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A study of the cytological effects of aster yellows virus on its insect vector
VIROLOGY
10.483-500
A Study
(1%@)
of the Cytological Effects of Aster Virus on Its Insect Vector’ VIR(:INIA
C.
IATTAIT
ds~
.4ccepted January
KARL
Yellows
RIARAMOROWH
6, 1960
c:yt,ological examination of viruliferous Macrosteles fascifrons St&l adults, which had fed for at least 3 weeks on p1ant.s infected with the Eastern strain of the ast,er yellows virus, showed that the characteristic microscopic morphology of the fat body had undergone certain changes. In extreme cases the nuclei had changed from round or irregular to sharply stellate in shape, while t.he cytoplasm became less homogeneous. The fat hody in the extreme viruliferous type was shown to have suffered an actual loss of tissue, alt#hough t,his very likely resulted from its having become more soluble (as a consequence of the presence of virus) in the preparative reagents used. The cytological changes were most clearly demonstrated in males, although females showed some slight effects. No virus-caused cytological effects were detected in M. fascifrons with a second strain of the ast,er yellows virus, in other leafhoppers with the Eastern strain, or with corn stunt virus and its vector. Q%ochemical tests for glvcogen, prot,ein, and lipid, which were used in order fully to describe the fat body cells, showed no fnrt.hcr difference hatween virrlliferons and virns-free inserts. IiVTROl>UCTIOn‘ Many plant viruses transmit,ted by leafhoppers (Homopt’era, Cicadellidae) are in reality both plant’ and animal viruses because they have been shown t’o multiply in an insect as well as in a plant. These viruses cause diseases in t,heir plant host,s, but no obvious ill effect.s in their vect’ors. (For a review of these viruses, see Black, 1959; Maramorosch, 1959h.) An investigation started in 1955 sought t,o det,ermiue whether such viruses produced changes iu t#heir vectors that, had been overlooked and, if so, to locate any changes in a part,icular tissue or orgau in order that further work might, elucidat,e whether the site of any change ‘This (irant
work was supported in part by ITnitetl States Pllblic Numtwr
E-1537.
Healt,h Hervicc
484
LITTAU
AND MARAMOROSCH
might be the site of virus multiplication. Brief reports on the results of some experiments were published (Littau and Maramorosch, 1956a, b, 1958). The present paper describes in detail cytological changes that occur in the fat body tissue of Macrosteles jascijrons fQ,&l, an insect vector of aster yellows virus. Dobroscky (1929, 1931), using standard histological methods, made a careful examination of t,he aliment,ary tract and it,s associated organs and the salivary glands of viruliferous and virus-free ast.er leafhoppers. She found no evidence t’hat t,he ast,er yellows virus had any cytological effect on the insect,. Reports of cytological differences between viruliferous and virus-free insects were published by Blattny (1932) for Myxus persicae (Sulz.) with potato leaf roll virus, by Hartzell (1937) for Macropsis trimacu2ata (Fitch) wit,h peach yellows virus, and by Sukhov (1940) for Delphax striatella Fallen infected with oat pseudo-rosette virus. Other workers observed no differences between vector and nonvector aphids (see Bawden, 1950; Day and Bennet@ 1954). The findings of Sukhov, of Blattny, and of Hartzell have not been confirmed. Recently Jensen (1958, 1959) reported a pat,hological effect of the peach yellow leaf roll virus on its leafhopper vector. Those insects that became transmitters lived for an average of 22 days, as cont’rasted with 55 days for nontransmitters. No cytological &udies have been reported on insects infe&ed wit,h this virus. Similarly Watson and Sinha (1959) found that the production of nymphs by female Delphacodes pellucida Fabricius was considerably lessened by the acquisition of European wheat, st,riate mosaic virus, for which this leafhopper act’s as a vector. On t,he other hand, Severin (1947) found that the mont,hly death rates of virus-free and viruliferous adult aster leafhoppers on barley plants did not differ significantly. Severin concluded that the ast’er yellows virus is neit)her beneficial nor injurious to infect’ed adult aster leafhoppers. MATERIALS
AND METHOTIS
Culture Methods All work concerned with the growing of plants and insects was carried out in greenhouses below 25” C and in lighted incubators maintained at 25”. Insects were kept on individually caged plants, uncaged test plants were kept in a different greenhouse, and caged control and experimental plants were kept next to each other to achieve similar growing condi-
tions. When t,ransfers were made, nonviruliferous insect’s were always handled first, to avoid cont,aminat’ion if any escaped. Transfers were tnade in specia1 rooms located outside of t.he greenhouses and ittcubator rooms. Both adults and nymphs are capable of acquiring and transmitt,ing virus, and kot,h were used. Various viruses and vectors were employed in experimettts designed to investigate possible cytological effects. Most of t’he work was carried out’ with the East)ern strain of aster yellows virus and t’he leafhopper vccfor, Macrosteles fascifrons, maintrained on China aster (Callislephus chinensis Nees) plants. Some observst.iotts mere madr on A/. .fascifTcu~~s kept) ott carrot plattt,s (Daucus car&a I,.) infected with aster yelloivs and on healt,hy carrot cottt,rol plants. Two other species of leafhoppers M‘ere included: t’he corn leafhopper Dalbulus m,aidis Del,. and Wol. and the clover leafhopper ,4gallia con,stricta vt~u Ihzce, both of jvhich wt’r~’ examined after a suitable period of time spent on yellons asters. ltt addition, D. maidis adults wit#h cot-11stunt virus (tnaittt,aitted in Zest Mays I,.) and Al. fasc
486
LITTAU
AND
MARAMOROSCH
reaction for arginine (Thomas, 1950). Boiling trichloroacetic acid (TCA) was used to extract deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) (Schneider, 1945). For the specific removal of RNA, sections were digested with ribonuclease (Worthington Biochemical Company, Freehold, New Jersey). The Feulgen reaction for DNA was employed, using a K&minute hydrolysis in normal hydrochloric acid. Determination of the relative volumes of various tissue and cellular components was made by the microscopic method of Chalkley (1943), which consists of scoring the number of hits on the different components made by randomly moved point’s. OBSERVATIONS
AN11 RESULTS
Cytological Observations on M. fascifrons wit.h Aster Yellows Virus All experiments were begun with adult insects to avoid complications arising from cytological changes in the fat body and other tissues during molting, until it was discovered that 18-28 days were required for the appearance of cytological effects (Littau and Maramorosch, 1958). Then, in order that losses from deaths due t,o aging might be kept to a minimum, nymphs were used at the beginning of experiments. The duration of the experiment’s was such that, the nymphs always had metamorphosed int’o adults at least a few days before they were killed and prepared for microscopical observation. The validity of this procedure was established when examination of t,he fat body cells of adults that had been hatched and allowed to mature on yellows asters showed them to be no different from those of insects that had spent 3-4 weeks, as adults, on diseased plants. The fat body in these insects is a diffuse tissue rather than a discrete organ. It may be found mainly in the head and abdomen, where t’he cells appear t,o fill any space not already occupied by other organs. Serial longitudinal sections through entire insects established that the cytological appearance of the fat body was the same in all locations within a single individual, although it might look quite different in another individual. FIG. 1. Fat body cells of male Macrosteles fuscifrons, “virus-free” type, showing round nuclei (N), dense cytoplasm with small vacuoles, and discrete cells. Stained with azure B aft,er Carnoy fixation; magnification: X 1600. FIG. 2. Fat body tissue of male Macrosteles fuscijrons, “extreme viruliferous” type. Note stellate nuclei (N), sparse cytoplasm, and the apparent lack of cell boundaries. Stained with azure B after Carnoy fixation; magnification: X 1600.
