Resistance of Galleria mellonella (Linnaeus) to the Tipula iridescent virus at high temperatures

JOURNAL

OF

INVERTEBRATE

PATHOLOGY

Resistance the

184-188

7,

of

(1965)

Gderia

Tipula

mellonella

Iridescent

Virus

(Linnaeus)

to

at High

Temperatures1 Y. Division

of Invertebrate

TANADA Pathology, ilccepted

AND

A. M.

University Jaizuary

TANABE

oj California,

Berkele.v,

California

25 i 2965

At 23” to 25”C, the larvae of the greater wax moth, Galleria mellonella (Linnaeus), which had been inoculated intrahemocoelically with the noninclusion virus of Tipula paludosa Meigen died of infection with the virus. At temperatures above 3O”C, the virusinoculated larvae survived to adults. Inasmuch as the virus was inoculated directly into the hemocoel, the resistance of the larvae at high temperatures was due to the destruction of the virus or the prevention of virus multiplication by immune and nonimmune host reactions, and not to the failure of the virus to penetrate into the hemocoel.

reported that some larvae of Pieris rapae High temperature is known to increase the (Linnaeus) survived infection from the granulosisvirus when reared at 36°C. The larvae of resistance or cause the disappearance of virus hercyniae (Hart&), when reared coninfections in plants and higher animals. In the Dip&n tinuously at 85°F (29.4’C) (Bird, 1955) and case of insects, there are only a few examples those of the cabbage looper, Trichoplusia ni of the effect of high temperature on virus in(Hiibner), and the corn earworm, Heliothis fections. Certain insect vectors lose their zea (Boddie) (Thompson, 1959), resisted the ability to transmit the viruses to plants when reared at high temperatures (Kunkel, 1937 ; infection by their respective nuclear-polyheMaramorosch, 1950). L’HCritier and Sigot drosis viruses. Aside from the study on the sigma virus of (1946) were apparently the first to report an Drosophila, all previous studies involved ininsect resisting infection from a virus insect viruses which were occluded in inclusion digenous for insects. They observed that at bodies.The present study was initiated, there30°C Drosophila lost the symptoms of infecfore, to determine whether high temperatures tion caused by the sigma virus. Later de also affected the pathogenicity of noninclusion Lestrange (1954; 1963) investigated this viruses, such as the Tip&a iridescent virus problem in detail and found that there was a continuous decreasein the sigmavirus content (TIV) , for insects. in flies maintained at 30°C. In 1953, Tanada MATERIALS AND METHODS A sample of the Tipula iridescent virus, originally found by K. M. Smith in Tip&a paludosa Meigen, was obtained on December 18, 1962, from Dr. F. T. Bird, Insect Pathology Research Institute, Canada Department of Agriculture, Sault Ste. Marie. Ontario, Canada. The virus was propagated in the

1 A portion of the study was conducted at the Laboratory of Sericulture, Tokyo University, Tokyo, Japan, where Y. Tanada spent his sabbatical year (October, 1962, to July, 1963) as a Fulbright Research Scholar. The study was supported, in part, by the Public Health Service Research Grant No. 03930, from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. 184

RESISTANCE

TOVIRUS

larvae of the greater wax moth, GalEeria mel(Linnaeus) . The virus inoculum was prepared in each test from a single wax-moth larva which had died of infection with TIV. The larva was triturated in a sterilized mortar and pestle and the homogenate was suspendedin sterile distilled water. The suspension was filtered through organdy (or cheesecloth) and the filtrate was centrifuged at 6,000 rev/min {3,600 g) for 15 minutes and the sediment discarded. The supernatant was then centrifuged at 12,000 rev/min (14,400 g) for 30 minutes, after which the supernatant was discarded and the virus pellet suspended with 100ml of sterile distilled water. This virus suspensionwas used as the stock inoculum in subsequentexperiments. In the first and third trials, about 1.0 ~1 of the virus suspensionwas inoculated into the hemocoel of each treated larva; for the second, fourth, and fifth trials, about 2.0 nl were used. In the latter three trials, the inoculum was first filtered through a 0.30-micron Millipore filter using a Swinny adaptera attached to a S-ml syringe. The control larvae in all trials were inoculated with equivalent volumes of sterile distilled water. The wax-moth larvae in the first and third trials were reared individually in sterilized test tubes stoppered with nonabsorbent cotton and fed beeswax. In the other three trials, they were reared individually or two each in a petri dish and fed an oligidic diet of mixed cereal, honey, and pollen.3 The temperatures of the cabinets in which the insects were reared were maintained constant at the various selected levels, but the humidity was not regulated except for an open lonella

2 Manufactured by the Millipore Filter Corp., Bedford, Massachusetts, U.S.A. 3 An oligidic diet composed of: honey, 100 ml; glycerine, lOOmI; water, 50ml; pollen, 50ml; Pablum Mixed Cereal, 1000 ml (manufactured by Edward Dalton Co., Evansville, Indiana, U.S.A.) ; Kellogg’s Special K, Z50ml (manufactured by Keliogg Co.. Battle Creek, Michigan, U.S.A.).

