in Table 1. Ten-day-old treated larvae were significantly smaller (73.5%) than untreated larvae (1% level of confidence); however, the weights of these larvae were less than if they had been reared at the normal rearing temperature of 29°C. (The insectary temperature was inadvertently held at 27°C during the first replicate, so larvae were held at this temperature in all subsequent tests to maintain uniformity. ) We did not observe any successive molts in infected larvae without increasesin body size as reported by Ignoffo and Adams (1966). The percentage of pupation in treated larvae was significantly less than in the untreated check (1% level) whether we counted the pupae that resulted from larvae living on day 7 of the test (42% reduction) or considered the rate of pupation of all larvae (51.4% reduction). Ignoffo and Adams (1966) reported
EFFECT
VIRUS
ON
Weight
(mg)
f
SD
Male
19.1
Untreated
6.8 -c 0.5
24.9 ?I 0.4
different different
20.8
-c 0.5
at 0.05 level at 0.01 level
LONGEVITY
in days
2 SD
Males
Diseased
13.9 k 3.2a
14.5 *
Healthy
26.9
27.0
at 0.05 level
All pupae
All larvae
57.0 2 6.6b
65.9 k 3.2”
22.2
98.2
96.3 + 1.4
66.7 t
t
TABLE 2 OF DLVEASED PINK BOLLWORM ( 5 REPLICATES )
1.2
k3.76 4.0
of confidence.
ADULTS
%
Females
5.9
% Emergence t SD from:
Table 2. The diseased adults of both sexes lived significantly fewer days ( 5% level), 48.3% and 46.3% less for males and females, respectively. Also diseased adult females laid 67% fewer eggs than healthy females. Thus, our data are in agreement with those of Neilson ( 1965) who reported that in four species of Lepidoptera, viral infection reduced the reproductive capacity of the hosts. He further reported that diseased adult fall cankerworms, AZsophikz pometaria, did not produce fertile eggs. In our tests, eggs from diseased pink bollworms did not have a reduced hatch. Jupin et al. (1968) have reported similar results with Drosophila infected with sigma virus: they found that sigma virus affected oogenesis in Drosophila but that the viability of laid eggs was not modified. Also, in our test, the incidence of disease in larvae hatched from eggs laid by diseased and healthy adults did not differ when they
Diagnosis
f
I? 4.8”
69.2 -r- 4.1
AND
of confidence. of confidence
AND FECTJND~Y Longevity
different
All larvae
Larvae living at 7 days
f- 0.9b 33.6
a retarded but not a reduced rate of pupation. Pupal weights of both males and females were significantly less in the treated than in the check (5% level for males and 1% level for females), and the male and female pupae from treated larvae weighed 17.3 and 23.3%, respectively, less than pupae from untreated larvae; these data are similar to those reported by Ignoffo and Adams ( 1966) for pupal weights (not differentiated by sex). Also, adult emergence was reduced in treated larvae whether the rate of emergence was based on number of pink bollworms reaching the pupal stage (31.6% less) or the number of larvae infested (66.6% less) compared with the untreated checks (1% level). Only 22% of treated larvae produced adults. Data on the results from the supplemental test of longevity and fecundity of diseased pink bollworms are presented in
0 Significantly
-C SD from:
Female
1.8 & 0.3b 17.2 & 0.5~
PUPATION,
% Pupation
Pupae
lo-day-old larvae
Treated ( 100 inclusions/mms)
Q Significantly b Significantly
111
BOLLWORM
TABLE 1 OF CYTOPLASMIC-POLYHEDROSIS VIRUS ON WEIGHT, EMERGENCE OF PINK BOLLWORM ( 6 Fhp~1cATEs )
EFFECT
Treatment
OF
3.1a
& 5.7
Eggs
hatched
Mated
No.
%
96
345
76.8
92
1037
79.3
112
BULLOCK ET AL.
fed on untreated diet, and there was no difference in mating between diseased and healthy adults. These data can be helpful in the construction of life tables to guide the management of insect cultures. Thus, one can predict the number of both adults and eggs that will be produced when the incidence of cytoplasmic disease is 85%. Changes in development and fecundity of the pink bollworm caused by cytoplasmic-polyhedrosis virus were therefore reflected by: (1) the weight of the larvae, (2) the rate of pupation and the weight of the pupae, (3) the rate of adult emergence and longevity, and (4) a reduction of about two-thirds in production of eggs. Mating and hatch were not affected by the virus in this experiment.
REFERENCES I~NOFFO, C. hl., AND ADAMS, J. R. 1966. A cytoplasmic-polyhedrosis virus, Smithiavirus pectinophorae, sp. n. of the pink bollworm, Pectinophora gossypiella ( Saunders ) . J. Invertebrate Pathol., 8, 59-66. JUPIN,
N., Action fertilite Pasteur,
PLUS, N., AND FLEUHIET, A. 1968. dune souche de virus sigma sur la des Drosophiles femelles. Ann. Inst.
114, 577-594.
NEILSON, M. M. 1965. Effects of a cytoplasmic polyhedrosis on adult Lepidoptera. 1. Invertebrate Pathol., 7, 306-314. OUYE,
M. T. 1962. Effects of antimicrobial agents on micro-organisms and pink bollworm development. J. &on. Entomol., 55, 854-857.
OUYE,
M. T., AND VANDERZANT, E. S. Vitamin requirements of the pink J. Econ. Entmol., 57, 427430.
1964. Bbollworm.