CYTOLOGICAL
EFFECTS
OF
ASTER
FIGS. l-2
YELLOWS
VIRUS
487
488
LITTAU
AND
MARhMOROSCH
Male and female leafhoppers differed in the ease with which cytological response to the virus could be detected. As the effect was much more pronounced in the males, they will be considered first and in greater detail. Characteristic features of the fat body tissue found in virus-free adult M. fascifrons males were the clearly seen cell boundaries, the homogeneity of the basophilic material of the cytoplasm, and the smooth contours of the almost-round nuclei (Fig. 1). The cells frequently were binucleate. The extreme type of cytological change associated with aster yellows virus in M. fascifrons, shown in Fig. 2, was encount,ered in an average of 40% of the viruliferous males. The cell boundaries were typically difficult to see, and the formed tissue elements were generally sparse. Nuclei were invariably sharply stellate, or very irregular, in outline, as may be seen in Fig. 3, which shows fat body tissue stained by the Feulgen reaction, so that only nuclei are colored. An intermediate condit,ionof the fat body occurred in bothviruliferous and nonviruliferous males (Fig. 4). The tissue usually was composed of easily distinguishable discrete cells. The cytoplasm, markedly less homogeneous than in the first t’ype, had the appearance of a network which exhibited a stronger basophilia than that of the extreme viruliferous type. Nuclei were irregular in shape, that is, intermediate between the smoothly rounded ones found only in virus-free males and the sharpely stellate ones. The relative distribut,ion of the three types of fat body is shown in Fig. 5, from which it may be seen that 2 % of the viruliferous males had fat, body of “virus-free type.” It is likely that this 2 % represenm the small number of inseem which either never become transmitters or become poor transmitters of the virus. The data for Fig. 5 were taken from those experimenm in which the viruliferous insects had been maintained for at least 3 weeks prior to fixation (in Carnoy’s fluid) on asters diseased wit,h the Eastern st’rain of aster yellows virus, and t,he virus-free controls had been kept on healt,hy asters. The general trend of the cytological changes in the fat body of the male vector might be described as follows: the cell membranes became FIG. 3. Feulgen staining for deoxyribonucleic acid, showing nuclei in fat body tissue of “extreme viruliferous” type in male Macrosteles fascifrons. The marked irregularity of the nuclear (N) outline makes a sharply focused picture almost impossible to achieve. Carnoy fixation; magnification: X 1600. FIG. 4. “Intermediate” type of fat body tissue in male Macrosteles jascijrons. Nuclei (N) are irregular, cytoplasm more vacuolate. Stained with azure B after Carnoy fixation; magnification: X 1600.
CYTOLOGICAL
EFFECTS
OF
ASTER
FIGS. 3-4
YELLOWS
VIRI%
48!)
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LITTAU
AND
MARAMOROSCH
Adult
males
Id
Virus-free insects (Nz144) Viruliferous ‘1 (W124)
2 8 50 3 pl
L 30%
0
FIG.
-
2%
Virus-free
Interme-
type
diate type
5. Distribution of fat body types in
Macrosteles
Viru-
lifemus
type fascifrons
adlIlt males.
difficult to discern, nuclei became increasingly irregular in shape until some were stellate, and large empty spaces appeared in the tissue (Table 1). In order to describe adequately the changed appearance and empty spaces of the extreme viruliferous type, relative volume measurement#s (Chalkley, 1943) were made on tissue of this type and also on fat body of the virus-free type. In each case, the following ratios were determined : cytoplasm: nucleus and cytoplasm plus empty space: nucleus. The results are given in Table 2. For each determination the cumulative ratio was plotted against the number of readings until it no longer fluctuated; this followed Chalkley’s original method to establish that sufficient readings had been made. The ratio of cytoplasmic to nuclear volume remained about the same in both sets of insects, but the relative volume of tissue space increased markedly in the extreme viruliferous type. If it is assumed that the total volume of fat body per
CYTOLOGICAL
EFFECTS
OF
TABLE OF THE THREE fi1acrosfele.s fascijrons,
COMPARISON
Nuclear shape Cytoplasmic appearance C!ell boundarles
Round Homogeneous with small vacuoles 1Xstinct
I&l ribul ion
In
invirus-free sects and a very few viruliferoux insects
Male virus-free t,ype Male ext,reme viruliferous
t,vue
YELLOM’S
191
VIIEI:S
1
FAT BODY TYPES FOI:NI) IX AI)~LT VIRUS-FREE AND VIRULIFEROUS
Virus-free
Type of fat body t,ixsrle
ASTER
MAI,E
Ext,reme viruliferons
Mermediate
Stellate Sparse, stringy, ragged Invariably difficult to discern
Irregular Reticulat,c with large vacuoles I)istinct ; or. somctimes, difficult to discern In both virulifcrous and virus-frrc irb sects
1Sxclusivcly virulifcrous insecf s
in
i
-
cytop1:ism: nrlclcw 4.1 0. 4
c)kJpl:rsm plus empt,v space: nucleus 9 .:< 28.5
insect was the same for both t’ypes (there is no reason to think they differed greatly), then it is apparent that, the viruliferous, with a greater proportion of empty space, must, have lost both cytoplasmic and nuclear subst#ance. A decrease in the amount’ of formed elements in the tissue has been observed in Carnoy-fixed material, but not in material frozensubst#ituted or fixed by McManus’ method. The reason for this decrease will be discussed below. Cytological effects in the females appeared to be similar to those in hhe males, but the changes did not progress so far (Littau, 1960). Nuclei became stellate, but there was less t’endency for spaces to appear in t,he tissue. That these processes were less marked in t’he females may have bee11a reflect’ion of fat body activity relat#ed to egg production.
492
LITTAU
AND
MARAMOROSCH
A few observations were made on M. fascifrons males and females that had acquired aster yellows virus from diseased carrot plants by a 12-day feeding period. Three out of 12 males had extreme cytological changes in the fat body, while the 15 females responded very much the same as on yellows asters. About a year later, some M. fascijmns which had been maintained for 4 weeks on healthy carrots (8 males and 16 females) were sectioned. Their fat body tissues appeared normal, that is, the same as those of insects maintained on healthy asters. In most of the experiments the viruliferous insects were kept on diseased asters for the duration of the experiment. A diseased aster, however, might be expected to differ chemically from a healthy one by the accumulation or reduction of substances associated with virus multiplication, in addition to the presence of the virus itself. To exclude the possible effect of such changes, acquired through feeding on diseased plants, insects were mechanically infected by needle inoculation. Healthy insects were injected with the juice of viruliferous ones; after a suitable period of time on immune rye and healthy aster plants, the insects were examined cytologically. First of all, it was found that none of the 6 male controls, which were injected with the juice of nonviruliferous insects, showed cytological effects. Of the injected viruliferous males, only 4 out of 12 were close to the extreme viruliferous condition of the fat body (Fig. 6); the rest were intermediate. This demonstrated that feeding on the diseased plant was not the sole cause of the changes, but it suggested t)hat such feeding might augment the effect of t’he virus. A possible source of error in this experiment might be the fact that a brei of whole diseased insects was used as the inoculum for injection. Since these “source” insects were taken from diseased asters, their bodies may have contained some plant products. Because of the instability in vitro of the aster yellows virus, it was not considered feasible to try to purify it. There was some evidence, besides that from injection experiments, that viruliferous insects which remained the entire time on a diseased plant showed more pronounced cytological changes than those which were allowed to acquire virus but were subsequently maintained on virus-free plants. In two experiments, insects that had been allowed to feed on diseased asters for a week or 10 days were then transferred to healthy asters for the customary 34 week period. Only 3% of these insects (1 out of 31 individuals) showed the extreme effects of the virus, whereas 38 % (15 out of 39 individuals) of a second group of insects
Flc:. 6. Fat body tissue of male Mucros~eles fasciJron.s which had acquired astrr yellows virus via injection. Note stellate nrlclei CN), fitringy cytoplnsm. and poorly visible cell houndaries, all characteristic of t,he “exutreme virulifr~wrls” type. Ht.ained with azure B aft,er Carnoy fixation; m:tgnifir:~tion: X 1600. FIGS. 7 and 8. A set, of control (Fig. 7) and rit,ollllclease-trelte(i (,Fig. 8) sectionn of male Macros/e/es Jasc
494
LI’ITAU
AND
MARAMOROSCH
studied during the same period but kept on diseased aster for the entire time, had the extreme cytological condition. If a con&ant supply of virus or virus by-products in the food is necessary for the full expression of the cytological effects, this could explain why they failed to develop in the first group. In connection with the above observations, it was thought desirable t,o establish if any correlat,ion exists between the appearance of t,he ext’reme viruliferous cytological condit,ion and ability to t,ransmit virus. Such a hypothesis assumes that a shorter feeding period results in t,he acquisition of a smaller amount, of virus, and postulat,es that both transmission ability and the severity of the cytological effects are direct)ly proportional to the amount, of virus received by the insect. For this purpose, records of transmitting ability and fat body appearance for each individual insect were required; therefore insects were allowed a feeding period of 4 days on diseased asters, after which groups of insects were transferred to healthy aster plants, and subsequently each insect was tested individually for transmitting ability for 1 week on each of two healthy aster plants. At the end of the experiment 34 males were collected, fixed in Carnoy’s fluid, and examined for cytological effects on their fat body cells. In this experiment, therefore, each test insect fed on a diseased aster for 4 days and then was kept on healthy asters until 4 weeks from the beginning of its virus acquisit,ion-feeding period. The control insects were kept throughout the experiment in groups on several healthy aster plants; 36 virus-free males were collected at the end. Transmission by 28 % of the viruliferous insects was recorded. However, all the viruliferous insects showed the intermediate condition of the fat body, rather than the extreme, and this was considered additional evidence that feeding on diseased plant,s furthers the expression of the extreme viruliferous condition. The virus-free controls also showed the intermediate condition. A similar experiment was tried using insects iujected with the juice of viruliferous insects: after the injection the insects were kept on rye plants for 16 days, then transferred to individual healthy aster plants for 7 days. Unfortunately only 6 males survived to the end of the experiment, and all these showed the intermediate fat body condition. None of them transmitted the virus. Of the 2 surviving virus-free injected controls, one had the intermediate and one the virus-free fat, body type. The number of insects in this injection experiment was small, but the results agreed with the other findings that, constant feeding on diseased plant’s intensifies the cytological effect of the virus.
