Indian Journal of Experimental Biology Vol. 40, July 2002, pp. 842-845

Entomopoxvirus of cotton bollworm, Helicoverpa armigera (Hbn.)

K Narayanan

Project Directorate of Biological Control, Hebbal, Bangalore 560 024, India Received 10 September 2001; revised 16 November 2001

Occurrence of an Entomopoxvirus (EPV) from a lepidopteran insect viz;. cotton bollworm, H. armigera (HaEPV) along with gross pathological symptoms is reported for the first time in India. Histopathological study revealed that the fat body being the most favoured site of infection followed by haemocytes and gut epithelium. HaEPV was found to be not cross infective to six of the agricultural lepidopteran insect pests except for the potato black cutworm, Agrotis segetum regis­tering 100% mortality showing typical symptom. Further, safety of HaEPV was shown against beneficial insect like mul­berry silkworm, Bombyx mori and an useful insect general predator, Chrysoperla camea.

Entomopoxviruses (EPV) are the most recently stud­ied group of occluded type insect viruses and these viruses have been used in trial to control insect pests. Though EPV was first discovered during 1963 (ref. 1 ) subsequent research has shown that they bear close morphological resemblances to Chordopox virus (pox viruses of vertebrate) except that they are distin­guished by being occluded in a proleinaceous matrix at the end of virus replication cycle, unlike that of amorphous proteinaceous structure of cowpox 'A' type inclusions. The genome of EPV is a linear double­stranded, large DNA molecule approximately 225kb in size replicating in the cytoplasm, unlike that of in­sect baculovirus which is a closed circular double­stranded DNA molecule and whose genome size var­ies from 100- 1 30 kb, replicating mainly in the nu­cleus. The occlusion body (OB) which has been called 'spheroid' because of the shape, stabilizes the virions within and provides protection against UV light, heat etc; like that of polyhedral occlusion in insect bacu­loviruses, thereby showing a remarkable example of unrelated group of DNA viruses independently evolv­ing same strategy for efficient horizontal transmission in insect hosts. In the recent years considerable inter­est in the basic biology, molecular biology including use of EPV as biopesticide for the control of insect pests has emerged in several laboratories. Though EPVs have been recorded nearly from 3 1 species of insects belonging to the order Coleoptera, Lepidop­tera, Orthoptera, Diptera2, there is no report on the

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occurrence of EPV on lepidopterans insects except the recent report of an EPV on a coleopteran insece in India. Hence an attempt has been made in this study to survey for the occurrence of EPV in lepidopteran insects.

During the course of our regular survey for bacu­lovirus in and around Bangalore, some dead larvae of Helicoverpa armigera were found to harbour ento­mopoxvirus. The virus from the cadavers were ex­tracted and partially purified by differential centrifu­gation and examined under light, phase and electron microscope. Counts of occlusion bodies (OBs) were made by using haemocytometer. This entomopoxvirus was characterized by presence of numerous, refractile, oval shaped bodies called "Spheroids" mostly contain­ing virus. In addition to the ovoid shaped inclusions another fusiform shaped cytoplasmic inclusion bodies called "Spindles" devoid of viruses are also present (Figs 1 and 2). The pathogenecity of the virus was tested in laboratory by artificial diet surface contami­nation technique using 2 x 1 06 EPV OBs/ml on third, fourth and fifth instar larvae. The larvae infected with EPV g��nerally become soft whitish after infection and become lethargic and exhibit partial paralysis of the abdomen, and difficulty in moulting during the later stages of infection. Dead larvae usually remain soft but intact. Towards the end, the larvae are motionless except for slight movement of feet and mouthparts. In some larvae hindgut is extroverted showing prolapse of rectal symptom (Figs 3 and 4). Mortality appears 1 2 to 1 6 days after inoculation depending upon the stage of insect. Several pupae that originated from HaEPV infected larvae were found to be incompletely

1

NOTES 843

pigmented in the first abdominal segment (Fig. 5) . No adult emerged from such pupae.

To study the site of infection the diseased larva was processed and fixed and sections were made using standard microtome procedure. The sections were

stained with Harris haematoxylin and counter stained with eosin. The virus showed a range of cell and tis­sue specificity, the fat bodies appeared to be the most severely affected (Fig. 6). In addition to the fat bodies, light virus infection is also noticed in other tissues l ike, haemocytes and gut epithelium.

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, >.

II

Figs 1 -6-( 1 ) Light micrograph of spheroids and spindles of HaEPY (x 100); (2) El�ctron micrograph of spindle and spheroids of Ha­EPY (x 6700); (3) Extrovertion of hind gut of H. armigera showing prolapse of rectum due to HaEPV infection; (4) Prolapse of rectum of H. annigera due to HaEPY infection: (5) HaEPY infected H. armigera larvae showing incomplete pigmentation in first abdominal segment; and (6) Section of larval fat body of H. armigera infected with HaEPY showing spindles and spheroids

