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Vector fecundity reduced by Western X-disease
JOL-It.\.\I.
OF
INVI~RTEliI1.\TI:
Vector
I’ITHOLOGY
Fecundity
17, 389.-394
Reduced D. D.
Division
of Entomology,
University lh-sived
(Igil)
by Western
X-Disease
JENSE$*~
of California, 12erkelt:y, December 1, 19W
Cdijorniu
947%
Western S-disease (WX), previously thought to be caused by a virus, appears to be due to a mycoplasma-like organism transmitted by certain leafhoppers. In the vector Collatlonlrs montanus, the WS agent produced lesions in a number of tissrles, reduced fecundity, and caused premature death. The fecundity of C’. wonlnrnzts leafhoppers was impaired by the WX agent when infection was acquired either 1)) injection with infectious leafhopper extracts or by feeding on diseased celery plants. The difference in fecundity between insects injected with the WS pathogen and thaw injected with noninfectious extracts remained approximately constant throrlgh time. In coutrast, in experiments with insects that had acquired WS infectivit) through feeding, the fecundity curve of infective insects declined rapidly during the last 4 a-eeks whereas that of the healthy controls rose and then remained almost constant throughout, this portion of the test period. The effect 011 fecuttditv was more extreme in insects that, fed on WS plants thau it. was in those leafhoppers that fed only on healthy plants and acquired WX infectivity by injectiou. These results suggest that. feeding on diseased plants reduces the fecundity of C’. montanus in some manner over and beyond that resulting from the action of WS per se 011 the reproductive system of this vector. The nutriGor derived by leafhoppers from diseased plants is adequate for normal longevity, but may be partially deficient in sc,mr constituents essential to normal fecundity.
Western X-disease (WX) of stone fruits and celery was previously thought to be caused by a virus. It now appears probable that the disorder results from infection by a mycoplasma-like organism (Nasu et, al., 1970; Amin and Jensen, 1971). The disease is of particular interest because, in addition to causing an important plant disease, the WX agent is lethal t,o its leafhopper vector, Collatlows montanus (Jensen, 1959; Jensen et al., 1967), causes lesions in several tissues of the body (Whitcomb et al., 1968), and affects celery plants in such a way that leafhoppers feeding on diseased plants develop 1 This investigation was supported by research grant. No. AI-03490 from the National Institut,e of Allergy and Infectious Diseases, National Institutes of Health, United States Public Health Service. 2 The author acknowledges the assistance of J. Richardson and B:. Ranft in portions of this investigation. 389
needlelike crystals in the alimentary tract (Lee and .Jensen, 1963). A preliminary report (Jensen, 196’2) also indicated that, the WX agent, reduced the fecundity of infective C. motltar~us leafhoppers. The present paper presents the results of several experiments in which a comparison was made of egg production in infetrtive C. worztanus leafhoppers and in noninfective cont.rols. Certain plant viruses have also been rcported to cause pathogenic effects in their leafhopper vectors, including impairment of fecundity. Nasu (1963) found that although t’he rice dwarf virus apparently did not, irnpair t,he viability of eggs laid by XepltoteftGz cincticeps, the virus did reduce the number of eggs laid by 32-72 %. Similar reductions in fecundity \vere also found to occur in other viruliferous vector species of t,his virus (Kasu, 1963). n’asu (1963) also report’ed that rice stripe virus caused :i2$ of thtl eggs,
390
JENSEN
which were laid by viruliferous Delphacodes striatella, to die before hatching. An earlier paper (Watson and Sinha, 1959) reported that’ Calligypona (Delphacodes)pellucida leafhoppers, aMer feeding on plants diseasedwith European wheat striate mosaic virus, had 40 % fewer progeny t.han did those fed on healthy plants. However, Kisimoto and Watson (1965), working with the same vector and virus, concluded that the reduction in progeny resulted from inbreeding rather than from virus infection.
