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An iridescent virus of the bollworm Heliothis zea (Lepidoptera:Noctuidae)
JOURNAL
OF
INVERTEBRATE
PATHOLOGY
An Iridescent
32, 71-76 (1978)
Virus of the Bollworm (Lepidoptera:Noctuidae)l
Heliothis
EARL A. STADELBACHER,* J. R. ADAMS,~ R. M. FAUST,? AND G.J.
zea TOMPKINS?
*U. S. Department of Agriculture, Science and Education Administration, Bioenvironmental Insect Control Laboratory, Stoneville. Mississippi 38776 and tU. S. Department of Agriculture, Science and Education Administration, Insect Pathology Laboratory, Beltsville, Maryland 20705 Received December 30, 1977 Larvae of Heliothis zea exhibiting iridescent lavender-blue, blue, blue-green color were obtained in spring field collections in host plants on a road right-of-way in Bolivar County, Mississippi. An iridescent virus was isolated and purified from these larvae. Size range of the virus was 131 to 160 nm, with a mean + SD of 145 ? 6.5. The nucleic acid content was: DNA, 13.97 2 1.58% (0.139 pg of DNA/mg of viral protein); RNA, none. Experimental infection was not achieved per OS, but the virus was highly virulent by intrahemocoelic injection. The virus from larvae killed in this way was of the same size and morphology as that obtained in the initial isolation. KEY WORDS: Heliothis zea, larvae; iridovirus
On April 21, 1977, 132 H. zea larvae were collected in crimson clover, Trifolium incarnatum, 19 in vetch, Viciu villosa, and 21 in a mixed stand of crimson clover and vetch along a 3.2-km section of road right-of-way in Bolivar County, Mississippi. In the laboratory, five of the larvae (three collected on crimson clover, one on vetch, and one on a mixed stand) turned an iridescent lavender-blue, blue, blue-green in color. We have never seen this symptom before in H. zea, nor has it been reported in the literature. Fukaya and Nasu (1966) were the first to report a natural infection of an iridescent virus in a lepidopteran larva- the rice borer, Chilo suppressalis. An unusually high percentage of parasitization by nematodes in these larval populations from Bolivar County, Mississippi, was noted and may be significant in relation to the virus.
INTRODUCTION
In the Delta of Mississippi, the generation of adult Heliothis zea that emerges from the overwintered pupal population and the subsequent F1 larval population are dependent for a period of about 1.5 months upon species of winter and early season annual host plants. These hosts, which occur primarily in areas such as field margins, ditch banks, and road right-of-ways, play a major role not only in the seasonal life history and population dynamics of H. zea but also, indirectly, in that of its parasites and pathogens. Since 1965, all early season species of plants that could potentially serve as hosts of Heliothis spp. have been swept with a standard 15-in insect sweepnet at approximately weekly intervals. The Heliothis spp. have been identified in the field, placed in 1-oz clear plastic cups on various artificial diets, indexed, identified in the laboratory, and then held at room temperature (26°C) for parasite and pathogen development and identification.
MATERIALS
AND METHODS
Laboratory-reared H. zea larvae were used in all infection tests. They were each held at 26°C in 1-oz clear plastic cups on an artificial diet (Brewer, 1976). The first inoculum was prepared from one
’ This research was conducted in cooperation with the Delta Branch, Mississippi Agricultural and Forestry Experiment Station. 71
0022-2011/78/0321-0071$01.00/0 Copyright All rights
Q 1978 by Academic Press, Inc. of reproduction in any form reserved.
72
STADELBACHER
ET AL.
FIG. 1. Electron micrograph of H. zea iridescent virus stained with 1% ammonium molybdate in H,O. ~62,750.
of the field-collected diseased larvae that had been kept at -20°C. A small portion of the diseased larva was ground and suspended in Ringer’s solution (Barbosa, 1974) and then filtered (Qualitative filter paper No. 7760, W. H. Curtin & CO.~) to remove large fragments. To determine whether the virus could be transmitted orally, 27 second to third instar larvae were provided with a diet that had been contaminated on the surface with the virus suspension. Similar check larvae (27) also were kept on a diet that had been treated with Ringer’s solution. Twenty-five fourth to fifth instar larvae were injected by hand syringe with 10 ~1 of the virus suspension in order to determine whether the virus could be transmitted by injection into the body cavity. The control consisted of 25 fourth to fifth instar larvae that were injected with 10 ~1 of Ringer’s solution. Isolation and purification
of the virus.
