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On the mechanism of persistence and distribution of barley yellow dwarf virus in an aphid vector
42, 668-680 (1970)
VIROLOGY
On
the
Mechanism
of Persistence Dwarf
Virus
and
in an Aphid
Y. C. PALIWAL Cell
Biology
Research
Institute,
Research
Distribution
Yellow
Vector’
AND R. C. SINHA
Branch, Accepted
of Barley
Canada July
Department
of Agriculture,
Ottawa,
Ontario
8, 19YO
Barley yellow dwarf virus (BYDV) was recovered, using infectivity bioassays, from the gut, hemolymph, and salivary glands but not from the brain (including ganglia) of aphids Macrosiphum avenue (Fabric&) that had an acquisition access period of 4 days. As a source of virus inoculum, salivary glands were the poorest and gut was the best. However, the virus was not detected in the gut of aphids that were
injected with a massive dose of virus, an observation suggesting that it does not pass from the hemolymph into the gut. The precipitin ring test detected virus antigens in the gut and hemolymph but not in the salivary glands and brain of aphids given a 4-day acquisition access period. Viruslike particles were observed dispersed in the lumen of the gut of exposed aphids, but their identity as BYDV particles could not be established, although large aggregations of BYDV particles were readily identified in the phloem cells of infected oat leaves. The relative virus concentration (as indicated by the percentage transmission of
injected insects) in the gut, when assayed at various times following a 6-hour acquisition access period, remained at about the same level between 6 and 24 hours and declined thereafter up to 96 hours. The virus apparently did not multiply in the gut. That the virus can accumulate in the gut was demonstrated by the increasing virus
concentration
in this organ with increasing
acquisition
access periods.
BYDV could not be maintained in the aphids by serial passage of virus from insect to insect. The ability of aphids to transmit the virus declined following acquisition or injection of the virus into aphids. Also, the period of retention of inoculativity and transmission efficiency of aphids was dependent on the dose of virus ingested or injected. These resldts suggest t,hat BYDV does not multiply in its vector M. aaenae.
the cells of salivary glands of t#he vector (Richardson and Sylvester, 1968; Sylvester and Richardson, 1969). Cytoplasmic infection of the vector tissues by lettuce necrotic yellows virus suggested multiplication of t)he virus in the vector aphids (O’Loughlin and Chambers, 1967). However, the evidence
INTRODUCTION
Although many viruses are known to be transmitted by aphids in a “persistent manner,” the mechanism of persistence of such viruses in their vector has been studied in only a few cases. In the case of at least 2 such viruses, both of which are bacilliform, the persistence can be explained on the basis
of virus multiplication in their vectors. Evidence for multiplication of sowthist’le yellow vein virus was provided by serial passageof the virus among successivegroups of virusfree aphids using hemolymph as inoculum and by demonstration of nuclear infection in 1 Contribution Research Institute.
No.
682 from
the
Cell
Biology
for multiplication of potato leaf roll and peaenat,ion mosaic viruses in their vectors is still conflicting or inconclusive (Stegwee and Ponsen, 1958; Harrison, 1958; Carter, 1963; Black, 1969; Sylvester, 1969). Both t,hese viruses are transmitted by their aphid vectors in a persistent manner and are spherical in shape. Barley yellow dwarf, a small spherical virus (BYDV), can persist in it.s aphid vec-
663
BARLEY
YELLOW
tor for a long period of time. Rochow (1959) found that single Macrosiphum avenae (Fabricius) , continued to transmit BYDV as long as they lived (11-12 days) after a 24-hour acquisition access period. Transstadial passageof the virus in 2M. averme has also been reported (Miller and Coon, 1964). The circulative nature of the virus in its aphid vectors was demonstrated by transmitting the virus through injection of hemolymph from viruliferous aphids into virusfree individuals (Mueller and Rochow, 1961). A latent period of BYDV in the vectors, although poorly defined in terms of a minimum, has been demonstrated as well (Rochow, 1963). We have studied certain aspects of BYDVvector relationship in order to understand the mechanism of persistence of the virus in its vector, $1. avenue. This paper reports on: (1) attempts to transmit BYDV serially from aphid to aphid, (2) effect of varying the length of t’he acquisition access period on transmission behaviour of aphids, (3) relationship of the dose of @us injected int’o aphids to the transmis$on efficiency and retention of inoculativity by the insects, (4) distribution of the virus, as determined by infectivity tests, and of virus antigens in the vector, and (5) relative virus concentration in the gut of aphids during the period following acquisition access. In addition ultrathin sections of internal organs of viruliferous aphids and of infected oat leaves were examined in an attempt to locate BYDV particles in situ. MATERIALS
AND
METHODS
Virus and aphids. A strain of BYDV specifically transmitted by M. avanae, and maintained in oat (Avena byzantina C. Koch var. Coast Black) plants by aphid inoculation was used. Coast Black oats were used as test plants in all experiments. Virus-free aphids were reared on oat plants in growth chambers at 20” with 12,000 lux of light for 16 hours per day. The plants used for rearing virus-free aphids were subsequently sprayed with nicotine sulphate and kept in the greenhouse for observation, so that experiments made from any supposedly virus-free aphids that produced an infected plant could be discarded. Also, as an additional control,
DWARF
VIRUS
669
about 100 aphids from the stock culture were always tested concurrently with every experiment. Apterous young adult aphids of about the same age (8 or 9 days old) were used in all experiments. All acquisition accessperiods were given at 15” on oat plants that had 18-21 days old infections of BYDV. The insects, either after an acquisition access or after injection were held, during the test feeding period, in a growth room with night-day temperature range of 19”-21” and 10,000 lux of light for 16 hours a day. When viruliferous aphids were required to be maintained in groups for more than a day, they were transferred to new plants every 24 hours because it has been shown that aphids can inoculate and reacquire BYDV from a plant in a minimum of 3 days (Gill, 1969). After the test feeding, inoculated plants were sprayed with nicotine sulphate, held in a greenhouse for 30 days and observed regularly for symptoms of BYDV. The test plants in the greenhouse were sprayed with nicotine sulphate every week. Certain terms used in this paper are defined as follows: Exposed aphids. Insects that had been confined to infected plants. Inoculativity. The ability of aphids to transmit the virus to plants. Inoculative aphid. An aphid that transmitted the virus to at least one plant when tested for its inoculativity on a seriesof successive test plants. Mean t+ansmission eficiency per aphid. Mean number of transmissions per inoculative aphid in a given number of successive inoculation feeding periods. It was calculated from the relationship cc-l, Xi,, where X is the proportion of plants to which an inoculative aphid transmitted the virus in a given number of successive 24-hour inoculation feeding periods, and N is the total number of inoculative aphids tested in each of the inoculation feeding periods. The t test was used to determine the significance of the difference between the values of this statistic for different doses of virus received by the aphids (either by injection or by acquisition access). The values of X (in percent) were transformed to angles (angle = arc sin per‘centage1J2)before using them to compute the t value.
670
PALIWAL
AND
Extracts of internal organs and hemolymph suspensions.Aphids were dissected in saline (0.85% NaCl) with the help of fine needles and forceps to obtain salivary glands, brain (including ganglia), and gut (Figs. l-3). First, the head along with most of the thorax was removed from the abdomen. The gut was then forced out of t’he abdomen by pushing the caudal end inward and pressing it gently. The connection of the hindgut to cauda was severed to free the gut. Salivary glands and brain were separated out carefully from the head-thorax portion. A known number of organs of t,he same kind were pooled, transferred into a tissue grinder, washed 2-3 times with saline and ground in a known volume of the latter. The hemolymph suspensionwas prepared by making a small incision in the abdomen of aphids and touching the oozing fluid into a known
SINHA
volume of saline on a depression slide. The extracts of organs and hemolymph suspension were clarified by low speed centrifugation before inject’ion into aphids. Details of the dissection, grinding and clarification procedures were the same as described previously by Sinha and Chiykowski (1967a). In experiments where an extract of guts of exposed aphids was used as a source of virus, the insects had an acquisition accessperiod of 4 days, the dilution of the inoculum being calculated on the basis of weight of the guts to the volume of solvent. The weight of a single gut, determined by weighing 2 batches of 100 guts each, was found to be about 0.05 mg. In all experiments at least 50 guts were used to prepare a base inoculum at a dilution of l/40 (for example, guts weighing 0.1 g ground in 3.9 ml of saline). Virus assay. The infectivity of the extracts
FIGS. 1 and 3. Some internal organs of an aphid, Macrosiphum avenue (Fabricius)-a vector of barley yellow dwarf virus. Fro. 1. The gut. Fg, foregut; C, crop; In, intestine; Hg, hindgut; Ca, cauda. X 48. FIG. 2. The nervous system. B, brain; G, ganglia; Mn, main abdominal nerve. X 52. FIG. 3. The salivary glands. Pg, principal gland; Ag, accessory gland; AC, acinus. X 48.
