Hereditary variation in the ability of a leafhopper to transmit two unrelated plant viruses

VIROLOGY

16, 152-162 (1962)

Hereditary

Variation Transmit

in the Two

Unrelated

A. N. NAGARAJ2 Department

of Botany,

Ability

Plant

AND

University

Accepted

of a Leafhopper

to

Viruses’

L. M. BLACK of Illinois,

Urbana, Illinois

October lY, 1961

Individuals of the leafhopper Agallia constricta (Van Duzee) were tested for their ability to transmit two unrelated plant viruses, wound-tumor virus (WTV) and the New Jersey strain of potato yellow-dwarf virus (PYDV). By selective breeding through six generations, four races of the leafhopper were obtained which differed in their relative transmitting abilities in relation to the two viruses. These races were designated WY, Wy, WY, and wy, the symbols representing the phenotypic responses of the races. The inbred races were difficult to maintain because of small numbers of progeny or slow growth or both. Races WY and WY were efficient and the races Wy and wy were inefficient in acquiring PYDV from, and transmitting it to, crimson clover. Insects of race WY were inefficient, whereas those of races Wy and WY were efficient, transovarial transmitters of WTV. These results indicate that hereditary mechanisms which determine the efficiency of the transfer of PYDV from plant to plant and the transovarial passage of WTV must be different. It is suggested that at least two alleles, if not two genes or more, are involved in determining the above responses. Results of a single experiment indicated that insects, freed of WTV and derived from ancestors which were selected for transmission of this virus in only one generation, could acquire WTV from, and transmit it to, crimson clover more efficiently than could unselected stock insects (Table 1 in the text). No transovarial passage of PYDV was observed in any races, whether they transmitted PYDV efficiently or inefficiently. This point remains to be conclusively established. However, high transovarial passage of WTV is probably associated with efficiency in acquiring the virus from and transmitting it to plants, though this remains to be demonstrated. Interference between WTV and PYDV was not detected in either the vector or in crimson clover plants. INTRODUCTION

In some species of leafhoppers, individuals have been found to vary in their ability to transmit plant viruses. Storey (1932) first demonstrated such variation in the leafhopper Cicadulina mbila (Naude) ; he found that the ability of individuals of C. l This investigation was supported in part by grants from the National Science Foundation and the National Institutes of Health. It represents data submitted in partial fulfillment of the requirements for the senior author’s Ph.D. degree in the Graduate College of the University of Illinois. o Present address : Virus Laboratory, University of California, Berkeley 4, California.

mbila to transmit

the maize-streak virus was controlled by a single sex-linked dominant gene. Since then, variation in virustransmitting ability has been demonstrated in the following leafhopper vectors of plant viruses: Nephotettix apicalis var. cincticeps (Uhl.), a vector of rice-dwarf virus (Fukushi 1933, 1940) ; Circulifer tenellus (Baker), the vector of sugarbeet curly-top virus (Bennett and Wallace, 1938) ; Aceratagillia sanguinolenta (Prov.), the vector of the New York strain of potato yellow-dwarf virus (Black, 1943a) ; Delphacodes striatella (Fallen), the vector of rice-stripe virus (Yamada and Yamamoto, 1955) ; and 152

