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Initial and subsequent sites of aster yellows virus infection in a leafhopper vector
vinoi,orx 33, 702-708 (1967)
Initial and Subsequent Sites of Aster Yellows Virus Infection in a Leafhopper Vector' R . C . SINHA AND L . N . CHIYKOWSKI Cell Biology Research Institute, Research Branch, Canada Department of Agriculture, Ottawa, Ontario Accepted September 1, 1967 Aster yellows virus (AYV) was recovered from hemclymph, alimentary canal, salivary glands and ovaries, but not from Malpighian tubules, mycetomes, fat body, testes, or brain of viruliferous leafhoppers, Macrosteles faseifttms (8thl) . The presence of virus in the tissues was determined by injecting their extracts into virus-free leafhoppers, then testing the latter singly for inoculativity on aster (Callistephus chinensis Nees .) seedlings . The virus was first recovered from the alimentary canal immediately after a 3-day acquisition access period and from the hemolymph and salivary glands on day 6 after the start of acquisition . The virus concentration, as indicated by the percentage of injected insects that became inoculative, increased rapidly in the alimentary canal, reached a peak by day 9, but then declined sharply by day 23 and remained at the same low level up to day 30, the longest time tested . The virus concentration in the hemolymph also increased rapidly, reached a peak by day 12, but remained at the same high level up to day 30 . In the salivary glands the virus concentration increased gradually, and reached a high level by day 30 . The increase of virus concentration in salivary glands was correlated with the increase in transmission by the source leafhoppers . The results suggest that the alimentary canal of the leafhopper is the initial site of virus multiplication, that hemocytes may be the main sites of virus multiplication, and that AYV must reach a certain concentration in the salivary glands before an insect can become inoculative . INTRODUCTION to multiply in the leafhopper vector, MacroThe distribution of propagative viruses steles fascifrons (Sthl) several years ago (Kunkel, 1937 ; Black, 1941 ; Maramoroseh, in their leafhopper vectors has been investi1952), little is known about the distribution . The particles of gated only in a few cases rice dwarf (Fukushi and Shikata, 1963 ; of the virus in the vector. Hirumi and Nasu, 1965) and wound-tumor (Shikata and Maramorosch (1963), using the injection Maramorosch, 1965) viruses were observed technique, recovered a large amount of aster in several tissues of their vectors . Wound- yellows virus from the salivary glands of tumor virus antigens were detected in hemo- leafhoppers removed 19 days after the start lymph and in several internal organs by of a 5-day acquisition access period . No serological techniques (Sinha and Black, virus was recovered from gut, Malpighian 1963 ; Sinha, 1965a) . Potato yellow dwarf tubules, ovaries, and testes at this time . The virus was recovered, using the injection salivary glands and gut removed immeditechnique, from the hemolymph and from ately after acquisition yielded only minute all the internal organs (Sinha, 1965b) . amounts of virus . The results of Hirumi and Although aster yellows virus was shown Maramorosch were based on only one experir Contribution No . 618 from the Cell Biology ment with each organ and the data are insufficient to draw any definite conclusions Research Institute . 702
ASTER YELLOWS VIRUS INFECTION with respect to the distribution or sites of virus multiplication in the insect . The experiments reported in this paper are concerned mainly with the distribution of a celery strain of aster yellows virus in viruliferous leafhoppers, M . fascifrons, and the concentration of the virus in specific tissues at different times after the insects acquire virus from infected plants . MATERIALS AND METHODS
Virus and leafhoppers . A celery-infecting strain of aster yellows virus (AYV) described by Chiykowski (1962) was used in all experiments . The virus was maintained in aster plants (Callistephus chinensis Nees .) infected by viruliferous leafhoppers, Macrosteles fascifcons (Strtl) . Virus-free leafhoppers were reared on oat (Arena saliva L .) plants. "Exposed" leafhoppers were obtained by caging virus-free insects on AYV-infected aster plants . Not all exposed insects trailsmitted the virus ; those that did are termed "inoculative ." Unless otherwise stated, all experiments were done in a greenhouse in which the temperature fluctuated between 22° and 28 °. Preparation of hemolymph suspensions and extracts of internal organs for virus assay. Leafhoppers were dissected in a physiological saline solution containing streptomycin and penicillin (Jones and Cunningham, 1961) by a method described earlier (Sinha and Black, 1963) . The internal organs tested for the presence of virus were brain, salivary glands, alimentary canal, Malpighian tubules, mycetomes, fat body, ovaries, and testes (Figs. 1-8) . Thirty organs of the same kind from exposed leafhoppers were pooled, washed once in physiological saline and twice in saline (0 .85 % NaCl) in a depression slide . They were then ground in 0 .1 ml saline in a 1-ml capacity tissue grinder 2 operated by an electric motor. The grinder was kept immersed in an icebath in a coldroom (4°) during grinding . The extracts were centrifuged in 2nil capacity tubes at 5000 rpm for 10 minutes in a Spinco Model L centrifuge in a No . 40 rotor with special adapters . Such "clarified extracts" were removed with a fine pipette 2
%orates Glass Company, Vineland, New Jersey .