Three of the infected larvae were ground in distilled water with a tissue grinder, and the suspension was filtered through several layers of cheesecloth and then centrifuged ’ Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
in three low-speed runs in a clinical centrifuge in order to remove debris. The supernatant fluid was removed, placed on a sucrose gradient of 20 to 50% (w/w, discontinuous), and centrifuged in a Beckman L-2 ultracentrifuge (SW 27.1 head) at 29,OOOg for 20 min. Several bands appeared in the tube. A large band, second from the top, contained viruslike particles which were washed several times by centrifugation in distilled water and used for further inoculations and for a nucleic acid analysis (Fig. 1). Face-to-face measurements were made of the negatively stained icosahedral virus particles, which were lying in a position that could be viewed as looking along an axis of threefold rotational symmetry according to Horne (1974). Infectivity of pursed virus. Each of 193 healthy fourth to fifth stage H. zea larvae was injected with 1 ~1 of the purified virus, and each of 15 larvae was injected with 2 ~1 of the purified virus. Twenty-five check larvae were punctured with the needle. The larvae in this and subsequent tests were injected with an automatic microapplicator because too many larvae were injured by the hand injection method. Finally, the stock suspension of purified virus was diluted lo-‘, 10w2, and lop3 with distilled water, and 14, 16, and 14 fourth
IRIDESCENT
VIRUS
instar H. zea larvae were injected with 1 yl of inoculum each. Fourteen fourth instar check larvae were each injected with 1 ~1 of distilled water. Color grading. With the assistance of Dr. Kent H. McKnight of the Mycology Laboratory, SEA, U. S. Department of Agriculture, Beltsville, Maryland, we attempted a more precise identification of the colors with Munsell’s (1966) notation and of the color names according to Kelly and Judd (1955). The iridescent colors seen in the pellet of purified virus were: strong blue (Centroid 178:near Munsell 6.25PB 4/12); brilliant green (Centroid 140:near Munsell 7.56 7/8); and deep reddish orange (Centroid 36:near 10R 4/12). Analysis for nucleic acid. The nucleic acids were extracted from duplicate samples each of 3 to 4 mg of lyophilized viral material as follows. The dried virus was suspended in 5 ml of a cold 10% solution of trichloroacetic acid (TCA) and centrifuged at 140,OOOg for 1 hr in a Beckman model LS-50 ultracentrifuge held at 4°C. The sediment was resuspended in 5 ml of cold 10% TCA and recentrifuged, and the pellet was resuspended twice in 5 ml of a 95% solution of ethanol and recovered each time by centrifugation at 140,OOOg for 1 hr. The nucleic acids were extracted by suspending the pellet in 3 ml of a 5% solution of TCA and heating for 1 hr at 90°C in a Dubnoff metabolic shaking incubator. These extractions with TCA were repeated twice, and the insoluble residues were removed by centrifugation at 50,OOOg for 20 min. Two extractions were adequate for complete removal of all nucleic acid. The nucleic acid in the two extracts was determined (in duplicate) by the colorimetric method of Schneider (1957), in which DNA is estimated by using the diphenylamine reagent (specific for purine-bound 2deoxyribose) and RNA is determined by using the orcinol reagent. Since the orcinol has a color reaction with both ribose and 2-deoxyribose, RNA was estimated by subtracting the predetermined amount of DNA
IN HELZOTHZS ZEA
73
from the OD6,,, developed in the orcinol reaction. The contributions of each nucleic acid to the optical density in this reaction are strictly additive. The ratio of protein to viral nucleic acid was determined by using the calorimetric method of Lowry et al. as described by Layne (1957). RESULTS AND DISCUSSION
All 27 of the larvae fed the virus suspension in Ringer’s solution and all 27 control larvae developed normally and produced healthy pupae and adults. Thus, under the conditions of this test, the probability of oral transmission of the virus is not great. Injection tests using crude preparations.
Ten of the twenty-five larvae injected with the crude preparation of virus and six of the check larvae died within 2 days of the treatment. Three of the dead inoculated larvae, however, had developed the iridescent color in the distal area of the abdomen, and 15 other inoculated larvae developed the iridescent lavender-blue, blue, bluegreen color within 4 days of inoculation. The iridescent color developed first in the membranous area at the base of the antennae, in the area between the anal prolegs and the suranal plate, and in the distal portion of the anal and abdominal prolegs just above the crotchets. This color then appeared in the labrum, the maxillolabialhypopharyngeal complex, and the dorsal thoracic region just posterior to the head. Once the color had developed in these areas, the larvae stopped feeding, burrowed into the diet, and formed abnormally large pupal cells. This was followed by a general paralysis of the larvae, intensification of the lavender-blue, blue, blue-green iridescence in the previously affected areas, and appearance of color in the mandibles, ocelli, the basal area of all prolegs, and the ventral area of the entire body. Nineteen of the check larvae (all except the six that died due to injury) developed normally and produced healthy pupae and adults.