TRANSMISSION

OF VIRUSES

BY A LEAFHOPPER

153

1)elphacodes pelEucicla Fabr., the vector of Plants European wheat-striate mosaic virus (WatTest plants for the viruses. Crimson son and Sinha, 1959). clover (Trifolium incarnatum L.) was used However, so far as the authors of this to test the infectivity of leafhoppers. The paper are aware, hereditary variation in the usual daily temperature range in which ability of a leafhopper vector to transmit crimson clover seed was germinated and two unrelated plant viruses has not yet grown as seedlings was 18-27” and that in been studied. The research reported in this which crimson clover was observed for dispaper is concerned with such variation in easedevelopment or held inside insect cages the ability of Agallia constricta (Van DU- during acquisition or inoculation of virus by zee) to transmit two plant viruses, namely, insect,a was 2430”. Seedlings started in wound-tumor virus (WTV) and the New sand were transplanted, when about 1 Tersey variety of potato yellow-dwarf vimonth old, into 21%inch pots filled with a JS (PYDV), which are serologically un- mixture of one part sand to two parts soil related (Black and Brakke, 1954; Wolcyrz that had been steamed to kill all weed seeds. and Black, 1956, and unpublished dat’a from For testing single insects, plants on which this laboratory) and have been shown by the first compound leaf was just unfolding the work of Black, Brakke, and Vatter to were used. For testing colonies of 3-5 inmorphologies (Black, possess distinctive sects, plants with 3-5 compound leaves were 1955j . A preliminary report of this work has used. These plants were fertilized weekly been published (Nagaraj, 1959). with a complete nutrient solution. After the inoculation period, the test plants were MATERIALS AND METHODS fumigated every 5-7 days with tet’raethyl pyrophosphate. Insects Host plants of the leafhopper. NTonviruliferous leafhoppers were reared on either Only one species of insect, A. constricta, was used in all the selection and trans- crimson clover or alfalfa plants (Medicago ovarial passage experiments. As far as pos- sativa L. var. Grimm) growing in 4-inch sible, in comparable experiments done at pots, each covered with a cage. To mainany one time, insects of the same age group tain viruliferous leafhoppers away from a were employed. Usually, when nymphs were source of virus they were reared similarly used, those in the second and third instars on Grimm alfalfa plants, which are known were preferred, because earlier experiments to be immune from both the viruses (Black and Brakke, 1952; Black, 1953). Alfalfa of Mr. Walter Atchison (unpublished data) had indicated that they were good trans- plants were grown from seed in the same mitters. Moreover, the use of such nymphs manner as the crimson clover. was preferable because, at the end of the test, period, they were usually adults and Symptoms of Wound-Tumor and Potato Yellow-Dwarf on Crimson Clover could be used in mating experiments. Both WTV and PYDV produce distincI’iruses tive symptoms on crimson clover (Black, Of the two speciesof viruses used in these 1944). According to Maramorosch (1950) studies WTV (Black, 1944) has an incuba- the incubation period of WTV in the plants tion period of 13-30 days at 25-30” in A. varies from 10 to 40 days. At 25-30” most constricta (Maramorosch, 1950). The in- plants show symptoms of disease in 2-4 cubation period of the New York strain of weeks. The principal symptoms of WTV on potato yellow-dwarf virus in A. sanguino- this host consist of irregular swellings on the lenta is 6-10 days (Black, 1943b). It was undersurface of veins with corresponding assumed that PYDV (Black, 1941) has a depressions on t,he upper surface. The incubation period of the New York strain of similar incubation period in A. constricta, and results of experiments are in accord potato yellow-dwarf virus in crimson clover plants is 2-3 weeks (Black, 1943b) ; PYDV with this assumption.

154

NAGARAJ

had a similar incubation period in our experiments. The symptoms of PYDV are usually noticed first on the older leaves; they consist of a rusty brown necrosis along the veins and veinlets. That the same crimson clover plant can be infected with both WTV and PYDV, by a single insect, was indicated by the work of Nagaich (1960) and was confirmed during the course of these studies. The test plants were observed for at least 6 weeks after the insects were removed from them and very few plants developed symptoms of either disease in the sixth week. Selection of Races Definitions. The term “family” as used here refers to the progeny of a single male and female pair. Wherever two or more families were bulked, this will be so indicated. The word “race” refers to a family, or a group of families, which differs from other such groups in ability to transmit WTV or PYDV. The designations “efficient race” and “inefficient race” denote the relative abilities of races to transmit a specified virus. The terms “transmitter” and “nontransmitter” are used to indicate insects that did or did not transmit the virus, respectively, during a particular test, regardless of their hereditary efficiency or inefficiency. Preliminary selection. Individual insects were tested for their ability to acquire and transmit WTV. After their feeding on test plants the insects were mated in pairs. Later when all transmission data were available it was found that only four of the families had one transmitter each as a parent; the other sixteen had none. In two families the transmitting parent was a male and in two others it was a female. These four families were bulked on alfalfa plants on which they were reared for three generations in order to free them from virus acquired through the egg. Twenty-five insects of the third generation progeny were divided into 5 colonies of 5; each colony was tested for 6 weeks (2 weeks on each of 3 successive crimson clover plants). None of the plants became infected, a result indicating that these insects had lost WTV during repro-