C’ytochemical Observations Since one of the chief effects of the aster yellows virus 011N. jascijrons was to cause a decrease in the amount of cytoplasm, test,s were made to identify some of the cytoplasmic components chemically. That the cytoplasm contained ribonucleic acid was shown hy digestion of et’hunol-fixed, frozen-subst,ituted sect,ions wit,h ribonuclease, which abolished t,he basophilia of the cytoplasm of the fat’ body cells’ (Figs. 7, 8). The removal of basophilia by a specific reagent such as t’he enzyme ribonuclease is evidence that RNA was t’he basophilic substance. This result, was checked by the use of hot trichloroacetic acid, which exfract,s bot,h DNA and Rh’A. Neither the nucleus nor cytoplasm of t’he fat body cells retained any basophilia after the treat#ment. The cytoplasm was also shown to contain prot.ein in approximat,ely the same location as t,he RNA, by means of naphthol yellow S staining and the Sakaguchi arginine reaction. At least, a part’ of the prot.ein and RXA probably occur as a ribonucleoprotein complex in the cells. The Feulgen reaction failed to reveal any further difference bet’wcen viruliferous and nonviruliferous aster leafhoppers with rrspcct to thn localization of DNA, which was exclusively nuclear. The insect fat body, like t,he vertebrat’e liver, is a storage organ as well as a metabolic organ. The localization and approximat,e relative amount,s of the stored subst.ances glycogen and lipid were compared in 1l011nu1and viruliferous M. juscijrons. It1 general, both the substances were present, in the cytoplasm of the fat body cells of all insects, where the amount’ and distribution varied greatly hut were independent of the insect’s condit.ion with respect to t.he virus. AIost of the insect material used for these t,ests was fixed in hot, ethanol after freeze-substitution OI by tiIcMnnus’ (1946a) special method for lipid. (.‘ytological r)bservations
(‘sing
Othrr I,wfhoppcw
and Other P’irt~.sc.s
Two other species of leafhoppers that acquired the aster yellows virus by feeding on diseased aster plants were studied. They were t,he corn leafhopper Dalbulus maidis and t,he clover leafhopper Agallia con~tricta. The virus can he recovered from them, aft,er a suitable period of time, by injection of the juices of t,hese insects int,o &f. fascifrons (Maramorosch, 1952), ahhough neit’hcr the corn nor t,he clover leafhopper becomes a transmitter of the virus. The fat body cells of -31 male aud female A. constricta adu1t.s that, had fed on yellows ast,ers for from 7 to 9 weeks were examined, and no difference was found betweeit them and the 35 controls on healthy asters.
496
LITTAU
AND
MARAMOROSCH
The experimental procedure for studying the effect of the yellows virus on D. maidis was somewhat different because this leafhopper ordinarily will not feed on healthy asters although it lives quite well on yellows asters (Maramorosch, 1958). Therefore, the insects acquired virus by feeding on diseased asters for 6 hours a day on 4 successive days, while controls were placed on healthy asters for the same time. All were kept on healthy corn plants between feeding times on asters and for the rest of the experiment (the total time was 4 weeks). There was no cytological difference between the 47 controls and the 51 male and female leafhoppers t,hat had had the opportunity to acquire virus. Also available were 7 males that had spent 35 days on yellows asters; their fat bodies were no different from the above-mentioned controls. A strain of the aster yellows virus that infects celery had no cyt,ological effect on M. fascijrons; this was found using 25 nonviruliferous males, 35 males with the celery-infecting strain, and 35 males with the Eastern strain, (of which 4, or II%, showed cytological changes), for comparison. This seemed all the more surprising because this strain causes much more severe symptoms in the plant host than does the Eastern strain used for the other experiments. Two strains of the corn stunt virus, Rio Grande and Mesa Central, were tested in one of their vect’ors, D. maidis. Both males and females were examined: 38 nonviruliferous, 44 with the Rio Grande strain, and 44 with the Mesa Central strain. Again no cyt’ological effect was seen in either males or females. In summary, so far, no cytological changes have been observed in any of the other leafhoppers infected with viruses that were studied, or in M. fascifrons with the celery-infecting strain of aster yellows virus. I)ISCUSSION
The findings of cytological changes, reported in detail in this paper, are the first known changes caused by a plant-pathogenic virus in an invertebrate animal. The lack of a completely clear distinction between plant and animal viruses has already been recognized for several years (Maramorosch, 1954; Enders, 1957), as the consequence of the vast amount of evidence for multiplication of certain plant viruses in insects. Although acute and chronic virus infections were known to be caused by yellows-type viruses in numerous species of plants, insects were usually considered as mere carriers and believed to suffer no ill effects. In the case of aster yellows virus, after the initial 4 days of the incubation
(‘YTOLOQICAL
EFFECTS
OF
ASTER
YELLON’S
VIRI’S
497
period, during which time virus cannot be recovered from the insect, t#heinfectlion becomes systemic and the virus can be recovered from t’hr hemolymph, although it cannot yet be transmitt,ed through t,he insect”s mouthparts. Transmission may begin after 9 days, but only after 18 or more days does the infection become expressed, as is evident’ from cytological changes t’hat appear at this t,ime. These changes seem to bc enhanced by constant feeding on diseased plants, which obviously favors the expression of the extreme condit,ion. Superinfection wit,h t’he virus is anot,her possible explanation for the findings. Such superinfect,ion probably t#akes place during prolonged feeding on diseased plants. The originally acquired virus may cause a systemic infection and establish an equilibrium with its invert~ebratc host, only rarely causing fat, body changes. When additional virus is acquired, this superinfection may upset the ecfuilibrium. Superinfection wit,h host,-specific viruses has been listed by Bergold (19%) as OW of the possible causes of activation of lat,ent viruses in insects. The possible toxic effect of diseased asters cannot, bc ruled out complet,ely, alt,hough no evidence for toxicit,y was found. On the contrary, astIer leafhoppers were reported to breed more prolitically on diseased plant’s (Severin, 194(i), which seems to deny the possibility of a iosic substance t’hat might, be acquired from diseased asters. There has been a tradiGona1 assumption that if vectors are affect,ed in any way at all by t,he virus t’hey carry, it, must bc pathologically, because viruses are pat,hological by their very nst,ure. Although t’here in no doubt t’hat! in diseased plants aster yellows virus is a pat,hogenic parasit)e, in t’he insect, host it may be more like a commensal or even a symbiont and changes caused by it are not necessarily pathogenic. In this connect~ion the work of Severin (1946) might again be recalled. Severin’s experiments indicated that vect,orship is a function of special biological significance, in t#hat, it indirectly aids t,hc insect’s multiplicatiotl. The contrary is t,rue for the infect,ed pIalIt, which becomes sterile in thr course of the disease. The problem of the underlying chemical events hchind tbe chattged appearance of the fat body of viruliferous insect8s remains ohscure, butf a general explanation may be suggest,ed. First of all, the decrease in actual Cssue subst(ance may only be apparent; some of the Gssue mttg have been, and indeed, probably was, lost during the Car&y fixation and the embedding process. The fact, t,hat frozen-substituted viruliferous ittsect s as well as t’hose fixed in McMnnus’ formalin-cobalt-c:Jrium fluid