74
STADELBACHER
Injection tests using purified virus. TWO days after treatment, all 15 of the larvae inoculated with 2 ~1 and all but nine of the 193 inoculated with 11.~1of the purified virus had died. These last nine larvae gradually developed the iridescent lavender-blue, blue, blue-green symptoms, and all except one died. The one that survived inoculation developed into a pupa which was grotesquely deformed in the head, ventral thoracic, and posterior regions of the body, and it died shortly after pupation. All 25 of the check larvae developed into healthy pupae and adults. The high percentage of mortality among the inoculated larvae indicated that the dosage used in this test might be too high. The results with serial dilution of the purified virus suspension are given in Table 1. At 7 days postinoculation, 38,90, and 100% of the larvae inoculated with the 10-l, 10P2, and 10U3dilutions, respectively, had formed abnormally large pupal cells in the diet; 0,30, and 13%, respectively, had died in the larval stage; and 0, 0, and 38%, respectively, had developed into deformed pupae and died. The larvae injected with the higher dilution formed greater numbers of abnormal pupal cells than those injected with the lower dilution because those injected with the lower dilution became paralyzed, remained in the larval stage for a prolonged period, and eventually died without forming pupal cells. TABLE ONSET
OF SYMETOMS AND LARVAL FROM INTRAHEMo~oELIC
ET AL.
The larvae injected with the higher dilutions developed at a more normal rate, however, and the disease symptoms developed gradually. Mortality in these larvae occurred when they molted or shortly after they formed pupal cells and attempted to pupate. Some of the larvae inoculated with the 10e2 and 10P3 dilutions that progressed to the prepupal and pupal stage retained the larval cuticle and the external larval characteristics but developed incomplete pupal characteristics in the abdominal region; some developed half larval and half pupal characters, with the larval cuticle splitting in the dorsal thoracic region so the pronotum of the incomplete pupa protruded; and some developed into pupae withgrotesque abnormalities in the head, thoracic, and posterior abdominal regions. A similar juvenile hormonelike effect on metamorphosis was observed with Chile iridescent virus (CIV) in Bombyx mori (Ono and Fukaya, 1969; Ono et al., 1972; Ishikawa and Muroga, 1976) and was attributed to the presence of CIV in the tissues. Nucleic acid analyses. The analyses of the iridescent virus from the H. zea larvae revealed that it contained DNA but no RNA and that DNA constituted 13.97 2 1.58% (0.139 pugDNA/mg viral protein) of the virus, which is similar to the iridescent viruses described by other investigators (Thomas, 1961; Allison and Burke, 1962; Day and Mercer, 1964; Bellett and Inman, 1967; Faust et al., 1
MORTALITY OF HELIOTHIS ZEA LARVAE INJECTIONS OF IRIDESCENT VIRUS
Percentage showing symptoms Inoculum dilution
Number of larvae”
2 days postinoculation
4 days postinoculation 100
RESULTING
18 days postinoculation
7 days postinoculation
10-l 10-Z 10-S
14 16 14
79 81 57
100
93
100 100 100
Control (Distilled H,O)
14
0
0
0
Larval mortality (%)
Dead deformed pupae (%I
100 80 50
0 20 50
0
0
D When exhibiting symptoms, six larvae from each dilution were taken for reisolation of the virus.
IRIDESCENT
VIRUS IN HELZUTHZS
1968; Kalmakoff and Tremaine, 1968; Kelly and Avery, 1974; Glitz et al., 1968). Size of virus. Electron microscopical examination of the virus particles obtained in the two isolations revealed the same size range and morphology (Fig. 1). Measurements of diameters of 100 negatively stained virus particles gave a mean and SD of 145 nm*6.5,witharangeof131to160nm.The size range is similar to that of other iridoviruses: bee,Apis mellifera IV, 150nm (Bailey et al., 1976); Chilo IV, 160 nm (Fukaya and Nasu, 1966), and Simulium IV, 140 to 160 nm (Weiser, 1968). The size and the nucleic acid analysis support the proposal that this iridescent virus should be classified in the Iridoviridae (Fenner, 1976). These tests have shown a causal relationship between the isolated virus and iridescent lavender-blue, blue, blue-green symptoms in H. zea larvae. Although transmission was not obtained per OS, intrahemocoelic injection of the diluted purified virus consistently produced the characteristic iridescent lavender-blue, blue, blue-green color symptoms and caused paralysis and death of all injected insects. Other investigators of the iridoviruses have experienced similar difficulty in obtaining per OS transmission (see Smith, 1967,1976; Stoltz and Summers, 1971; Carter, 1973a,b; Bailey et al., 1976). Carter (1973a,b) proposed that natural transmission probably occurs by cannibalism, since he obtained negative results in tests for transovum transmission, confinement of healthy with diseased Tipula oleracea larvae, and contamination of the spiracles. Later, however, he was able to achieve infection by feeding and maintaining the insects according to the techniques he described (Carter, 1974). Stoltz and Summers (1971) found that, in Aedes taeniorhynchus larvae, ingested mosquito iridescent virus was degraded shortly after entering the midgut and was unable to penetrate the peritrophic membrane. Possible route of transmission in thejield.
High percentages of the larvae collected in the field were parasitized by a nematode
ZEA
75
belonging to the family Mermithidae and the genus Hexamermis (39.4% in crimson clover, 42.1% in vetch, and 47.6% in the mixed stand of crimson clover and vetch). Also, parasitic nematodes emerged in the laboratory from two of the five H. zea larvae that developed the iridescent lavender-blue, blue, blue-green virus symptoms. This high incidence of parasitism of this H. zea population and the occurrence of the iridescent virus in the same population suggest that infection could be carried by nematodes when they invade the body cavity. ACKNOWLEDGMENT We are grateful to Mr. Russell Travers of the Insect Pathology Laboratory for technical assistance.
