Development of resistance in the silkworm, Bombyx mori, to peroral infection of a cytoplasmic-polyhedrosis virus

JOURNAL

OF

INVERTEBRATE

PATHOLOGY

9,

Development Bombyx

of

474-479

( 1967)

Resistance

in

to Peroral

tori,

Laboratory

of

Sericulture,

of

a

Virus1

WATANABE

Faculty Bunkyo-ku, Accepted

Silkworm,

Infection

Cytoplasmic-Polyhedrosis HITOSHI

the

of Agriculture, Tokyo, Japan October

20,

University

of

Tokyo,

1966

By selecting survivors from v;rus-fed larvae for eight generations, resistance to cytoplasmic-polyhedrosis virus was developed in the silkworm, Bombyx mori. The resistance increased up to the fifth generation and subsequently reached a plateau. The highest degree of developed resistance, based on the ratio of ED, values of selected and unselected strains, was about l&fold. A selection pressure greater than 60% mortality was effective in inducing and retaining the resistance of the selected generations. Hybrids from orosses between two selected strains were more resistant than those from crosses of unselected strains.

INTRODUCTION

Although there are a large number of studies in various insect species on the development of resistance to insecticides, only a few cases have been established of the development of the resistance of insects to pathogens. One well-authenticated case was reported by Harvey and Howell (1965) on the resistance of the house fly, Musca domestica, to Bacillus thuringiens.:s. They studied the induction of resistance in the house fly by selecting survivors of 50 generations reared on larval medium treated with B. thuringiemis. From such selection, they obtained resistant house flies. As to resistance to viruses, Sidor (1959) and Rivers (1959) stated that the resistance to granulosis infection in Pieris brassicae increased after breeding for several 1 This work was supported in Aid for Scientific Research Education in Japan.

in part by a Grant from the Ministry of

474

generations the survivors of a granulosisvirus outbreak. Subsequently, David and Gardiner (1960) also obtained a stock of P. brassicae resistant to the granulosis virus following laboratory selections. Yamafuji et al. (195Sa,b) attempted to select silkworms resistant to virus induction by chemicals, but they were unsuccessful. However, Aizawa et al. (1961) succeeded in selecting a strain of silkworm resistant to virus induction by cold. Recently, Uzigawa and Aruga (1966) investigated the selection of silkworms which were resistant to the infectious flacherie virus, and a resistant strain was obtained after five generations. These experimental results are of significant value in silkworm breeding in Japan, because the resistance of the silkworm larvae to virus diseasesis one of the important commercial aspects of sericulture. The present paper reports on the development of resistance in the silkworm to the cytoplasmic-polyhedrosis virus and on

DEVELOPMENT

the resistance of hybrids from tween two selected strains. MATERIALS

SiZkzsorm

AND

crosses

OF

be-

METHODS

Strains

Two strains of the silkworm which are highly susceptible to oral infection by the cytoplasmic-polyhedrosis virus were used in the present study. The first strain, SN, was derived from a Japanese ecological race, and the second, SC, possessed characteristics of the Chinese race. Both strains were selected for resistance against the virus. The strains from SN and SC, which were selected for resistance to the virus, were designated as RN and RC, respectively. Vi?US Two strains of the Bombyx cytoplasmicpolyhedrosis virus were used, i.e., a strain forming polyhedra tetragonal (TC) in outline and another forming hexagonal polyhedra ( HC ). Properties of these two virus strains and the shapes of their polyhedra have been described previously by Hukuhara and Hashimoto ( 1966). The virus inocula contained polyhedra which were freshly produced and purified each rearing season and stored in the refrigerator until ready for use. A uniform suspension of the polyhedra was fed to the larvae. Selection

Procedures

The larvae were reared at 25” to 28°C throughout the experiments. The control strains were reared under the same conditions as the selected strains but not exposed to virus. Immediately after ecdysis, the fifth-instar larvae, which were under selection, were fed the virus for 20 hours on mulberry leaves smeared with the polyhedra suspension. For each selected strain, three rearing units containing about 150 larvae were maintained in each genera-