498
LITTAU
AND
MARAMOROSCH
did not show the loss of tissue supports this hypothesis. This means that one effect of the virus was to render the tissue more easily extractable by histological reagents, a condition that might have resulted from a change in the solubility properties of either the nucleic acids or proteins or both, in the cytoplasm and nuclei. It is also possible that only the cytoplasm was involved; if the cytoplasm surrounding a nucleus were dissolved out, the nucleus might be carried along with it. It, is impossible at present to relate either a qualitat#ive or quantit,ative change in fat body RKA directly to virus multiplication, the actual sit,e of which is not known. The fat body occupies a position of considerable importance in the metabolism of the insect, with the result that events taking place in other parts of its body undoubtedly have an effect on these cells. However, the fact that a great deal of synthetic activity normally occurs in the fat body also makes it a not unlikely place for virus synthesis as well. In any case, the length of time required for the cytological changes would seem to rule out their immediate relation t’o virus multiplication: the incubation period of the virus in the insect was only 9-12 days, whereas from 18 to 28 days had to elapse after acqui&ion of virus before changes were seen in the fat body. Finally, mention should be made of the interesting results with the celery-infecting strain of aster yellows. This strain, as was already noted, caused much more severe stunting and chlorosis of the diseased asters than the East,ern strain, but had no cytological effect on the vector, M. fascifrms. In this connection, Maramorosch (1959a) has observed that the celery-infecting strain also failed to condition corn leafhoppers to live on asters, unlike the Eastern strain. Both sets of facts imply that! the two virus strains differ by at least some chemical processes they cause in the plan@ or possibly also by their direct effects on the insect. REFERENCES F. C. (1959). Plant Viruses and Vz’rus Diseases, 3rd ed. Chronica BoPress, Waltham, Massachusetts. BERGOLD, G. H. (1958). Viruses of insects. In Handbuch der VGusforschung, Vol. 4, pp. 60-142. BLACK, L. M. (1959). Biological cycles of plant viruses in insect vectors. In The Viruses (F. M. Burn& and W. M. Stanley, eds.), Vol 2, pp. 157-185. Academic Press, New York. BLATTNY, C. (1932). Lzezjistiti pisitomnost viru pfisobiciho nekter6 ohoroby Brambord v jejich pfen&ReEi, mgicich? (Can the viruses that cause certain potato diseases be det,ected in their aphid vectors?) VbtnZk kr(lZov. Ceske spoleSnr,sti nauk 1931, No. 4, 7 pp. (Czech, wit,h English summary.) BAWDEN,
tanica
CYTOLOGICAL
EFFECTS
OF
ASTER
YELLOWS
VIRUS
4%
H. W. (1943). Method for the quantitative morphologic analysis of tissues. J. Natl. Cancer Inst. 4, 47-53. DAY, M. F., and BENNETTS, M. J. (1954). A review of problems of specificity in arthropod vectors of plant and animal viruses. C.Y.I.R.O., Canberra, Australia. 171 pp. (Mimeographed.) LIEITCH, A. D. (1955). Microspectrophotometric study of the binding of the &IIionic dye, Naphthol Yellow S, by tissue sections and by purified proteins. Lu6. Invest. 4, 32+351. I)OBROSCKY, I. 1). (1929). Is the aster-yellows virus detectable in its insect. vect,or? Phytopalhclogy 19, 1009-1015. DOBROSCKY, I. 1). (1931). Morphological and cytological studies on thr salivur?
CHALKLEY,
glands and alimentary tract of Cicadxla sernolata (Fallen), the carrier of ast (‘I >rellows virus. Co&ribs. Royce Thompson Inst. 3, 3’3-58. KNDERS, J. F. (1957). The future of virus studies in tissue cult urp. J. l$‘tct(. Cancer
Inst. 19, 735-742.
FLAX, H. M., and HIMER, M. H. (1952). Microxpeotrophot,ometric analyses of metachromatic staining of nucleic acids. Physiol. Zoiil. 26, 297-311. HARTZELI,, A. (1937). Movement of intracellular bodies associaf rd with peach yellows. (“ontribs. Boyce l’hompson Inst. 8, 375-388.
HOX,HKISS, R. I>. (1948). A microchemical react,ion result,ing in t,he staining of polysaccharide structures in fixed tissue preparations. .Irch. Biochet~. 16, 131. 111, JEW&N, I). I). (1058). Reduction of longevity of leafhoppers (xrrging prach yellow leaf roll virus. Phytopalhology 48, 3Q4. (Abstract. ) JENSEN, I). 1). (,lQ59). A plant virus lethal to its insect vector. Virolqq 8, 164-175. JOHASREK, I). A. (1940). Plan1 Microlechniqcte, pp. 13&lX~. M~(;rawHill, N~u York. IATTAC, V. C. (1960). Cyt,ological dif-fcrences between the fat body tissue of adult male and female leafhoppers (Homoptera, Cicadellidae). J. Morphology, in press. IATT.~I., V. C., and MARAMOROSCH, K. (1956ai. Cytological changes in Macrosteles ,fasci~rons fed on plants infected with aster >-ellows virus. I’h,~~topathwlogy 46, 468. (Abstract.) LITTA~~, L’. C., and MARAM~ROSCH, K. (1956b). Cytologiral effeptx of aster-qlloas virus on its insect, vector. Virology 2, 128-130. LITTAL-, V. C., and MARAMOROSCH, K. (1958). Cytopathogenic effects of the astrt yellows virus on its insect vector. Phytopath~ology 48. 263. (Abstract..) hl~~hl~~us, J. F. A. (1946a). 1)emonstration of cert.ain fatty subsfames in paraffin sections. J. Pafhol. Bacterial. 68, 93-95. MCWIANLTS, J. F. A. (1946b). Histological demonstration of mucin after periodic: acid. AJature 168, u)2. (Abstract.) MARAMOROSCH, K. (1952). Studies on the nature of the specific transmission of aster-yellows and corn-stunt viruses. Phytopathology 42, 663-668. MARAMOROSCH, K. (1954). Biological transmission of plant viruses by animal vectors. Trans. N. Y. Acad. Sci. 12116, 189-195. ~~ARAMoROS(‘H, K. (1956). Semiautomatic equipment. for injecting insects with measured amom~ts of liquids containing viruses or toxic substances. Ph?/topathology
46,
188-190.
500
LITTAU
AND
MAHhMOHOSCH
MARAMOROSCH, K. (1953). Beneficial effect of virus diseased plants on non-vector
insects. Tijdschr. Planienziekten 64, 383-391. MARAMOROSCH, K. (1959a). Further tests on the beneficial effects of aster yellows on corn leafhoppers. Phytopathology 49, 545. (Abstract.) MARAMOROSCH, K. (195913).Leafhoppers (Cicadellidae) as vectors and reservoirs of phytopathogenic viruses. Horn. Vol. Savulescu, Rumanian Acad. Sci., Bu-
charest, pp. 403-421. SCHNEIDER, W. C. (1945). Phosphorus compounds in animal tissues. I. Extraction
and estimation
of desoxypentose nucleic acid and of pentoae nucleic acid. J.
