Nucleopolyhedrosis of Heliothis: Activity of isolates from Heliothis zea

JOUHc\AL

OF

IXVEHTEMHATE

PATHOLOGY

Nucleopolyhedrosis

M. Interwtional

Minerals

16,

107-111

of Heliothk from Heliothis SHAPLRO

and

AND

Chemicul

Receiced

C.

Activity zea M.

of Isolates

IGNOFF~

Corporutiorr, October

( 1979)

Lihertyuille,

11hoi.s

600&Y

17 > 1969

Activity of 34 isolates of the Heliothis nucleopolyhedrosis virus were characterized. Levels of LD,, activity varied 56-fold from 0.7 to 39.0 polyhedral inclusion bodies (PIB)/mm of surface-treated diet. At least two and possibly three to eight activity classes were delineated. Activity of one selected isolate increased -80% after 5 serial passes through Heliothis zea and remained unchanged during 11 additional passes. No change in activity was detected when another isolate was serially passed sever, times.

to minimize variations in activity due to differences in larval body weight. All test insects were maintained at 31 + 1°C and 5060% relative humidity. Isolate technique. Isolates of the Ii. ZeLl NPV were collected over a period of 3 vcars from both laboratory and field diseased larvae. All isolates were prepared from diseased larvae using the same protocol (Ignoffo, 1966a). Freeze-dried preparations, standardized asto polyhedral inclusion bod\r (PIB) counts, were stored at -20°C until tested for activity. Inclusion-body counts of all isolates were made by the same individual to minimize variation between counts on different isolates. Final counts were averages of counts made on at least three separate samples using 160 mg PIB-preparation/lOO ml water. Activity, characterizc~tl as LDSo, was determined using a die( Ig11offo. surface-treatment technique 1966b). Three to five dosages, 50-109 neonate larvae/dosage, were used to obtairi a dosage-response line and an LD,,, for each viral isolate. Larval mortalitv was r(‘corded after 5-6 days of continuous exposure on treated diets. Serial passage procedure. Two virus isolates (DB-116501: LD-50. 6.6 PIB/tnm”:

Experimental data on differences in activity between isolates of the same insect viral species are beginning to accumulate. Ossowski ( 1958, 1960), Smirnoff ( 1961), and Chauthani et al. (1968) reported that they could detect differences in activity although no differences were detected by Schuster (1965) and Tanada et al. (1960). Several workers also reported that serial passage through the primary or alternate hosts may (Veber, 1962; Smirnoff, 1963; Woodward and Chapman, 1968) or may not (Woodward and Chapman, 1968) increase viral activitv. While these authors refer to virus “strains” we prefer to use the term “isolate” until the existence of strains can be supported by additional characterization (specificity. serology, chemistry, etc. ) . In onr study variation in activity of 34 isolates of the nucleopolyhedrosis virus (NPV) of the cotton bollworm. Heliothis zea, and effects of serial passage on viral activity were explored. MATERIALS

AND METHODS

Ce,lercrl. technique. Neonate bollwoims wc>rc used for all studies discussed herein 197

108

SHAPXRO AND IGNOFFO

DB-066501: 3.3 PIB/mm”) were selected for serial passage through neonate, cotton bollworm larvae. Mortality was first observed 2 days after exposure. Larvae dying 35 days postexposure were pooled, suspended in a total volume of 50 ml sterile distilled water, and blended at 23,000 rpm for 2 min (Model A-l Hispeed Blending and Mixing Corp., Hillside, N. J. ) . After blending, the virus suspension was passed through fine muslin to remove large particles and debris. The number of inclusion bodies was determined, using a Levy double-line hemacytometer with improved Neubauer ruling and phase microscopy ( magnification 630X ) . For evaluation of 1 6 12 10 24 30 36 activity, virus obtained from each passage was always adjusted to the original PIB/ D-50: PIB/mm2 mm3 dose. About 1000 virus-treated and FE. 1. Frequency distribution of LD,, values 200 untreated larvae were used for each of from 34 isolates of the nwleopolyhedrosis virus of passagel-12 inclusive and 900 virus-treated Fieliotliis zea larvae. and 100 untreated larvae were used for each of passage 13-16 inclusive. having an LDjO 5 SD’ of 2.5 t 0.6 PIB/ mm?, was more active than two other isolates, i.e., an Ivory Coast and a Sudan RESULTS AND DISCUSSION isolate. It is probable that Biotrol, Ivory Activity of isohztes. The LDBo of the 34 Coast, and Sudan isolates are part of the isolates of Heliothis NPV ranged from 0.7- same activity-class since the dose-mortality 39.0 PIB/mm2, a difference of about 56-fold. slope and LDso of all three isolates were At least 2, and possibly 3, distinct activity nearly identical (2) value difference of 0.047 groups can be formed from the 34 isolates. alld a LDno ratio of 1.7 between the least The 3 groups would consist of isolates hav- and most active isolate), the 95% confiing LDso values of 0.7-9, 9-21, and 3039 dence limits for the LDso were coincident PIB/mm” respectively (Fig. 1). If one as- for all three isolates, and it is not uncomsumesa 99% confidence interval of 3.6 FIB/ mon to have a 60% spread between the mm2 (Ignoffo, 196613) and that 0.7 PIB/ highest and lowest values from a series of mm? represents the most active isolate avail- PIB counts (Ignoffo, unpublished). able, then 8 classesof activity could be difPaschke et al. (1968) detected a 3.5 and ferentiated among the 34 isolates. 3.7-fold difference in activity within a single Approximately 60% of the isolates had an isolate of the cabbage looper ( Trichoplusin LDB,, activity of 0.7-11.7 PIB/mm?. None ni) nucleopolyhedrosis and granulosis viof the 34 isolates had an LDx of 19.2-30.2 ruses, respectively. Schuster (1965) did not PIB/mm” and only 13% of the isolates had detect any difference in activity between a an LDso greater than 19.2 PIB/mm?. California and a Texas isolate of TrichoChauthani et al. (1968) reported that an @U&Z nucleopolyhedrosis; however, both isolate of Heliothis NPV named Biotrol, originally obtained from one of us and 1 Standard deviation.