time bet,ween reaching the adult stage and the onset of oviposition averaged 9.4 days in these experiments. Female leafhoppers from the infective and from the healthy control groups were then caged singly on healthy celery plants for egg laying. Colladonusmantams prefers to lay eggs in tissues of the petiole and in the leaf margins of older rather than the youngest leaves. To make the counting of eggseasier, the test celery plants were cut back prior to caging leaving only one older leaf and the youngest developing leaf. Nearly all eggs were laid in the single MATERIALS AND METHODS older leaf. Each leafhopper was transferred The Colladonusmontanus leafhoppers used to a new celery plant at weekly intervals, and in this study were from st,ock colonies main- the survival of the adult insects was also tained on celery in the greenhouse. The checked mid-weekly. After exposure to egg inoculum was the peach yellow leafroll strain laying, t,he test p1ant.swere held in the greenof Western X-disease that is maintained in house for subsequent counting of the eggs the greenhouse by frequent passagethrough and development of WX symptoms. As the celery. The test plants, Utah type green eggs incubate within the leaf tissue, they celery (Apium graveolens), were grown in change in color from a translucent, pale 4-inch clay pots after transplant,ing from green (inconspicuous in the leaf t’issue) to seed pans. They were used when t.he older light yellow which makes them easily deleaves were approximately 3 inches in length. tected. Insect,s were confined to t,he individual test When a population of C. montanus feeds on diseased celery, only part of the group plants by means of cages consisting of acquires WX infectivity in any given day or wooden frames (745” X 5%” X 5%“) havweek. An extended acquist.ion accessperiod ing organdy cloth on three sidesand glasson the fourt,h. of several weeks is therefore necessary for a The infective insects used in each experi- high percentage of the individuals to become ment and their noninfective controls always infective. In contrast, if leafhoppers receive originat,ed from a single healthy stock cul- WX inoculum by inject’ion all acquire infecture. One group of small nymphs (usually tivity the same day. Extracts containing WX inoculum were 200-400) was removed from t.he st,ock colony and caged on a diseased plant’. A control prepared by grinding infective adult insects group was caged on a healthy plant,. Since in either 0.85% NaCl or in tissue culture diseased celery plants decline and die much medium, using 100 adults for each 1.5 ml of more rapidly than heahhy celery, the infec- fluid. The extract was then clarified by centive colonies were transferred at 5-lo-day trifuging for 10 minutes at 8000 g, and passintervals to new diseased plants until the ing it through a Millipore filter with 0.45 p insects had fed on diseasedplants for 30-35 pore size which also removed any contamidays. rlt that time all were adults and the nating bacteria. All steps in bhe preparation incubation period of WX (ea. 30 days) had of WX extract were carried out at approximately 4°C. Control insects were injected been completed in most of the infective individuals. Also, by this time the females with extract.s made from healthy leafhophad mated and could lay fertile eggs. The pers. The injection procedure and equipment
TVESTERK
used were f't
al.,
described
previously
X-DISEASE
(Whit comb
1SMi).
RESULTS
Females Fed OR.WX Plants Five experiments were conducted in which t,he females acquired infectivity by feeding 011 diseased celery plants. Healthy controls, from the same source colony, fed only on healthy plants. The number of females tested singly for egg laying from the diseased and healthy stocks were 270 and 159, respectively. The tot’al number of eggs laid by females from the infective stock was only approsimat,ely one-third of that laid by an equal number of healthy controls. Horn-ever, part of this difference resulted from the fact, that infective females did not live as long as did the noninfect’ive controls. This is consistent with earlier experiments which demonstrated that the WX agent kills C. montams premnt.urely (*Jensen, 1959; Jensen et, al., 1967). Thr mean longevity of the females from the infective stock \vas 40 days from the date they were caged singly for egg laying compared to 67 days for the cont,rols. The problem is complicated further by the fact. that, not a11the females that fed on a diseased plant, acquired WX infectivity. If all had been infective, the total egg production would have been even less. In one experiment, involving 50 females from an infect ive stock, $1 of them proved t,o be infective :U evidenced by transmission of the WX agent to celery. Their mean longevity was 37 days compared lo 7X days for the S now t,r:msmitt ws. Wwtcrn S-disease results in sterility in sonw individuals and in lowered fecundity in others. In the present investigations, of the individuals that had fed on WX celery, 205 out of 270 (77%) laid one or more eggs during adult life compared to 130 of 159 (xl. “l’ ) for the healthy controls. Although this difference in the number of compIet.eIy sterile females was not’ great, the total effect of’ W-X on the diseased stock was very sig-