BARLEY
YELLOW
was assayed by injecting them into virusfree aphids and testing the latter for their inoculativity. Aphids were anesthetized in batches of 10 with COZ and were injected in their abdominal hemocoele (Sinha and Chiykowski, 1967a). Needles for injection were drawn from glass capillaries (outer diameter, 2 mm) using a pulling device (Roberts and Granados, 1968). The volume of inoculum injected into each aphid was estimated to be about 0.04 ~1. Insects were injected in the late afternoon and held overnight at 15” on detached oat leaves in a petri dish. Next morning, they were caged singly (unless stated otherwise) for 4 days on oat plants to test their inoeulativity. Only those aphids that survived for all 4 days were used to calculate the percentage transmission. The day of injection was numbered 0, the next day 1, and so on throughout the test feeding. Xerology. To prepare an antiserum, BYDV was partially purified, using a procedure described by Rochow and Brakke (1964) but without density gradient centrifugation, from 300 g of infected oat leaves (15-18 days old infection). Five injections of such a preparation were given at weekly intervals into a rabbit after being emulsified wit,h an equal volume of Freund’s incomplete adjuvant. The precipitin ring test was used in all serological reactions. The antiserum had a titer of l/80 against an extract of guts (at a dilution of l/20, w/v) obtained from exposed aphids but showed no reaction against extracts of guts, salivary glands, brain, and hemolymph suspensions prepared from virus-free aphids. Also, the preimmune serum showed no reaction with extracts of organs from exposed aphids. Electron microscopy. Oat leaves, and gut, salivary glands and brain of M. avenue were processed and sectioned following the procedures described earlier (Paliwal, 1970; Sinha and Paliwal, 1970), and examined in a Siemens Elmiskop I. RESULTS
Attempts to Transmit the Virus Serially from Aphid to Aphid by Hemolymph Inoculations A method described by Sylvester and Richardson (1969) was used to collect hemo-
DWARF
VIRUS
671
lymph from an aphid directly in the injecting. needle. Each aphid from a group of insects,. that was allowed a 4-day acquisition access period, was used as a donor of hemolymph for the inoculation of one virus-free aphid (1st passage). All injected aphids were then tested singly for their inoculativity (1 day on each of 2 successive plants). Each surviving insect again served as a donor of hemolymph to a virus-free aphid (2nd passage). After doing one more such hemolymph transfer (3rd passage) the whole experiment was repeated. A total of 36 aphid lines completed the 3 passages. In the first passage, 14 out of 36 aphids transmitted the virus but no transmission occurred in the 2nd or 3rd passage, suggesting that BYDV does not multiply in aphids that are injected with the virus. Rochow (1969), in a review article, also reported that attempts in his laboratory to transmit BYDV serially from aphid t.o aphid were unsuccessful. Distribution leaves
of the virus in vector and in oat
The gut, salivary glands, brain, and hemolymph suspensions from aphids, that had a 4-day acquisition access period, were tested for the presence of BYDV. One hundred organs of each kind (in 0.1 ml saline) were used to prepare the extracts of various organs. The hemolymph suspension was prepared also from 100 aphids in 0:l ml of saline. Aphids injected wit.h different inocula were caged in groups of 5 on oat plants for 4 days. The virus was recovered from the gut, salivary glands and hemolymph but not from the brain (Table 1). When these extracts and hemolymph suspension were tested against BYDV antiserum (dilution l/20), the virus antigens were detected in the gut and hemolymph but not in the salivary glands and brain. It is evident from the results that as a source of virus inoculum, the salivary glands were the poorest and gut was the best. Ultrathin sections of infected oat leaves showed large aggregations of spherical virus particles, 22-25 nm in diameter, in the phloem cells only (Figs. 4 and 5). No such particles were found in healthy leaves. While this work was in progress, Jensen (1969) reported the occurrence of large con-
672
PALIWAL TABLE
1
AND SINHA
and the latter were tested singly for their inoculativity for 8 days (1 day each on 8 avenae successiveplants). The survival of injected aphids (Fig. 7) during the g-day test feeding vinls antigens period appears to have been affected by the Source of inoculuma z&$&b % ine;F concentration of tissue constituents in the inoculum injected as the insect mortality tractsC was comparatively higher when the more concentrated inoculum was used. The numGut 34/37 92 ++ ber of insects that became inoculative after Hemolymph suspension 12/23 52 + Salivary glands 13128 46 being injected with the inocula at dilutions Brain (including ganglia) o/20 0 of l/40 and l/320 were 140/166 (84%) and 55/120 (46 %), respectively. a One hundred organs of the same kind, obThe daily transmission by inoculative tained from aphids that had an acquisition of 4 aphids during the test feeding period showed days, were pooled, ground in 0.1 ml of saline, (Table 2) that all insects did not transmit their clarified extracts injected into virus-free simultaneously on any one day. On day 1, aphids, and the latter tested for their inoculativity. Hemolymph suspensions were also prepared 80% of the inoculative insects transmitted from 100 exposed aphids in 0.1 ml of saline. the virus when dilution of the inoculum was b Combined results of two experiments; numerl/40 but only 49 % of the insects transmitted ator is the number of plants that became infected; on this day in case of l/320 dilution. Also, denominator is the number of plants inoculated the percentage transmission always remained by 5 injected insects per plant. comparatively higher when the aphids rec The precipitin ring test was used to detect the virus antigens; the antiserum was used at a ceived the higher dose. The ability of aphids to transmit the virus declined following indilution of l/20. The number of plus signs indicates the relative thickness of the ring after 90 jection with either dose of virus while the period of retention of inoculativity appears minutes; a minus sign means that the results were to be dependent on the dose of virus. The negative. mean transmission efficiency per inoculative centration of BYDV particles in the phloem aphid, based on transmission by those incells of barley leaves infected with a strain of sects that survived for at least 7 days, was the virus transmitted by Rhopalosiphum significantly higher for the aphids injected with the higher dose of virus. These results padi (Linneaus) . Examinations of 5 internal organs of each also suggest a lack of virus multiplication in kind from exposed (4-day acquisition access aphids that were injected with the virus. period) as well as virus-free aphids did not reveal aggregations of virus particles, such Transmission of BYDV by Aphids after Various Acquistion Accessl’eriods as those observed in plants, in any of the We wanted to determine wllether or not tissues examined. However, viruslike particles, some poorly defined, were found dis- the transmission behaviour of aphids after persed in the lumen of the guts of several feeding on infected plants was the same as that observed with insects that became inocexposed aphids, (Fig. 6), but it was difficult to identify BYDV virions among these ulative after injection with the virus. The particles because a few poorly defined par- virus dosage in aphids can be varied by ticles were occasionally found scattered in varying the length of acquisition access the lumen of the gut of virus-free aphids. period (Rochow, 1963). Virus-free aphids were given acquisition Transmission of BYDV by Aphids Injected accessperiods of 3, 6, 12, and 24 hours. They with Di$erent Dosesof Virus were caged singly immediately thereafter An extract, prepared from guts of exposed on oat plants to test their inoculativity. All aphids, at a dilution of l/40 and its 8-fold insects were transferred to new plants at dilution were injected into virus-free aphids 24-hour intervals. The insects that had an DISTRIBUTION
OF BYDV Macrosiphum
IN THE VECTOR
FIG. 4. BYDV particles in a phloem cell of oat leaf. X 25,500. FIG. 5. A mass of BYDV particles at a higher magnification. X 45,000. FIG. 6. Viruslike particles in the lumen of the gut of M. avenae. Aphids were exposed to BYDV fected plants for 4 days, and then their guts were removed and processed for electron microscopy. difficult to identify BYDV virions among these particles because a few scattered, somewhat similar, poorly defined particles were occasionally seen in the lumen of healthy controls also. X 46,500. ri7.1
inIt is but
674
PALIWAL
AND SINHA