AT HIGH

185

TEMPERATURE

dish of water kept in each cabinet. In the first trial, the virus-inoculated and control larvae were held at 25” and 38°C; in the second, at 23’, 30”, 32”, and 35°C; in the third? at 25”. 37”, and 37°C after 3 days at 25°C; in the fourth, at 25’, 35”, and at 35°C after 1, 2, and 3 days at 25°C; in the fifth trial, at 25”, 35”, and at 25°C after 1, 2, 3, 6?and 10 days at 35°C. The presenceof the virus in the dead larva was determined by dissecting and examining its internal organs for the bluish iridescent color which is characteristic for infection by TIV. If no iridescence was observable, the larva was triturated, the body contents suspended in sterile distilled water, filtered through organdy, and centrifuged and purified as described previously. When TIV was present, the final purified pellet had a bluish iridescence. RESULTS

The wax-moth larvae inoculated with TIV and reared at 23’ to 25’C died from virus infection, but at 30°C and higher temperatures, most of them survived to adults (Table 1). Virus-inoculated larvae which died at the high temperatures had secondary septicemia from the bacteria present as a contaminant in the virus inoculum. When reared at 25°C for 1 day and then transferred to 37”C, 8 out of 10 virus-inoculated larvae survived to adults and two died with septicemia (Table 2). When reared over 1 day at 25°C and transferred to the high temperature, the larvae died with virus infection. During the 2 days at 25”C, the virus seemed to have multiplied sufficiently to cause the death of the larvae which had been subsequently placed at the higher temperature. The larvae did not survive virus infection when reared up to 10 days at 35°C and then transferred to 25°C. DISCUSSION

Thompson (1959) from his tests with ni and Heliothis zea and their respective nuclear-polyhedrosis viruses conTrichoplusia

186

TANADA

AND

TABLE EFFECT

II

9 0 0 0

1 5 3 3

Virus Virus Virus Virus Control Control Control Control

18 18 18 18 18 18 18 18

18 0 0 0 0 0 0 0

0 0 0 0 0 4 1 0

at at at at

10

23°C 30°C 32°C 35°C at 23°C at 30°C at 32°C at 35°C

in test tubes

TABLE LARVAE

INOCULATED

TEMPERATURE

INTRAHEMOCOELICALLY

Treatment

IV

v

a In

AT A HIGH

on beeswax;

Trial

III,

18 18 14 17 18

II,

they

were

reared

ON

THE

OF Galleria

SURVIVAL

TIME Tibula

WITH

IRIDESCENT

No.

dead

mellonella VIRUS~

from

No. adults

larvae

Virus

Others

Virus at 25°C Virus at 37°C Virus at 37°C after 3 days at 25°C Control at 25°C Control at 37°C Control at 37°C after 3 days at 25°C

10 10 10 10 10 10

10 0 6

0 8 4

0 2 0

1

1 5

8 5

3

7

Virus Virus Virus Virus Virus Control Control Control Control Control

10 10 10 10 10 10 10 10 10 10

10 0

0 0

0

0

2 8 1 3 1 0

10 10 10 10 10 10 10 10 10

10 0 10 9 8 9 9 0 0

at at at at at

25°C 35°C 35°C after 1 35°C after 2 35°C after 3 at 25°C at 35°C at 35°C after at 35°C after at 35°C after

Virus at 25°C Virus at 35°C Virus at 25°C after 1 Virus at 25°C after Virus at 25°C after Virus at 25°C after Virus at 25°C after Control at 25OC Control at 35°C individually

in Trial

0 18 18

2

NO.

Trial no.

reared

DURATION

No. adults

Others

10 10 10

OF THE

III

dead from

Virus

Virus at 25°C Virus at 38°C Control at 25°C Control at 38°C

a In Trial I, the larvae were reared individually two each in petri dishes containing an oligidic diet.

EFFECT

No.

No. larvae

Treatment

I

1

TEMPERATURES ON THE SURVIVAL OF Galleria mellonella LARVAE INTRAHEMOCOELICALLY WITH THE T&da IRIDESCENT VIRUS~

OF HIGH INOCULATED

Trial no.