AND

BLACK

duction for three generations on alfalfa. The insects were then put together on alfalfa, and their progeny ((‘partially selected insects”) were used for selecting an efficient race for transmission of WTV. These “partially selected insects” were compared with an equal number of insects from the virusfree stock culture “stock insects” for their ability to transmit WTV and PYDV. This generation of insects will be referred to as the first generation, their progeny as the second generation, and so on. Selection through six generations. In the first generation the “partially selected” and (‘stock insects” were fed separately on WTV source plants for 4 days, then on PYDV source plants for 4 days; they were subsequently tested individually for 6 weeks (1 week on each of 6 successive test plants). From the second generation onward the acquisition period was increased to 7 days on each of the WTV and PYDV sources in order to obtain better transmission. Each family was fed on a single virus source plant unless otherwise stated. The source plants used in any one experiment were carefully selected for uniformity. The criteria used in doing so were (1) vigor of the diseased plant, (2) age of infection (as far as possible plants which showed first symptoms of disease during the same week were used), and (3) incubation period of the virus in the plant (an effort was made to select infected plants with comparable incubation periods). After acquisition on virus sources the insects were individually tested for infectivity for 6 weeks (2 weeks on each of 3 successive test plants), unless stated otherwise. According to their transmission record the insects were placed in four groups: (1) efficient transmitters of both viruses (group WY), (2) efficient transmitters of WTV but not PYDV (group WY), (3) efficient transmitters of PYDV but not WTV (group WY), and (4) inefficient transmitters of both viruses (group WY). By selective breeding within these four groups, it was expected that four races designated WY, Wy, WY, and wy respectively, would be obtained. These symbols are not intended to represent genes concerned, the capital letters

TRANSMISSION

OF VIRUSES

W and Y indicating the phenotypic responses, efficiency in transmitting WTV and PYDV, respectively, but not dominance of factors determining transmission. Similarly, small letters w and y indicate inefficiency in transmitting WTV and PYDV, respectively. At the end of each testing period insects which could be classified in one of the four groups were mated in pairs within the same group. Each pair was caged on an alfalfa plant. Insects that could not be classified, owing to lack of complete data, were considered for purposes of mating to belong to the group to which their parents belonged, and were mated in pairs within each group. Later, when complete transmission data were available these insects were reclassified according to their transmission record. Inbreeding by mating individuals within a group or even a family was employed, except in the first and second generations where outbreeding between members of similar transmissive performance in different groups was also used. During the first and second generations differences between groups were not marked and could not be regarded as fixed. From the third generation onward t,he families in the four groups WY, Wy, wY, and wy showed clear differences in ability to t,ransmit the two viruses, and were regarded as races WY, Wy, WY, and wy, respectively. Insects that died on the first test plant without transmitting are not included in the data presented on infectivity tests. Insects t.hat died on test plants other than the first one without transmitting either virus are considered as nontransmitters. In most experiments few insects died before the end of the 6-week t,est period and therefore these considerations do not appreciably affect final results.

BY A LEAFHOPPER

155

Statistical Analyses

Wherever necessary, the statistical significance of data obtained was determined by the use of the fourfold table to ascertain chi-square values. Yates’ correction (Fisher and Yates, 1948) was employed in all cases. From these values the probabilities were determined by referring to tables in Fisher and Yates. RESULTS

Selection Experiments

Records of performance by single insects were maintained, and those considered to be the best performers in each of the four groups (WY, Wy, WY, and wy) were used to produce progeny for further selection. Details of results during first and second generations will be presented for each family tested to illustrate why particular insects or families were chosen to continue the lines. Results of infectivity tests during generations 3 through 6 will be presented in summarized form. First generation. Forty-eight “partially selected insects” and an equal number of “stock insects” were fed in groups of twelve on 8 WTV plants and then on 8 PYDV source plants and subsequently were tested individually for 6 weeks (a week on each of 6 successive plants). The results of these tests (Table 1) show that the “partially selected insects” as a group were better transmitters of WTV (21%) than the “stock insects” (4%) _ These differences between the two groups in percentages of insects transmitting WTV, are significant at the 5% level of probability. The differences in transmission of PYDV by “partially selected insects” (13%) and lLstock insects” (35%) are also statistically significant (P value between 0.02 and O.Ol), indicating that the former were unintentionally seTests of Transovarial Passage lected for inefficient transmission of PYDV. Twenty-eight pairings were made at the Tests for transovarial passage of the viend of the test period as described earlier. ruses were done only in the 5th, 6th, and 7th Insects for further selection in the Wy generations. Pairs of insects were confined group were obtained by matings among to alfalfa and the progeny were individually selected insects.” Those in WY tested for infectivity for at least 6 weeks, “partially each insect being transferred to a fresh and wy groups were obtained by matings among either “partially selected” or “stock test plant at intervals of l-3 weeks.