REFERENCES A. C., AND BURKE, D. C. 1962. The nucleic acid content of viruses. J. Cm. Microbial.. 27, 181194. BAILEY, L., BALL, B. V., AND WOODS, R. D. 1976. An iridovirus from bees. J. Gen. Viral., 31,459-461. BARBOSA, P. 1974. “Manual of Basic Techniques in Insect Histology,” 253 pp. Autumn publishers. Amherst, Mass. BELLETT, A. J. D., AND INMAN, R. B. 1967. Some properties of deoxyribonucleic acid preparations from Chile. Sericesthis and Tipula iridescent viruses. .I. ALLISON,
Mol.
Biol.,
25, 425-432.
BREWER, F. D. 1976. Development of the sugarcane borer on various artificial diets. U. S. Dept. Agric., Agric. Res. Serv., ARS-S-116, 6 pp. CARTER, J. B. 1973a. The mode of transmission of Tipula iridescent virus: I, Source of infection. J. Invertebr. Pathol., 21, 123-130. CARTER, J. B. 1973b. The mode of transmission of Tip&a iridescent virus: II, Route of infection. J. Invertebr. Pathol., 21, 136- 143. CARTER, J. B. 1974. Tip&a iridescent virus infection in the developmental stages of Tipula oleruceo. J. Invertebr. Parhol.. 24, 211-281. DAY, M. F., AND MERCER, E. H. 1964. Properties of an iridescent virus from the beetle. Seric~esthis pruinosu. Amt. 1. Biol. Sri., 17, 892-902. FAUST, R. M., DOUGHERTY, E. M., AND ADAMS, J. R. 1968. Nucleic acid in the blue-green and orange mosquito iridescent viruses (MIV) isolated from larvae of Aedes tueniorhynchus. J. lnvertebr. Pathol. 10, 160. FENNER, F. 1976. “Classification and Nomenclature of Viruses: Second Report of the International Com-
76
STADELBACHER
mittee on Taxonomy of Viruses,” 115 pp. S. Karger, Basel. FUKAYA, M., AND NASU, S. 1966. A Chilo iridescent virus (CIV) from the rice stem borer, Chilo suppressalis, Walker (Lepidoptera: Pyralidae). Appl. Entomol.
2001..
1, 69-12.
GLITZ, D. G., HILLS, G. J., AND RIVERS, C. F. 1968. A comparison of the Tip&a and Sericesthis iridescent viruses. J. Gen. Virol., 3, 209-220. HORNE, R. W. 1974. “Virus Structure,” 52 pp. Academic Press, New York. ISHIKAWA, K., AND MUROGA, M. 1976. The influence of Chile iridescent virus (CIV) infection on metamorphosis of host insects. I, Abnormality of metamorphosis with the virus infection. Japan. J. Appl. Entomol. Zool., 20, 61-68. KALMAKOFF, J., AND TREMAINE, J. H. 1968. Physiochemical properties of Tip&a iridescent virus. J. Virol.
2, 738-744.
KELLY, D. C., AND AVERY, R. J. 1974. The DNA content offour small iridescent viruses: Genome size, redundancy, and homology determined by renaturation kinetics. Virology, 57, 425-435. KELLY, D. L., AND JUDD, D. B. 1955. The ISCCNBS method of designating colors and a dictionary of color names. Nat. Bur. Standards Circ. 533, U. S. Government Printing Office, Washington, D. C. LAYNE, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. In “Methods in
ET AL.
Enzymology,” Vol3, pp. 447-450. Academic Press, New York. MUNSELL, A. H. 1966. “Munsell book ofcolor. Glossy Finish Collection,” 2 Vol. Munsell Color Co., Baltimore, Md. ONO, M., AND FUKAYA, M. 1969. The juvenile-hormone-like effect of Chile iridescent virus (CIV) on the metamorphosis of the silkworm, Bombyx mori L. Appl. Entomol. Zool, 9, 211-212. ONO, M., YAGI, S., AND FUKAYA, M. 1972. Chilo iridescent virus. Bull. Seric. Exp. Sm., 25, 77- 102. SCHNEIDER, W. C. 1957. Determination ofnucleic acid in tissues by pentose analysis. In “Methods in Enzymology,” Vol. 3, pp. 680-684. Academic Press, New York. SMITH, K. M. 1967. “Insect Virology,” 256 pp. Academic Press, New York. SMITH, K. M. 1976. “Virus Insect Relationships,” 291 pp. Longman, New York. STOLTZ, D. B., AND SUMMERS, M. D. 1971. Pathway of infection of mosquito iridescent virus: I, Preliminary observations on the fate of ingested virus. J. Virol..
8, 900-909.
THOMAS, R. S. 1961. The chemical composition and particle weight of Tip&a iridescent virus. Virology, 14, 240-252. WEISER, J. 1968. Iridescent virus from the blackfly Simulium ornatum Meigen in Czechoslovakia. J. Invertebr.