the 15 surviving inoculative insects had ceased to transmit in case of the shorter acquisition access,whereas 6 out of 13 were 80 still transmitting when the insects had the longer acquisition accessperiod. On day 20, of 5 and 9 aphids that survived after the 3and 24-hour acquisition access periods respectively, only 1 insect was still transmitting from the group that had the longer acquisition access.This insect, incidentally, transmitted the virus to all of the 20 test plants. There were two aphids in the 3-hour acquisition accessgroup which transmitted to 12 consecutive test plants. In addition to 100 a general decline in the ability of insects to 2 3 4 5 6 7 a transmit the virus, the period of retention of DAYS AFTER INJECTION inoculativity was dependent on the dose of FIG. 7. Survival curves for adult M. avenue on virus acquired by the insects. Also, the mean oat plants after being injected with two doses of transmission efficiency per inoculative aphid, virus. A total of 166 and 120 insects injected with based on transmission by those insects that virus inocula at dilutions of l/40 and l/320, respecsurvived for 11 days, was significantly higher tively, were tested. for insects that had a longer acquisition acacquisition access of either 3 or 24 hours cessperiod (Table 3). The transmission of BYDV had similar were tested for 20 days but those that were attributes whether the aphids received the allowed a 6- or la-hour acquisition access virus through injection or by feeding on inwere tested for only 6 days. The results show jected plants, except that aphids that bethat no new transmission occurred beyond day 6, after either 3- or 24-hour acquisition came inoculative after injection lost their access periods. The proportion of aphids ability to transmit in a shorter period of that became inoculative after various ac- time, as compared to those that became quisition accessperiods had a linear re!ation- inoculative after an acquisition accessperiod. ship with the length of acquisition access Attempts to Detect the Virus in Guts of Injected period (Fig. 8). Aphids The survival of exposed aphids during the The distribution of BYDV in the aphids lo-day period following 3- or 24-hour acquisition accessis shown in Fig. 9 and trans- (Table 1) suggeststhat when insects acquire mission results (up to day 11-the day when virus by feeding on infected plants, the virus passesfrom the gut into the hemocoele, is about 50% of the aphids were surviving) for aphids that became inoculative after the carried to the salivary glands via hemotwo acquisition accessperiods are given in lymph, and is introduced into the plants Table 3. Although all inoculative insects through the salivary secretions; whereas never transmitted the virus simultaneously when the insects are injected, the virus is on any one day either after 3- or 24-hour directly introduced into the hemocoele. It acquisition access, the percentage transmis- can be argued, therefore, that longer retension on all days was higher when the aphids tion of BYDV by aphids after an acquisition had a longer acquisition accessperiod. For accesscould be due to multiplication of the virus in the gut. If this is true then it must be example, on day 11, 52 % of the inoculative insects transmitted the virus in case of 24- presumed that the virus in the injected inhour acquisition access but only 25 % did sects cannot passinto the gut. Attempts were made, therefore, to detect when the insects had a 3-hour acquisition access (Table 3). However, by day 15, all the virus in the guts of aphids that were
BARLEY
YELLOW
DWARF
TABLE TR.4NSMISSION
PATTERN
OF APHIDS DIFFERENT
675
VIRUS
2
THAT WERE INJECTED WITH CONCENTRATIONS OF BYDV
INOCULA
CONTAINING
Daily transmission by inoculative aphids Based only on those aphids that survived for 7 days
Based on the aphids surviving on a given day Days Dilution of the virus inoculaa
--. 1 2 3 4 5 6 7 8
l/JO Transmissionb
%
l/320 Transmission
%
112/140 113/138 (2Dc 94/131 (7) 60/114 35181 9151 3/24 l/12
80 82 72 53 43 18 12 8
27/55 28/55 (17) 21/54 (10) wo (1) 5149 2/47 O/42 O/36
49 51 39 18 10 4 0 0
Mean transmission efficiency per inoculative aphidd
-
-
1140
l/320
7. Transmission 75 75 71 42 33 17 12 -
45 48 40 21 12 5 0 -
44.3”
23.6
a To prepare an inoculum at a dilution of l/40 (w/v), 50 guts (obtained from aphids that had an acquisition access of 4 days on infected plants) were ground in saline and the extract was clarified. This inoculum was diluted with saline to l/320 and both dilutions were injected into virus-free aphids. b Injected aphids were caged singly on oat plants and transferred daily to a new plant for 8 days. Numerator is the number of aphids that transmitted the virus on a given day; denominator is the number of inoculative aphids surviving on that day. Combined results af two experiments. c The figures in parentheses represent the number of new transmissions on that day. d Calculated from the daily transmission data of individual inoculative aphids surviving for 7 days, expressed in percents. e Difference between the values for the two doses of virus are highly significant; t value at 64 degrees of freedom = 10.09, P < 0.001.
C
3
6 12 ACQUISITION ACCESS PERIOD IN HOURS
24
FIG. 8. Transmission of BYDV by M. auenae (tested singly) after various acquisition access periods. The percentages of transmission for acquisition access periods of 3,6,12, and 24 hours are based on transmission by 71, 61, 70, and 105 exposed insects, respectively.
injected with a virus inoculum. An inoculum prepared from 100 guts (dilution l/20) of aphids that had an acquisition accessperiod of 4 days, was injected into virus-free aphids. In one experiment, 2 days after, and in another, 6 days after injection, extracts (dilution l/20) prepared from 100 guts of injected aphids in each case, were assayed for the presence of virus. None of the 70 and 85 test insects in the first and secondexperiments, respectively, transmitted the virus showing that the virus was not detected in the guts of injected aphids. In the casesof wound-tumor and wheat striate mosaic viruses also, the virus was not detected in the gut of their leafhopper vectors following
676
PALIWAL
yj
AND
SINHA
50-
65 p 40.---.3 30-
HOUR ACPlJlSlTlON ACCESS 24 HOUR ACQIJISITION ACCESS
-
ZOIO
I 10
I 5
1
DAYS AFTER
FIG. 9. Survival curves for adult access period of 3- or 24-hours (based
TRANSMISSION
PATTERN
1 20
I 15
ACQUISITION
ACCESS
PERIOD
M. avenue on oat plants, after the insects had on 93 and 74 insects in the two cases, respectively).
TABLE AFTER
BY APHIDS
Daily
3 V.*RIOUS
ACQUISITION
ACCESS
transmission
by inoculative
aphids
Days Acquisition
1 2 3 4 5 6 7 8 9 10 11 Mean transmission efficiency per inoculative aphid* 0 Numerator is the number number of inoculative aphids b Calculated from the daily expressed as percentages. e Difference between these value at 53 degrees of freedom
%
4135 7135 18/35 16134 16/31 17/29 12129 10128 111% 9/26 6124
11 20 51 47 52 59 41 36 42 35 25
access period
24 Transmissiona
-
PERIODS
in hours 3 24 To Transmission
%
30165 45/65 46164 56/64 56164 51/63 41158 30147 32/43 21/38 16/31
an acquisition
Based only on those aphids that survived for 11 days
Based on the number of aphids surviving on a given day
3 Transmissiona
had
46 69 72 87 87 81 71 63 74 55 52 -
17 12 46 37 54 58 42 37 46 37 25
45 61 65 90 74 74 74 61 71 48 52
36.3c
70.1
of aphids that transmitted the virus on a given day; denominator tested on that day. Combined results of two experiments. transmission data of individual inoculative aphids surviving for values for the two acquisition = 4.23, P < 0.001.
access
periods
are
highly
is the 11 days
significant;
1
BARLEY
YELLOW
DWARF
677
VIRUS
injection with a massive dose of virus (Sinha, unpublished results). It appears that the movement of BYDV between gut and hemocoele is unidirectional. Relative Concentration of BYDV in the Gut of Aphids after Ingestion of the Virus
In an attempt to determine whether the virus multiplies in the gut, virus-free aphids were given an acquisition access period of 6 hours and were then maintained on healthy plants. At various time intervals, an extract (at a dilution of l/40) prepared from the guts of exposed aphids was assayed for its infectivity. The results show (Fig. 10) that the relative virus concentration, as indicated by the percentage of insects that became inoculative, remained at about the same level between 6 and 24 hours and then decreased to a low concentration by 96 hours. The following experiment demonstrated that the percentage transmission by injected
0 ( 0
I 6
24
I 40
7i
I 96
TIME OF ASSAY IN HOURS
FIG. 10. Relative BYDV concentration in the gut of M. avenae at various times after a 6-hour acquisition access period. After the acquisition access period, aphids were maintained on healthy plants and at various times (from the start of acquisition access) an extract (dilution l/40) of 50 guts of exposed aphids was injected into virus-free aphids and the latter tested singly for their incculativity. Percentages of transmission by aphicis injected with gut extracts made at 6,24,48, and 96 hours from the start of acquisition access are calculated from a total of 125, 128, 97, 109, and 113 aphids, respectively (combined results of two experiments).