156

NAGARAJ

AND

BLACK

TABLE INFECTIVITY TESTS

1

OF THE FIRST

GENERATION

Insects Group “Partially “Stock”

Generations

Total tested

Family

selected”

1, 2, 3, 4 Mixed

“Partially

Plants

48 48

Transmitters of WTV

PYDV

10 2

6 17

Both 0 0

Selected Insects”

(1)wY 0,13,0 ---

(5Wfy

13,3,3 -

12,1,1 -

4~42

‘QJ

(5Wy lO,O,O yd

(~WY

(1)wY

v3,o

3,W ----

(2)wY 10,2,1

11,10,7 -

4,J5

(5Wy WV

WV 29,&l

37

(VW

W’,O

PYDV

Both

15 3

15 38

0 0

2,17,0

(4)W

(6)W 1,0,0 ----

WTV

“‘Stock Insects”

10,6,0

16,2,0 -

Infected with

Total used ~-284 285

(I)6,7,3

(2)wY

3,4,1

WY Not tested

W,O

12,0,0

6,16,3

(3)wY 2,12,2

l\

@)m 6,15,2

(3)wY

11,2,0

11,2,0

ww

(4)wY

(7)wy

0,390 6

v,o

ww

7,

2

(4M 0,3,0

WY

WY

0,30,0 37

Not tested

FIG. 1. Pedigree of the races of Agallia constricta. The three numbers of the numerator indicate numbers of insects in each family infected with WTV, PYDV, and both viruses, respectively. Denominator represents total number of insects tested. Numbers in parentheses are family numbers. Lines above each family number point upward toward the parent family or families in the previous generation. For example, families (4) and (5) in generation 3 were both obtained from individuals of families (4) and (71, and (4) and (6), respectively, in generation 2; family (1) in generation 3 was obtained from individuals of families (5) and (2) in generation 2.

TRANSMISSION

OF VIRUSES

BY A LEAFHOPPER

157

TABLE 2 INFECTIVITY TESTS OF THE SECOND GENERATION Plants

Insects

Group

Family number

Transmitters

Total tested

WTV

PYDV

of

Infected

Both

Total used

WTV

with

PYDV

Both -__

WY WY WY

1 2 3

15 5 9

0 2 8

13 4 0

0 1 0

60 20 35

0 13 24

40 4 0

0 2 0

WY

4 5

22 14

18 11

2 10

0 7

83 50

50 23

3 13

2 6

6 7

15 15

12 11

0 2

0 0

50

59

32 24

0 2

0 1

WY WY WJ

insects.” Since none of the insects transmitted both viruses, no selection for the WY group was possible at this time. Subsequent generations. Progenies of seven selected matings in the first generation were used for testing and further selection during the second generation (Fig. 1). Three of the 7 families were from parents in the WY group, two were from parents in the Wy group, and two from parents in the wy group. After acquisition on WTV and PYDV source plants, insects in these families were tested individually for 8 weeks (2 weeks on each of 4 successive plants). The data (Table 2) show that among three families tested in the WY group there was only family 1, in which all insects that transmitted PYDV failed to transmit WTV. Insects from this family were inbred to produce progeny for further selection in the WY group. Both families in the Wy group were good transmitters of WTV. Insects of family 5, in addition, proved to be good transmitters of PYDV. In the wy group, both the families tested were good transmitters of WTV but poor transmitters of PYDV. Some of the individuals of these families which were good transmitters of WTV and nontransmitters of PYDV were mated with similar individuals in families 4 and 5 to provide progeny for further selection in the Wy group (Fig. 1). Nontransmitters from families 6 and 7 were mated with each other to obtain progeny for selection of the wy

group. Insects in families 2 and 5 which were good transmitters of both viruses were mated with each other to provide progeny in the WY group. In generations 3 through 5, pairs of insects having similar transmissive performance within each group were mated. For example, in generation 3 matings within families 4 and 5, 2 and 1, 3, and 6 provided progeny for further selection in the groups Wy, WY, WY, and wy, respectively. In the fifth generation all the families within each race were bulked together in an attempt to counteract debility due to inbreeding. Summarized results. The results of tests on transmission of WTV by the four races of A. constricta, during generations 3, 4, 5, and 6 are summarized in Table 3. It is evident from these results that in races WY and Wy a higher proportion of insects TABLE