Pathol..
12, 36-39.
OF
INVERTEBRATE
PATHOLOGY
An Iridescent
32, 71-76 (1978)
Virus of the Bollworm (Lepidoptera:Noctuidae)l
Heliothis
EARL A. STADELBACHER,* J. R. ADAMS,~ R. M. FAUST,? AND G.J.
zea TOMPKINS?
*U. S. Department of Agriculture, Science and Education Administration, Bioenvironmental Insect Control Laboratory, Stoneville. Mississippi 38776 and tU. S. Department of Agriculture, Science and Education Administration, Insect Pathology Laboratory, Beltsville, Maryland 20705 Received December 30, 1977 Larvae of Heliothis zea exhibiting iridescent lavender-blue, blue, blue-green color were obtained in spring field collections in host plants on a road right-of-way in Bolivar County, Mississippi. An iridescent virus was isolated and purified from these larvae. Size range of the virus was 131 to 160 nm, with a mean + SD of 145 ? 6.5. The nucleic acid content was: DNA, 13.97 2 1.58% (0.139 pg of DNA/mg of viral protein); RNA, none. Experimental infection was not achieved per OS, but the virus was highly virulent by intrahemocoelic injection. The virus from larvae killed in this way was of the same size and morphology as that obtained in the initial isolation. KEY WORDS: Heliothis zea, larvae; iridovirus
On April 21, 1977, 132 H. zea larvae were collected in crimson clover, Trifolium incarnatum, 19 in vetch, Viciu villosa, and 21 in a mixed stand of crimson clover and vetch along a 3.2-km section of road right-of-way in Bolivar County, Mississippi. In the laboratory, five of the larvae (three collected on crimson clover, one on vetch, and one on a mixed stand) turned an iridescent lavender-blue, blue, blue-green in color. We have never seen this symptom before in H. zea, nor has it been reported in the literature. Fukaya and Nasu (1966) were the first to report a natural infection of an iridescent virus in a lepidopteran larva- the rice borer, Chilo suppressalis. An unusually high percentage of parasitization by nematodes in these larval populations from Bolivar County, Mississippi, was noted and may be significant in relation to the virus.
INTRODUCTION
In the Delta of Mississippi, the generation of adult Heliothis zea that emerges from the overwintered pupal population and the subsequent F1 larval population are dependent for a period of about 1.5 months upon species of winter and early season annual host plants. These hosts, which occur primarily in areas such as field margins, ditch banks, and road right-of-ways, play a major role not only in the seasonal life history and population dynamics of H. zea but also, indirectly, in that of its parasites and pathogens. Since 1965, all early season species of plants that could potentially serve as hosts of Heliothis spp. have been swept with a standard 15-in insect sweepnet at approximately weekly intervals. The Heliothis spp. have been identified in the field, placed in 1-oz clear plastic cups on various artificial diets, indexed, identified in the laboratory, and then held at room temperature (26°C) for parasite and pathogen development and identification.
MATERIALS
AND METHODS
Laboratory-reared H. zea larvae were used in all infection tests. They were each held at 26°C in 1-oz clear plastic cups on an artificial diet (Brewer, 1976). The first inoculum was prepared from one
’ This research was conducted in cooperation with the Delta Branch, Mississippi Agricultural and Forestry Experiment Station. 71
0022-2011/78/0321-0071$01.00/0 Copyright All rights
Q 1978 by Academic Press, Inc. of reproduction in any form reserved.
72
STADELBACHER
ET AL.
FIG. 1. Electron micrograph of H. zea iridescent virus stained with 1% ammonium molybdate in H,O. ~62,750.
of the field-collected diseased larvae that had been kept at -20°C. A small portion of the diseased larva was ground and suspended in Ringer’s solution (Barbosa, 1974) and then filtered (Qualitative filter paper No. 7760, W. H. Curtin & CO.~) to remove large fragments. To determine whether the virus could be transmitted orally, 27 second to third instar larvae were provided with a diet that had been contaminated on the surface with the virus suspension. Similar check larvae (27) also were kept on a diet that had been treated with Ringer’s solution. Twenty-five fourth to fifth instar larvae were injected by hand syringe with 10 ~1 of the virus suspension in order to determine whether the virus could be transmitted by injection into the body cavity. The control consisted of 25 fourth to fifth instar larvae that were injected with 10 ~1 of Ringer’s solution. Isolation and purification
of the virus.