ASD
VECTOR
I”ECUKDITT
:
nificant. Many females from the diseased stock laid very fen- eggs-especially during t,he last half of the test period. In order 10 evaluate the effect of feeding on WX plants on the production of fertilized eggs1 only egg-laying females were compared from the two stocks. Also, in order to eliminate diffrrences in t’otal egg production, due to differences in longevity, only the mean number:: of eggs laid/femttle,/week of life are (VII. sidered (Fig. 1). The experiments svcre carried out, over a i--week period. The mean egg production per female among the cl&eased insects was two-thirds that of the healt#hy stock during the first 3 \\wks. Thereafter the curves diverge strongly. The fecundity of the infective females declined rapidly to almost zero during the last 3 week;: (Fig. 1). In cont’rast’, the control females xew still laying more eggs after 7 weeks than the WX individuals did at their maximum \\hich n-as at 2 Lveeks. Fecundity
qf Iyjecfed Females
Experiments effect of WX 011 hoppers, all of celery planf~. _--_-
were carried out 011 the the fecundity of female leafn-hi& fed only on health\ Thr possibly complicating o,PtlstO SlOCK lZOS991
FJG. 1. The effect of the Western Ldiseuse agent on the fecundity of CuEla&n~s rtrontnrr,cs leafhoppers. The time period shown hegalL at the completion of SO-35-day feeding periods on diseased or healthy celery.
392
JEiWEN
effects of nutritional differences between diseased and healthy celery plants were thus eliminated. Infective extracts were made from adult leafhoppers that had fed for the previous 30-40 days on WX diseased plants. The WX extract was injected into healthy, last-instar nymphs. Control insects were injected with extracts made from healthy leafhoppers. Injected nymphs became adults within a few days after inject,ion. Three weeks after injection, w-hen the adults were sexually mature, had mated, and were laying eggs, the females were caged singly for oviposition. Each female was transferred to a new plant weekly. A record was kept of the number of eggs laid by each female each week and also of her longevity. The experiments extended over a period of 10 weeks after the insects were injected and for 7 weeks after the females were caged singly and egg counts were recorded. The incubat,ion period of WX is completed in most injected leafhoppers in 14-21 days compared to approximately 30 days in leafhoppers which acquire infectivity by feeding on diseased plants. The egg-laying data for 77 females inected with healthy extract and 63 females njected with WX ext.ract demonstrate (Fig.
2) that. the infective insects lvere consistent, in laying fewer eggs than did the healthy controls. The curves run essentially parallel t)hroughout the test period. The results are given in terms of the mean number of eggs laid per female per week of life, thus eliminating differences in egg production due to differences in longevity. DISCUSSION
In the present experiments, C. nzo?2tanus leafhoppers that acquired the WX agent, either by feeding on diseased plants or by injection with Wx ext,racts from diseased leafhoppers, laid fewer eggs per week of life than did the noninfective controls. In the experiment,s involving injected insects, the difference in fecundity between the test and cont,rol insects remained approximately constant through time. There was a peak at the fourth week after injection and then both curves gradually declined, with t,hat of the infective insects being slightly steeper during the final 6 weeks. In other work (Amin and Jensen, 1971) we have also shown that C. montanus leafhoppers that received the WX agent by injection and subsequently fed on a solution of achromycin retained a fecundity equal to that of healthy controls. H
Diseased
-
Healthy
FIG. 2. The effect of the Western X-disease agent on the fecundity of had been injected with infectious leafhopper extract. The control leafhoppers leafhopper extract.