703
and injected into virus-free adult leafhoppers (Sinha and Chiykowski, 1967) . The injected insects were caged in groups on healthy aster plants for 3 weeks (1 week on each of 3 successive plants) and were then tested singly for inoculativity for 2 weeks on aster seedlings . In most experiments more than 50% of the injected insects survived . Hemolymph suspensions were prepared by removing the heads of 30 exposed leafhoppers and dipping the open portions of the bodies in 0 .1 nil saline . The suspensions were then clarified by low speed centrifugation and tested for the presence of virus as described above . Injection technique . Leafhoppers were injected by a method described earlier (Sinha and Chiykowski, 1967) . During injection, groups of adult insects were kept immobilized by a continuous flow of CO2 at a pressure of 2 .5 pounds per square inch controlled by a flowmeter attached to a regulator on a CO s cylinder . Preliminary experiments on the effect of C0 2 on the survival of leafhoppers showed that under the conditions employed all insects survived a 10-minute exposure . However, exposures of 20 minutes resulted in only 55 % survival, and, as the exposure time increased, percentage survival of the leafhoppers decreased . Therefore, during injection, leafhoppers were never exposed to C0 2 for more than 10 minutes . RESULTS
Distribution of the Virus in the Vector To determine the distribution of AYV in leafhoppers, nymphs (2nd or 3rd instar) or adults were given an acquisition access period of 7` days and were then transferred weekly to healthy aster plants . Three to four weeks after the start of the acquisition access period, hcmolymph suspensions and extracts of various internal organs were assayed for AYV . The virus was recovered from the alimentary canal, hemolymph, salivary glands and ovaries, but not from Malpighian tubules, mycetomes, fat body, testes, or brain (Table 1) . The age of insects at the time of acquisition access period had no effect on the subsequent distribution of AYV in the leafhoppers . However, more virus was recovered from the ovaries of insects that had acquisition access as adults
d
408 2
Fros. 1-8 . Internal anatomy of the leafhopper, Macrosteles fascifroe .s . Magnification : Figs . 1, 3, 6, 7 and 8, X 24 ; Figs . 2, 4, and 5, X 3d . Frc . 1 . Brain : og, abdominal ganglion ; aneig, metathoraeie ganglion ; mtg, mesothoracie ganglion : d, deuto-cerebrium ; ol, optic lobe . FIG . 2 . Salivary glands : al, anterior lobe ; pl, posterior lobe ; ad, salivary duet . FIG. 3 . Fat body tissue . Fro. 4 . Mycetume . FIG. 5 . Alimentary canal : oe, oesophagus ; fe, filter chamber ; v, ventriculus ; mi, mid-intestine ; font, tubular portion of mid-intestine ; hi, hind-intestine . FIG. 6 . Malpighian tubules : pr, proximal region ; -rut, Malpighian tubules ; fr, filter chamber of the alimentary canal showing the origin of the Malpighiaa tubules . Fin- 7 Male reproductive organs : aeg, accessory gland ; f, follicle (6 follicles compose each testis) ; ad, vas deferens ; as, vesicula seminalis . Frc . 8 . Female reproductive organs : on, ovariole ; mo, main oviduct . 704
ASTER
YELLOWS
TABLE 1
7~
DISTRIBUTION OF ASTER YELLOWS VIRUS IN THE LEAFHOPPER VECTOR Macrosteles fascifrons AFTER AcQuisncoN ACCESS As NYMPHS OR ADULTS'
Transmission by insects injected with the extracts of tissues from leafhoppers that had acquisition access as : Source of extract' Nymphs
Adults
Transmission , Alimentary Canal Hemolymph Salivary glands Ovaries Testes Malpighian tubules Fat body Myeetomes Brain
4/50
Transmission,
72/86
8 84
29/35
23/88 2/105 0/94 0/SO
26 2 0 0
20/60 24/74 0/61 0/98
83 33 32 0 0
0/90
0
0/40 0/69 0/71
0 0 0
NT 0189
0
4/100
/c
4
a Insects were given an acquisition access period of 7 days and then were maintained on healthy aster plants for 3 weeks . 6 Thirty organs of the same kind were pooled, washed 3 times, ground in 0.1 ml saline, their clarified extracts injected into virus-free insects, and the latter tested singly for their inoculativity . Hemolymph suspensions were prepared from 30 exposed leafhoppers in 0.1 ml saline . Combined results of two experiments ; nuinerator is the number of injected insects that transmitted the virus ; denominator is the number of insects tested . NT means not tested .