Biol. Chem. 161, 293-303. SEVERIN, H. H. P. (1946). Longevity,
or life histories, of leafhopper species on 17, 121-137. virus-infected and on healthy plants. Hilgardia SEVERIN, H. H. I’. (1947). Longevity of noninfect)ive and infective leafhoppers on a plant, nonsusceptible to a virus. Hilgardia 17, 54-543. SUKHOV, K. S. (1940). X-Bodies in salivary glands of Delphax striate&z Fallen, the carrier of zakuklivanie. Compt. rend. acnd. sci. U. R. S. S. 27, 377-379. THOMAR, L. E. (1950). An improved arginine histochemical method. Stain Technol. 26, 143-14s. WATSON, M. A., and SINHA, R. C. (1959). Studies on the transmission of European wheat striate mosaic virus by Delphacodes pellucida Fabricius. Virology 8, 139-163. Woons, P. S., and P'OLLISTER, A. W. (1955). An ice-solvent method of drying frozen tissue for plant cytology. Stain Technol. 30, 123-131.
10.483-500
A Study
(1%@)
of the Cytological Effects of Aster Virus on Its Insect Vector’ VIR(:INIA
C.
IATTAIT
ds~
.4ccepted January
KARL
Yellows
RIARAMOROWH
6, 1960
c:yt,ological examination of viruliferous Macrosteles fascifrons St&l adults, which had fed for at least 3 weeks on p1ant.s infected with the Eastern strain of the ast,er yellows virus, showed that the characteristic microscopic morphology of the fat body had undergone certain changes. In extreme cases the nuclei had changed from round or irregular to sharply stellate in shape, while t.he cytoplasm became less homogeneous. The fat hody in the extreme viruliferous type was shown to have suffered an actual loss of tissue, alt#hough t,his very likely resulted from its having become more soluble (as a consequence of the presence of virus) in the preparative reagents used. The cytological changes were most clearly demonstrated in males, although females showed some slight effects. No virus-caused cytological effects were detected in M. fascifrons with a second strain of the ast,er yellows virus, in other leafhoppers with the Eastern strain, or with corn stunt virus and its vector. Q%ochemical tests for glvcogen, prot,ein, and lipid, which were used in order fully to describe the fat body cells, showed no fnrt.hcr difference hatween virrlliferons and virns-free inserts. IiVTROl>UCTIOn‘ Many plant viruses transmit,ted by leafhoppers (Homopt’era, Cicadellidae) are in reality both plant’ and animal viruses because they have been shown t’o multiply in an insect as well as in a plant. These viruses cause diseases in t,heir plant host,s, but no obvious ill effect.s in their vect’ors. (For a review of these viruses, see Black, 1959; Maramorosch, 1959h.) An investigation started in 1955 sought t,o det,ermiue whether such viruses produced changes iu t#heir vectors that, had been overlooked and, if so, to locate any changes in a part,icular tissue or orgau in order that further work might, elucidat,e whether the site of any change ‘This (irant
work was supported in part by ITnitetl States Pllblic Numtwr
E-1537.
Healt,h Hervicc
484
LITTAU
AND MARAMOROSCH
might be the site of virus multiplication. Brief reports on the results of some experiments were published (Littau and Maramorosch, 1956a, b, 1958). The present paper describes in detail cytological changes that occur in the fat body tissue of Macrosteles jascijrons fQ,&l, an insect vector of aster yellows virus. Dobroscky (1929, 1931), using standard histological methods, made a careful examination of t,he aliment,ary tract and it,s associated organs and the salivary glands of viruliferous and virus-free ast.er leafhoppers. She found no evidence t’hat t,he ast,er yellows virus had any cytological effect on the insect,. Reports of cytological differences between viruliferous and virus-free insects were published by Blattny (1932) for Myxus persicae (Sulz.) with potato leaf roll virus, by Hartzell (1937) for Macropsis trimacu2ata (Fitch) wit,h peach yellows virus, and by Sukhov (1940) for Delphax striatella Fallen infected with oat pseudo-rosette virus. Other workers observed no differences between vector and nonvector aphids (see Bawden, 1950; Day and Bennet@ 1954). The findings of Sukhov, of Blattny, and of Hartzell have not been confirmed. Recently Jensen (1958, 1959) reported a pat,hological effect of the peach yellow leaf roll virus on its leafhopper vector. Those insects that became transmitters lived for an average of 22 days, as cont’rasted with 55 days for nontransmitters. No cytological &udies have been reported on insects infe&ed wit,h this virus. Similarly Watson and Sinha (1959) found that the production of nymphs by female Delphacodes pellucida Fabricius was considerably lessened by the acquisition of European wheat, st,riate mosaic virus, for which this leafhopper act’s as a vector. On t,he other hand, Severin (1947) found that the mont,hly death rates of virus-free and viruliferous adult aster leafhoppers on barley plants did not differ significantly. Severin concluded that the ast’er yellows virus is neit)her beneficial nor injurious to infect’ed adult aster leafhoppers. MATERIALS
AND METHOTIS
Culture Methods All work concerned with the growing of plants and insects was carried out in greenhouses below 25” C and in lighted incubators maintained at 25”. Insects were kept on individually caged plants, uncaged test plants were kept in a different greenhouse, and caged control and experimental plants were kept next to each other to achieve similar growing condi-
tions. When t,ransfers were made, nonviruliferous insect’s were always handled first, to avoid cont,aminat’ion if any escaped. Transfers were tnade in specia1 rooms located outside of t.he greenhouses and ittcubator rooms. Both adults and nymphs are capable of acquiring and transmitt,ing virus, and kot,h were used. Various viruses and vectors were employed in experimettts designed to investigate possible cytological effects. Most of t’he work was carried out’ with the East)ern strain of aster yellows virus and t’he leafhopper vccfor, Macrosteles fascifrons, maintrained on China aster (Callislephus chinensis Nees) plants. Some observst.iotts mere madr on A/. .fascifTcu~~s kept) ott carrot plattt,s (Daucus car&a I,.) infected with aster yelloivs and on healt,hy carrot cottt,rol plants. Two other species of leafhoppers M‘ere included: t’he corn leafhopper Dalbulus m,aidis Del,. and Wol. and the clover leafhopper ,4gallia con,stricta vt~u Ihzce, both of jvhich wt’r~’ examined after a suitable period of time spent on yellons asters. ltt addition, D. maidis adults wit#h cot-11stunt virus (tnaittt,aitted in Zest Mays I,.) and Al. fasc
486
LITTAU
AND
MARAMOROSCH
reaction for arginine (Thomas, 1950). Boiling trichloroacetic acid (TCA) was used to extract deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) (Schneider, 1945). For the specific removal of RNA, sections were digested with ribonuclease (Worthington Biochemical Company, Freehold, New Jersey). The Feulgen reaction for DNA was employed, using a K&minute hydrolysis in normal hydrochloric acid. Determination of the relative volumes of various tissue and cellular components was made by the microscopic method of Chalkley (1943), which consists of scoring the number of hits on the different components made by randomly moved point’s. OBSERVATIONS
AN11 RESULTS
Cytological Observations on M. fascifrons wit.h Aster Yellows Virus All experiments were begun with adult insects to avoid complications arising from cytological changes in the fat body and other tissues during molting, until it was discovered that 18-28 days were required for the appearance of cytological effects (Littau and Maramorosch, 1958). Then, in order that losses from deaths due t,o aging might be kept to a minimum, nymphs were used at the beginning of experiments. The duration of the experiment’s was such that, the nymphs always had metamorphosed int’o adults at least a few days before they were killed and prepared for microscopical observation. The validity of this procedure was established when examination of t,he fat body cells of adults that had been hatched and allowed to mature on yellows asters showed them to be no different from those of insects that had spent 3-4 weeks, as adults, on diseased plants. The fat body in these insects is a diffuse tissue rather than a discrete organ. It may be found mainly in the head and abdomen, where t’he cells appear t,o fill any space not already occupied by other organs. Serial longitudinal sections through entire insects established that the cytological appearance of the fat body was the same in all locations within a single individual, although it might look quite different in another individual. FIG. 1. Fat body cells of male Macrosteles fuscifrons, “virus-free” type, showing round nuclei (N), dense cytoplasm with small vacuoles, and discrete cells. Stained with azure B aft,er Carnoy fixation; magnification: X 1600. FIG. 2. Fat body tissue of male Macrosteles fuscijrons, “extreme viruliferous” type. Note stellate nuclei (N), sparse cytoplasm, and the apparent lack of cell boundaries. Stained with azure B after Carnoy fixation; magnification: X 1600.