monlanus which were injected with healthy
Colladonus
\I-ESTERN
S-DISEASE
AND
ln experiments with insects that acquired WX infectivity t,hrough feeding, the fecundity curve of infective insects declined rapidly during t’he last four weeks whereas that of the healthy controls rose and t,hen remainecl almost at the same level throughout the test, period. Insects fed on celery plants infected with the WX agent are different in two respects from control insects fed on healt.hy plants. First, most. of t.he former acquire WX infeetivity if given access to diseased plants for 30 clays or more. The WX agent itself has a profound effect 011 the vital life systems of C. m~)~fmus, causing lesions in many of its tissues, impairing fecundity, and killing the leafhopper prematurely. Second, since WX diseased plants are st rongl? altered physiologically, being dwarfed and chlorotic, the composit’ion of the plant sap imbibed by the leafhoppers must be different from that of healthy celery-. The nutritional quality of plant sap from diseased celery apparently is equal to that of healthy celery if the ody criterion is that of inwct longevity. Repeated experiments have demonstrated that C. montanus that feed on diseased celery for several weeks without acquiring WX infectivity live as lorlg as do the healthy controls. However, if they acquire WX their life span is conspicuously shortened. Although insects that feed 011 diseased plants from 1 to 4 weeks without acquiring WX do not’ suffer nutritionally in terms of longevity, it is nevertheless probable that the nutritional difference of sap from diseased plants may have an effect on some of the leafhopper’s vital functions, including reproduct,ion. WP do know, for example, that feeding 011 a diseased plant results in the accumulation of needlelike crystals in the st,omach (Lee and Jensen, 1963) and that, this phenomenon apparently is not a direct result of WX infection. When infective insects feed on healthy celery plants the tryst als gradually disappear. Moreover, in-
VECTOR
FEC’USDITT
393
sects acquiring WX by injection never tievelop such crystals. Present experimental evidence thus tends to indicate that, the fecundity of female C’. motrtunu~ le&oppers is impaired by infcction wit,11the WX agent, and that, in addition, the feeding on WX diseased plants further reduces the number of eggs laid b> this insect. The eff rcts of WX on the reproductive capacity of male leafhoppers ll:ts not, been investigated directly. Ho\vever, there did not, appear to be a significant8difference in the fertility of eggs laid by infwt ivc females, mated lvith infective males, :ultl t,hat of eggs laid by noninfect ive t’t~malt~~ mated \vifh noninfwtive males. I
AMIN, P. W.,
ANI) JI,:NSI,:S. 1). I). l!Pl. ICffects of tet,racycline on the transmission and pat ho. genicity of the Western X-disease agent iu its insert and plant hosts. I’h!//r,pnlho/og!/, iilr press), JENSI~:S, I). 1). 1!)59. .4 plant virtls lethal to iI+ insect, vector. I’irolog!/, 8, lM-175.
JENSEN, .I). I)., WHLTCUMH, 1:. F., .LND I~ICTL~ISON, J. 1967. Lethality of injected peach Western X-disease virus to its IeafhoppPr vector. Virology, 14, 5X-538. KISIMOTO, It., .\NL) W.\wo~. 31. .4. 1965. Abnormal development of embryos indwed 1, imbreeding in DeZphacorles pdlucida Fabricills and Delphacodes dubiu Kirschhaum (Araeckpidae, Homoptera), vectors of Rlwopenn wheat striate mosaic virus. J. Inwrtt~hr. Pathol.,
7, 297-305.
LEE, P. El., .~ND JP:NSI~:N, 1). 1).
1963. (‘ryst:11Iine inclusions in Colladonw n~ontan~s (Van Tjuzee), a vector of Western S-disease virus. Virology, 20, 328-332. NASU, S. 1963. Studies on some leafhoppers and planthoppers which transmiT virus diseases of rice plant in Japan. Btrll. K!/zcshtr Agr. Exp. Sk., 8, 153-349.
NASU, S., JENSF:N, II. I)., AND EICH.\HDSOX, .f. 1970. Electron microscopy like bodies associated with
of mycoplasmaiusect and pl:illt
394
JENSEN
hosts of peach Western X-disease. Virology, 41, 583-595. WATSON, M. A., AND SINHA, R. C. 1959. Studies on the transmission of European wheat striate mosaic virus by Delphacodes pellucida Fabricius. Vi’irology, 8, 139-163. WHITCOMB, R. F., JENSEN, D. D., AND RICHARDSON, J. 1966. The infection of leafhoppers
by Western S-disease. I. Frequency of transmission after injection or acquisition feeding. Virology, 28, 448353. WHITCOMB, R. F., JENSEN, D. D., AND RICHARUSON, J. 1968. The infection of leafhoppers by Western X-disease virus, VI. Cytopathological interrelationship. J. Invertebr. Pathol., 12, 202-221.