RESISTANCE

TO

475

VIRUS

tion and fed different dosage levels of virus. Vigorous moths were collected from the unit where the selection was most rigorous and in which sufficient numbers of pupae were present. These moths were used to produce the subsequent generation. The intensity of selection varied between 30% and 92% mortalities. In Tables 1 and 2. each strain selected with HC virus and TC virus is designated with the letter I-I and T in parentheses, respectively. The number of each generation is also illeluded in the parentheses. Testing

Procedures

The susceptibility of the larvae of each strain to the virus was based on the log EDso that was estimated from the log dosage-infectivity tests. The virus inocula fed to each strain were suspensions of the polyhedra at five different concentrations; lo”, 105.s. lo”, 10”.:, lo’, lo’.“, and lo8 polyhedra of TC per ml. Each concentration of the virus was administered perorally by feeding the fourth-instar larvae for 20 hours on mulberry leaves smeared with the polyhedra suspension. All larvae were dissected 6 days after they had fed on the virus and their body contents examined under the microscope to determine thr number infected with the virus. RESULTS

Development Strains

AND

of

Drscussro~

Resistance

irr SeletM

Differences in the susceptibility to irtfection with the cytoplasmic-polyhedrosis virus between unselected SN and selected RN strains at each generation were ohserved (Table 1). The results indicated that the resistance of the selected strain developed by the fifth generation, and selections made in earlier generations did not possess significant resistance. The lack of resistance in the early generations may

476

WATANABE

be partly caused by the application of a low percent of mortality. There was no essential difference in the development of resistance between selection with the TC virus and the HC virus because both viruses are equal in virulence for the silkworm ( Watanabe, I966 ) . After resistance had developed in the selected strains, the slopes of the regression lines of both selected strains became steeper than those of the control unselected strains. Table 2 summarizes the results of log EDso values estimated from five dosageinfectivity tests that compared the control (SC) with the two selected strains from the second to the eighth generation. The TABLE DIFFERENCE

1964

Summer,

Fall,

Spring,

Fall,

Strain

1965

Spring,

1964

1964

1965

1965

1

IN SUS~EPTIBILITYT~ INFECTION WITH THE CYT~PL.~~MIC-P~LY~EDR~~~~ UNSELECTED (SN) AND SELECTED (RN) STRAINS OF THE FOURTH-INSTAR

Season

Fall,

data revealed that, after the fifth generation, the log EDGO values of the selected strains were significantly higher than comparable log EDsO’s of the SC strain. In this case, the intensity of selection greater than about 60% mortality seemed to be effective in inducing and retaining the resistance of the selected strains. In addition, as in the case of the RN strains previously described, the RC strains that had significant resistance generally showed a little steeper slopes of regression lines than those of their control SC strains. This might indicate that the genetic variability in resistance became small in the selected strains during the course of rigorous selections.

Log

EDa,

959,

c. L.

Vr~us SILKWORM

Slope0

BETWEEN

Pelectionb intensity

652’3

SN RN(TQc RN(HQd

5.7? 5.90 6.06

5.38-6.06 5.5Y-6.!28 5.57-6.55

0.72 0.60 0.86

56yc

SN RN(W) RN(HS)

6.73 6.44 6.66

6.44-7.0" 6.26-6.6" 6.78-7.14

1.43 1.24 1.30

34% 48%

SN RN(T4) RN(H4)

5.50 6.14 6.50

5.28-5.7‘2 5.96-6.28 6.36-6.64

0.64 0.82 1.20

SN RN(T5) RN(H5)

5.32 6.75 6.55

4.91-5.73 G.S8-7.l? 6.12-6.98

0.59 1.20 1.70

SN RN(T6) RN(H6)

5.56 6.60 6.88

5.37-5.85 6.38-6.88 6.6G7.02

1.04 1.24 1.38

SN RN(T6) RN(H8)

5.50 6.67 6.80

5.1s5.88 6.43-6.91 6.5.5-7.05

0.88 1.38 0 8"

D Slope of log dosage-probit mortality line. * Mortality of virus-fed larvae in the previous generation of selection. ted Each strain selected with HC virus and TC virus is noted with the letter spectively. The number of each generation is also included in the parentheses.