CYTOLOGICAL
EFFECTS
OF
ASTER
FIGS. l-2
YELLOWS
VIRUS
487
488
LITTAU
AND
MARhMOROSCH
Male and female leafhoppers differed in the ease with which cytological response to the virus could be detected. As the effect was much more pronounced in the males, they will be considered first and in greater detail. Characteristic features of the fat body tissue found in virus-free adult M. fascifrons males were the clearly seen cell boundaries, the homogeneity of the basophilic material of the cytoplasm, and the smooth contours of the almost-round nuclei (Fig. 1). The cells frequently were binucleate. The extreme type of cytological change associated with aster yellows virus in M. fascifrons, shown in Fig. 2, was encount,ered in an average of 40% of the viruliferous males. The cell boundaries were typically difficult to see, and the formed tissue elements were generally sparse. Nuclei were invariably sharply stellate, or very irregular, in outline, as may be seen in Fig. 3, which shows fat body tissue stained by the Feulgen reaction, so that only nuclei are colored. An intermediate condit,ionof the fat body occurred in bothviruliferous and nonviruliferous males (Fig. 4). The tissue usually was composed of easily distinguishable discrete cells. The cytoplasm, markedly less homogeneous than in the first t’ype, had the appearance of a network which exhibited a stronger basophilia than that of the extreme viruliferous type. Nuclei were irregular in shape, that is, intermediate between the smoothly rounded ones found only in virus-free males and the sharpely stellate ones. The relative distribut,ion of the three types of fat body is shown in Fig. 5, from which it may be seen that 2 % of the viruliferous males had fat, body of “virus-free type.” It is likely that this 2 % represenm the small number of inseem which either never become transmitters or become poor transmitters of the virus. The data for Fig. 5 were taken from those experimenm in which the viruliferous insects had been maintained for at least 3 weeks prior to fixation (in Carnoy’s fluid) on asters diseased wit,h the Eastern st’rain of aster yellows virus, and t,he virus-free controls had been kept on healt,hy asters. The general trend of the cytological changes in the fat body of the male vector might be described as follows: the cell membranes became FIG. 3. Feulgen staining for deoxyribonucleic acid, showing nuclei in fat body tissue of “extreme viruliferous” type in male Macrosteles fascifrons. The marked irregularity of the nuclear (N) outline makes a sharply focused picture almost impossible to achieve. Carnoy fixation; magnification: X 1600. FIG. 4. “Intermediate” type of fat body tissue in male Macrosteles jascijrons. Nuclei (N) are irregular, cytoplasm more vacuolate. Stained with azure B after Carnoy fixation; magnification: X 1600.
CYTOLOGICAL
EFFECTS
OF
ASTER
FIGS. 3-4
YELLOWS
VIRI%
48!)
490
LITTAU
AND
MARAMOROSCH
Adult
males
Id
Virus-free insects (Nz144) Viruliferous ‘1 (W124)
2 8 50 3 pl
L 30%
0
FIG.
-
2%
Virus-free
Interme-
type
diate type
5. Distribution of fat body types in
Macrosteles
Viru-
lifemus
type fascifrons
adlIlt males.
difficult to discern, nuclei became increasingly irregular in shape until some were stellate, and large empty spaces appeared in the tissue (Table 1). In order to describe adequately the changed appearance and empty spaces of the extreme viruliferous type, relative volume measurement#s (Chalkley, 1943) were made on tissue of this type and also on fat body of the virus-free type. In each case, the following ratios were determined : cytoplasm: nucleus and cytoplasm plus empty space: nucleus. The results are given in Table 2. For each determination the cumulative ratio was plotted against the number of readings until it no longer fluctuated; this followed Chalkley’s original method to establish that sufficient readings had been made. The ratio of cytoplasmic to nuclear volume remained about the same in both sets of insects, but the relative volume of tissue space increased markedly in the extreme viruliferous type. If it is assumed that the total volume of fat body per
CYTOLOGICAL
EFFECTS
OF
TABLE OF THE THREE fi1acrosfele.s fascijrons,
COMPARISON
Nuclear shape Cytoplasmic appearance C!ell boundarles
Round Homogeneous with small vacuoles 1Xstinct
I&l ribul ion
In
invirus-free sects and a very few viruliferoux insects
Male virus-free t,ype Male ext,reme viruliferous
t,vue
YELLOM’S
191
VIIEI:S
1
FAT BODY TYPES FOI:NI) IX AI)~LT VIRUS-FREE AND VIRULIFEROUS
Virus-free
Type of fat body t,ixsrle
ASTER
MAI,E
Ext,reme viruliferons
Mermediate
Stellate Sparse, stringy, ragged Invariably difficult to discern
Irregular Reticulat,c with large vacuoles I)istinct ; or. somctimes, difficult to discern In both virulifcrous and virus-frrc irb sects
1Sxclusivcly virulifcrous insecf s
in
i
-
cytop1:ism: nrlclcw 4.1 0. 4
c)kJpl:rsm plus empt,v space: nucleus 9 .:< 28.5
insect was the same for both t’ypes (there is no reason to think they differed greatly), then it is apparent that, the viruliferous, with a greater proportion of empty space, must, have lost both cytoplasmic and nuclear subst#ance. A decrease in the amount’ of formed elements in the tissue has been observed in Carnoy-fixed material, but not in material frozensubst#ituted or fixed by McManus’ method. The reason for this decrease will be discussed below. Cytological effects in the females appeared to be similar to those in hhe males, but the changes did not progress so far (Littau, 1960). Nuclei became stellate, but there was less t’endency for spaces to appear in t,he tissue. That these processes were less marked in t’he females may have bee11a reflect’ion of fat body activity relat#ed to egg production.