OF
INVI~RTEliI1.\TI:
Vector
I’ITHOLOGY
Fecundity
17, 389.-394
Reduced D. D.
Division
of Entomology,
University lh-sived
(Igil)
by Western
X-Disease
JENSE$*~
of California, 12erkelt:y, December 1, 19W
Cdijorniu
947%
Western S-disease (WX), previously thought to be caused by a virus, appears to be due to a mycoplasma-like organism transmitted by certain leafhoppers. In the vector Collatlonlrs montanus, the WS agent produced lesions in a number of tissrles, reduced fecundity, and caused premature death. The fecundity of C’. wonlnrnzts leafhoppers was impaired by the WX agent when infection was acquired either 1)) injection with infectious leafhopper extracts or by feeding on diseased celery plants. The difference in fecundity between insects injected with the WS pathogen and thaw injected with noninfectious extracts remained approximately constant throrlgh time. In coutrast, in experiments with insects that had acquired WS infectivit) through feeding, the fecundity curve of infective insects declined rapidly during the last 4 a-eeks whereas that of the healthy controls rose and then remained almost constant throughout, this portion of the test period. The effect 011 fecuttditv was more extreme in insects that, fed on WS plants thau it. was in those leafhoppers that fed only on healthy plants and acquired WX infectivity by injectiou. These results suggest that. feeding on diseased plants reduces the fecundity of C’. montanus in some manner over and beyond that resulting from the action of WS per se 011 the reproductive system of this vector. The nutriGor derived by leafhoppers from diseased plants is adequate for normal longevity, but may be partially deficient in sc,mr constituents essential to normal fecundity.
Western X-disease (WX) of stone fruits and celery was previously thought to be caused by a virus. It now appears probable that the disorder results from infection by a mycoplasma-like organism (Nasu et, al., 1970; Amin and Jensen, 1971). The disease is of particular interest because, in addition to causing an important plant disease, the WX agent is lethal t,o its leafhopper vector, Collatlows montanus (Jensen, 1959; Jensen et al., 1967), causes lesions in several tissues of the body (Whitcomb et al., 1968), and affects celery plants in such a way that leafhoppers feeding on diseased plants develop 1 This investigation was supported by research grant. No. AI-03490 from the National Institut,e of Allergy and Infectious Diseases, National Institutes of Health, United States Public Health Service. 2 The author acknowledges the assistance of J. Richardson and B:. Ranft in portions of this investigation. 389
needlelike crystals in the alimentary tract (Lee and .Jensen, 1963). A preliminary report (Jensen, 196’2) also indicated that, the WX agent, reduced the fecundity of infective C. motltar~us leafhoppers. The present paper presents the results of several experiments in which a comparison was made of egg production in infetrtive C. worztanus leafhoppers and in noninfective cont.rols. Certain plant viruses have also been rcported to cause pathogenic effects in their leafhopper vectors, including impairment of fecundity. Nasu (1963) found that although t’he rice dwarf virus apparently did not, irnpair t,he viability of eggs laid by XepltoteftGz cincticeps, the virus did reduce the number of eggs laid by 32-72 %. Similar reductions in fecundity \vere also found to occur in other viruliferous vector species of t,his virus (Kasu, 1963). n’asu (1963) also report’ed that rice stripe virus caused :i2$ of thtl eggs,
390
JENSEN
which were laid by viruliferous Delphacodes striatella, to die before hatching. An earlier paper (Watson and Sinha, 1959) reported that’ Calligypona (Delphacodes)pellucida leafhoppers, aMer feeding on plants diseasedwith European wheat striate mosaic virus, had 40 % fewer progeny t.han did those fed on healthy plants. However, Kisimoto and Watson (1965), working with the same vector and virus, concluded that the reduction in progeny resulted from inbreeding rather than from virus infection.