3

TRANSMISSION OF WTV BY FOUR RACES OF Agallia constricta Gener ation

3 4 5 6 -_ Total

I

Insects

tested,

(To)

Race WY

Race Wy

Race wY

Race wy ___---

10/14, 13/19, 14/45,

71 68 31

25/26, 12/14, 7/10, 29/X, -----

96 86 70 78

6/21, 29 2/18, 11 O/4,0 o/37, 0

11/23, 48 O/23, 0 4/44, 9 -> ---

47

73/8i.

84

S/80,

15/90.

_-

transmitting/insects

37/B,

10

17

158

NAGARAJ

AND

transmitted WTV to plants than in races WY and wy, the differences being quite pronounced in generations 4 and 5. Among all the insects tested in generations 3 through 6, 47%, 84%, lo%, and 17% were transmitters of WTV in races WY, Wy, WY, and wy, respectively. TABLE TRANSMISSION

Generation

4

OF PYDV BY FOUR OF Agallia constricta

Insects

transmitting/insects

RACES

tested,

(To)

Race Wy

Race WY

3 4 5 6

6/14, 5/19, 22/45, -,

43 26 49 -

4/26, O/14, O/10, 2/37, __-

15 0 0 5

16/21, 15/l& 314, 29/37,

76 83 75 78

2123, 2/23, 2/M, -, __-

9 9 5 -

Total

33/78,

42

6/87,

7

63/80,

79

6/90,

7

INFECTION OF TEST PLANTS WITH BY DIFFERENT RACES OF Agallia constricta

WTV

Gener ation

Total

Plants Race WY

Race Wy

20/38,53 20/38,53 19/88,22 -, _-~--

50/60,83 20/28,71 13/17,76 55/104,53

59/N,

3 4 5 6 Total

18/65, O/46, 4/87, -, --__

28 0 5 -

22/198,

11

10/189,5

6

TEST PLANTS WITH PYDV RACES OF Agallia constricta

Plants Race WY

infected/plants

7/60, 12 O/28, 0 o/17,0 3/104, 3

25 10/209,

5

BY

used, (70) Race WY

Race Wy

g/38,24 5/38, 13 27/88,31 -> 41/M,

Race wy

8/54, 15 2/36,6 O/8,0 o/91,0

TABLE

Gener ation

used, (%) Race WY

36 138/209, 66

INFECTION OF DIFFERENT

Race wy

5

infected/plants

Race wy

30/54,56 21/36, 58 4/8, 50 50/91,55 -__-__-105/189,

4/65, 3/46, 2/87, ->

56 g/198,5

Similarly, among all the insects tested in generations 3 through 6,42%, 7%, 79%, and 7% in the races WY, Wy, WY, and wy transmitted PYDV, respectively (Table 4). These data show that the difference between efficient races WY and WY, and the inefficient races Wy and wy, in ability to transmit PYDV, were quite apparent even during the third generation. Performance of Transmitters in Eficient and Inefficient Races

Race WY

TABLE

BLACK

6 7 2 -

During generations 3 though 6, the numbers of WTV transmitters in races WY, Wy, WY, and wy were 37, 73, 8, and 15, respectively (Table 3) and they infected 59, 138, 10, and 22 plants, respectively (Table 5). A comparison of the frequencies of transmissions by transmitter insects in races WY and Wy on one hand and WY and wy on the other, showed that the transmitters in races WY and Wy transmitted WTV to a greater proportion of plants than did such insects in the races WY and wy, but the difference was not statistically significant. Similarly, the numbers of PYDV transmitters during generations 3 through 6 in races WY, Wy, WY, and wy were 33, 6, 63, and 6, respectively (Table 4), and they infected 41, 10, 105, and 9 plants, respectively (Table 6). There was no significant difference between the ratios of plants infected by transmitters in efficient and those in the inefficient races. Black (1943a) reported significant differences between transmitters of efficient and inefficient races of A. sanguinolenta in regard to the frequency with which they transmitted the New York variety of potato yellowdwarf virus. Our failure to obtain such significant differences could be due to the smaller numbers of test plants employed in our experiments. Lack of Interference between WTV PYDV in the Insect

and

Among the four races, race WY was selected for its high transmission of both viruses. A study of the record of transmission of the two viruses by this race may provide a basis for determining whether WTV and