844 INDIAN J EXP BIOL, JULY 2002

Though most of the baculoviruses are specific to their respective hosts4, there is not much information on the cross infectivity of EPVs. Hence a study has been initiated to find out whether HaEPV is cross in­fective to other lepidopterans. The following insects were subjected to cross infectivty test namely; to­bacco caterpiller, Spodoptera litura, cabbage looper, Trichoplusi ni, potato black cutworm, Agrotis segetum; Bihar hairy caterpillar, Spilarctia (Diacri­sia) obfiqua; castor semilooper, Achoea janata; greater wax moth, Galleria mellonella and rice flour moth, Corcyra cephalonica. In S. filura; H. armigera, A. segetum and G. mellon ella respective artificial di­ets surface were contaminated with 0. 1 ml of HaEPV containing 2 X 106 OBs/ml and in the case of S. obli­qua and A. janata; castor leaf was contaminated with HaEPV containing 2 x 106 OBs Iml and in C. cephalo­nica sorghum grains were impregnated with HaEPV containing 2 X 106 OBs/ml. In all these cases 30 sec­ond instar larvae of respective insects were exposed to virus contaminated substrate for 48 hr and later on they were allowed to feed with normal dietlleaf till death or pupation. Larvae were observed daily for mortality. Smear from the dead was examined in phase-contrast microscope to ascertain the cause of death. The result of above cross-infective study showed that except A. segetum which showed 100% mortality showing typical symptom including prolapse of the rectum; none of the other insects was found to be susceptible to HaEPV (Table 1 ).

The safety of HaEPV was studied on mulberry silkworm Bombyx mori and a common predator Chrysoperla camea. Six days old B. mari (pM x NB4D2)

6 larvae were exposed to HaEPV at 2 x 10 OBs/ml and BmNPV 3.6 x 107 POBs/ml mulberry leaf by surface contaminant technique along with control. The larvae were allowed to feed the virus contaminated leaf for 48 hr. Thereafter, the larvae were transferred and fed with uncontaminated foliage. Mortality of larvae was recorded daily and their body weight was recorded at an interval of 48 hr. In predator, four day old C.

6 camea grubs were exposed to HaEPV at 2 x 10 OBslml by way of treating the Corcyra eggs with HaEPV and allowed the C. camea grubs to feed the virus con­taminated eggs of C. cephalonica for 48 hr and there­after allowed to feed the uncontaminated Corcyra eggs continuously till pupation. Mortality as well as body weight for both treated and untreated larvae was recorded every 24 hr. No death could be recorded ei­ther in control or HaEPV treated B. mori larvae. But 1 00% death of B. mori larvae occurred when they were treated with its own virus viz; BmNPV. Death of larvae started from 72 hr after treatment (Table 2). Body weight gain in B.mori larvae treated with

Table I -Cross infectivity of H. armigera EPV

Name of the insect Number of Mortality

Tobacco caterpillar, Spodoptera litura

Cabbage looper, Trichoplusia ni Potato cutworm, Agrotis segetum

Bihar hairy caterpillar, Spilarctia

obliqua Castor semilooper, Achoea janata

Greater wax moth, Galleria mellonella

Rice flour moth, Corcyra cephalonica

insects (%, tested

30 30 30 30

30 30 30

100

Table 2-Body weight (mg) of B. mori larvae treated with HaEPV

Treatment

HaEPV BmNPV Control Mean · F test

S. Em.

CD at 5%

0

1 3.46 1 2.69 12.56 1 2.90

··Significant at 1 % level

Hours after treatments 48 96 1 44 1 92 Mean

24. 1 3 8 1 . 1 9 1 50.34 300.24 1 13.87 27.47 49.60 10 1 .45 1 55.56 69.35 28.82 85.34 1 49.30 305.48 1 16.30 26.81 72.04 1 33.70

Period ••

Treatment ••

Period x Treatment ••

Period 1 .33 14 Treatment 1 .03 1 3 Period x Treatment 2.3061 Period 3.8444 Treatment 2.9779 Period x Treatment 6.6587

1

NOTES 845

HaEPV did not show any significant difference from control larvae. However, body weight of BmNPV treated larva was significantly lower than the control as well as that of treated larva. Mean body weight of the larva during this 1 92 hr was 1 1 3 .87, 69.35 and 1 16.30 mg for HaEPV, BmNPV treated and control larva respectively. HaEPV was also innocuous to C.carnea grubs. Treated grubs showed no sign of dis­ease and completed their life cycle. Cent per cent adult emergence was recorded. Body weight of the treated grubs (3 . 125 mg) did not differ significantly from that of control grubs (3 . 123 mg) suggesting that HaEPV is safe to both B. mori and C. carnea.

Several attributes of pox viruses in general and en­tomopoxviruses in particular, have led to their exten­sive use as expression vectors. The availability of in­sect permissive cell lines5; isolation of gene coding for spheroidin viz; sph and the described spheroidin gene may be one of the strongest known Eukaryotic promoter6 for the expression of foreign genes. Recent studies on the enhancement of insect baculoviruses pathogenecity through the administration of EPV in vivo? as well as through the expression of enhancing factor gene into the rice plants in order to increase the susceptibility of paddy cutworm, Pseudalatia sepa­rata to its nucleopolyhedrosis virus8 has shown its future potential. Though there are reports on the oc­currence of Nuclear Polyhedrosis Virus (NPV), Cyto­plasmic Polyhedrosis Virus (CPV) and Granulosis Virus (GV) on H. armigera, this is the first report of

EPV on a lepidopteran insect viz. H. armigera from India. Further studies are under progress.

Thanks are due to Dr S. S. Indi, Microbiology and Cell B iology Department, IISc. for electron micro­scope facility.

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275. 2 Entwistle P F, in Insect viruses and pest management, edited

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8 Hukuhara T, Hayakawa T & Wijonarko, Increased baculovirus susceptibility of armyworm larvae feeding on transgenic rice plants expressing an entomopox virus gene, Nat Biotechnol, 17 ( 1 999) 1 122.