RECIPROCAL
LOG DILUTIONS OF INOCULd
FIG. 11. Transmission
of BYDV by M. avenae injected with inocula containing different concentrations of the virus. An inoculum at a base dilution of l/40 (w/v) was prepared from 50 guts of aphids that had an acquisition access period of 4 days. This inoculum and its 4 serial dilutions were injected into virus-free aphids and the latter tested singly for their inoculativity. The percentages of transmission for inoculum dilutions of l/40, l/80, l/160, l/320, and l/640 are based on transmission by 146, 83, 114, 175, and 109 injected insects, respectively (combined results of two experiments). The dilution curve shows that percentage transmission can be considered as indicative of relative virus concentration in an inoculum.
aphids could be considered indicative of relative virus concentration in various inocula. A virus inoculum at a dilution of l/40 was prepared from the guts of exposed aphids. This inoculum and its 4 serial dilutions (2fold) were assayed for their infectivity. The combined results of two such experiments are shown graphically in Fig. 11. Our results have suggestedt,hat the persistence of BYDV in aphids, as judged by their ability to transmit, depends on the length of the acquisition accessperiod (Table 3). To find out whether the virus concentration in the gut would continue to increase with increasing acquisition accessperiods, virus-free aphids were caged on infected plants and, at various times, their gut extracts (dilution l/40) were assayed for their infectivity. The combined results of two such experiments
678
PALIWAL
AND
SINHA
any time (Fig. lo), suggesting that the virus did not multiply in this organ. However, with increasing acquisition access periods, the virus continued t’o accumulate in the gut of aphids (Fig. 12). The virus occurs also in large concentrations in the phloem cells of oat plants (Fig. 4). The amount of virus that can be recovered from the gut of aphids, therefore, may simply depend on the period of time the insects feed on cells carrying the virus. This, in turn, seemsto determine the g IO period for which an aphid remains inoculaP tive. It is interesting to note t’hat the relationTIME OF ASSAY IN HOURS ship of BYDV to its vector M. avenue reFIG. 12. Relative BYDV concentration in the semblesin many respects that of pea enation gut of M. avenae after various acquisition access mosaic virus (PEMV) to its aphid vector periods. Virus-free aphids were caged on infected Acyrthosiphon pisum (Harris) (Simons, 1954; plants and at various times 50 insects were reKyriakopoulou and Sylvester, 1969). Sylmoved and their gut extract (dilution l/40) was vester (1969) suggestedthat several features injected into virus-free aphids. Injected aphids of PEMV transmission could be used as were then tested singly for their inoculativity. evidence for a hypothesis that’ the virus is The percentages of transmission by aphids injected with gut extracts made at 3,6,24,48, and 96 propagative in its aphid vector. Many of hours after the start of acquisition access are these features are also exhibited in the case based on a total of 123, 104, 113, 117, and 128 inof BYDV and these are: (1) the occurrence sects, respectively (combined results of two of transstadial passage, (2) the detection of experiments). virus in the hemolymph, (3) the existence of a latent period, (4) the length of the latent period being dependent on the dose of show (Fig. 12) that the virus accumulated in the gut with increasing acquisition access inoculum, (5) the persistence of inocu1ativit.y of vector for a long period of time, and (6) periods. t,he retention of inoculativity being indepenDISCUSSION dent, of the presenceof detectable virus in the gut. The following results suggest that BYDV We believe t,hat the features of transmisdoes not multiply in its aphid vector M. sion outlined above can be explained wit,hout avenae: (1) the failure to transmit the virus assuming BYDV multiplication in the serially from aphid to aphid, (2) the eventual vector. Transstadial passage and presence of loss of inoculativity of most aphids, (3) the period of retention of inoculativity and virus in hemolymph show that the virus is transmission efficiency of aphids being de- circulative but not necessarily propagative pendent on the dose of virus injected or in- in the vector. The latent period of the virus in vector may simply represent the time gested. It should be realized, however, that required for the virus to t’ravel to the salithese results may not be incompatible with virus multiplication in the vector (Sinha vary glands and be inoculated into the and Chiykowski, 1967b; Sinha, 1968), and plants. As the dose of virus is increased, the chancesfor the virus to arrive in the salivary the possibility that the virus may multiply to a limited extent in certain tissues of the glands in a shorter period of time would also increase. As the persist’enceof inoculativity vector is difficult to eliminate. The results of assays of relative BYDV of aphids depends on the dose of virus, it concentrations in the gut of aphids at various cannot be considered as indicative of BYDV times after a 6-hour acquisition access, did multiplicat’ion in the vector. The last feature not show increasesin virus concentration at is a rather weak argument in favour of virus
OL6
BARLEY
YELLOW
multiplication in the vector, unless one presumes that the virus multiplies in an organ(s) other than the gut. BYDV was not detected in the gut of aphids that were injected with a massive dose of virus, suggesting that t’he virus does not pass into this organ from hemolymph. The aphids that, became inoculative after injection with the virus retained their inoculativity for a shorter period of time as compared to those that had fed on infected plants. Furthermore, the virus could not be maintained in the aphids through serial passage. Therefore, it would fat bodies, and appear, that hemocytes, salivary glands do not support BYDV multiplication. Most of the features of PEMV transmission which are considered indicative of a lack of virus multiplication in the vector (Sylvester, 1969) are also exhibited in case of BYDV and are out.lined earlier in the discussion. We, therefore, favour the hypothesis that BYDV does not mult’iply in its aphid vector. The use of aphid tissue culture techniques, recently developed by Peters and Black (1970), may help to resolve the questlion of multiplication of small spherical viruses in their aphid vectors. ACKNOWLEDGMENTS The able technical assistance of Mrs. Brenda Winchester in transmission tests and maintenance of insect cultures is greatly appreciated. We are thankful to Dr. W. F. Rochow, U.S.D.A.-Cornell University, Ithaca, New York, and the California Department of Agriculture, for supplying seed of Coast Black oats and to Dr. C. C. Gill, Canada Department of Agriculture, Winnipeg, Manitoba, for supplying the strain of virus used in this study. The helpful suggestions of Mr. M. R. Binns of the Statistical Research Service, Canada Department of Agriculture, in analysis of the transmission efficiency data are gratefully acknowledged. REFERENCES Black, L. M. (1969). Insect tissue cultures as tools in plant virus research. Annu. Rev. Phytopathol. 7, 73-100. CARTER, W. (1963). “Insects in Relation to Plant Disease,” p. 559. Wiley (Interscience), New York. GILL, C. C. (1969). Cyclical transmissibility of barley yellow dwarf virus from oats with increasing age of infection. Phytopathology 69, 2328.
DWARF
VIRUS
679
HaRRISON, B. D. (1958). Studies on the behaviour of potato leaf roll and other viruses in the body of their aphid vector Myzus persicae (Sula.). Virology 6, 265-277. JENSEN, S. G. (1969). Occurrence of virus particles in the phloem tissue of BYDVinfected barley. Virology 38, 83-91. KYRIAKOPOULOU, P. E., and SYLVESTER, E. S. (1969). Vector age and length of acquisition and inoculation access periods as factors in transmission of pea enation mosaic virus by the pea aphid. J. Econ. Entomol. 62, 1423-1427. MILLER, J. W., and COON, B. F. (1964). The effect of barley yellow dwarf virus on the biology of its vector the English grain aphid, Macrosiphum granarium. J. Econ. Entomol. 67,970-974. MUELLER, W. C., and ROCHOW, W. F. (1961). An aphid-injection method for the transmission of barley yellow dwarf virus. Virology 14, 253-258. O'LOUGHLIN, G.T.,and CHAMBERS, T. C. (1967). The systemic infection of an aphid by a plant virus. Virology 33, 262-271. PALIWAL, Y. C. (1970). Electron microscopy of bromegrass mosaic virus in infected leaves. J. Ultrastruct. Res. 30, 491602. PETERS, D., and BLACK, L. M. (1970). Infection of primary cultures of aphid cells with a plant virus. Virology 40, 847-853. RICHARDSON, J., and SYLVESTER, E. S. (1968). I’urther evidence of multiplication of sowthistle yellow vein virus in its aphid vector, Hyperontyzus lactucae. Virology 36,347-355. ROBERTS, D. W., and GR.4N.4DOS, R. R. (1968). An inexpensive device for pulling glass microneedles for injection of small arthropods. Ann. Entomol. Sot. Amer. 61, 1042-1043. ROCHOW, W. F. (1959). Transmission of strains of barley yellow dwarf virus by two aphid species. Phytopathology 49, 744-748. I
PALIWAL Multiplication of aster yellows virus in a nonvector leafhopper. virology 31, 461-466. SINHA, R. C., and CHIYKOWSKI, L. N. (1967b). Multiplication of wheat striate mosaic virus in its leafhopper vector Endria inimica. Virology 32,402405. SINHA, R. C., and PALIWAL, Y. C. (1970). Localization of a Mycoplasma-like organism in tissues of a leafhopper vector carrying clover phyllody agent. Virology 40.665-672. STEGWEE, D., and PONSEN, M. B. (1958). Multi-
AND
SINHA
plication of potato leaf roll virus in the aphid Myzus persicae (Sulz.). Entomol. Exp. Appl. 1, 291-300. SYLVESTER, E. S. (1969). Virus transmission by aphids-a viewpoint. In “Viruses, Vectors and Vegetation” (K. Maramorosch, ed.), pp. 159173. Wiley (Interscience), New York. SYLVESTER, E. S., and RICHARDSON, J. (1969). Additional evidence of multiplication of the sowthistle yellow vein virus in an aphid vectorserial passage. Virology 37, 26-31.