TRANSMISSION

OF VIRUSES

PYDV interfere with transmission of each other by the insect, under the experimental conditions. Such data on transmission by insects of race WY, in generations 3,4, and 5 (Table 7) show that the actual number of insects which transmit both viruses (11 of 78) is not significantly different statistically from the expected number (16 of 78) calculated from the frequencies of WTV and PYDV transmission actually obtained. This indicates that under the experimental conditions there is little or no interference between the two viruses in the insects of race WY. However, it should be pointed out that these results do not rule out the possibility of interference between the two viruses under other experimental conditions (such as allowing one virus to establish itself thoroughly in the insect before allowing t.he latter to acquire the second virus, a condition not existing in our experiments). Parenthetically, an examination of the data on the number of plants infected by insects of the race WY during generations 3 through 6, shows that the number infected by both viruses (9 of 164) is not significantly different statistically from the expected number (14 of 164) calculated from the frequencies of plants infected with either W7TV or PYDV. Under the experimental conditions no interference between the two viruses in the plants was detected. Transovarinl

Passage Experiments

Wound-tumor virus. The experiments described above were designed on the assumption of a low transovarial passage of WTV and PYDV as was indicated by Black’s (1953) earlier work showing 1.8% transovarinl passage of WTV through Agalliopsis ?aovella (Say) and 0.8% transovarial passage of PYDV through A. constricta. After ;I. co?bricta had been selected for five generations, Watson and Sinha (1959) reported that efficient races of D. pellucida allowed higher amounts of transovarial passage of European wheat striate mosaic virus than the inefficient races of the vector. The four selected races of A. constricta were therefore tested for the relative amounts of transovarial passage of WTV and PYDV. For testing transovarial passage of WTV

139

BY A LEAFHOPPER

TABLE 7 LACK OF INTERFERENCE BETWEEN WTV PYDV IN INSECTS OF RACE WY OF Agallia constricta

Insects transmitting

Viruses WTV PYDV WTV and PYDV WTV and PYDV

AXLJ

(observed) (expected)

Ratioa

Per cent

37/78 33178 11/78 X/78

47 42 14 20

a Numerator represents insects transmitting; denominator represents total insects tested.

in different races, infective females were paired with infective or noninfective males on alfalfa. The progeny from these matings were tested individually for 6-8 weeks (2 weeks on each of 3 or 4 successive test plants). In all, 190 insects from 12 families were tested. The results of such tests are summarized in Table 8. The minimum transmission through the egg in races WY and Wy was 50% and the maximum was 100%. In the only family tested in race wY, transovarial passage was 14%. In experiment 1, Table 8, odds that the differences between the ratios of transovarially infective insects in races WY and WY are not due to chance alone are greater than 1000: 1. When ratios of transovarially infective insects among all the WY and Wy insects tested were separately compared with the ratio among WY insects, odds that the differences were not due to chance alone were also greater than 1000: 1. Potato yellow-dwarf virus. All insects tested for transovarial passage of PYDV TABLE 8 SUMMARIZED DATA ON TRANSOVARIAL PASSAGE OF WTV IN ngallia constricta Race