time bet,ween reaching the adult stage and the onset of oviposition averaged 9.4 days in these experiments. Female leafhoppers from the infective and from the healthy control groups were then caged singly on healthy celery plants for egg laying. Colladonusmantams prefers to lay eggs in tissues of the petiole and in the leaf margins of older rather than the youngest leaves. To make the counting of eggseasier, the test celery plants were cut back prior to caging leaving only one older leaf and the youngest developing leaf. Nearly all eggs were laid in the single MATERIALS AND METHODS older leaf. Each leafhopper was transferred The Colladonusmontanus leafhoppers used to a new celery plant at weekly intervals, and in this study were from st,ock colonies main- the survival of the adult insects was also tained on celery in the greenhouse. The checked mid-weekly. After exposure to egg inoculum was the peach yellow leafroll strain laying, t,he test p1ant.swere held in the greenof Western X-disease that is maintained in house for subsequent counting of the eggs the greenhouse by frequent passagethrough and development of WX symptoms. As the celery. The test plants, Utah type green eggs incubate within the leaf tissue, they celery (Apium graveolens), were grown in change in color from a translucent, pale 4-inch clay pots after transplant,ing from green (inconspicuous in the leaf t’issue) to seed pans. They were used when t.he older light yellow which makes them easily deleaves were approximately 3 inches in length. tected. Insect,s were confined to t,he individual test When a population of C. montanus feeds on diseased celery, only part of the group plants by means of cages consisting of acquires WX infectivity in any given day or wooden frames (745” X 5%” X 5%“) havweek. An extended acquist.ion accessperiod ing organdy cloth on three sidesand glasson the fourt,h. of several weeks is therefore necessary for a The infective insects used in each experi- high percentage of the individuals to become ment and their noninfective controls always infective. In contrast, if leafhoppers receive originat,ed from a single healthy stock cul- WX inoculum by inject’ion all acquire infecture. One group of small nymphs (usually tivity the same day. Extracts containing WX inoculum were 200-400) was removed from t.he st,ock colony and caged on a diseased plant’. A control prepared by grinding infective adult insects group was caged on a healthy plant,. Since in either 0.85% NaCl or in tissue culture diseased celery plants decline and die much medium, using 100 adults for each 1.5 ml of more rapidly than heahhy celery, the infec- fluid. The extract was then clarified by centive colonies were transferred at 5-lo-day trifuging for 10 minutes at 8000 g, and passintervals to new diseased plants until the ing it through a Millipore filter with 0.45 p insects had fed on diseasedplants for 30-35 pore size which also removed any contamidays. rlt that time all were adults and the nating bacteria. All steps in bhe preparation incubation period of WX (ea. 30 days) had of WX extract were carried out at approximately 4°C. Control insects were injected been completed in most of the infective individuals. Also, by this time the females with extract.s made from healthy leafhophad mated and could lay fertile eggs. The pers. The injection procedure and equipment
TVESTERK
used were f't
al.,
described
previously
X-DISEASE
(Whit comb
1SMi).
RESULTS
Females Fed OR.WX Plants Five experiments were conducted in which t,he females acquired infectivity by feeding 011 diseased celery plants. Healthy controls, from the same source colony, fed only on healthy plants. The number of females tested singly for egg laying from the diseased and healthy stocks were 270 and 159, respectively. The tot’al number of eggs laid by females from the infective stock was only approsimat,ely one-third of that laid by an equal number of healthy controls. Horn-ever, part of this difference resulted from the fact, that infective females did not live as long as did the noninfect’ive controls. This is consistent with earlier experiments which demonstrated that the WX agent kills C. montams premnt.urely (*Jensen, 1959; Jensen et, al., 1967). Thr mean longevity of the females from the infective stock \vas 40 days from the date they were caged singly for egg laying compared to 67 days for the cont,rols. The problem is complicated further by the fact. that, not a11the females that fed on a diseased plant, acquired WX infectivity. If all had been infective, the total egg production would have been even less. In one experiment, involving 50 females from an infect ive stock, $1 of them proved t,o be infective :U evidenced by transmission of the WX agent to celery. Their mean longevity was 37 days compared lo 7X days for the S now t,r:msmitt ws. Wwtcrn S-disease results in sterility in sonw individuals and in lowered fecundity in others. In the present investigations, of the individuals that had fed on WX celery, 205 out of 270 (77%) laid one or more eggs during adult life compared to 130 of 159 (xl. “l’ ) for the healthy controls. Although this difference in the number of compIet.eIy sterile females was not’ great, the total effect of’ W-X on the diseased stock was very sig-