TANABE

the larvae in petri

day at 25°C days at 25°C days at 25°C

1 day at 25°C 2 days at 25°C 3 days at 2S”C

day at 35°C 2 days at 3 days at 6 days at 10 days at

35°C 35°C 35°C 35°C

were reared individually in test tubes dishes containing an oligidic diet.

on beeswax;

0 0

2 9 0 0 0 0 0

in Trials

10 8 0 0 9 10

1 1

9 9

0 2 0 1 2 1

0 8 0 0 0 0

1

0

0 0

10 10

IV and V, they

were

RESISTANCE

T O VIRUS

AT

eluded: “In the insects in which high temperatures do prevent virus infection, it appears that the mechanism or mode of invasion [after feeding on the virus] is the factor affected, since disease is not inhibited if the insects are exposed to high temperatures after infection is accomplished.” On the other hand, in the study with Pieris sapae and its granulosis virus, Tanada (1953) found that somelarvae which had been fed virus and reared at room temperatures (20-25°C) for 2 to 5 days and transferred to 36°C survived to adults. Moreover, in the case of Drosophila and its sigma virus (de Lestrange, 1954; 1963) and in the present study with Galleria mellonella and TIV, the virus was already present in the insect or had been introduced into the hemocoel at the time of the high-temperature treatment. In these cases,it was apparent that the effect of the high temperature was not on the mechanismor mode of penetration of the virus into the insect hemocoel, but more likely on other factors such as the destruction of the virus, a reduction or cessationof virus multiplication, and the action of immune and nonimmune host responses. de Lestrange ( 1963) proposed three hypotheses to explain the continuous decrease of the sigma virus in Drosophila reared at high temperatures. (1) There is a “single crop” of virus particles, and at high temperatures they are gradually destroyed, but when the Drosophila is returned to a low temperature, the surviving virus particles multiply and begin the new cycle. (2) A dynamic equilibrium exists between virus formation and destruction. High temperatures cause the equilibrium to move towards the direction of destruction and low temperatures in the direction of virus multiplication. (3) The Drosophila fly has two types of virus particles: virus particles without protein envelope which are noninfectious and complete particles with envelope which are infectious. During the entire life of the fly, there

HIGH

187

TEMPERATURE

would be a transformation from one to the other. At 2O”C, an equilibrium is reached between the two processes.The heat, whether by slowing the first reaction or accelerating the second, or acting on them simultaneously would cause a decreasein the yield of infectious virus. When the flies are placed again at 25”C, the relative importance of the two processeswould be reversed until a new equilibrium is attained. de Lestrange (1963) believed that the third hypothesis was more applicable to the results obtained with the sigma virus in Drosophila. Whether the hypotheses presented by de Lestrange are applicable to other insect viruses has not been established.However, we believe that these hypotheses do not sufficiently emphasize the role of the immune and nonimmune host reactions which may govern or effect the formation and destruction of the virus particles at high temperatures. Such host reactions may be the production of interferon (Isaacs, 1963) or the febrile response (Baron, 1963).

We wish to express our grateful appreciation to Professor H. Aruga and his staff, especially Dr. H. Watanabe, for their kind cooperation in the portion of the study that was conducted at the Tokyo University, to the Board of Foreign Scholarships, U. S. Department of State, and to the United States Educational Commission (Fulbright Commission) in Japan for providing the research fellowship to Y. Tanada. REFERENCES

S. 1963. Mechanism of recovery from viral infection. Advan. Virus Res., 10, 39-64. BIRD: F. T. 1955. Virus diseases of sawflies. Can. Entomologist, 87, 124-127. DE LESTRANGE, M. TH. 1954. Action de la temperature sur le virus responsable de la sensibilitt a l’anhydride carbonique chez la Drosophile. Compt. Rend., 239, 1159-1162. DE LESTRANGE, M. TH. 1963. Contribution a l’etude du virus hereditaire de la Drosophile: action de l’hyperthermie sur le contenu en virus des tissus somatiques de l’hbte. Ann. G&t., 6, 39-96. ISAACS, A. 1963. Interferon. Advan. Virus Res., 10, 1-38. BARON,

188

TANADA

AND

1937. Effect of heat on ability of sennotata (Fall.) to transmit aster Am. J. Botany, 24, 316-327. L’HBRITIER, PH., AND SIGOT, A. 1946. Contribution a l’etude du phenomene de la sensibilite au CO, chez la Drosophile. Influence du chauffage aux differents stades au developpement sur la manifestation de la sensibilite chez I’imago. Bull. Biol. France et Belg., 80, 171-227. KUNKEL,

L.

Cicadula yellows.

0.

TANABE

K. 1950. Influence of temperature on incubation and transmission of the woundtumor virus. Phytopathology, 40, 1071-1093. TANADA, Y. 1953. Description and characteristics of a granulosis virus of the imported cabbageworm. PYOC. Hawaiian Entomol. Sot., 15, 235-260. THOMPSON, C. G. 1959. Thermal inhibition of certain polyhedrosis virus diseases. J. Insect Patkol., 1, 189-190. MARAMOROSCH,