A nuclear polyhedrosis virus infective to the pink bollworm, Pectinophora gossypiella

JOURNAL

OF

INVERTERKATE

A Nuclear

PATHOLOGY

Polyhedrosis

124-128 (1972)

20,

Virus

Infective

Pectinophora P. hMnnology

Research

Division,

V.

Quality

Research

Research

Plant

Protection

and Quarantine

Bollworm,

JAY

Service,

USDA,

Phoenix,

Arizona

860,$0

HUNTER

Agricultural AND

D. L.

VAIL,

Agricultural

Division,

Pink

gossypiellal

D.K. Market

to the

Research

Service,

USDA,

Fresno,

California

93727

R. T. STATEN

Programs, Animal and Plant Phoenix, Arizona 86040 Received

February

Health

Service,

USDA,

I, 197.2

A nuclear polyhedrosis virus isolated from the alfalfa looper, Autographa californica, infected larvae of the pink bollworm, Pectinophora gossypiella. Transmission was confirmed by light and electron microscopy and by feeding polyhedra obtained from pink bollworm cadavers to larvae of the cabbage looper, Trichoplusia ni. Preliminary studies were made of the infectivity, symptomatology, and histopathology of the virus in pink bollworm larvae.

the infectivity of the alfalfa looper virus to the pink bollworm, and to study the sympThe pink bollworm, Pectirlophwa gossytomatology and histopathology of infect*ion. pie&Z, iS a SWiOUS pest of CothJn ill nlany areas of the world, but only bacteria and &lETHODS AND RESULTS protozoa

were

known

to

bc

pat’llogenic

t’o

t’his speck until recently when Ignoffo and Adams (19%) isolated a cytoplasmic poly hedrosis virus from infected laborat’oryreared insects and proved it to be crossinfective t,o several other species of Lepidoptera. Subsequently, Vail cxt al. (1971b) described a virus isolated from the alfalfa looper, 9 utographa califomica, \\-hkh iI1fected caterpillar speciesfrom sctvcral families of Lepidoptera. In addition, primaq passageof this virus through two alternate hosts, the beet armyvorm, Spodopl’e,a e.Ggua, and the saltmarsh caterpillar, Bsfigmerle acrea, had no meai;urable effects on pathogrnicity to the cabbage looper, Tm'choplusia 7/i, larvae. Preliminary test’s wcw t,hereforr made in 1971 to dctcrmine 1 In cooperation tixperiment Station, Copyright All rights

wit,h University Tucson, Arizona

@ 1972 by Academic Press, of reproduction in any form

Inc. reserved.

of Arizona 85721.

I0fectizlity

The original alfalfa looper virus was obtained from a single diseased larva (Vail ct al., 1971a), propagated on the original host,, and found to be of the type of virus in which the virions are embedded multiply (:\IEV) in the polyhedral matrix. Polyhedra (NBS) obtained after passage through the original host, and PIBs obtained by infecting two alternate hosts, the beet armyworm and the saltmarsh caterpillar, were checked for purity

of

virus

type

by

ctxamining

thin

sections of at least 500 PIBs with the elecat,ron microscope. When it was determined that only t,hc KIEV-type virions n-erc present, the PIBx were assayed and found to be as infective to nconate larvae of the cabbage looper, Trichoplusia HZ’,as those obt’ained from the original host.

WV OF Pertinopho~a gossypiella Ta4BLE PERCENTIGE SECOND-INST.~R POLYHEDR.~

Dose (PIB/mm?)