than as nymphs . In both cases, hemolymph was the best source of virus inoculum . Age and Sex of Insect in Relation to Virus Transmission To find whether the age of male and female leafhoppers at the time of acquisition access period had any effect on their subsequent ability to transmit the virus, nymphs (2nd and 3rd instar) and adults were caged for 7 days on the same infected plant . The exposed nymphs and adults were then maintained separately on healthy aster plants for 2 weeks (1 week on each of 2 successive plants) . Under these conditions there was no danger that the insects that had not acquired AYV while feeding on infected plants might acquire it from plants on which they
VIRUS INFECTION
705
were subsequently maintained together with leafhoppers which were carrying the virus (Maramorosch, 1953) . The exposed insects were then tested singly for their inoculativity for 2 weeks on healthy aster seedlings . The sex of each insect was recorded . The results of two such experiments showed that the total number of male and female leafhoppers that became inoculative after they had acquired the virus as nymphs or as adults, were : females as nymphs 23/40 (58%) ; males as nymphs 23/40 (58%) ; females as adults 29/40 (73%), and males as adults 25/40 (63%) . The results suggest that the ability of leafhoppers to transmit AYV is Hot affected significantly by sex or age of the insect at the time of the acquisition access period . Percentage Transmission by Injected Insects as a Measure of Relative Virus Concentration In these experiments virus-free adult insects were Caged for 3 days on infected plants and 28 days after the start of the acquisition access period the exposed insects were ground in saline . The extracts were then clarified by low speed centrifugation and passed through a Millipore filter (HA 0 .45 p, 13 mm) as described earlier (Sinha and Chiykowski, 1967) . Four dilutions of clarified extracts were then injected into virus-free insects and the latter tested singly for their inoculativity . In Calculating the dilution of the extracts, it was assumed that a group of leafhoppers weighing 1 g had a volume of 1 ml and that I g of tissue in 9 ml of saline equalled a 1/10 dilution . The number of insects that became inoculative after being injected with various dilutions were : 36/42 (86 7c) at 1/10 ; 40/108 (37 7c) at 1/50 ; 7/83 at 1/200 ; 1/107 (1%) at 1/800 . These results show that the percentage transmission by injected insects is indicative of relative virus concentration in various inocula . Initial and Subsequent Sites of Infection in Leafhoppers after Ingestion of Virus The sequential infection of the internal organs of a leafhopper vector has only been studied with wound-tumor virus (Sinha, 1965a) . By the use of immunofluorescent
SINIIA AND CHIYKOWSKI
706 TABLE 2
VIRUS
FROM
RECOVERY OF ASTER YELLOWS ALIMENTARY CAS AL, HEMOLYMPH AND SALIVARY GLANDS OF Macrosteies fascifrons AT VARIOUS TIMES AFTER ACQUISITION ACCESS PERIOD nay w hich -
Leafhoppers that transmitted the virus after being injected with extracts of : Alimentary
tracts were
pre-
Transmissions
rO
3 6 9
7/119 58/105 94/111
6 55 85
12 16 19
36/68 52/106 38/100 12/100
53 49 38 12
10/92
11
23 30
Hemolymph`
Salivary glandsi
bansmission s
'0
Trans-, mlssiond
%
0/108 2/106 42/57 94/102
0 2 74 92
0/79 1/114 2/91 1/114
0 1 2 1
77/93 52/58 83/91
83 91 91
60/69
87
10/114 31/102 50/88 79/91
9 30 57 87
At various intervals after the start of the acquisition access period of 3 days, hemolymph suspensions and extracts of alimentary canals and salivary glands of the leafhoppers were injected into virus-free insects . The injected insects were then tested singly for their inoculativity on aster seedlings . o Each time, 30 organs of the same kind were pooled, washed 3 times, ground in 0 .1 ml saline, and their clarified extracts were tested for virus . ° Each time, hemolymph suspensions were prepared from 30 leafhoppers in 0 .1 ml saline and tested for virus . a Combined results of two experiments ; numerator is the number of injected insects thatt transmitted the virus ; denominator is the number of insects tested . techniques it was shown that the alimentary canal was the initial site of virus multiplication . As the incubation period of the virus progressed the virus spread to other susceptible sites succh as hemocytes, fat body tissues, mycetomes, and salivary glands . Our experiments were designed to study AYV concentration in alimentary canal, hemolymph, and salivary glands of leafhoppers at various times after ingsetion of virus, and to determine the sequence in which these tissues become infected . Virusfree adult leafhoppers were caged for 3 days on infected plants and were then maintained on healthy plants for 30 days (5 days on each of 6 successive plants) at 27 ° . The day on which insects were caged on infected plants
was numbered 0, the next day 1, and so on throughout the incubation period . At various times, hemolymph suspensions and extracts of alimentary canals and of salivary glands were assayed for AYV . Each time, alimentary canals and salivary glands of 30 exposed lcafhoppers in 0 .1 inn saline were used to prepare the inocula . hemolymph suspensions were prepared in 0 .1 ml saline from another 30 leafhoppers of the same group . The results of two such experiments arc summarized in Table 2 and are shown graphically in Fig . 9 . The virus was first detected in the alimentary canal on day 3, in hemolymph and in salivary glands on day 6 . As the incubation period of the virus in leafhoppers progressed, the virus concentration in the alimentary canal increased rapidly and reached a peak by day 9, but then decreased sharply by day 23, and remained at the same level up to day 30 . These results show that the alimentary canal, or some part of it, is the initial site of virus multiplication . The reason for the eventual decrease in virus concentration in this tissue is not known . In the hemolymph, the virus concentration also increased rapidly and reached a peak by day 12, but remained at the same level up to day 30, which suggests that hemocytes may be the main site of virus multiplication . In the salivary glands only small amounts of virus were recovered up to day 12 and then the virus concentration increased gradually, reaching a high level by day 30 . The incubation period of the virus in source leafhoppers (insects of the group which were used to prepare the extracts of various tissues) was also studied . In these experiments insects were transferred singly to fresh aster seedlings on the same days on which the extracts of the tissues were assayed for AYV. The results of two such experiments showed that the number of insects that were inoculative on days 12, 16, 19, 23, and 30 were 0/55 (0%), 2/55 (4/'o), 4/55 (7%), 21/53 (40%), and 33/53 (62%), respectively (Fig . 9) . The virus was recovered from salivary glands, although in very small amounts, before the source insects started to transmit . The increase in virus concentration in salivary glands was correlated with an increase in percentage transmission by the
707
ASTER YELLOWS VIRUS INFECTION
/00 90 60 70 02
40
so yy` 50 ~a 40,1 SALIVARY GLANDS
30 / $ 3 6 9 /2 16 /9 23 /NOCULUM PREPARED OND4Y
3 6 9 /2 /6 /9 23 DAYS AFTER ACOU/S/T/ON
O 4 30
FIG . 9 . Recovery of AYV from alimentary canal, hemolymph and salivary glands of M . fascifrons at various times after the start of acquisition access period of 3 days . Hemolymph suspensions, and extracts of alimentary canals and of salivary glands were injected into virus-free insects and the latter were tested singly on aster seedlings for their inoculativity . The percentage transmission by the source insects, on days when the inocula of various tissues were assayed for AYV, was also recorded .