VIROLOGY
On
the
Mechanism
of Persistence Dwarf
Virus
and
in an Aphid
Y. C. PALIWAL Cell
Biology
Research
Institute,
Research
Distribution
Yellow
Vector’
AND R. C. SINHA
Branch, Accepted
of Barley
Canada July
Department
of Agriculture,
Ottawa,
Ontario
8, 19YO
Barley yellow dwarf virus (BYDV) was recovered, using infectivity bioassays, from the gut, hemolymph, and salivary glands but not from the brain (including ganglia) of aphids Macrosiphum avenue (Fabric&) that had an acquisition access period of 4 days. As a source of virus inoculum, salivary glands were the poorest and gut was the best. However, the virus was not detected in the gut of aphids that were
injected with a massive dose of virus, an observation suggesting that it does not pass from the hemolymph into the gut. The precipitin ring test detected virus antigens in the gut and hemolymph but not in the salivary glands and brain of aphids given a 4-day acquisition access period. Viruslike particles were observed dispersed in the lumen of the gut of exposed aphids, but their identity as BYDV particles could not be established, although large aggregations of BYDV particles were readily identified in the phloem cells of infected oat leaves. The relative virus concentration (as indicated by the percentage transmission of
injected insects) in the gut, when assayed at various times following a 6-hour acquisition access period, remained at about the same level between 6 and 24 hours and declined thereafter up to 96 hours. The virus apparently did not multiply in the gut. That the virus can accumulate in the gut was demonstrated by the increasing virus
concentration
in this organ with increasing
acquisition
access periods.
BYDV could not be maintained in the aphids by serial passage of virus from insect to insect. The ability of aphids to transmit the virus declined following acquisition or injection of the virus into aphids. Also, the period of retention of inoculativity and transmission efficiency of aphids was dependent on the dose of virus ingested or injected. These resldts suggest t,hat BYDV does not multiply in its vector M. aaenae.
the cells of salivary glands of t#he vector (Richardson and Sylvester, 1968; Sylvester and Richardson, 1969). Cytoplasmic infection of the vector tissues by lettuce necrotic yellows virus suggested multiplication of t)he virus in the vector aphids (O’Loughlin and Chambers, 1967). However, the evidence
INTRODUCTION
Although many viruses are known to be transmitted by aphids in a “persistent manner,” the mechanism of persistence of such viruses in their vector has been studied in only a few cases. In the case of at least 2 such viruses, both of which are bacilliform, the persistence can be explained on the basis
of virus multiplication in their vectors. Evidence for multiplication of sowthist’le yellow vein virus was provided by serial passageof the virus among successivegroups of virusfree aphids using hemolymph as inoculum and by demonstration of nuclear infection in 1 Contribution Research Institute.
No.
682 from
the
Cell
Biology
for multiplication of potato leaf roll and peaenat,ion mosaic viruses in their vectors is still conflicting or inconclusive (Stegwee and Ponsen, 1958; Harrison, 1958; Carter, 1963; Black, 1969; Sylvester, 1969). Both t,hese viruses are transmitted by their aphid vectors in a persistent manner and are spherical in shape. Barley yellow dwarf, a small spherical virus (BYDV), can persist in it.s aphid vec-
663
BARLEY
YELLOW
tor for a long period of time. Rochow (1959) found that single Macrosiphum avenae (Fabricius) , continued to transmit BYDV as long as they lived (11-12 days) after a 24-hour acquisition access period. Transstadial passageof the virus in 2M. averme has also been reported (Miller and Coon, 1964). The circulative nature of the virus in its aphid vectors was demonstrated by transmitting the virus through injection of hemolymph from viruliferous aphids into virusfree individuals (Mueller and Rochow, 1961). A latent period of BYDV in the vectors, although poorly defined in terms of a minimum, has been demonstrated as well (Rochow, 1963). We have studied certain aspects of BYDVvector relationship in order to understand the mechanism of persistence of the virus in its vector, $1. avenue. This paper reports on: (1) attempts to transmit BYDV serially from aphid to aphid, (2) effect of varying the length of t’he acquisition access period on transmission behaviour of aphids, (3) relationship of the dose of @us injected int’o aphids to the transmis$on efficiency and retention of inoculativity by the insects, (4) distribution of the virus, as determined by infectivity tests, and of virus antigens in the vector, and (5) relative virus concentration in the gut of aphids during the period following acquisition access. In addition ultrathin sections of internal organs of viruliferous aphids and of infected oat leaves were examined in an attempt to locate BYDV particles in situ. MATERIALS
AND
METHODS
Virus and aphids. A strain of BYDV specifically transmitted by M. avanae, and maintained in oat (Avena byzantina C. Koch var. Coast Black) plants by aphid inoculation was used. Coast Black oats were used as test plants in all experiments. Virus-free aphids were reared on oat plants in growth chambers at 20” with 12,000 lux of light for 16 hours per day. The plants used for rearing virus-free aphids were subsequently sprayed with nicotine sulphate and kept in the greenhouse for observation, so that experiments made from any supposedly virus-free aphids that produced an infected plant could be discarded. Also, as an additional control,
DWARF
VIRUS
669
about 100 aphids from the stock culture were always tested concurrently with every experiment. Apterous young adult aphids of about the same age (8 or 9 days old) were used in all experiments. All acquisition accessperiods were given at 15” on oat plants that had 18-21 days old infections of BYDV. The insects, either after an acquisition access or after injection were held, during the test feeding period, in a growth room with night-day temperature range of 19”-21” and 10,000 lux of light for 16 hours a day. When viruliferous aphids were required to be maintained in groups for more than a day, they were transferred to new plants every 24 hours because it has been shown that aphids can inoculate and reacquire BYDV from a plant in a minimum of 3 days (Gill, 1969). After the test feeding, inoculated plants were sprayed with nicotine sulphate, held in a greenhouse for 30 days and observed regularly for symptoms of BYDV. The test plants in the greenhouse were sprayed with nicotine sulphate every week. Certain terms used in this paper are defined as follows: Exposed aphids. Insects that had been confined to infected plants. Inoculativity. The ability of aphids to transmit the virus to plants. Inoculative aphid. An aphid that transmitted the virus to at least one plant when tested for its inoculativity on a seriesof successive test plants. Mean t+ansmission eficiency per aphid. Mean number of transmissions per inoculative aphid in a given number of successive inoculation feeding periods. It was calculated from the relationship cc-l, Xi,, where X is the proportion of plants to which an inoculative aphid transmitted the virus in a given number of successive 24-hour inoculation feeding periods, and N is the total number of inoculative aphids tested in each of the inoculation feeding periods. The t test was used to determine the significance of the difference between the values of this statistic for different doses of virus received by the aphids (either by injection or by acquisition access). The values of X (in percent) were transformed to angles (angle = arc sin per‘centage1J2)before using them to compute the t value.
670
PALIWAL
AND
Extracts of internal organs and hemolymph suspensions.Aphids were dissected in saline (0.85% NaCl) with the help of fine needles and forceps to obtain salivary glands, brain (including ganglia), and gut (Figs. l-3). First, the head along with most of the thorax was removed from the abdomen. The gut was then forced out of t’he abdomen by pushing the caudal end inward and pressing it gently. The connection of the hindgut to cauda was severed to free the gut. Salivary glands and brain were separated out carefully from the head-thorax portion. A known number of organs of t,he same kind were pooled, transferred into a tissue grinder, washed 2-3 times with saline and ground in a known volume of the latter. The hemolymph suspensionwas prepared by making a small incision in the abdomen of aphids and touching the oozing fluid into a known
SINHA
volume of saline on a depression slide. The extracts of organs and hemolymph suspension were clarified by low speed centrifugation before inject’ion into aphids. Details of the dissection, grinding and clarification procedures were the same as described previously by Sinha and Chiykowski (1967a). In experiments where an extract of guts of exposed aphids was used as a source of virus, the insects had an acquisition accessperiod of 4 days, the dilution of the inoculum being calculated on the basis of weight of the guts to the volume of solvent. The weight of a single gut, determined by weighing 2 batches of 100 guts each, was found to be about 0.05 mg. In all experiments at least 50 guts were used to prepare a base inoculum at a dilution of l/40 (for example, guts weighing 0.1 g ground in 3.9 ml of saline). Virus assay. The infectivity of the extracts
FIGS. 1 and 3. Some internal organs of an aphid, Macrosiphum avenue (Fabricius)-a vector of barley yellow dwarf virus. Fro. 1. The gut. Fg, foregut; C, crop; In, intestine; Hg, hindgut; Ca, cauda. X 48. FIG. 2. The nervous system. B, brain; G, ganglia; Mn, main abdominal nerve. X 52. FIG. 3. The salivary glands. Pg, principal gland; Ag, accessory gland; AC, acinus. X 48.