100 5.0 1.5 0.5 0.15 0.05 0.01 0 (control)

MORT.~LITY

I .\ND

EMERGENCE

OF

PISK BOLLW-ORM L.ARV~E FED ORTAINED FROM THE ALF:~LF.~ LOOPKR

,

56 Mortality

on indicated

day

9

28-y. adult mergence

83.3 31.5 8.3 11.1

100 63.1

0.0 0.0

-

8.ti 4.3

0.0 31.5 58.3 X2.6 83.3 01.3 100.0 91.G

The initial assays of the susceptibility of pink bollworm larvae wrc made by sprcading various concentrations of PIBs collected from alfalfa loopers on the surface of a shandard artificia.1 diet in 1-oz plastic cups by the methods of Ignoffo (1965). Because of its antiviral nature, formaldehydr was spccitically cxcludcd from all die& (Vail et al., 196s). Aft.er the surface of the diet had dried sufficiently, each cup was infested with a second instar larva from the laboratory colon)-. Twenty-five cups per dose \\cre used, and the cups were held at 267°C unt.il mot,h emergence. As showi in Table 1, 916 ‘; of t,hc control larvae emerged as moths during a Z-day period, but no moths wcrc obt#aincd from larvae that fed diet, t,rcattd lvith 100 PIBs/nun2 of diet surface area; S:j.:<‘5:’ of these larvae were dead nit8hin ,5 days. Also, cmcrgcncc was decreased by a dose as Ion as 0.15 I’IBs/nnn”. Positrive diagnosis of nuclear polyhedrosis \vas confirmed from cadavers of larvae treated at conccntrat,ions as low as 0.05 polyhedra/ 1111n?. Both tht light and clcctron microscope examinations of cadavers contirmcd the prescnw of the ;\II?V-type polyhedra. In addition, several larvxc ww found to

135

conta,in cytoplasmic polghedrosis virus inclusion bodies typical of the virus reported by Ipoffo and Adams (1966). In t,he second assay, I’IBs obt,ained from the t,wo alternatr hosts were incorporated int’o hot artificial diet) (50%); the diet was poured into the l-ox plastic cups, cooled, and each cup )%-asinfested with one neonate pink bollwom~ larva. Doses, expressed as the number of polyhedra/ml of diet, ranged from 20,500 t’o 2.05. The cups were maintained a,t room tcmpcrature (cn. 3°C) until “4 days after infestation, and total mortality \vas recorded. Blank cups (no larva found) or cups containing larvae that died within 24 hr after infestation were discardrd and not included in the data. Positive diagnosis of nuclrar polylredrosis virus was confirmed for almost all doses testrd (Table 2). ,I’o large differences in the susceptibility of the larvae to the virus passed t,hrough the alternate hcJstS \vcrc noted. TABLE 2 SUSCEPTIBILITY

OF

NEON.\TI'.

T O ALF~LF.\

LIRVAE

HISJROSIS ALTERNaTE

LOOPER WHEN

Vrrms

HOSTS

POR,~TICD

ISTO

PINK

~JOLLXVORM

NUCLEAI~ POI.YPIBs FROM 2 WERE INCOI
THF:

I)IIGT Dose

No.

(polyhedra/ml medium)

PIBs 2.05 2.05 2.05 2.05 2.05

from

x

10’

33

x

103

2-l

x 10” X 10

2x 31 3-l PIBs

2.05 2.05 2.05 2.05 2.05

of

larvae

X X X x

lo4 lo3 lo2 10

Contr.01

* Held

from 32 29 32 31 33

Total

for

y0 Mortality due to NPVa

Spodoptera ezigua 9G.O 54.2 21.4 19.3 14.7

51.5 203 0.0 3.2 0.0

Esiigmene ao‘en 96.9 58.0 39.0 29.0 33.3 20.7

"9

at 24°C

%

mortality*

24 dsp

after

59.4 37.9 ti 2 3.2 3.0 0.0 infesting.

126

VAIL

ET

AL.