W% ‘33%

w% 76%

II and T in parentheses,

re-

DEVELOPhIENT

OF

In order to illustrate more clearly the rate of increase in the resistance of the selected strains, the results of Tables 1 and 2 are presented graphically in Fig. 1. The degree of resistance is presented in the figure as a logarithmic ratio obtained by dividing the log ED%,, of the selected strain by the log ED,” of the unselected strain. As shown in Fig. 1, the selected strains showed no increase in resistance prior to the fourth generation, but subsequently developed a sudden increase in resistance. Thus, the increase in resistance apparently resulted from the higher intensity of selection present in the fourth but not in thy earlier generations. Feigin ( 3963 ) attempted to select house flies resistant to B. tlmringiensis, hut obtained no rrsistunt house flies even after 27 generations. Since the selection pressure in her studv was about 40% mortality, she be-

Season

F:tll,

1963

Sprmg,

Fall,

1964

1964

Spring,

l’all,

Strain

1965

1965

Lop

mjo

RESISTANCE

TO

47:

VIRUS

lieved that the house fly did not become resistant because the selection pressurr was too low. ln the present study, higher than 60% mortality seemed to be required as a selection pressure in order to inducts and retain the resistance of the silkworm to virus infection. Despite rigorous+ continued selection. there seemed to be no further incn~asc~ in resistance after attaining a certain plateau. At this plateau the degree of rtbsistance for the selected strains generalI\; varied from 1.0 to 1.2 in logarithmic ratio; ED,,, values of the selected strains illcreased to about lo- to 16-fold values of the unselected strains. The simplest CCplanation for the plateau is that th(, g(‘netic factors concerned with resistance are homozygous. On this point, however, four-ther studies are necessary to determine whether or not the selected strains IOW

95(‘> (‘. 1,.

SC RC(T4) RC(H?Z)

5.32 5.48

4.79-B.

05

5 14--5.

X?

5.27

4. M--5.

6X

SC RC (T3) RC(H3)

5.88

5.66-6.10

5.96 6.38

5 53-6 ti 07 -6

SC RC(T4) RC(H4)

5.03

0.65

5.11

0 BU 0 50

u .x9 39 69

5.50

SC RC(T5) RC(H5)

5.00

SC RC(T6)

6. 06 7.“7

lIC(H6)

7.18

SC RC(T8) RC(H8)

6 25

5.58 6. “5

7.39

7 ‘3

II,66

Il. HO

4.84-5.lti 5.38-5.78 5.97-6.53 5.7X-6.34

7.5’ ; 83 ;

7 O-L-7 .47 6. !U-7 .&a 5 W-6 58 7.19-7.59 Ii 49-7.57

0.90 1 .07 0.64

tie”; 65“;

478

WATANABE

their resistance when released selection pressure of the virus. Resistance between

in Hybrids from Selected Strains

from

the

Crosses

At the seventh generation, RN and RC selected with TC or HC viruses were crossed, and the F1 hybrids were compared in their resistance with the control hybrids from crosses between unselected strains. The results, including those of the parental strains, are illustrated in Fig. 2. The log EDso values of hybrids from

7-

crosses between selected strains were larger than those of the control hybrids. However, when the comparison between the two hybrids was made on the manifestation of heterosis in the resistance, several different features were observed. The resistance of the control hybrids appeared greater than that of their unselected parental strains, and this indicated a marked heterosis. On the other hand, none of the hybrids from the crosses of selected strains was more resistant than either of their parental strains. The differences in heterosis observed in the two hybrids may be explained as follows: The different genetic combination of the two control strains is more likely to develop marked heterosis in resistance, while in the selected strains the integrations of highly resistant genes and yet of similar genetic constitution for resistance have occurred during the course of selection, and the