BARLEY
YELLOW
was assayed by injecting them into virusfree aphids and testing the latter for their inoculativity. Aphids were anesthetized in batches of 10 with COZ and were injected in their abdominal hemocoele (Sinha and Chiykowski, 1967a). Needles for injection were drawn from glass capillaries (outer diameter, 2 mm) using a pulling device (Roberts and Granados, 1968). The volume of inoculum injected into each aphid was estimated to be about 0.04 ~1. Insects were injected in the late afternoon and held overnight at 15” on detached oat leaves in a petri dish. Next morning, they were caged singly (unless stated otherwise) for 4 days on oat plants to test their inoeulativity. Only those aphids that survived for all 4 days were used to calculate the percentage transmission. The day of injection was numbered 0, the next day 1, and so on throughout the test feeding. Xerology. To prepare an antiserum, BYDV was partially purified, using a procedure described by Rochow and Brakke (1964) but without density gradient centrifugation, from 300 g of infected oat leaves (15-18 days old infection). Five injections of such a preparation were given at weekly intervals into a rabbit after being emulsified wit,h an equal volume of Freund’s incomplete adjuvant. The precipitin ring test was used in all serological reactions. The antiserum had a titer of l/80 against an extract of guts (at a dilution of l/20, w/v) obtained from exposed aphids but showed no reaction against extracts of guts, salivary glands, brain, and hemolymph suspensions prepared from virus-free aphids. Also, the preimmune serum showed no reaction with extracts of organs from exposed aphids. Electron microscopy. Oat leaves, and gut, salivary glands and brain of M. avenue were processed and sectioned following the procedures described earlier (Paliwal, 1970; Sinha and Paliwal, 1970), and examined in a Siemens Elmiskop I. RESULTS
Attempts to Transmit the Virus Serially from Aphid to Aphid by Hemolymph Inoculations A method described by Sylvester and Richardson (1969) was used to collect hemo-
DWARF
VIRUS
671
lymph from an aphid directly in the injecting. needle. Each aphid from a group of insects,. that was allowed a 4-day acquisition access period, was used as a donor of hemolymph for the inoculation of one virus-free aphid (1st passage). All injected aphids were then tested singly for their inoculativity (1 day on each of 2 successive plants). Each surviving insect again served as a donor of hemolymph to a virus-free aphid (2nd passage). After doing one more such hemolymph transfer (3rd passage) the whole experiment was repeated. A total of 36 aphid lines completed the 3 passages. In the first passage, 14 out of 36 aphids transmitted the virus but no transmission occurred in the 2nd or 3rd passage, suggesting that BYDV does not multiply in aphids that are injected with the virus. Rochow (1969), in a review article, also reported that attempts in his laboratory to transmit BYDV serially from aphid t.o aphid were unsuccessful. Distribution leaves
of the virus in vector and in oat
The gut, salivary glands, brain, and hemolymph suspensions from aphids, that had a 4-day acquisition access period, were tested for the presence of BYDV. One hundred organs of each kind (in 0.1 ml saline) were used to prepare the extracts of various organs. The hemolymph suspension was prepared also from 100 aphids in 0:l ml of saline. Aphids injected wit.h different inocula were caged in groups of 5 on oat plants for 4 days. The virus was recovered from the gut, salivary glands and hemolymph but not from the brain (Table 1). When these extracts and hemolymph suspension were tested against BYDV antiserum (dilution l/20), the virus antigens were detected in the gut and hemolymph but not in the salivary glands and brain. It is evident from the results that as a source of virus inoculum, the salivary glands were the poorest and gut was the best. Ultrathin sections of infected oat leaves showed large aggregations of spherical virus particles, 22-25 nm in diameter, in the phloem cells only (Figs. 4 and 5). No such particles were found in healthy leaves. While this work was in progress, Jensen (1969) reported the occurrence of large con-
672
PALIWAL TABLE
1
AND SINHA
and the latter were tested singly for their inoculativity for 8 days (1 day each on 8 avenae successiveplants). The survival of injected aphids (Fig. 7) during the g-day test feeding vinls antigens period appears to have been affected by the Source of inoculuma z&$&b % ine;F concentration of tissue constituents in the inoculum injected as the insect mortality tractsC was comparatively higher when the more concentrated inoculum was used. The numGut 34/37 92 ++ ber of insects that became inoculative after Hemolymph suspension 12/23 52 + Salivary glands 13128 46 being injected with the inocula at dilutions Brain (including ganglia) o/20 0 of l/40 and l/320 were 140/166 (84%) and 55/120 (46 %), respectively. a One hundred organs of the same kind, obThe daily transmission by inoculative tained from aphids that had an acquisition of 4 aphids during the test feeding period showed days, were pooled, ground in 0.1 ml of saline, (Table 2) that all insects did not transmit their clarified extracts injected into virus-free simultaneously on any one day. On day 1, aphids, and the latter tested for their inoculativity. Hemolymph suspensions were also prepared 80% of the inoculative insects transmitted from 100 exposed aphids in 0.1 ml of saline. the virus when dilution of the inoculum was b Combined results of two experiments; numerl/40 but only 49 % of the insects transmitted ator is the number of plants that became infected; on this day in case of l/320 dilution. Also, denominator is the number of plants inoculated the percentage transmission always remained by 5 injected insects per plant. comparatively higher when the aphids rec The precipitin ring test was used to detect the virus antigens; the antiserum was used at a ceived the higher dose. The ability of aphids to transmit the virus declined following indilution of l/20. The number of plus signs indicates the relative thickness of the ring after 90 jection with either dose of virus while the period of retention of inoculativity appears minutes; a minus sign means that the results were to be dependent on the dose of virus. The negative. mean transmission efficiency per inoculative centration of BYDV particles in the phloem aphid, based on transmission by those incells of barley leaves infected with a strain of sects that survived for at least 7 days, was the virus transmitted by Rhopalosiphum significantly higher for the aphids injected with the higher dose of virus. These results padi (Linneaus) . Examinations of 5 internal organs of each also suggest a lack of virus multiplication in kind from exposed (4-day acquisition access aphids that were injected with the virus. period) as well as virus-free aphids did not reveal aggregations of virus particles, such Transmission of BYDV by Aphids after Various Acquistion Accessl’eriods as those observed in plants, in any of the We wanted to determine wllether or not tissues examined. However, viruslike particles, some poorly defined, were found dis- the transmission behaviour of aphids after persed in the lumen of the guts of several feeding on infected plants was the same as that observed with insects that became inocexposed aphids, (Fig. 6), but it was difficult to identify BYDV virions among these ulative after injection with the virus. The particles because a few poorly defined par- virus dosage in aphids can be varied by ticles were occasionally found scattered in varying the length of acquisition access the lumen of the gut of virus-free aphids. period (Rochow, 1963). Virus-free aphids were given acquisition Transmission of BYDV by Aphids Injected accessperiods of 3, 6, 12, and 24 hours. They with Di$erent Dosesof Virus were caged singly immediately thereafter An extract, prepared from guts of exposed on oat plants to test their inoculativity. All aphids, at a dilution of l/40 and its 8-fold insects were transferred to new plants at dilution were injected into virus-free aphids 24-hour intervals. The insects that had an DISTRIBUTION
OF BYDV Macrosiphum
IN THE VECTOR
FIG. 4. BYDV particles in a phloem cell of oat leaf. X 25,500. FIG. 5. A mass of BYDV particles at a higher magnification. X 45,000. FIG. 6. Viruslike particles in the lumen of the gut of M. avenae. Aphids were exposed to BYDV fected plants for 4 days, and then their guts were removed and processed for electron microscopy. difficult to identify BYDV virions among these particles because a few scattered, somewhat similar, poorly defined particles were occasionally seen in the lumen of healthy controls also. X 46,500. ri7.1
inIt is but
674
PALIWAL
AND SINHA