source insects . These results suggest that AYV also multiplies in the salivary glands and that the virus must reach a certain concentration before an insect can transmit the virus to plants . DISCUSSION Hirunu and Maramorosch (1963) used the incubation period of AYV in injected leafhoppers to estimate the relative virus concentration in extracts prepared from salivary glands and alimentary canals of viruliferous insects. Whitcomb et al . (1966) developed a quantification of the incubation period method for assaying the concentration of Western X-disease virus in hemolymph of leafhoppers during the early stages of infection . In the current study percentage transmission by injected insects was used as a measure of relative AYV concentration in various inocula . In these experiments approximately 2400 dissections were made, 5300 insects were injected of which 3800
survived and were tested singly for their inoculativity . Laborious and time-consuming as it may be, the injection technique is the only method available at present to detect AYV in an inoculum . AYV was detected in the alimentary canal, hemolymph, salivary glands, and ovaries of viruliferous leafhoppers, M . fascifrons . Failure to recover the virus from fat body, testes, mycetomes, or brain, however, does not rule out the possibility that AYV was present at undetected concentrations in these tissues . Recovery of the virus from ovaries was unexpected because transovarial transmission of AYV has never been shown . The transmission of viruses to progeny insects probably depends on the ability of the virus to penetrate the ovum. How a virus enters an ovum is not known, but there is a suggestion, based on electron microscopic work on rice dwarf virus (Nasu, 1965), that the virus may be transported through the symbiotes of mycetocytes into the ooc;ytes of the vector .
708
SINHA AND CHIYAOWSKI
It is possible that although AYV was present in the ovaries it cannot reach the oocytes and therefore no transovarial transmission can occur . It is not known at present whether the virus accumulates or multiplies in the ovaries . The virus was recovered in high concentrations from alimentary canal, hemolymph and salivary glands, but the time required to reach such concentrations was different for each tissue . The virus concentration in inocula prepared from the three tissues, at the time when maximum virus was re. Howcovered from them, was about equal ever, it should he noted that our results do not permit comparison of AYV concentration among the different tissues themselves . The inocula from various tissues were prepared on the basis of number rather than weight . Since there is a considerable difference in size and weight among the different tissues, the actual dilutions of the inocula prepared from them were not the same . ACKNOWLEDGMENTS We wish to thank Mr . J . E . Lendvay-Zwickl for his excellent assistance in transmission experiments and care of the cultures . REFERENCES BLACK, L . M . (1941) . Further evidence for multiplication of the aster yellows virus in the aster leafhopper . Phytopathology 31, 120-135 . CaiyKowsKi, L . N . (1962) . Scaphytopius acutus (Say), a newly discovered vector of celery-infecting aster-yellows virus . Can . J . Botany 40, 799-801 . Fuxasm, T ., and SancATA, E . (1963) . Localiza-
tion of rice dwarf virus in its insect vector . Virology 21, 503-505 . Hinuan, H ., and MAixaronoscn, K . (1963) . Recovery of aster yellows virus from various organs of the insect vector, Macrosteles fascifrons . Contrib . Boyce Thompson Inst . 22, 141-152 . JoyEs, B . M ., and CUNNINGHAM, I . (1961), Growth by cell division in insect tissue culture . Exptl . Cell Res . 23, 386-401 . KUNKEL, L . O . (1937) . Effect of heat on ability of Cicadula sexnotata (Fall .) to transmit aster yellows . Am . J . Botany 24, 316-327 . MAlAMOaosea, K . (1952) . Direct evidence for the multiplication of aster yellows virus in its insect vector . Phytopathotogy 42, 59-64 . MAaAMOROSCH, K . (1953) . Incubation period of aster-yellows virus . Am . J . Botany 40, 797-809 . NASU, S . (1965) . Electron microscopic studies on transovarial passage of rice dwarf virus . Japan . J . Appl . Entomol . Zeal . 9, 225-237 . SHLKATA, E ., and MARAMOaoscn, K . (1965), Electron microscopic evidence for the systemic invasion of an insect host by a plant pathogenic virus . Virology 27, 461-475 . SINHA, R . C . (1965a) . Sequential infection and distribution of wound-tumor virus in the internal organs of a vector after ingestion of virus . Virology 26, 673-686 . SINHA, R. C . (1965b) . Recovery of potato yellow dwarf virus from hemolymph and internal organs of an insect vector . Virology 27, 118-119 . SINIA, It. C ., and BLACK, L . M . (1963) . Woundtumor virus antigens in the internal organs of an insect vector . Virology 21, 183-187 . SINHA, It . C ., and CmYxowsK :, L . N . (1967) . Multiplication of aster yellows virus in a nonvector leafhopper. Virology 31, 461 466. WmTCorm, R . F ., JENSEN, D . D ., and RICnARDsoN, J . (1966) . The infection of leafhoppers by Western X-disease virus . II . Fluctuation of virus concentration in the hemolymph after injection, Virology 28, 454-458 .