ing the liquefied internal contents of the dead larva to be released. Such symptoms were observed as early as 3 days after larvae HEDROSIS VIRUS WHEN PIBs FROM 2 ALwere placed on contaminated diet, and TERNATE HOSTS WERE INCORPORMTD mortality occurred on or before the 5th day INTO THE ARTIFICIAL DIET post-treatment depending on the age and Dose No. of Total y0 y0 Mortality stage of the larva when exposed. (polyhedra/ml larvae mortality4 due to NPV” medium) For the histopathological studies, polyhedra obtained from the beet armyworm PIBs from Spodoptera exigua were incorporated into larval diet at a con40 90.0 77.5 8.2 x 103 centration of 2.05 X lo4 PIBs/ml, the diet 8.2 x 102 39 53.8 25.6 was poured into petri dishes and cooled, and 8.2 X 10 40 20.0 2.5 each petri dish was infested with 30 larvae 40 15.0 5.0 8.2 of different ages. (Control diets were not 0.82 39 25.6 0.0 contaminated with polyhedra.) When PIBs from Estigmene acrea treated larvae showed maximum symptoms, 39 84.6 71.8 8.2 x 103 after approximately 7 days of feeding, they 40 30.0 15.0 8.2 x 102 were cut into two portions and fixed in 8.2 x 10 40 30.0 17.5 39 41.0 30.8 8.2 alcoholic Bouin’s fixative for 72 hr; control 26.3 0.82 38 55.3 larvae were fixed simultaneously. Then the 35 17.1 0.0 Control tissues were placed successively for 24 hr in 50, 70, 90, and 100% alcohol, 50/50 alcohol (1 Held at 24°C for 39 days after infesting. xylene, 100% xylene, 50/50 xylene paraffin, The third assay was similar to the second and 3 changes of paraffin before they lucre except that doses ranged from X.2 X lo3 finally embedded. After sectioning at 4 p, to 0.82 PIBs/ml of diet, and 4-day-old the tissues were stained by the methods of larvae were used (Table 3). Mort’ality due Hamm (1966). to nuclear polyhedrosis virus was confirmed No polyhedra were observed in sections at all but one dose, 0.82 PIBs obtained from of the control larvae (Figs. 1, 3). Sections of beet armyworm larvae. No positive diagnosis infected larvae revealed typical hypertrophy of nuclear polyhedrosis was confirmed in of the nuclei (Fig. 2) and the presence of the controls. Mortalit’y ranged from 90.0 to virogenic stroma in the cells of the fat body, 15.0% among larvae fed diet containing hypodermis, and tracheal matrix (Figs. polyhedra, and 77.5 % of the larvae fed the 2, 4); also, other hypertrophied cells contained large numbers of polyhedra. In addilargest dosefrom X. ezigua contained typical polyhedra. Some cadavers could not be tion, cells of the ventral nerve cord were found, which may explain the difference infected. Polyhedra were not observed in between the total mortality and the per- other tissues of this insect. centage confirmed to have nuclear polyDISCUSSION hedrosis. The nuclear polyhcdrosis virus originally Symptomatology awl Histopathology isolated from the alfalfa looper was deterPink bollworm larvae infected with the mined to infect larvae of the pink bollworm. nuclear polyhedrosis virus become flaccid This virus is now known to infect the caband gradually turn an off-white color. Soon bage looper, t’he beet armyworm, the saltafter death, t’he integument ruptures, allow- marsh caterpillar, the diamondback moth, TABLE

SUSCEPTIBILITY OF ~-DAY-OLD LARVAE T O ALFALFA LOOPER

3

PINK BOLLWORM NUCLIG.~R POLY-

NW OF Pectinophma

gossypiella

1. Hypodermal (H) and fat body (FB) cells from healthy larva. 3. Infected fat body cells (IFB) showing nuclei containing polyhedra. hypertrophied nuclei and displaying virogenic stroma (VS). Infected hypodermal polyhedra. FIG. 3. Trachea (T) from healthy larvae. FIG. 4. Infected tracheal matrix cells (IT) surrounded by infected FIG. FIG.

127

Cells also present with cell (IH) containing

fat

body.