492
LITTAU
AND
MARAMOROSCH
A few observations were made on M. fascifrons males and females that had acquired aster yellows virus from diseased carrot plants by a 12-day feeding period. Three out of 12 males had extreme cytological changes in the fat body, while the 15 females responded very much the same as on yellows asters. About a year later, some M. fascijmns which had been maintained for 4 weeks on healthy carrots (8 males and 16 females) were sectioned. Their fat body tissues appeared normal, that is, the same as those of insects maintained on healthy asters. In most of the experiments the viruliferous insects were kept on diseased asters for the duration of the experiment. A diseased aster, however, might be expected to differ chemically from a healthy one by the accumulation or reduction of substances associated with virus multiplication, in addition to the presence of the virus itself. To exclude the possible effect of such changes, acquired through feeding on diseased plants, insects were mechanically infected by needle inoculation. Healthy insects were injected with the juice of viruliferous ones; after a suitable period of time on immune rye and healthy aster plants, the insects were examined cytologically. First of all, it was found that none of the 6 male controls, which were injected with the juice of nonviruliferous insects, showed cytological effects. Of the injected viruliferous males, only 4 out of 12 were close to the extreme viruliferous condition of the fat body (Fig. 6); the rest were intermediate. This demonstrated that feeding on the diseased plant was not the sole cause of the changes, but it suggested t)hat such feeding might augment the effect of t’he virus. A possible source of error in this experiment might be the fact that a brei of whole diseased insects was used as the inoculum for injection. Since these “source” insects were taken from diseased asters, their bodies may have contained some plant products. Because of the instability in vitro of the aster yellows virus, it was not considered feasible to try to purify it. There was some evidence, besides that from injection experiments, that viruliferous insects which remained the entire time on a diseased plant showed more pronounced cytological changes than those which were allowed to acquire virus but were subsequently maintained on virus-free plants. In two experiments, insects that had been allowed to feed on diseased asters for a week or 10 days were then transferred to healthy asters for the customary 34 week period. Only 3% of these insects (1 out of 31 individuals) showed the extreme effects of the virus, whereas 38 % (15 out of 39 individuals) of a second group of insects
Flc:. 6. Fat body tissue of male Mucros~eles fasciJron.s which had acquired astrr yellows virus via injection. Note stellate nrlclei CN), fitringy cytoplnsm. and poorly visible cell houndaries, all characteristic of t,he “exutreme virulifr~wrls” type. Ht.ained with azure B aft,er Carnoy fixation; m:tgnifir:~tion: X 1600. FIGS. 7 and 8. A set, of control (Fig. 7) and rit,ollllclease-trelte(i (,Fig. 8) sectionn of male Macros/e/es Jasc
494
LI’ITAU
AND
MARAMOROSCH
studied during the same period but kept on diseased aster for the entire time, had the extreme cytological condition. If a con&ant supply of virus or virus by-products in the food is necessary for the full expression of the cytological effects, this could explain why they failed to develop in the first group. In connection with the above observations, it was thought desirable t,o establish if any correlat,ion exists between the appearance of t,he ext’reme viruliferous cytological condit,ion and ability to t,ransmit virus. Such a hypothesis assumes that a shorter feeding period results in t,he acquisition of a smaller amount, of virus, and postulat,es that both transmission ability and the severity of the cytological effects are direct)ly proportional to the amount, of virus received by the insect. For this purpose, records of transmitting ability and fat body appearance for each individual insect were required; therefore insects were allowed a feeding period of 4 days on diseased asters, after which groups of insects were transferred to healthy aster plants, and subsequently each insect was tested individually for transmitting ability for 1 week on each of two healthy aster plants. At the end of the experiment 34 males were collected, fixed in Carnoy’s fluid, and examined for cytological effects on their fat body cells. In this experiment, therefore, each test insect fed on a diseased aster for 4 days and then was kept on healthy asters until 4 weeks from the beginning of its virus acquisit,ion-feeding period. The control insects were kept throughout the experiment in groups on several healthy aster plants; 36 virus-free males were collected at the end. Transmission by 28 % of the viruliferous insects was recorded. However, all the viruliferous insects showed the intermediate condition of the fat body, rather than the extreme, and this was considered additional evidence that feeding on diseased plant,s furthers the expression of the extreme viruliferous condition. The virus-free controls also showed the intermediate condition. A similar experiment was tried using insects iujected with the juice of viruliferous insects: after the injection the insects were kept on rye plants for 16 days, then transferred to individual healthy aster plants for 7 days. Unfortunately only 6 males survived to the end of the experiment, and all these showed the intermediate fat body condition. None of them transmitted the virus. Of the 2 surviving virus-free injected controls, one had the intermediate and one the virus-free fat, body type. The number of insects in this injection experiment was small, but the results agreed with the other findings that, constant feeding on diseased plant’s intensifies the cytological effect of the virus.
C’ytochemical Observations Since one of the chief effects of the aster yellows virus 011N. jascijrons was to cause a decrease in the amount of cytoplasm, test,s were made to identify some of the cytoplasmic components chemically. That the cytoplasm contained ribonucleic acid was shown hy digestion of et’hunol-fixed, frozen-subst,ituted sect,ions wit,h ribonuclease, which abolished t,he basophilia of the cytoplasm of the fat’ body cells’ (Figs. 7, 8). The removal of basophilia by a specific reagent such as t’he enzyme ribonuclease is evidence that RNA was t’he basophilic substance. This result, was checked by the use of hot trichloroacetic acid, which exfract,s bot,h DNA and Rh’A. Neither the nucleus nor cytoplasm of t’he fat body cells retained any basophilia after the treat#ment. The cytoplasm was also shown to contain prot.ein in approximat,ely the same location as t,he RNA, by means of naphthol yellow S staining and the Sakaguchi arginine reaction. At least, a part’ of the prot.ein and RXA probably occur as a ribonucleoprotein complex in the cells. The Feulgen reaction failed to reveal any further difference bet’wcen viruliferous and nonviruliferous aster leafhoppers with rrspcct to thn localization of DNA, which was exclusively nuclear. The insect fat body, like t,he vertebrat’e liver, is a storage organ as well as a metabolic organ. The localization and approximat,e relative amount,s of the stored subst.ances glycogen and lipid were compared in 1l011nu1and viruliferous M. juscijrons. It1 general, both the substances were present, in the cytoplasm of the fat body cells of all insects, where the amount’ and distribution varied greatly hut were independent of the insect’s condit.ion with respect to t.he virus. AIost of the insect material used for these t,ests was fixed in hot, ethanol after freeze-substitution OI by tiIcMnnus’ (1946a) special method for lipid. (.‘ytological r)bservations
(‘sing
Othrr I,wfhoppcw
and Other P’irt~.sc.s
Two other species of leafhoppers that acquired the aster yellows virus by feeding on diseased aster plants were studied. They were t,he corn leafhopper Dalbulus maidis and t,he clover leafhopper Agallia con~tricta. The virus can he recovered from them, aft,er a suitable period of time, by injection of the juices of t,hese insects int,o &f. fascifrons (Maramorosch, 1952), ahhough neit’hcr the corn nor t,he clover leafhopper becomes a transmitter of the virus. The fat body cells of -31 male aud female A. constricta adu1t.s that, had fed on yellows ast,ers for from 7 to 9 weeks were examined, and no difference was found betweeit them and the 35 controls on healthy asters.
496
LITTAU
AND
MARAMOROSCH
The experimental procedure for studying the effect of the yellows virus on D. maidis was somewhat different because this leafhopper ordinarily will not feed on healthy asters although it lives quite well on yellows asters (Maramorosch, 1958). Therefore, the insects acquired virus by feeding on diseased asters for 6 hours a day on 4 successive days, while controls were placed on healthy asters for the same time. All were kept on healthy corn plants between feeding times on asters and for the rest of the experiment (the total time was 4 weeks). There was no cytological difference between the 47 controls and the 51 male and female leafhoppers t,hat had had the opportunity to acquire virus. Also available were 7 males that had spent 35 days on yellows asters; their fat bodies were no different from the above-mentioned controls. A strain of the aster yellows virus that infects celery had no cyt,ological effect on M. fascijrons; this was found using 25 nonviruliferous males, 35 males with the celery-infecting strain, and 35 males with the Eastern strain, (of which 4, or II%, showed cytological changes), for comparison. This seemed all the more surprising because this strain causes much more severe symptoms in the plant host than does the Eastern strain used for the other experiments. Two strains of the corn stunt virus, Rio Grande and Mesa Central, were tested in one of their vect’ors, D. maidis. Both males and females were examined: 38 nonviruliferous, 44 with the Rio Grande strain, and 44 with the Mesa Central strain. Again no cyt’ological effect was seen in either males or females. In summary, so far, no cytological changes have been observed in any of the other leafhoppers infected with viruses that were studied, or in M. fascifrons with the celery-infecting strain of aster yellows virus. I)ISCUSSION