Initial and Subsequent Sites of Aster Yellows Virus Infection in a Leafhopper Vector' R . C . SINHA AND L . N . CHIYKOWSKI Cell Biology Research Institute, Research Branch, Canada Department of Agriculture, Ottawa, Ontario Accepted September 1, 1967 Aster yellows virus (AYV) was recovered from hemclymph, alimentary canal, salivary glands and ovaries, but not from Malpighian tubules, mycetomes, fat body, testes, or brain of viruliferous leafhoppers, Macrosteles faseifttms (8thl) . The presence of virus in the tissues was determined by injecting their extracts into virus-free leafhoppers, then testing the latter singly for inoculativity on aster (Callistephus chinensis Nees .) seedlings . The virus was first recovered from the alimentary canal immediately after a 3-day acquisition access period and from the hemolymph and salivary glands on day 6 after the start of acquisition . The virus concentration, as indicated by the percentage of injected insects that became inoculative, increased rapidly in the alimentary canal, reached a peak by day 9, but then declined sharply by day 23 and remained at the same low level up to day 30, the longest time tested . The virus concentration in the hemolymph also increased rapidly, reached a peak by day 12, but remained at the same high level up to day 30 . In the salivary glands the virus concentration increased gradually, and reached a high level by day 30 . The increase of virus concentration in salivary glands was correlated with the increase in transmission by the source leafhoppers . The results suggest that the alimentary canal of the leafhopper is the initial site of virus multiplication, that hemocytes may be the main sites of virus multiplication, and that AYV must reach a certain concentration in the salivary glands before an insect can become inoculative . INTRODUCTION to multiply in the leafhopper vector, MacroThe distribution of propagative viruses steles fascifrons (Sthl) several years ago (Kunkel, 1937 ; Black, 1941 ; Maramoroseh, in their leafhopper vectors has been investi1952), little is known about the distribution . The particles of gated only in a few cases rice dwarf (Fukushi and Shikata, 1963 ; of the virus in the vector. Hirumi and Nasu, 1965) and wound-tumor (Shikata and Maramorosch (1963), using the injection Maramorosch, 1965) viruses were observed technique, recovered a large amount of aster in several tissues of their vectors . Wound- yellows virus from the salivary glands of tumor virus antigens were detected in hemo- leafhoppers removed 19 days after the start lymph and in several internal organs by of a 5-day acquisition access period . No serological techniques (Sinha and Black, virus was recovered from gut, Malpighian 1963 ; Sinha, 1965a) . Potato yellow dwarf tubules, ovaries, and testes at this time . The virus was recovered, using the injection salivary glands and gut removed immeditechnique, from the hemolymph and from ately after acquisition yielded only minute all the internal organs (Sinha, 1965b) . amounts of virus . The results of Hirumi and Although aster yellows virus was shown Maramorosch were based on only one experir Contribution No . 618 from the Cell Biology ment with each organ and the data are insufficient to draw any definite conclusions Research Institute . 702
ASTER YELLOWS VIRUS INFECTION with respect to the distribution or sites of virus multiplication in the insect . The experiments reported in this paper are concerned mainly with the distribution of a celery strain of aster yellows virus in viruliferous leafhoppers, M . fascifrons, and the concentration of the virus in specific tissues at different times after the insects acquire virus from infected plants . MATERIALS AND METHODS
Virus and leafhoppers . A celery-infecting strain of aster yellows virus (AYV) described by Chiykowski (1962) was used in all experiments . The virus was maintained in aster plants (Callistephus chinensis Nees .) infected by viruliferous leafhoppers, Macrosteles fascifcons (Strtl) . Virus-free leafhoppers were reared on oat (Arena saliva L .) plants. "Exposed" leafhoppers were obtained by caging virus-free insects on AYV-infected aster plants . Not all exposed insects trailsmitted the virus ; those that did are termed "inoculative ." Unless otherwise stated, all experiments were done in a greenhouse in which the temperature fluctuated between 22° and 28 °. Preparation of hemolymph suspensions and extracts of internal organs for virus assay. Leafhoppers were dissected in a physiological saline solution containing streptomycin and penicillin (Jones and Cunningham, 1961) by a method described earlier (Sinha and Black, 1963) . The internal organs tested for the presence of virus were brain, salivary glands, alimentary canal, Malpighian tubules, mycetomes, fat body, ovaries, and testes (Figs. 1-8) . Thirty organs of the same kind from exposed leafhoppers were pooled, washed once in physiological saline and twice in saline (0 .85 % NaCl) in a depression slide . They were then ground in 0 .1 ml saline in a 1-ml capacity tissue grinder 2 operated by an electric motor. The grinder was kept immersed in an icebath in a coldroom (4°) during grinding . The extracts were centrifuged in 2nil capacity tubes at 5000 rpm for 10 minutes in a Spinco Model L centrifuge in a No . 40 rotor with special adapters . Such "clarified extracts" were removed with a fine pipette 2
%orates Glass Company, Vineland, New Jersey .