NUCLEOPOLYHEDROSIS

isolates produced 100% mortality within 4 days and only a limited number of larvae were used ( 12 larvae/isolate). Activity of serially passed isolates. Serial passage of isolate DB-116501: LDjo, 6.6 PIB/mmZ was made 16 times using neonate, cotton bollworm larvae. During the first five pn~scs no changes were observed in either the LDsO or LTjo (Table 1) , At the sixth pass, however, the LT,,, was reduced from 6 to 4 days and the cumulative percent mortality at Day 6 increased from a 1 to 5 serial-pass average of 46 to 81% (P < 0.01). NO further increase in activity occurred during the nest 10 passes, and activity stabilized at an average of 72.5% larval nrortalitv (range: 59-81%). Untreated larval mortality averaged 3% during the entire test period and never exceeded 7%. Although the increase was significant (P < 0.01)) it did not shift isolate DB-116501 into another activitv class. Gregory (personal communication) obtained a similar increase in activity from an isolate of Heliothis nuclcopolvhedrosis after four passes through the tobacco budworm, Heliothis zjirescens. Vebcr ( 1962) obtained an increase in activity after only two passes of the nucleopolyhedrosis of Galleria mellonella. The activity of another isolate (DB066501: LD;,,,, 3.3 PIB/mm”) did not increase during seven serial passes ( Table 1). In addition, the variabilitv in activity of isolate DB-066501 (Table. 1) was greater than that encountered in isolate DB-116501, possibly indicating a less stable host-pathogen svstem. Similar results were obtained by tioodward and Chapman (1968) who reported that the infectivity of “the regular VIosquito Iridescent Virus (MIV) was enhanced bv passage through Aecles taeniorhynclu.” while activity of a “blue” isolate of MIV did not change after 30 serial passes. Serial passage apparently selects the most active in a natural isolate of a heterogeneous population of isolates. It also rcsnlts in a more homogeneous population

OF

He/iothis

PEHCEIVT

I.()9

TABLE I ~~~OHTALITY" OF i%~:~.\Al.k: nea LARVAE EXPOSED TO Two OF SEH~ALL~ PASSED He1inthi.v

LAHVAL

Heliothis ISOLATES

NUCLEoPoI.Y~I-fEI~IIOSIS

Serial pass number .__~ 1 2 3 4 s 0 7 h: 9 10 II 12 1.3 14 15 16

VnuJs

Isolate DH-116501 Isolate DB-066501 .-.. I_---~.-------_ -__~.~.-.-_---Larval LZllWl mortality Esthnattd mortality Estimated (a,. ( 5% 1 L-l-s,, L7’,,, ,_-. ~-_~~__ ~~ ._._ ~~__ ._~_ _ 41.6 A.4 4.9 02.9 17.4 5.6 44.0 -7.0 :3.x: 41.4 6.4 73.7 53.3 S.K 48.7 7.6 45.4 0.2 71.3 $.I :3.:3 3.2 81.4 58.2 AS.2 :3.9 39.1 - 9.0 :3.:3 75.9 :3.-I 78.4 70.0 4.4 58.9 5.0 66.0 ‘1.2 70.0 3.9 78.0 :3. 7 7Fj.5 4.6 77.2 4.6

‘1 MortaIit~

rccwkl

6 dn!.s

after

.

test initiated.

of isolates. A similar opinion was esprcssed by Veber (1962) who felt that increases in activitvr were due to selection of more virulent isolates. The nmnber of passesreyuired to achieve stabilization apparently depends upon the host-pathogen system under study. Vebci ( 1962) achieved stabilization after two passes( Galleria nucleopoIyhedrosis ) , Smirnoff (1963) after three passes (NPV ol Trichiocamps uirninalis adapted to T. irregdaris) , Gregory (personal communication) after four passes (HeZiothis NV\’ in N. virescens) and activity reached a platcnu after five passesin this study. Machay and Lovas ( 19S7) suggested that an incrcasc in nctivitv could br due to an increase in the number of virions/inclusion body. This possibiliv was not excluded in the present study, but if it did occur, the number of virions/PIB would have to increase about 80% from an average 26.447.5 virions/inclusion body. A maximum of