the 15 surviving inoculative insects had ceased to transmit in case of the shorter acquisition access,whereas 6 out of 13 were 80 still transmitting when the insects had the longer acquisition accessperiod. On day 20, of 5 and 9 aphids that survived after the 3and 24-hour acquisition access periods respectively, only 1 insect was still transmitting from the group that had the longer acquisition access.This insect, incidentally, transmitted the virus to all of the 20 test plants. There were two aphids in the 3-hour acquisition accessgroup which transmitted to 12 consecutive test plants. In addition to 100 a general decline in the ability of insects to 2 3 4 5 6 7 a transmit the virus, the period of retention of DAYS AFTER INJECTION inoculativity was dependent on the dose of FIG. 7. Survival curves for adult M. avenue on virus acquired by the insects. Also, the mean oat plants after being injected with two doses of transmission efficiency per inoculative aphid, virus. A total of 166 and 120 insects injected with based on transmission by those insects that virus inocula at dilutions of l/40 and l/320, respecsurvived for 11 days, was significantly higher tively, were tested. for insects that had a longer acquisition acacquisition access of either 3 or 24 hours cessperiod (Table 3). The transmission of BYDV had similar were tested for 20 days but those that were attributes whether the aphids received the allowed a 6- or la-hour acquisition access virus through injection or by feeding on inwere tested for only 6 days. The results show jected plants, except that aphids that bethat no new transmission occurred beyond day 6, after either 3- or 24-hour acquisition came inoculative after injection lost their access periods. The proportion of aphids ability to transmit in a shorter period of that became inoculative after various ac- time, as compared to those that became quisition accessperiods had a linear re!ation- inoculative after an acquisition accessperiod. ship with the length of acquisition access Attempts to Detect the Virus in Guts of Injected period (Fig. 8). Aphids The survival of exposed aphids during the The distribution of BYDV in the aphids lo-day period following 3- or 24-hour acquisition accessis shown in Fig. 9 and trans- (Table 1) suggeststhat when insects acquire mission results (up to day 11-the day when virus by feeding on infected plants, the virus passesfrom the gut into the hemocoele, is about 50% of the aphids were surviving) for aphids that became inoculative after the carried to the salivary glands via hemotwo acquisition accessperiods are given in lymph, and is introduced into the plants Table 3. Although all inoculative insects through the salivary secretions; whereas never transmitted the virus simultaneously when the insects are injected, the virus is on any one day either after 3- or 24-hour directly introduced into the hemocoele. It acquisition access, the percentage transmis- can be argued, therefore, that longer retension on all days was higher when the aphids tion of BYDV by aphids after an acquisition had a longer acquisition accessperiod. For accesscould be due to multiplication of the virus in the gut. If this is true then it must be example, on day 11, 52 % of the inoculative insects transmitted the virus in case of 24- presumed that the virus in the injected inhour acquisition access but only 25 % did sects cannot passinto the gut. Attempts were made, therefore, to detect when the insects had a 3-hour acquisition access (Table 3). However, by day 15, all the virus in the guts of aphids that were
BARLEY
YELLOW
DWARF
TABLE TR.4NSMISSION
PATTERN
OF APHIDS DIFFERENT
675
VIRUS
2
THAT WERE INJECTED WITH CONCENTRATIONS OF BYDV
INOCULA
CONTAINING
Daily transmission by inoculative aphids Based only on those aphids that survived for 7 days
Based on the aphids surviving on a given day Days Dilution of the virus inoculaa
--. 1 2 3 4 5 6 7 8
l/JO Transmissionb
%
l/320 Transmission
%
112/140 113/138 (2Dc 94/131 (7) 60/114 35181 9151 3/24 l/12
80 82 72 53 43 18 12 8
27/55 28/55 (17) 21/54 (10) wo (1) 5149 2/47 O/42 O/36
49 51 39 18 10 4 0 0
Mean transmission efficiency per inoculative aphidd
-
-
1140
l/320
7. Transmission 75 75 71 42 33 17 12 -
45 48 40 21 12 5 0 -
44.3”
23.6
a To prepare an inoculum at a dilution of l/40 (w/v), 50 guts (obtained from aphids that had an acquisition access of 4 days on infected plants) were ground in saline and the extract was clarified. This inoculum was diluted with saline to l/320 and both dilutions were injected into virus-free aphids. b Injected aphids were caged singly on oat plants and transferred daily to a new plant for 8 days. Numerator is the number of aphids that transmitted the virus on a given day; denominator is the number of inoculative aphids surviving on that day. Combined results af two experiments. c The figures in parentheses represent the number of new transmissions on that day. d Calculated from the daily transmission data of individual inoculative aphids surviving for 7 days, expressed in percents. e Difference between the values for the two doses of virus are highly significant; t value at 64 degrees of freedom = 10.09, P < 0.001.
C
3
6 12 ACQUISITION ACCESS PERIOD IN HOURS
24
FIG. 8. Transmission of BYDV by M. auenae (tested singly) after various acquisition access periods. The percentages of transmission for acquisition access periods of 3,6,12, and 24 hours are based on transmission by 71, 61, 70, and 105 exposed insects, respectively.
injected with a virus inoculum. An inoculum prepared from 100 guts (dilution l/20) of aphids that had an acquisition accessperiod of 4 days, was injected into virus-free aphids. In one experiment, 2 days after, and in another, 6 days after injection, extracts (dilution l/20) prepared from 100 guts of injected aphids in each case, were assayed for the presence of virus. None of the 70 and 85 test insects in the first and secondexperiments, respectively, transmitted the virus showing that the virus was not detected in the guts of injected aphids. In the casesof wound-tumor and wheat striate mosaic viruses also, the virus was not detected in the gut of their leafhopper vectors following
676
PALIWAL
yj
AND
SINHA
50-
65 p 40.---.3 30-
HOUR ACPlJlSlTlON ACCESS 24 HOUR ACQIJISITION ACCESS
-
ZOIO
I 10
I 5
1
DAYS AFTER
FIG. 9. Survival curves for adult access period of 3- or 24-hours (based
TRANSMISSION
PATTERN
1 20
I 15
ACQUISITION
ACCESS
PERIOD
M. avenue on oat plants, after the insects had on 93 and 74 insects in the two cases, respectively).
TABLE AFTER
BY APHIDS
Daily
3 V.*RIOUS
ACQUISITION
ACCESS
transmission
by inoculative
aphids
Days Acquisition
1 2 3 4 5 6 7 8 9 10 11 Mean transmission efficiency per inoculative aphid* 0 Numerator is the number number of inoculative aphids b Calculated from the daily expressed as percentages. e Difference between these value at 53 degrees of freedom
%
4135 7135 18/35 16134 16/31 17/29 12129 10128 111% 9/26 6124
11 20 51 47 52 59 41 36 42 35 25
access period
24 Transmissiona
-
PERIODS
in hours 3 24 To Transmission
%
30165 45/65 46164 56/64 56164 51/63 41158 30147 32/43 21/38 16/31
an acquisition
Based only on those aphids that survived for 11 days
Based on the number of aphids surviving on a given day
3 Transmissiona
had
46 69 72 87 87 81 71 63 74 55 52 -
17 12 46 37 54 58 42 37 46 37 25
45 61 65 90 74 74 74 61 71 48 52
36.3c
70.1
of aphids that transmitted the virus on a given day; denominator tested on that day. Combined results of two experiments. transmission data of individual inoculative aphids surviving for values for the two acquisition = 4.23, P < 0.001.
access
periods
are
highly
is the 11 days
significant;
1
BARLEY
YELLOW
DWARF
677
VIRUS
injection with a massive dose of virus (Sinha, unpublished results). It appears that the movement of BYDV between gut and hemocoele is unidirectional. Relative Concentration of BYDV in the Gut of Aphids after Ingestion of the Virus
In an attempt to determine whether the virus multiplies in the gut, virus-free aphids were given an acquisition access period of 6 hours and were then maintained on healthy plants. At various time intervals, an extract (at a dilution of l/40) prepared from the guts of exposed aphids was assayed for its infectivity. The results show (Fig. 10) that the relative virus concentration, as indicated by the percentage of insects that became inoculative, remained at about the same level between 6 and 24 hours and then decreased to a low concentration by 96 hours. The following experiment demonstrated that the percentage transmission by injected
0 ( 0
I 6
24
I 40
7i
I 96
TIME OF ASSAY IN HOURS
FIG. 10. Relative BYDV concentration in the gut of M. avenae at various times after a 6-hour acquisition access period. After the acquisition access period, aphids were maintained on healthy plants and at various times (from the start of acquisition access) an extract (dilution l/40) of 50 guts of exposed aphids was injected into virus-free aphids and the latter tested singly for their incculativity. Percentages of transmission by aphicis injected with gut extracts made at 6,24,48, and 96 hours from the start of acquisition access are calculated from a total of 125, 128, 97, 109, and 113 aphids, respectively (combined results of two experiments).