703
and injected into virus-free adult leafhoppers (Sinha and Chiykowski, 1967) . The injected insects were caged in groups on healthy aster plants for 3 weeks (1 week on each of 3 successive plants) and were then tested singly for inoculativity for 2 weeks on aster seedlings . In most experiments more than 50% of the injected insects survived . Hemolymph suspensions were prepared by removing the heads of 30 exposed leafhoppers and dipping the open portions of the bodies in 0 .1 nil saline . The suspensions were then clarified by low speed centrifugation and tested for the presence of virus as described above . Injection technique . Leafhoppers were injected by a method described earlier (Sinha and Chiykowski, 1967) . During injection, groups of adult insects were kept immobilized by a continuous flow of CO2 at a pressure of 2 .5 pounds per square inch controlled by a flowmeter attached to a regulator on a CO s cylinder . Preliminary experiments on the effect of C0 2 on the survival of leafhoppers showed that under the conditions employed all insects survived a 10-minute exposure . However, exposures of 20 minutes resulted in only 55 % survival, and, as the exposure time increased, percentage survival of the leafhoppers decreased . Therefore, during injection, leafhoppers were never exposed to C0 2 for more than 10 minutes . RESULTS
Distribution of the Virus in the Vector To determine the distribution of AYV in leafhoppers, nymphs (2nd or 3rd instar) or adults were given an acquisition access period of 7` days and were then transferred weekly to healthy aster plants . Three to four weeks after the start of the acquisition access period, hcmolymph suspensions and extracts of various internal organs were assayed for AYV . The virus was recovered from the alimentary canal, hemolymph, salivary glands and ovaries, but not from Malpighian tubules, mycetomes, fat body, testes, or brain (Table 1) . The age of insects at the time of acquisition access period had no effect on the subsequent distribution of AYV in the leafhoppers . However, more virus was recovered from the ovaries of insects that had acquisition access as adults
d
408 2
Fros. 1-8 . Internal anatomy of the leafhopper, Macrosteles fascifroe .s . Magnification : Figs . 1, 3, 6, 7 and 8, X 24 ; Figs . 2, 4, and 5, X 3d . Frc . 1 . Brain : og, abdominal ganglion ; aneig, metathoraeie ganglion ; mtg, mesothoracie ganglion : d, deuto-cerebrium ; ol, optic lobe . FIG . 2 . Salivary glands : al, anterior lobe ; pl, posterior lobe ; ad, salivary duet . FIG. 3 . Fat body tissue . Fro. 4 . Mycetume . FIG. 5 . Alimentary canal : oe, oesophagus ; fe, filter chamber ; v, ventriculus ; mi, mid-intestine ; font, tubular portion of mid-intestine ; hi, hind-intestine . FIG. 6 . Malpighian tubules : pr, proximal region ; -rut, Malpighian tubules ; fr, filter chamber of the alimentary canal showing the origin of the Malpighiaa tubules . Fin- 7 Male reproductive organs : aeg, accessory gland ; f, follicle (6 follicles compose each testis) ; ad, vas deferens ; as, vesicula seminalis . Frc . 8 . Female reproductive organs : on, ovariole ; mo, main oviduct . 704
ASTER
YELLOWS
TABLE 1
7~
DISTRIBUTION OF ASTER YELLOWS VIRUS IN THE LEAFHOPPER VECTOR Macrosteles fascifrons AFTER AcQuisncoN ACCESS As NYMPHS OR ADULTS'
Transmission by insects injected with the extracts of tissues from leafhoppers that had acquisition access as : Source of extract' Nymphs
Adults
Transmission , Alimentary Canal Hemolymph Salivary glands Ovaries Testes Malpighian tubules Fat body Myeetomes Brain
4/50
Transmission,
72/86
8 84
29/35
23/88 2/105 0/94 0/SO
26 2 0 0
20/60 24/74 0/61 0/98
83 33 32 0 0
0/90
0
0/40 0/69 0/71
0 0 0
NT 0189
0
4/100
/c
4
a Insects were given an acquisition access period of 7 days and then were maintained on healthy aster plants for 3 weeks . 6 Thirty organs of the same kind were pooled, washed 3 times, ground in 0.1 ml saline, their clarified extracts injected into virus-free insects, and the latter tested singly for their inoculativity . Hemolymph suspensions were prepared from 30 exposed leafhoppers in 0.1 ml saline . Combined results of two experiments ; nuinerator is the number of injected insects that transmitted the virus ; denominator is the number of insects tested . NT means not tested .