110

SHAPLRO

AND

38 virionslinclusion body was reported for Heliothis NPV (Gregory et al., 1969). The LDsO of an Alabama isolate of Heliothis NPV (collected from H. virescens by Dr. Don Canerday) is of the same magnitude (4.0 PIB/mm2) as DB-066501 (3.3 PIB/ mm2) and yet differences in virions/inclusion were not found between the less active isolate DB-116501 (6.6 PIB/mm”) and the more active Alabama isolate. The variation in average size of inclusion bodies from six isolates (Table 2) was <20% and yet the range of activity was >SOO%. Factors other than size and/or number of virions/inclusion bodies must account for differences in activity between isolates of insect viruses. Since variabiIity due to host susceptibility (H. zea used has been continuously bred since 1963 on semisynthetic diet), host weight (use of neonate larvae), and technique (standardized dietsurface) was minimized, the differences in activity must be attributed to some characteristic of the inclusion body and/or its occluded virions. Although it is logical to assume that the differences observed are virion-induced differences, we can not exclude the possibility that some other factor such as solubility of inclusions and/or availability of occluded virions may account for TABLE

2

ACTIVITY, AVERAGE, AND RANGE IN DIAMETERS OF INCLUSION BODIES COLLECTED FROM He&this zea AND H. virescens

Isolate DB-116501 Alabama H. zea-1 Alabama H. virescens Alabama H. zea-2 DB-066501 DB-096628 a Based bodies/isolate.

upon

Inclusionbody

LD6,,

diametera

( p)

Average

Range

PIB/ mm?

1.18 1.30 1.35 1.33 1.25 1.20

0.90-1.55 0.65-1.93 1.05-2.70 1.03-1.93 0.6s1.55 0.90-1.81

6.6 6.3 4.0 3.4 3.3 1.0

measurements

of

100

inclusion

IGNOFFO

the differences observed. Additional study is required to further clarify these points. REFERENCES CHAXJTHANI, A. R., CLAUSSEN, D., AND REHNBORG, C. S. 1968. Dosage-mortality data on a nuclear-polyhedrosis virus of the bollworm, He&this zea. J. Invertebr. Path& 12, 335 338. GREGORY, B., IGNOFFO, C. M., AND SHAPIRO, M. 1969. Nucleopolyhedrosis of Heliothis: Morphological description of inclusion bodies and virions. J. Invertebr. Pathol., 14, 186193. IGNOFFO, C. M. 1966a. Insect Viruses, In “Insect Colonization and Mass Production” ( C. N. Smith, ed. ) pp. 501-530. Academic Press, New York. IGNOFFO, C. M. 1966b. Susceptibility of the bollworm, Heliothis zeu, and the tobacco budworn, Heliothis virescens, to Heliothis nuclear-polyhedrosis virus. J. Invertebr. Pathol., 8531-536. MACHAY, M. L. AND LOVAS, B. 1957. Histological, electromicroscopic and epizootical studies on a virus isolated from the larvae of Hyphantria mnea Drury. Biol. kiinlem. 5, 7-16. OSSOWSKI, L. L. J. 1958. Occurrence of strains of the nuclear polyhedral virus of the wattle bagworm. Nature X81:648. OSSOWSKI, L. L. J. 1960. Variation in virulence of a wattle bagworm virus. J. Insect Pathol., 2, 3543. PASCHKE, J. D., LOWE, R. E. AND GIESE, R. L. 1968. Bioassay of the nucleopolyhedrosis and granulosis viruses of Trichoplusia ni. I. Znvertebr. Pathol., 10, 327-334. SCHUSTER, M. 1965. Comparative virulence of Texas and California polyhedrosis virus of the cabbage-looper. I. Rio Grande Val. Hart. Sot., 19, 87-88. SMIRNOFF, W. A. 1961. A virus disease of Neodiprion swainei Middleton. J. Insect Pathol., 3, 2946. SMIRNOFF, W. A. 1963. Adaptation of a nuclear-polyhedrosis virus of Trichiocampus viminalis (Fallon) to larvae of Trichiocampus ilregularis (Dyar). J. Insect Pathol., 5, 104110. TANADA, Y., H UKUHARA, T., AND CHANG, C. Y. 1969. A strain of nuclear-polyhedrosis virus causing extensive cellular hypertrophy. J. fnvertebr. Pathol., 13, 394-409. VEBER, J. 1962. Virulence of an insect virus in-

NUCLEOPOLYHEDROSIS

creased by repeated passages. Co&q. Int. Pathol. Insectes, Paris., 2, 403-405. WOODWARD. D. B. AKD CHAPMAS. H. C. 1968.

OF

~$diOtil

i.7

111

Laboratory studies with the mosquito iridrl; cent virus (MN). 1. Inwrtehr. Patho!., 1 I. 296401.