5.5 I

LOG 6 I

ED5o 6.6 I

7 I

7.5

SN

Fl GENERATION FIG. 1. Development of resistance in the fourthinstar larvae to infection with the cytoplasmicpolyhedrosis virus in successive generations of the silkworm selected with the virus. RN(H) and RN(T) are selected strains with HC virus and TC virus, respectively, from SN control strain. RC( H) and RC( T) are selected strains with HC virus and TC virus, respectively, from SC control strain. The degree of resistance is presented as a logarithmic ratio obtained by dividing the log ED, of the selected strain by the log EDso of the unselected strain.

Fl

FIG. 2. Comparative resistance in instar larvae to the cytoplasmic-polyhedrosis among selected (RN, RC), unselected strains and their hybrids.

the

fourthvirus (SN, SC)

DEVELOPhlENT

OF

genetic combination of the two selected strains tends to show poor heterosis.

The author wishes to thank Professor H. Aruga for his helpful advice during this investigation, and Dr. Y. Tanada, Department of Entomology and Parasitology, University of California for criticizing the manuscript. The author is also indebted to Miss H. Nngano for her technical assistance. RRFEHISNCEY

K., FUIWTA, Y., AND NAKAMURA, K. 1961. Selection of a resistant strain to virus induction in the silkworm, Bon&q mori. J. Sericult. Sci. Jupan, 30, 405412 (in Japanese with English summary). DAVII,, W. A. L., AND GARDINER, B. 0. C. 1960. A Pieris brassicue (Linnaeus) culture resistant to a granulosis. J. Insect Pathol., 2, 106-144. E‘E:IGlN, J. M. 1963. Exposure of the house fly to selection by Rucilltcs thuringiensis. AWL Entornol. Sot. Ant., 56, 87X-879. HAHV~Y, T. I,.. ANI> HOWELL, D. E. 1965. ResistATLAWA,

RESISTANCE

TO

479

VIRUS

ante of the house fly to Bacillus thuringiensis Berliner. J. Inuertebrute Pathol., 7, 92-100. HUKUHANA, T., AND HASHIMOTO, Y. 1966. Studies of two strains of cytoplasmic-polyhedrosis virus. J. Invertebrate Puthol., 8, 184-192. RISERS, C. F. 1959. Virus resistance in larvae of Picris brrdcae ( L). Trans. Intern. Con/. Insect Pathol. Biol. Control. kt., Prague. lS9)s8 pp. ‘05-210. SIDOR, C. 1959. Susceptibility of larvae of the large, white butterfly ( Picris hrassicuc, L. 1 to two virus diseases. Ann. Appl. Biol.. 47, 1W-f-1 I:?. UZIGAWA, K., AND AHUCA, H. 1966. On thr sc+c~tion of resistard strains to the infectioti\ flacherie virus in the silkworm, Bonth!/x p)rori L. I. Sericldf. Svi. Japm, 25. 28-W i 111 Japanese ) . WATANABE, H. 1966. Relative virulerlcc of pulphedrosis viruses and host-resistance in t.jkC silkworm, Bomhyx mori L. ( Lepidoptera: Bombycidae). Ap)11. Entomol. Zool.. 1. 19!1144. YAMAFUJI,

h.,

SATO,

ht.,

AXD

Chemical virogenesis and in silkworm. Enzymologia, YAhlAFUJI,

Chemical silkworm.

K..

NATO,

bf.,

virogenesis En;f/mologia,

AND

and

~'AGATA,

J.

1W-h

virogenic trratmcnt 19, 48-52. hHIIiA\Vh,

J. 1%8h.

remote infection 19, 151-156.

in