ASD
VECTOR
I”ECUKDITT
:
nificant. Many females from the diseased stock laid very fen- eggs-especially during t,he last half of the test period. In order 10 evaluate the effect of feeding on WX plants on the production of fertilized eggs1 only egg-laying females were compared from the two stocks. Also, in order to eliminate diffrrences in t’otal egg production, due to differences in longevity, only the mean number:: of eggs laid/femttle,/week of life are (VII. sidered (Fig. 1). The experiments svcre carried out, over a i--week period. The mean egg production per female among the cl&eased insects was two-thirds that of the healt#hy stock during the first 3 \\wks. Thereafter the curves diverge strongly. The fecundity of the infective females declined rapidly to almost zero during the last 3 week;: (Fig. 1). In cont’rast’, the control females xew still laying more eggs after 7 weeks than the WX individuals did at their maximum \\hich n-as at 2 Lveeks. Fecundity
qf Iyjecfed Females
Experiments effect of WX 011 hoppers, all of celery planf~. _--_-
were carried out 011 the the fecundity of female leafn-hi& fed only on health\ Thr possibly complicating o,PtlstO SlOCK lZOS991
FJG. 1. The effect of the Western Ldiseuse agent on the fecundity of CuEla&n~s rtrontnrr,cs leafhoppers. The time period shown hegalL at the completion of SO-35-day feeding periods on diseased or healthy celery.
392
JEiWEN
effects of nutritional differences between diseased and healthy celery plants were thus eliminated. Infective extracts were made from adult leafhoppers that had fed for the previous 30-40 days on WX diseased plants. The WX extract was injected into healthy, last-instar nymphs. Control insects were injected with extracts made from healthy leafhoppers. Injected nymphs became adults within a few days after inject,ion. Three weeks after injection, w-hen the adults were sexually mature, had mated, and were laying eggs, the females were caged singly for oviposition. Each female was transferred to a new plant weekly. A record was kept of the number of eggs laid by each female each week and also of her longevity. The experiments extended over a period of 10 weeks after the insects were injected and for 7 weeks after the females were caged singly and egg counts were recorded. The incubat,ion period of WX is completed in most injected leafhoppers in 14-21 days compared to approximately 30 days in leafhoppers which acquire infectivity by feeding on diseased plants. The egg-laying data for 77 females inected with healthy extract and 63 females njected with WX ext.ract demonstrate (Fig.
2) that. the infective insects lvere consistent, in laying fewer eggs than did the healthy controls. The curves run essentially parallel t)hroughout the test period. The results are given in terms of the mean number of eggs laid per female per week of life, thus eliminating differences in egg production due to differences in longevity. DISCUSSION
In the present experiments, C. nzo?2tanus leafhoppers that acquired the WX agent, either by feeding on diseased plants or by injection with Wx ext,racts from diseased leafhoppers, laid fewer eggs per week of life than did the noninfective controls. In the experiment,s involving injected insects, the difference in fecundity between the test and cont,rol insects remained approximately constant through time. There was a peak at the fourth week after injection and then both curves gradually declined, with t,hat of the infective insects being slightly steeper during the final 6 weeks. In other work (Amin and Jensen, 1971) we have also shown that C. montanus leafhoppers that received the WX agent by injection and subsequently fed on a solution of achromycin retained a fecundity equal to that of healthy controls. H
Diseased
-
Healthy
FIG. 2. The effect of the Western X-disease agent on the fecundity of had been injected with infectious leafhopper extract. The control leafhoppers leafhopper extract.