The findings of cytological changes, reported in detail in this paper, are the first known changes caused by a plant-pathogenic virus in an invertebrate animal. The lack of a completely clear distinction between plant and animal viruses has already been recognized for several years (Maramorosch, 1954; Enders, 1957), as the consequence of the vast amount of evidence for multiplication of certain plant viruses in insects. Although acute and chronic virus infections were known to be caused by yellows-type viruses in numerous species of plants, insects were usually considered as mere carriers and believed to suffer no ill effects. In the case of aster yellows virus, after the initial 4 days of the incubation
(‘YTOLOQICAL
EFFECTS
OF
ASTER
YELLON’S
VIRI’S
497
period, during which time virus cannot be recovered from the insect, t#heinfectlion becomes systemic and the virus can be recovered from t’hr hemolymph, although it cannot yet be transmitt,ed through t,he insect”s mouthparts. Transmission may begin after 9 days, but only after 18 or more days does the infection become expressed, as is evident’ from cytological changes t’hat appear at this t,ime. These changes seem to bc enhanced by constant feeding on diseased plants, which obviously favors the expression of the extreme condit,ion. Superinfection wit,h t’he virus is anot,her possible explanation for the findings. Such superinfect,ion probably t#akes place during prolonged feeding on diseased plants. The originally acquired virus may cause a systemic infection and establish an equilibrium with its invert~ebratc host, only rarely causing fat, body changes. When additional virus is acquired, this superinfection may upset the ecfuilibrium. Superinfection wit,h host,-specific viruses has been listed by Bergold (19%) as OW of the possible causes of activation of lat,ent viruses in insects. The possible toxic effect of diseased asters cannot, bc ruled out complet,ely, alt,hough no evidence for toxicit,y was found. On the contrary, astIer leafhoppers were reported to breed more prolitically on diseased plant’s (Severin, 194(i), which seems to deny the possibility of a iosic substance t’hat might, be acquired from diseased asters. There has been a tradiGona1 assumption that if vectors are affect,ed in any way at all by t,he virus t’hey carry, it, must bc pathologically, because viruses are pat,hological by their very nst,ure. Although t’here in no doubt t’hat! in diseased plants aster yellows virus is a pat,hogenic parasit)e, in t’he insect, host it may be more like a commensal or even a symbiont and changes caused by it are not necessarily pathogenic. In this connect~ion the work of Severin (1946) might again be recalled. Severin’s experiments indicated that vect,orship is a function of special biological significance, in t#hat, it indirectly aids t,hc insect’s multiplicatiotl. The contrary is t,rue for the infect,ed pIalIt, which becomes sterile in thr course of the disease. The problem of the underlying chemical events hchind tbe chattged appearance of the fat body of viruliferous insect8s remains ohscure, butf a general explanation may be suggest,ed. First of all, the decrease in actual Cssue subst(ance may only be apparent; some of the Gssue mttg have been, and indeed, probably was, lost during the Car&y fixation and the embedding process. The fact, t,hat frozen-substituted viruliferous ittsect s as well as t’hose fixed in McMnnus’ formalin-cobalt-c:Jrium fluid
498
LITTAU
AND
MARAMOROSCH
did not show the loss of tissue supports this hypothesis. This means that one effect of the virus was to render the tissue more easily extractable by histological reagents, a condition that might have resulted from a change in the solubility properties of either the nucleic acids or proteins or both, in the cytoplasm and nuclei. It is also possible that only the cytoplasm was involved; if the cytoplasm surrounding a nucleus were dissolved out, the nucleus might be carried along with it. It, is impossible at present to relate either a qualitat#ive or quantit,ative change in fat body RKA directly to virus multiplication, the actual sit,e of which is not known. The fat body occupies a position of considerable importance in the metabolism of the insect, with the result that events taking place in other parts of its body undoubtedly have an effect on these cells. However, the fact that a great deal of synthetic activity normally occurs in the fat body also makes it a not unlikely place for virus synthesis as well. In any case, the length of time required for the cytological changes would seem to rule out their immediate relation t’o virus multiplication: the incubation period of the virus in the insect was only 9-12 days, whereas from 18 to 28 days had to elapse after acqui&ion of virus before changes were seen in the fat body. Finally, mention should be made of the interesting results with the celery-infecting strain of aster yellows. This strain, as was already noted, caused much more severe stunting and chlorosis of the diseased asters than the East,ern strain, but had no cytological effect on the vector, M. fascifrms. In this connection, Maramorosch (1959a) has observed that the celery-infecting strain also failed to condition corn leafhoppers to live on asters, unlike the Eastern strain. Both sets of facts imply that! the two virus strains differ by at least some chemical processes they cause in the plan@ or possibly also by their direct effects on the insect. REFERENCES F. C. (1959). Plant Viruses and Vz’rus Diseases, 3rd ed. Chronica BoPress, Waltham, Massachusetts. BERGOLD, G. H. (1958). Viruses of insects. In Handbuch der VGusforschung, Vol. 4, pp. 60-142. BLACK, L. M. (1959). Biological cycles of plant viruses in insect vectors. In The Viruses (F. M. Burn& and W. M. Stanley, eds.), Vol 2, pp. 157-185. Academic Press, New York. BLATTNY, C. (1932). Lzezjistiti pisitomnost viru pfisobiciho nekter6 ohoroby Brambord v jejich pfen&ReEi, mgicich? (Can the viruses that cause certain potato diseases be det,ected in their aphid vectors?) VbtnZk kr(lZov. Ceske spoleSnr,sti nauk 1931, No. 4, 7 pp. (Czech, wit,h English summary.) BAWDEN,
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H. W. (1943). Method for the quantitative morphologic analysis of tissues. J. Natl. Cancer Inst. 4, 47-53. DAY, M. F., and BENNETTS, M. J. (1954). A review of problems of specificity in arthropod vectors of plant and animal viruses. C.Y.I.R.O., Canberra, Australia. 171 pp. (Mimeographed.) LIEITCH, A. D. (1955). Microspectrophotometric study of the binding of the &IIionic dye, Naphthol Yellow S, by tissue sections and by purified proteins. Lu6. Invest. 4, 32+351. I)OBROSCKY, I. 1). (1929). Is the aster-yellows virus detectable in its insect. vect,or? Phytopalhclogy 19, 1009-1015. DOBROSCKY, I. 1). (1931). Morphological and cytological studies on thr salivur?
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glands and alimentary tract of Cicadxla sernolata (Fallen), the carrier of ast (‘I >rellows virus. Co&ribs. Royce Thompson Inst. 3, 3’3-58. KNDERS, J. F. (1957). The future of virus studies in tissue cult urp. J. l$‘tct(. Cancer
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FLAX, H. M., and HIMER, M. H. (1952). Microxpeotrophot,ometric analyses of metachromatic staining of nucleic acids. Physiol. Zoiil. 26, 297-311. HARTZELI,, A. (1937). Movement of intracellular bodies associaf rd with peach yellows. (“ontribs. Boyce l’hompson Inst. 8, 375-388.
HOX,HKISS, R. I>. (1948). A microchemical react,ion result,ing in t,he staining of polysaccharide structures in fixed tissue preparations. .Irch. Biochet~. 16, 131. 111, JEW&N, I). I). (1058). Reduction of longevity of leafhoppers (xrrging prach yellow leaf roll virus. Phytopalhology 48, 3Q4. (Abstract. ) JENSEN, I). 1). (,lQ59). A plant virus lethal to its insect vector. Virolqq 8, 164-175. JOHASREK, I). A. (1940). Plan1 Microlechniqcte, pp. 13&lX~. M~(;rawHill, N~u York. IATTAC, V. C. (1960). Cyt,ological dif-fcrences between the fat body tissue of adult male and female leafhoppers (Homoptera, Cicadellidae). J. Morphology, in press. IATT.~I., V. C., and MARAMOROSCH, K. (1956ai. Cytological changes in Macrosteles ,fasci~rons fed on plants infected with aster >-ellows virus. I’h,~~topathwlogy 46, 468. (Abstract.) LITTA~~, L’. C., and MARAM~ROSCH, K. (1956b). Cytologiral effeptx of aster-qlloas virus on its insect, vector. Virology 2, 128-130. LITTAL-, V. C., and MARAMOROSCH, K. (1958). Cytopathogenic effects of the astrt yellows virus on its insect vector. Phytopath~ology 48. 263. (Abstract..) hl~~hl~~us, J. F. A. (1946a). 1)emonstration of cert.ain fatty subsfames in paraffin sections. J. Pafhol. Bacterial. 68, 93-95. MCWIANLTS, J. F. A. (1946b). Histological demonstration of mucin after periodic: acid. AJature 168, u)2. (Abstract.) MARAMOROSCH, K. (1952). Studies on the nature of the specific transmission of aster-yellows and corn-stunt viruses. Phytopathology 42, 663-668. MARAMOROSCH, K. (1954). Biological transmission of plant viruses by animal vectors. Trans. N. Y. Acad. Sci. 12116, 189-195. ~~ARAMoROS(‘H, K. (1956). Semiautomatic equipment. for injecting insects with measured amom~ts of liquids containing viruses or toxic substances. Ph?/topathology
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MARAMOROSCH, K. (1953). Beneficial effect of virus diseased plants on non-vector
insects. Tijdschr. Planienziekten 64, 383-391. MARAMOROSCH, K. (1959a). Further tests on the beneficial effects of aster yellows on corn leafhoppers. Phytopathology 49, 545. (Abstract.) MARAMOROSCH, K. (195913).Leafhoppers (Cicadellidae) as vectors and reservoirs of phytopathogenic viruses. Horn. Vol. Savulescu, Rumanian Acad. Sci., Bu-
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