128

VAIL

Plufella xylostella, the corn carworm, Heliothis xea, the cotton leaf perforator, Bucculatrix thwberiella, and the pink bollworm. The JJD50 for the pink bollworm is probably between 5.0 and 1.5 polyhedra/mm2 when second-instar larva? are fed 011 diet COINtaminated with polyhedra. Thus, this species is probably less susceptible than neonate cabbage looper larvae (Vail et al. 1971a). However, the burrowing habits of the pink bollworm cause them to be exposed to the virus for only a short period when orlly the surfaw of the diet is contaminat’ed. When neonate pink boll\\orm larvae were fed diet in which polyhedra were incorporated, t’he LL)s~ was between 2.05 X lo3 and 2.05 X 10” PIBs/ml, regardless of whclthcr the polyhedra were passed through the beet army\vorm or saltmarsh caterpillar. The I11150 for &day-old larvae is probably bet’wcen S.2 X lo3 and S.2 X 10’ l’IRs/ml of diet). The data give INJ indication that passage through alternate hosts had any influence OII the infectivity of th(l alfalfa lOcIper virus to pink bollworm larvae. Also, the> gross s~iiiptoIuat,ol(~g~r was similar to that reported for this virus in other inswts, and major differcnccs in suswptibility of tissues were not noted bctwwn infcctc,d pink bollworm larvae and alfalfa looper larvae. The virus can thcreforc replicate in the fat body, tracheal matrix, n(‘rw cord, and hypodermis, which arc’ tlicl thrw classic major sites of rcxplication of nuclear polyhcdrosis viruses. The ability of the alfalfa looper virus to infwt pink bollworm larvae makw microbial control \\-ith this agent, much mow feasible. Thcl pink bollworm, which is notoriously difficult’ to war, would not haw to b(> used for virus propagation. Any ow of wwral easily warc>d hosts could bc used and w~ultl also probably giw llighc~r yic>lds per unit8 timcx and moray ospc~ndcd. Also, since thcl virus infcc%s most, major lcpidoptc~rous pests of c~otton, \Y(’ nwtl not uw a inultiplicit)of

ET

AL.

viruses; which means that problems of formulation, standardization, and production need be solved for only the one material. In addition, application of this virus over a wide area to cotton, which is a major alternate host of many serious pests affecting other crops, for example, the cabbagcl looper, might drastically reduce populations of these pest species. Field test,s will be conducted in 1972 to establish the efficacy of th(> alfalfa looper virus to CcJIltrOl the pink bollwwm and other lcpidopterous pests of (*otton. In addition, more precise definition of the susceptibility of pink boll\vorm larvae \vill bo attempted. Methods of increasing yields and decreasing the cost of production of tlte virus will also be sought. REFERENCES H~MM, J. J. 1966. A modified Azan staining technique for inclusion body virrlses. J. Inverleb,. Pathol., 8, 125-126. HEIMPEL, A. M., AND ADIMS, J. IL. 1966. A new nuclear polyhedrosis of the cabbage looper, Trichoplusia. ni. J. Invert&. Ptathol., 8, 340346. IGKOFFO, C. M. 1965. The nuclear polyhedrosis virus of Heliothis zea (Boddie) and Heliothis vilescens (Fabricius). IV. Bioassay of virus activity. J. Znvertebr. Pathol., 7, 3X-319. IGNOFFO, C. NI., SD ADAMS, J. I:. 1966. B cytoplasmic-polyhedrosis virw, Smithiavirus pectinophorae sp. II. of the pink bollworm, Pectinophora gossypiella (Saunders). J. Invertebr. Pafhol., 8, 59-66. X7.111,, P. V., HWNXEXSICRRY, T. J., KISH.\IU, A. S.. AA-D AR.X.LJV~Y, IX. Y. 190X. Sodium hypochlorite and formalin as antiviral agents against nuclear polyhedrosis virus in larvae of the cabbage looper. J. Invertebr. Pathol., 10, 84-93. VAIL, P. \‘., &TTER, c:., J-iv, 11. L., \ND CkJUOH, D. 1971a. lteciprocal infectivity of cabbage looper and alfalfa looper nuclear polyhedrosis viruses. .J. Znaertebr. Pathol., 17, 3X3-388. VAIL, P. V., J.\-r, I). I,., AND HL-ZTEII, I). K. 197lb. Cross infectivit,y of a nllclcar polyhedrosis virus isolated from the alfalfa looper, .I I(tographa Inswl

“9-304.

californiw. Path&.,

f’rrv. College

Park,

4th .\lrl.

Cotloy.

In/. 1971,

pp.