monlanus which were injected with healthy
Colladonus
\I-ESTERN
S-DISEASE
AND
ln experiments with insects that acquired WX infectivity t,hrough feeding, the fecundity curve of infective insects declined rapidly during t’he last four weeks whereas that of the healthy controls rose and t,hen remainecl almost at the same level throughout the test, period. Insects fed on celery plants infected with the WX agent are different in two respects from control insects fed on healt.hy plants. First, most. of t.he former acquire WX infeetivity if given access to diseased plants for 30 clays or more. The WX agent itself has a profound effect 011 the vital life systems of C. m~)~fmus, causing lesions in many of its tissues, impairing fecundity, and killing the leafhopper prematurely. Second, since WX diseased plants are st rongl? altered physiologically, being dwarfed and chlorotic, the composit’ion of the plant sap imbibed by the leafhoppers must be different from that of healthy celery-. The nutritional quality of plant sap from diseased celery apparently is equal to that of healthy celery if the ody criterion is that of inwct longevity. Repeated experiments have demonstrated that C. montanus that feed on diseased celery for several weeks without acquiring WX infectivity live as lorlg as do the healthy controls. However, if they acquire WX their life span is conspicuously shortened. Although insects that feed 011 diseased plants from 1 to 4 weeks without acquiring WX do not’ suffer nutritionally in terms of longevity, it is nevertheless probable that the nutritional difference of sap from diseased plants may have an effect on some of the leafhopper’s vital functions, including reproduct,ion. WP do know, for example, that feeding 011 a diseased plant results in the accumulation of needlelike crystals in the st,omach (Lee and Jensen, 1963) and that, this phenomenon apparently is not a direct result of WX infection. When infective insects feed on healthy celery plants the tryst als gradually disappear. Moreover, in-
VECTOR
FEC’USDITT
393
sects acquiring WX by injection never tievelop such crystals. Present experimental evidence thus tends to indicate that, the fecundity of female C’. motrtunu~ le&oppers is impaired by infcction wit,11the WX agent, and that, in addition, the feeding on WX diseased plants further reduces the number of eggs laid b> this insect. The eff rcts of WX on the reproductive capacity of male leafhoppers ll:ts not, been investigated directly. Ho\vever, there did not, appear to be a significant8difference in the fertility of eggs laid by infwt ivc females, mated lvith infective males, :ultl t,hat of eggs laid by noninfect ive t’t~malt~~ mated \vifh noninfwtive males. I
AMIN, P. W.,
ANI) JI,:NSI,:S. 1). I). l!Pl. ICffects of tet,racycline on the transmission and pat ho. genicity of the Western X-disease agent iu its insert and plant hosts. I’h!//r,pnlho/og!/, iilr press), JENSI~:S, I). 1). 1!)59. .4 plant virtls lethal to iI+ insect, vector. I’irolog!/, 8, lM-175.
JENSEN, .I). I)., WHLTCUMH, 1:. F., .LND I~ICTL~ISON, J. 1967. Lethality of injected peach Western X-disease virus to its IeafhoppPr vector. Virology, 14, 5X-538. KISIMOTO, It., .\NL) W.\wo~. 31. .4. 1965. Abnormal development of embryos indwed 1, imbreeding in DeZphacorles pdlucida Fabricills and Delphacodes dubiu Kirschhaum (Araeckpidae, Homoptera), vectors of Rlwopenn wheat striate mosaic virus. J. Inwrtt~hr. Pathol.,
7, 297-305.
LEE, P. El., .~ND JP:NSI~:N, 1). 1).
1963. (‘ryst:11Iine inclusions in Colladonw n~ontan~s (Van Tjuzee), a vector of Western S-disease virus. Virology, 20, 328-332. NASU, S. 1963. Studies on some leafhoppers and planthoppers which transmiT virus diseases of rice plant in Japan. Btrll. K!/zcshtr Agr. Exp. Sk., 8, 153-349.
NASU, S., JENSF:N, II. I)., AND EICH.\HDSOX, .f. 1970. Electron microscopy like bodies associated with
of mycoplasmaiusect and pl:illt
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hosts of peach Western X-disease. Virology, 41, 583-595. WATSON, M. A., AND SINHA, R. C. 1959. Studies on the transmission of European wheat striate mosaic virus by Delphacodes pellucida Fabricius. Vi’irology, 8, 139-163. WHITCOMB, R. F., JENSEN, D. D., AND RICHARDSON, J. 1966. The infection of leafhoppers
by Western S-disease. I. Frequency of transmission after injection or acquisition feeding. Virology, 28, 448353. WHITCOMB, R. F., JENSEN, D. D., AND RICHARUSON, J. 1968. The infection of leafhoppers by Western X-disease virus, VI. Cytopathological interrelationship. J. Invertebr. Pathol., 12, 202-221.