than as nymphs . In both cases, hemolymph was the best source of virus inoculum . Age and Sex of Insect in Relation to Virus Transmission To find whether the age of male and female leafhoppers at the time of acquisition access period had any effect on their subsequent ability to transmit the virus, nymphs (2nd and 3rd instar) and adults were caged for 7 days on the same infected plant . The exposed nymphs and adults were then maintained separately on healthy aster plants for 2 weeks (1 week on each of 2 successive plants) . Under these conditions there was no danger that the insects that had not acquired AYV while feeding on infected plants might acquire it from plants on which they
VIRUS INFECTION
705
were subsequently maintained together with leafhoppers which were carrying the virus (Maramorosch, 1953) . The exposed insects were then tested singly for their inoculativity for 2 weeks on healthy aster seedlings . The sex of each insect was recorded . The results of two such experiments showed that the total number of male and female leafhoppers that became inoculative after they had acquired the virus as nymphs or as adults, were : females as nymphs 23/40 (58%) ; males as nymphs 23/40 (58%) ; females as adults 29/40 (73%), and males as adults 25/40 (63%) . The results suggest that the ability of leafhoppers to transmit AYV is Hot affected significantly by sex or age of the insect at the time of the acquisition access period . Percentage Transmission by Injected Insects as a Measure of Relative Virus Concentration In these experiments virus-free adult insects were Caged for 3 days on infected plants and 28 days after the start of the acquisition access period the exposed insects were ground in saline . The extracts were then clarified by low speed centrifugation and passed through a Millipore filter (HA 0 .45 p, 13 mm) as described earlier (Sinha and Chiykowski, 1967) . Four dilutions of clarified extracts were then injected into virus-free insects and the latter tested singly for their inoculativity . In Calculating the dilution of the extracts, it was assumed that a group of leafhoppers weighing 1 g had a volume of 1 ml and that I g of tissue in 9 ml of saline equalled a 1/10 dilution . The number of insects that became inoculative after being injected with various dilutions were : 36/42 (86 7c) at 1/10 ; 40/108 (37 7c) at 1/50 ; 7/83 at 1/200 ; 1/107 (1%) at 1/800 . These results show that the percentage transmission by injected insects is indicative of relative virus concentration in various inocula . Initial and Subsequent Sites of Infection in Leafhoppers after Ingestion of Virus The sequential infection of the internal organs of a leafhopper vector has only been studied with wound-tumor virus (Sinha, 1965a) . By the use of immunofluorescent
SINIIA AND CHIYKOWSKI
706 TABLE 2
VIRUS
FROM
RECOVERY OF ASTER YELLOWS ALIMENTARY CAS AL, HEMOLYMPH AND SALIVARY GLANDS OF Macrosteies fascifrons AT VARIOUS TIMES AFTER ACQUISITION ACCESS PERIOD nay w hich -
Leafhoppers that transmitted the virus after being injected with extracts of : Alimentary
tracts were
pre-
Transmissions
rO
3 6 9
7/119 58/105 94/111
6 55 85
12 16 19
36/68 52/106 38/100 12/100
53 49 38 12
10/92
11
23 30
Hemolymph`
Salivary glandsi
bansmission s
'0
Trans-, mlssiond
%
0/108 2/106 42/57 94/102
0 2 74 92
0/79 1/114 2/91 1/114
0 1 2 1
77/93 52/58 83/91
83 91 91
60/69
87
10/114 31/102 50/88 79/91
9 30 57 87
At various intervals after the start of the acquisition access period of 3 days, hemolymph suspensions and extracts of alimentary canals and salivary glands of the leafhoppers were injected into virus-free insects . The injected insects were then tested singly for their inoculativity on aster seedlings . o Each time, 30 organs of the same kind were pooled, washed 3 times, ground in 0 .1 ml saline, and their clarified extracts were tested for virus . ° Each time, hemolymph suspensions were prepared from 30 leafhoppers in 0 .1 ml saline and tested for virus . a Combined results of two experiments ; numerator is the number of injected insects thatt transmitted the virus ; denominator is the number of insects tested . techniques it was shown that the alimentary canal was the initial site of virus multiplication . As the incubation period of the virus progressed the virus spread to other susceptible sites succh as hemocytes, fat body tissues, mycetomes, and salivary glands . Our experiments were designed to study AYV concentration in alimentary canal, hemolymph, and salivary glands of leafhoppers at various times after ingsetion of virus, and to determine the sequence in which these tissues become infected . Virusfree adult leafhoppers were caged for 3 days on infected plants and were then maintained on healthy plants for 30 days (5 days on each of 6 successive plants) at 27 ° . The day on which insects were caged on infected plants
was numbered 0, the next day 1, and so on throughout the incubation period . At various times, hemolymph suspensions and extracts of alimentary canals and of salivary glands were assayed for AYV . Each time, alimentary canals and salivary glands of 30 exposed lcafhoppers in 0 .1 inn saline were used to prepare the inocula . hemolymph suspensions were prepared in 0 .1 ml saline from another 30 leafhoppers of the same group . The results of two such experiments arc summarized in Table 2 and are shown graphically in Fig . 9 . The virus was first detected in the alimentary canal on day 3, in hemolymph and in salivary glands on day 6 . As the incubation period of the virus in leafhoppers progressed, the virus concentration in the alimentary canal increased rapidly and reached a peak by day 9, but then decreased sharply by day 23, and remained at the same level up to day 30 . These results show that the alimentary canal, or some part of it, is the initial site of virus multiplication . The reason for the eventual decrease in virus concentration in this tissue is not known . In the hemolymph, the virus concentration also increased rapidly and reached a peak by day 12, but remained at the same level up to day 30, which suggests that hemocytes may be the main site of virus multiplication . In the salivary glands only small amounts of virus were recovered up to day 12 and then the virus concentration increased gradually, reaching a high level by day 30 . The incubation period of the virus in source leafhoppers (insects of the group which were used to prepare the extracts of various tissues) was also studied . In these experiments insects were transferred singly to fresh aster seedlings on the same days on which the extracts of the tissues were assayed for AYV. The results of two such experiments showed that the number of insects that were inoculative on days 12, 16, 19, 23, and 30 were 0/55 (0%), 2/55 (4/'o), 4/55 (7%), 21/53 (40%), and 33/53 (62%), respectively (Fig . 9) . The virus was recovered from salivary glands, although in very small amounts, before the source insects started to transmit . The increase in virus concentration in salivary glands was correlated with an increase in percentage transmission by the
707
ASTER YELLOWS VIRUS INFECTION
/00 90 60 70 02
40
so yy` 50 ~a 40,1 SALIVARY GLANDS
30 / $ 3 6 9 /2 16 /9 23 /NOCULUM PREPARED OND4Y
3 6 9 /2 /6 /9 23 DAYS AFTER ACOU/S/T/ON
O 4 30
FIG . 9 . Recovery of AYV from alimentary canal, hemolymph and salivary glands of M . fascifrons at various times after the start of acquisition access period of 3 days . Hemolymph suspensions, and extracts of alimentary canals and of salivary glands were injected into virus-free insects and the latter were tested singly on aster seedlings for their inoculativity . The percentage transmission by the source insects, on days when the inocula of various tissues were assayed for AYV, was also recorded .