RECIPROCAL
LOG DILUTIONS OF INOCULd
FIG. 11. Transmission
of BYDV by M. avenae injected with inocula containing different concentrations of the virus. An inoculum at a base dilution of l/40 (w/v) was prepared from 50 guts of aphids that had an acquisition access period of 4 days. This inoculum and its 4 serial dilutions were injected into virus-free aphids and the latter tested singly for their inoculativity. The percentages of transmission for inoculum dilutions of l/40, l/80, l/160, l/320, and l/640 are based on transmission by 146, 83, 114, 175, and 109 injected insects, respectively (combined results of two experiments). The dilution curve shows that percentage transmission can be considered as indicative of relative virus concentration in an inoculum.
aphids could be considered indicative of relative virus concentration in various inocula. A virus inoculum at a dilution of l/40 was prepared from the guts of exposed aphids. This inoculum and its 4 serial dilutions (2fold) were assayed for their infectivity. The combined results of two such experiments are shown graphically in Fig. 11. Our results have suggestedt,hat the persistence of BYDV in aphids, as judged by their ability to transmit, depends on the length of the acquisition accessperiod (Table 3). To find out whether the virus concentration in the gut would continue to increase with increasing acquisition accessperiods, virus-free aphids were caged on infected plants and, at various times, their gut extracts (dilution l/40) were assayed for their infectivity. The combined results of two such experiments
678
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any time (Fig. lo), suggesting that the virus did not multiply in this organ. However, with increasing acquisition access periods, the virus continued t’o accumulate in the gut of aphids (Fig. 12). The virus occurs also in large concentrations in the phloem cells of oat plants (Fig. 4). The amount of virus that can be recovered from the gut of aphids, therefore, may simply depend on the period of time the insects feed on cells carrying the virus. This, in turn, seemsto determine the g IO period for which an aphid remains inoculaP tive. It is interesting to note t’hat the relationTIME OF ASSAY IN HOURS ship of BYDV to its vector M. avenue reFIG. 12. Relative BYDV concentration in the semblesin many respects that of pea enation gut of M. avenae after various acquisition access mosaic virus (PEMV) to its aphid vector periods. Virus-free aphids were caged on infected Acyrthosiphon pisum (Harris) (Simons, 1954; plants and at various times 50 insects were reKyriakopoulou and Sylvester, 1969). Sylmoved and their gut extract (dilution l/40) was vester (1969) suggestedthat several features injected into virus-free aphids. Injected aphids of PEMV transmission could be used as were then tested singly for their inoculativity. evidence for a hypothesis that’ the virus is The percentages of transmission by aphids injected with gut extracts made at 3,6,24,48, and 96 propagative in its aphid vector. Many of hours after the start of acquisition access are these features are also exhibited in the case based on a total of 123, 104, 113, 117, and 128 inof BYDV and these are: (1) the occurrence sects, respectively (combined results of two of transstadial passage, (2) the detection of experiments). virus in the hemolymph, (3) the existence of a latent period, (4) the length of the latent period being dependent on the dose of show (Fig. 12) that the virus accumulated in the gut with increasing acquisition access inoculum, (5) the persistence of inocu1ativit.y of vector for a long period of time, and (6) periods. t,he retention of inoculativity being indepenDISCUSSION dent, of the presenceof detectable virus in the gut. The following results suggest that BYDV We believe t,hat the features of transmisdoes not multiply in its aphid vector M. sion outlined above can be explained wit,hout avenae: (1) the failure to transmit the virus assuming BYDV multiplication in the serially from aphid to aphid, (2) the eventual vector. Transstadial passage and presence of loss of inoculativity of most aphids, (3) the period of retention of inoculativity and virus in hemolymph show that the virus is transmission efficiency of aphids being de- circulative but not necessarily propagative pendent on the dose of virus injected or in- in the vector. The latent period of the virus in vector may simply represent the time gested. It should be realized, however, that required for the virus to t’ravel to the salithese results may not be incompatible with virus multiplication in the vector (Sinha vary glands and be inoculated into the and Chiykowski, 1967b; Sinha, 1968), and plants. As the dose of virus is increased, the chancesfor the virus to arrive in the salivary the possibility that the virus may multiply to a limited extent in certain tissues of the glands in a shorter period of time would also increase. As the persist’enceof inoculativity vector is difficult to eliminate. The results of assays of relative BYDV of aphids depends on the dose of virus, it concentrations in the gut of aphids at various cannot be considered as indicative of BYDV times after a 6-hour acquisition access, did multiplicat’ion in the vector. The last feature not show increasesin virus concentration at is a rather weak argument in favour of virus
OL6
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YELLOW
multiplication in the vector, unless one presumes that the virus multiplies in an organ(s) other than the gut. BYDV was not detected in the gut of aphids that were injected with a massive dose of virus, suggesting that t’he virus does not pass into this organ from hemolymph. The aphids that, became inoculative after injection with the virus retained their inoculativity for a shorter period of time as compared to those that had fed on infected plants. Furthermore, the virus could not be maintained in the aphids through serial passage. Therefore, it would fat bodies, and appear, that hemocytes, salivary glands do not support BYDV multiplication. Most of the features of PEMV transmission which are considered indicative of a lack of virus multiplication in the vector (Sylvester, 1969) are also exhibited in case of BYDV and are out.lined earlier in the discussion. We, therefore, favour the hypothesis that BYDV does not mult’iply in its aphid vector. The use of aphid tissue culture techniques, recently developed by Peters and Black (1970), may help to resolve the questlion of multiplication of small spherical viruses in their aphid vectors. ACKNOWLEDGMENTS The able technical assistance of Mrs. Brenda Winchester in transmission tests and maintenance of insect cultures is greatly appreciated. We are thankful to Dr. W. F. Rochow, U.S.D.A.-Cornell University, Ithaca, New York, and the California Department of Agriculture, for supplying seed of Coast Black oats and to Dr. C. C. Gill, Canada Department of Agriculture, Winnipeg, Manitoba, for supplying the strain of virus used in this study. The helpful suggestions of Mr. M. R. Binns of the Statistical Research Service, Canada Department of Agriculture, in analysis of the transmission efficiency data are gratefully acknowledged. REFERENCES Black, L. M. (1969). Insect tissue cultures as tools in plant virus research. Annu. Rev. Phytopathol. 7, 73-100. CARTER, W. (1963). “Insects in Relation to Plant Disease,” p. 559. Wiley (Interscience), New York. GILL, C. C. (1969). Cyclical transmissibility of barley yellow dwarf virus from oats with increasing age of infection. Phytopathology 69, 2328.
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HaRRISON, B. D. (1958). Studies on the behaviour of potato leaf roll and other viruses in the body of their aphid vector Myzus persicae (Sula.). Virology 6, 265-277. JENSEN, S. G. (1969). Occurrence of virus particles in the phloem tissue of BYDVinfected barley. Virology 38, 83-91. KYRIAKOPOULOU, P. E., and SYLVESTER, E. S. (1969). Vector age and length of acquisition and inoculation access periods as factors in transmission of pea enation mosaic virus by the pea aphid. J. Econ. Entomol. 62, 1423-1427. MILLER, J. W., and COON, B. F. (1964). The effect of barley yellow dwarf virus on the biology of its vector the English grain aphid, Macrosiphum granarium. J. Econ. Entomol. 67,970-974. MUELLER, W. C., and ROCHOW, W. F. (1961). An aphid-injection method for the transmission of barley yellow dwarf virus. Virology 14, 253-258. O'LOUGHLIN, G.T.,and CHAMBERS, T. C. (1967). The systemic infection of an aphid by a plant virus. Virology 33, 262-271. PALIWAL, Y. C. (1970). Electron microscopy of bromegrass mosaic virus in infected leaves. J. Ultrastruct. Res. 30, 491602. PETERS, D., and BLACK, L. M. (1970). Infection of primary cultures of aphid cells with a plant virus. Virology 40, 847-853. RICHARDSON, J., and SYLVESTER, E. S. (1968). I’urther evidence of multiplication of sowthistle yellow vein virus in its aphid vector, Hyperontyzus lactucae. Virology 36,347-355. ROBERTS, D. W., and GR.4N.4DOS, R. R. (1968). An inexpensive device for pulling glass microneedles for injection of small arthropods. Ann. Entomol. Sot. Amer. 61, 1042-1043. ROCHOW, W. F. (1959). Transmission of strains of barley yellow dwarf virus by two aphid species. Phytopathology 49, 744-748. I
PALIWAL Multiplication of aster yellows virus in a nonvector leafhopper. virology 31, 461-466. SINHA, R. C., and CHIYKOWSKI, L. N. (1967b). Multiplication of wheat striate mosaic virus in its leafhopper vector Endria inimica. Virology 32,402405. SINHA, R. C., and PALIWAL, Y. C. (1970). Localization of a Mycoplasma-like organism in tissues of a leafhopper vector carrying clover phyllody agent. Virology 40.665-672. STEGWEE, D., and PONSEN, M. B. (1958). Multi-
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plication of potato leaf roll virus in the aphid Myzus persicae (Sulz.). Entomol. Exp. Appl. 1, 291-300. SYLVESTER, E. S. (1969). Virus transmission by aphids-a viewpoint. In “Viruses, Vectors and Vegetation” (K. Maramorosch, ed.), pp. 159173. Wiley (Interscience), New York. SYLVESTER, E. S., and RICHARDSON, J. (1969). Additional evidence of multiplication of the sowthistle yellow vein virus in an aphid vectorserial passage. Virology 37, 26-31.