Experiment

WY WY WY WY

1 1 all all

27143 3/21 83/119 33150

63 14 i0 (iii

160

NAGARAJ

AND

BLACK

siderations indicate that the racial differences in ability to transmit PYDV are genetic. As regards variation in the ability of A. constricta to transmit WTV, the existence of a high transovarial passage in races WY and Wy complicates interpretation of data on transmission by the four races. Under the experimental conditions there was obviously a great difference in the proportions of transmitting insects in races WY and Wy on the one hand, and races WY and DISCUSSION wy on the other (Table 3). Transmission experiments in generation 6, and transVariation in Transmitting Ability ovarial passage experiments, clearly indiThe results of selection experiments con- cated that efficient transmission of WTV clusively demonstrated hereditary variaby races WY and Wy was largely due to tion in the ability of A. constricta to acquire persistence of virus by transovarial passage. and transmit PYDV. Among the four races It is not definitely known whether insects selected, races WY and WY can be con- of these races, when freed from virus, can sidered to be efficient transmitters and races acquire and transmit virus more efficiently Wy and wy inefficient transmitters of than those of races WY and wy. However, there is experimental evidence, in the rePYDV. If one assumes that the presence of WTV sults of infectivity tests in generation 1, inin an insect reduces chances of transmission dicating that insects selected once for efof PYDV, it is conceivable that inefficient ficient transmission of WTV, when freed transmission of PYDV in race Wy (which is from virus, can acquire WTV from infected efficient in transmitting WTV) might be plants and transmit it more efficiently than due to the presence of WTV in the insect unselected stock insects. Therefore it is (obtained either transovarially or during likely that insects of races WY and Wy are acquisition feeding on the WTV source). more efficient both in acquiring and transThere is evidence that this is probably not mitting WTV than insects of races WY and so, because insects of race wy which trans- wy. To demonstrate conclusively that races mitted little WTV to plants were inefficient WY and Wy are efficient in acquiring WTV in transmitting PYDV. However, it is pos- from infected plants as well as in transsible that insects of race wy may carry mitting it, attempts were made to free these WTV without transmitting it, and that this cultures from this virus. This proved to be virus interferes with transmission of PYDV. a difficult problem. Because of high transIf this were the case, one might logically ovarial passage in these two races very few expect a similar interference in race WY, insects were found to be virus-free and because of debility due to inbreeding still the insects of which were tested in exactly the same way as the others. But race WY is fewer survived through a test period of 6 known to be efficient in transmitting PYDV. weeks. When such insects were mated most Moreover, a study of the transmission rec- of them did not produce any progeny. In ord of race WY in generations 3 through 6 some cases very few nymphs were obtained shows that the actual number of insects and these grew slowly and had not become which transmitted both the viruses is not adults 3 months after the date of hatching. When viruliferous colonies of race WY, significantly different statistically from the expected number calculated by assuming after 6 generations of selection, were mainlack of interference. This indicates that tained on alfalfa without further selection under the experimental conditions there is for seven additional generations, progeny little or no interference between the two vi- of the thirteenth generation were almost ruses in insects of the race WY. These con- free from WTV (1 of 38 insects tested

were progeny of transmitter females, the male parent being either a transmitter or nontransmitter of PYDV. In all, 139 insects in 13 families were tested. No transovarial passage of PYDV was observed in any of these families tested in the four races. However, it should be pointed out that the number of insects tested may not have been large enough to detect the very small amount of transovarial passage (about 0.8%) earlier reported by Black (1953).

TRANSMISSION

OF

VIRUSES

transmitted the virus). In one experiment these insects were individually compared for acquisition and transmission of WTV with similarly maintained virus-free insects of race WY. The insects were tested for 6 weeks, each insect being transferred to a fresh plant every 2 weeks. Of 35 individuals of race WY thus tested, 19 transmitted WTV to 34 of 57 plants on which they fed; whereas, of 29 insects of race WY, 14 transmitted WTV to 17 of 42 test plants. Odds are only 4: 1 that this difference in the number of plants infected by transmitters of the two races are due to inherent differences in them. The unexpectedly high percentage of transmitters among individuals of race WY obtained in this experiment might be due to recombination and natural selection for more vigorous progeny with an associated unplanned selection toward the transmitting efficiency for WTV possessed by the original heterogeneous stock insects with which the experiments were started. Variation sage

in Extent

of Transovarial

Pas-

The differences in transovarial passage of WTV between races WY and Wy on one hand, and race WY on the other (Table 8), are statistically significant and indicate that the extent of transovarial passage of WTV is a genetically determined trait in A. cons tricta.

Black (1953) had earlier found that in unselected insects of A. novella there was a very small amount of transovarial passage of WTV (1.8%). He obtained evidence indicating that in some individuals it might be as high as 10.7%. The fact that different species of vectors, A. novella and A. constricta, allow different amounts of transovarial passage indicates that the extent of transovarial passage is determined not merely by the virus but also by the genetic constitution of the vector. It is interesting to note that insects of race WY, which were efficient in transmitting both the viruses, allowed a high transovarial passage of WTV but not of PYDV. This observation emphasizes that the virus species can be important in determining extent of transovarial passage.