source insects . These results suggest that AYV also multiplies in the salivary glands and that the virus must reach a certain concentration before an insect can transmit the virus to plants . DISCUSSION Hirunu and Maramorosch (1963) used the incubation period of AYV in injected leafhoppers to estimate the relative virus concentration in extracts prepared from salivary glands and alimentary canals of viruliferous insects. Whitcomb et al . (1966) developed a quantification of the incubation period method for assaying the concentration of Western X-disease virus in hemolymph of leafhoppers during the early stages of infection . In the current study percentage transmission by injected insects was used as a measure of relative AYV concentration in various inocula . In these experiments approximately 2400 dissections were made, 5300 insects were injected of which 3800
survived and were tested singly for their inoculativity . Laborious and time-consuming as it may be, the injection technique is the only method available at present to detect AYV in an inoculum . AYV was detected in the alimentary canal, hemolymph, salivary glands, and ovaries of viruliferous leafhoppers, M . fascifrons . Failure to recover the virus from fat body, testes, mycetomes, or brain, however, does not rule out the possibility that AYV was present at undetected concentrations in these tissues . Recovery of the virus from ovaries was unexpected because transovarial transmission of AYV has never been shown . The transmission of viruses to progeny insects probably depends on the ability of the virus to penetrate the ovum. How a virus enters an ovum is not known, but there is a suggestion, based on electron microscopic work on rice dwarf virus (Nasu, 1965), that the virus may be transported through the symbiotes of mycetocytes into the ooc;ytes of the vector .
708
SINHA AND CHIYAOWSKI
It is possible that although AYV was present in the ovaries it cannot reach the oocytes and therefore no transovarial transmission can occur . It is not known at present whether the virus accumulates or multiplies in the ovaries . The virus was recovered in high concentrations from alimentary canal, hemolymph and salivary glands, but the time required to reach such concentrations was different for each tissue . The virus concentration in inocula prepared from the three tissues, at the time when maximum virus was re. Howcovered from them, was about equal ever, it should he noted that our results do not permit comparison of AYV concentration among the different tissues themselves . The inocula from various tissues were prepared on the basis of number rather than weight . Since there is a considerable difference in size and weight among the different tissues, the actual dilutions of the inocula prepared from them were not the same . ACKNOWLEDGMENTS We wish to thank Mr . J . E . Lendvay-Zwickl for his excellent assistance in transmission experiments and care of the cultures . REFERENCES BLACK, L . M . (1941) . Further evidence for multiplication of the aster yellows virus in the aster leafhopper . Phytopathology 31, 120-135 . CaiyKowsKi, L . N . (1962) . Scaphytopius acutus (Say), a newly discovered vector of celery-infecting aster-yellows virus . Can . J . Botany 40, 799-801 . Fuxasm, T ., and SancATA, E . (1963) . Localiza-
tion of rice dwarf virus in its insect vector . Virology 21, 503-505 . Hinuan, H ., and MAixaronoscn, K . (1963) . Recovery of aster yellows virus from various organs of the insect vector, Macrosteles fascifrons . Contrib . Boyce Thompson Inst . 22, 141-152 . JoyEs, B . M ., and CUNNINGHAM, I . (1961), Growth by cell division in insect tissue culture . Exptl . Cell Res . 23, 386-401 . KUNKEL, L . O . (1937) . Effect of heat on ability of Cicadula sexnotata (Fall .) to transmit aster yellows . Am . J . Botany 24, 316-327 . MAlAMOaosea, K . (1952) . Direct evidence for the multiplication of aster yellows virus in its insect vector . Phytopathotogy 42, 59-64 . MAaAMOROSCH, K . (1953) . Incubation period of aster-yellows virus . Am . J . Botany 40, 797-809 . NASU, S . (1965) . Electron microscopic studies on transovarial passage of rice dwarf virus . Japan . J . Appl . Entomol . Zeal . 9, 225-237 . SHLKATA, E ., and MARAMOaoscn, K . (1965), Electron microscopic evidence for the systemic invasion of an insect host by a plant pathogenic virus . Virology 27, 461-475 . SINHA, R . C . (1965a) . Sequential infection and distribution of wound-tumor virus in the internal organs of a vector after ingestion of virus . Virology 26, 673-686 . SINHA, R. C . (1965b) . Recovery of potato yellow dwarf virus from hemolymph and internal organs of an insect vector . Virology 27, 118-119 . SINIA, It. C ., and BLACK, L . M . (1963) . Woundtumor virus antigens in the internal organs of an insect vector . Virology 21, 183-187 . SINHA, It . C ., and CmYxowsK :, L . N . (1967) . Multiplication of aster yellows virus in a nonvector leafhopper. Virology 31, 461 466. WmTCorm, R . F ., JENSEN, D . D ., and RICnARDsoN, J . (1966) . The infection of leafhoppers by Western X-disease virus . II . Fluctuation of virus concentration in the hemolymph after injection, Virology 28, 454-458 .