Three of the infected larvae were ground in distilled water with a tissue grinder, and the suspension was filtered through several layers of cheesecloth and then centrifuged ’ Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
in three low-speed runs in a clinical centrifuge in order to remove debris. The supernatant fluid was removed, placed on a sucrose gradient of 20 to 50% (w/w, discontinuous), and centrifuged in a Beckman L-2 ultracentrifuge (SW 27.1 head) at 29,OOOg for 20 min. Several bands appeared in the tube. A large band, second from the top, contained viruslike particles which were washed several times by centrifugation in distilled water and used for further inoculations and for a nucleic acid analysis (Fig. 1). Face-to-face measurements were made of the negatively stained icosahedral virus particles, which were lying in a position that could be viewed as looking along an axis of threefold rotational symmetry according to Horne (1974). Infectivity of pursed virus. Each of 193 healthy fourth to fifth stage H. zea larvae was injected with 1 ~1 of the purified virus, and each of 15 larvae was injected with 2 ~1 of the purified virus. Twenty-five check larvae were punctured with the needle. The larvae in this and subsequent tests were injected with an automatic microapplicator because too many larvae were injured by the hand injection method. Finally, the stock suspension of purified virus was diluted lo-‘, 10w2, and lop3 with distilled water, and 14, 16, and 14 fourth
IRIDESCENT
VIRUS
instar H. zea larvae were injected with 1 yl of inoculum each. Fourteen fourth instar check larvae were each injected with 1 ~1 of distilled water. Color grading. With the assistance of Dr. Kent H. McKnight of the Mycology Laboratory, SEA, U. S. Department of Agriculture, Beltsville, Maryland, we attempted a more precise identification of the colors with Munsell’s (1966) notation and of the color names according to Kelly and Judd (1955). The iridescent colors seen in the pellet of purified virus were: strong blue (Centroid 178:near Munsell 6.25PB 4/12); brilliant green (Centroid 140:near Munsell 7.56 7/8); and deep reddish orange (Centroid 36:near 10R 4/12). Analysis for nucleic acid. The nucleic acids were extracted from duplicate samples each of 3 to 4 mg of lyophilized viral material as follows. The dried virus was suspended in 5 ml of a cold 10% solution of trichloroacetic acid (TCA) and centrifuged at 140,OOOg for 1 hr in a Beckman model LS-50 ultracentrifuge held at 4°C. The sediment was resuspended in 5 ml of cold 10% TCA and recentrifuged, and the pellet was resuspended twice in 5 ml of a 95% solution of ethanol and recovered each time by centrifugation at 140,OOOg for 1 hr. The nucleic acids were extracted by suspending the pellet in 3 ml of a 5% solution of TCA and heating for 1 hr at 90°C in a Dubnoff metabolic shaking incubator. These extractions with TCA were repeated twice, and the insoluble residues were removed by centrifugation at 50,OOOg for 20 min. Two extractions were adequate for complete removal of all nucleic acid. The nucleic acid in the two extracts was determined (in duplicate) by the colorimetric method of Schneider (1957), in which DNA is estimated by using the diphenylamine reagent (specific for purine-bound 2deoxyribose) and RNA is determined by using the orcinol reagent. Since the orcinol has a color reaction with both ribose and 2-deoxyribose, RNA was estimated by subtracting the predetermined amount of DNA
IN HELZOTHZS ZEA
73
from the OD6,,, developed in the orcinol reaction. The contributions of each nucleic acid to the optical density in this reaction are strictly additive. The ratio of protein to viral nucleic acid was determined by using the calorimetric method of Lowry et al. as described by Layne (1957). RESULTS AND DISCUSSION
All 27 of the larvae fed the virus suspension in Ringer’s solution and all 27 control larvae developed normally and produced healthy pupae and adults. Thus, under the conditions of this test, the probability of oral transmission of the virus is not great. Injection tests using crude preparations.
Ten of the twenty-five larvae injected with the crude preparation of virus and six of the check larvae died within 2 days of the treatment. Three of the dead inoculated larvae, however, had developed the iridescent color in the distal area of the abdomen, and 15 other inoculated larvae developed the iridescent lavender-blue, blue, bluegreen color within 4 days of inoculation. The iridescent color developed first in the membranous area at the base of the antennae, in the area between the anal prolegs and the suranal plate, and in the distal portion of the anal and abdominal prolegs just above the crotchets. This color then appeared in the labrum, the maxillolabialhypopharyngeal complex, and the dorsal thoracic region just posterior to the head. Once the color had developed in these areas, the larvae stopped feeding, burrowed into the diet, and formed abnormally large pupal cells. This was followed by a general paralysis of the larvae, intensification of the lavender-blue, blue, blue-green iridescence in the previously affected areas, and appearance of color in the mandibles, ocelli, the basal area of all prolegs, and the ventral area of the entire body. Nineteen of the check larvae (all except the six that died due to injury) developed normally and produced healthy pupae and adults.