BY

lril

A LEAFHOPPER

Hereditary Relationships the Two Viruses

of the Vector

to

This study of variation in ability of different races of A. constricta to acquire and transmit WTV and PYDV, and variation in the extent of transovarial passage of WTV provides evidence on the hereditary relationships between the insect and the two viruses. Race WY, which was efficient in transmitting PYDV, allowed a high transovarial passage of WTV, but race WY, which was also efficient in transmitting PYDV, did not allow a high transovarial passage of WTV. On the other hand, race Wy, which was inefficient in transmitting PTDV, allowed a high transovarial passage of WTV. These facts indicate that races WY and Wy on the one hand, and race WY on the other, differ in hereditary ability for transovarial passage of WTV in a manner independent of their ability to acquire and

transmit

PYDV efficiently. Race wY, which

was efficient in transmitting PYDV, permitted a low incidence of transovarial passage of WTV whereas race Wy, Tvhich permitted a high incidence of trnnsovarial passage of WTV, was inefficient in transmitt’ing PYDV. This indicates that at least two different alleles, if not two different

genes, must be involved

in determining

the

ability of A. constricta to acquire PYDV from and transmit it to plants on t,he one hand, and its ability to allow transovarial passageof WTV on the other. REFERENCES BEXNETT,

C. W.,

and

W.~LLACE,

H.

E. (1938).

Re-

lation of the curly-top virus to its vector. J. Agr. Research 56,31-51. BLACK, L. M. (1941). Specific

transmissionof varietiesof potato yellow-dwarf virus by related insects.Am. Potato J. 18, 231-233. BLACK, L. M. (1943a). Genetic variation in the clover leafhopper’sability to transmit potato yellow-dwarfvirus. Genetics28, 200-209. BLACK, L. M. (194313). Somerelationshipsbetween potato yellow-dwarf virus and the clover leafhopper.Phy2opathology33,363-371. BLACK, L. M. (1944). Somevirusestransmittedby Agallian leafhoppers.Proc. Am. Phil. SOC. 88, 132-144. BLACK, L. M. (1953). Occasionaltransmissionof someplant viruses through eggs of their insect vectors. Phytopathology 43, Q-10.

162

NAGARAJ

L. M. (1955). Concepts and problems concerning purification of labile insect-transmitted plant viruses. Phytopathology 45,208216. BLACK, L. M., and BRAKKE, M. K. (1952). Multiplication of wound-tumor virus in an insect vector. Phytopathology 42,269-273. BLACK, L. M., and BRAKKE, M. K. (1954). Serological reactions of a plant virus transmitted by leafhoppers. (Abstract.) Phytopathology 44, 482. FISHER, R. A., and YATES, F. (1948). “Statistical Tables for Biological, Agricultural, and Medical Research,” 3rd ed. Haffner, New York. FUKUSHI, T. (1933). Transmission of the virus through the eggs of an insect vector. Proc. Imp. Acad. (Tokyo) 9,457-460. FUKUSHI, T. (1940). Further studies on the dwarf disease of the rice plant. J. Fat. Agr. Hokkaido Univ. 40,83-154. MARAMOROSCH, K. (1950). Influence of temperature on incubation and transmission of wound-tumor virus. Phytopathology 40,1071-1093. NAGAICH, B. B. (1960). Simultaneous transmission BLACK,

AND BLACK of wound-tumor and potato yellow-dwarf viruses by Agallia constricta Van Duzee. Sci. and Culture (Calcutta) 25,591-592. NAGARAJ, A. N. (1959). Genetic variation in the ability of the leafhopper Agallia constricta Van Duzee to transmit viruses. (Abstract). Phytopathology 49,546-547. STOREY, H. H. (1932). The inheritance by an insect vector of the ability to transmit a plant virus. Proc.Roy. Soc.B112,41%30. WATSON, M. A., and SINHA, R. C. (1959). Studies on the transmission of European wheat striate mosaic virus by Delphacodes pellucida Fabricius. l%oZogy 8,139-U% WOLCYRZ,S. and BLACK, L. M. (1956). Serology of potato yellow-dwarf virus. (Abstract.) Phytopathology 46, 32. YAMADA, M., and YAMAMOTO, H. (1955). Studies on the stripe disease of the rice plant. I. On the virus transmission by an insect, Delphacodes stnbtella Fallen. Okayama Agr. Expt. Sta. Spec. Bull. 52,93-112.