74
STADELBACHER
Injection tests using purified virus. TWO days after treatment, all 15 of the larvae inoculated with 2 ~1 and all but nine of the 193 inoculated with 11.~1of the purified virus had died. These last nine larvae gradually developed the iridescent lavender-blue, blue, blue-green symptoms, and all except one died. The one that survived inoculation developed into a pupa which was grotesquely deformed in the head, ventral thoracic, and posterior regions of the body, and it died shortly after pupation. All 25 of the check larvae developed into healthy pupae and adults. The high percentage of mortality among the inoculated larvae indicated that the dosage used in this test might be too high. The results with serial dilution of the purified virus suspension are given in Table 1. At 7 days postinoculation, 38,90, and 100% of the larvae inoculated with the 10-l, 10P2, and 10U3dilutions, respectively, had formed abnormally large pupal cells in the diet; 0,30, and 13%, respectively, had died in the larval stage; and 0, 0, and 38%, respectively, had developed into deformed pupae and died. The larvae injected with the higher dilution formed greater numbers of abnormal pupal cells than those injected with the lower dilution because those injected with the lower dilution became paralyzed, remained in the larval stage for a prolonged period, and eventually died without forming pupal cells. TABLE ONSET
OF SYMETOMS AND LARVAL FROM INTRAHEMo~oELIC
ET AL.
The larvae injected with the higher dilutions developed at a more normal rate, however, and the disease symptoms developed gradually. Mortality in these larvae occurred when they molted or shortly after they formed pupal cells and attempted to pupate. Some of the larvae inoculated with the 10e2 and 10P3 dilutions that progressed to the prepupal and pupal stage retained the larval cuticle and the external larval characteristics but developed incomplete pupal characteristics in the abdominal region; some developed half larval and half pupal characters, with the larval cuticle splitting in the dorsal thoracic region so the pronotum of the incomplete pupa protruded; and some developed into pupae withgrotesque abnormalities in the head, thoracic, and posterior abdominal regions. A similar juvenile hormonelike effect on metamorphosis was observed with Chile iridescent virus (CIV) in Bombyx mori (Ono and Fukaya, 1969; Ono et al., 1972; Ishikawa and Muroga, 1976) and was attributed to the presence of CIV in the tissues. Nucleic acid analyses. The analyses of the iridescent virus from the H. zea larvae revealed that it contained DNA but no RNA and that DNA constituted 13.97 2 1.58% (0.139 pugDNA/mg viral protein) of the virus, which is similar to the iridescent viruses described by other investigators (Thomas, 1961; Allison and Burke, 1962; Day and Mercer, 1964; Bellett and Inman, 1967; Faust et al., 1
MORTALITY OF HELIOTHIS ZEA LARVAE INJECTIONS OF IRIDESCENT VIRUS
Percentage showing symptoms Inoculum dilution
Number of larvae”
2 days postinoculation
4 days postinoculation 100
RESULTING
18 days postinoculation
7 days postinoculation
10-l 10-Z 10-S
14 16 14
79 81 57
100
93
100 100 100
Control (Distilled H,O)
14
0
0
0
Larval mortality (%)
Dead deformed pupae (%I
100 80 50
0 20 50
0
0
D When exhibiting symptoms, six larvae from each dilution were taken for reisolation of the virus.
IRIDESCENT
VIRUS IN HELZUTHZS
1968; Kalmakoff and Tremaine, 1968; Kelly and Avery, 1974; Glitz et al., 1968). Size of virus. Electron microscopical examination of the virus particles obtained in the two isolations revealed the same size range and morphology (Fig. 1). Measurements of diameters of 100 negatively stained virus particles gave a mean and SD of 145 nm*6.5,witharangeof131to160nm.The size range is similar to that of other iridoviruses: bee,Apis mellifera IV, 150nm (Bailey et al., 1976); Chilo IV, 160 nm (Fukaya and Nasu, 1966), and Simulium IV, 140 to 160 nm (Weiser, 1968). The size and the nucleic acid analysis support the proposal that this iridescent virus should be classified in the Iridoviridae (Fenner, 1976). These tests have shown a causal relationship between the isolated virus and iridescent lavender-blue, blue, blue-green symptoms in H. zea larvae. Although transmission was not obtained per OS, intrahemocoelic injection of the diluted purified virus consistently produced the characteristic iridescent lavender-blue, blue, blue-green color symptoms and caused paralysis and death of all injected insects. Other investigators of the iridoviruses have experienced similar difficulty in obtaining per OS transmission (see Smith, 1967,1976; Stoltz and Summers, 1971; Carter, 1973a,b; Bailey et al., 1976). Carter (1973a,b) proposed that natural transmission probably occurs by cannibalism, since he obtained negative results in tests for transovum transmission, confinement of healthy with diseased Tipula oleracea larvae, and contamination of the spiracles. Later, however, he was able to achieve infection by feeding and maintaining the insects according to the techniques he described (Carter, 1974). Stoltz and Summers (1971) found that, in Aedes taeniorhynchus larvae, ingested mosquito iridescent virus was degraded shortly after entering the midgut and was unable to penetrate the peritrophic membrane. Possible route of transmission in thejield.
High percentages of the larvae collected in the field were parasitized by a nematode
ZEA
75
belonging to the family Mermithidae and the genus Hexamermis (39.4% in crimson clover, 42.1% in vetch, and 47.6% in the mixed stand of crimson clover and vetch). Also, parasitic nematodes emerged in the laboratory from two of the five H. zea larvae that developed the iridescent lavender-blue, blue, blue-green virus symptoms. This high incidence of parasitism of this H. zea population and the occurrence of the iridescent virus in the same population suggest that infection could be carried by nematodes when they invade the body cavity. ACKNOWLEDGMENT We are grateful to Mr. Russell Travers of the Insect Pathology Laboratory for technical assistance.
REFERENCES A. C., AND BURKE, D. C. 1962. The nucleic acid content of viruses. J. Cm. Microbial.. 27, 181194. BAILEY, L., BALL, B. V., AND WOODS, R. D. 1976. An iridovirus from bees. J. Gen. Viral., 31,459-461. BARBOSA, P. 1974. “Manual of Basic Techniques in Insect Histology,” 253 pp. Autumn publishers. Amherst, Mass. BELLETT, A. J. D., AND INMAN, R. B. 1967. Some properties of deoxyribonucleic acid preparations from Chile. Sericesthis and Tipula iridescent viruses. .I. ALLISON,
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BREWER, F. D. 1976. Development of the sugarcane borer on various artificial diets. U. S. Dept. Agric., Agric. Res. Serv., ARS-S-116, 6 pp. CARTER, J. B. 1973a. The mode of transmission of Tipula iridescent virus: I, Source of infection. J. Invertebr. Pathol., 21, 123-130. CARTER, J. B. 1973b. The mode of transmission of Tip&a iridescent virus: II, Route of infection. J. Invertebr. Pathol., 21, 136- 143. CARTER, J. B. 1974. Tip&a iridescent virus infection in the developmental stages of Tipula oleruceo. J. Invertebr. Parhol.. 24, 211-281. DAY, M. F., AND MERCER, E. H. 1964. Properties of an iridescent virus from the beetle. Seric~esthis pruinosu. Amt. 1. Biol. Sri., 17, 892-902. FAUST, R. M., DOUGHERTY, E. M., AND ADAMS, J. R. 1968. Nucleic acid in the blue-green and orange mosquito iridescent viruses (MIV) isolated from larvae of Aedes tueniorhynchus. J. lnvertebr. Pathol. 10, 160. FENNER, F. 1976. “Classification and Nomenclature of Viruses: Second Report of the International Com-
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mittee on Taxonomy of Viruses,” 115 pp. S. Karger, Basel. FUKAYA, M., AND NASU, S. 1966. A Chilo iridescent virus (CIV) from the rice stem borer, Chilo suppressalis, Walker (Lepidoptera: Pyralidae). Appl. Entomol.
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GLITZ, D. G., HILLS, G. J., AND RIVERS, C. F. 1968. A comparison of the Tip&a and Sericesthis iridescent viruses. J. Gen. Virol., 3, 209-220. HORNE, R. W. 1974. “Virus Structure,” 52 pp. Academic Press, New York. ISHIKAWA, K., AND MUROGA, M. 1976. The influence of Chile iridescent virus (CIV) infection on metamorphosis of host insects. I, Abnormality of metamorphosis with the virus infection. Japan. J. Appl. Entomol. Zool., 20, 61-68. KALMAKOFF, J., AND TREMAINE, J. H. 1968. Physiochemical properties of Tip&a iridescent virus. J. Virol.
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KELLY, D. C., AND AVERY, R. J. 1974. The DNA content offour small iridescent viruses: Genome size, redundancy, and homology determined by renaturation kinetics. Virology, 57, 425-435. KELLY, D. L., AND JUDD, D. B. 1955. The ISCCNBS method of designating colors and a dictionary of color names. Nat. Bur. Standards Circ. 533, U. S. Government Printing Office, Washington, D. C. LAYNE, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. In “Methods in
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Enzymology,” Vol3, pp. 447-450. Academic Press, New York. MUNSELL, A. H. 1966. “Munsell book ofcolor. Glossy Finish Collection,” 2 Vol. Munsell Color Co., Baltimore, Md. ONO, M., AND FUKAYA, M. 1969. The juvenile-hormone-like effect of Chile iridescent virus (CIV) on the metamorphosis of the silkworm, Bombyx mori L. Appl. Entomol. Zool, 9, 211-212. ONO, M., YAGI, S., AND FUKAYA, M. 1972. Chilo iridescent virus. Bull. Seric. Exp. Sm., 25, 77- 102. SCHNEIDER, W. C. 1957. Determination ofnucleic acid in tissues by pentose analysis. In “Methods in Enzymology,” Vol. 3, pp. 680-684. Academic Press, New York. SMITH, K. M. 1967. “Insect Virology,” 256 pp. Academic Press, New York. SMITH, K. M. 1976. “Virus Insect Relationships,” 291 pp. Longman, New York. STOLTZ, D. B., AND SUMMERS, M. D. 1971. Pathway of infection of mosquito iridescent virus: I, Preliminary observations on the fate of ingested virus. J. Virol..
8, 900-909.
THOMAS, R. S. 1961. The chemical composition and particle weight of Tip&a iridescent virus. Virology, 14, 240-252. WEISER, J. 1968. Iridescent virus from the blackfly Simulium ornatum Meigen in Czechoslovakia. J. Invertebr.
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