Centro de Ciencias Medioambientales, CSIC, Serrano 115 dpo, 28006 Madrid, Spain1
Station de Recherches de Pathologie Comparée, INRA-CNRS-UM II, 30380 Saint Christol-les-Alès, France2
Author for correspondence: Alberto Fereres. Fax +34 91 5640800. e-mail afereres{at}ccma.csic.es
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Abstract |
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Introduction |
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CaMV uses the helper strategy for the transmission process (Pirone & Blanc, 1996 ). The helper strategy implies that a non-structural viral protein (helper component, HC) mediates the interaction for attachment, and thus retention, of viral particles in the cuticle of the aphid stylet. For CaMV, the HC is the viral protein P2, which binds with one domain to a non-identified attachment site in the stylet and with another domain to the viral protein P3 associated with the virus capsid (Blanc et al., 2001
; Leh et al., 1999
, 2001
; Woolston et al., 1987
). In order to be transmitted, a P2P3virion complex must form. However, in infected plant cells P2 and P3virions are mainly sequestered in two different viral inclusion bodies. While electron-dense inclusion bodies (edIBs) contain 96% of the total number of virions associated with P3 and no P2, the electron-lucent inclusion bodies (elIBs) contain all of P2, some P3, and the remaining 4% of virus particles (Drucker et al., 2002
; Espinoza et al., 1991
). Consequently, it has been suggested that transmissible complexes do not predominantly form in plant cells but in the aphid mouthparts, where the P2 and P3-virion pools are bound during the aphids feeding activities.
There are at least 27 aphid species listed as known vectors of CaMV (Kennedy et al., 1962 ), but the main vectors in the field are Brevicoryne brassicae L. and Myzus persicae Sulzer (Broadbent, 1957
). Markham et al. (1987)
demonstrated that CaMV is transmitted in a semipersistent manner, although conflicting results were obtained in earlier work: Some authors have considered CaMV to be an atypical non-persistently (Hamlyn, 1955
; van Hoof, 1958
) or bimodally transmitted virus (Bouchery et al., 1990
; Chalfant & Chapman, 1962
). The term bimodal was proposed to describe the transmission rate of CaMV by B. brassicae as a curve with two peaks associated with optimum acquisition-access times of around 5 min and 8 h. This bimodal type of transmission has been described for only one other plant virus, the potyvirus Pea seed-borne mosaic virus (Lim & Hagedorn, 1977
). However, the term bimodal is somehow misleading, because the optimum acquisition peaks of CaMV may vary and exhibit a bi- or multiphasic pattern depending on the species of vector used for the transmission experiments (Markham et al., 1987
).
Electrical systems for monitoring insect probing and feeding behaviour provide detailed analysis of aphid stylet penetration and the vectorplant interactions involved in the transmission of plant viruses (see reviews by Fereres & Collar, 2001 ; Tjallingii & Prado, 2001
). Using DC-amplifier monitoring systems, researchers can correlate recorded waveform patterns (so-called electrical penetration graphs, EPG) with specific stylet positioning and feeding activities of the insect associated with the transmission plant viruses. Acquisition of typical non-persistent viruses by aphids occurs during the last subphase (II-3) of superficial intracellular stylet puncture (Martin et al., 1997
; Powell et al., 1995
), while persistently transmitted viruses are acquired during the phloem ingestion phase (Prado & Tjallingii, 1994
). However, information on specific probing or feeding behavioural events (or EPG waveforms) associated with the acquisition of semipersistent viruses by aphids is lacking.
Semipersistently transmitted viruses include members of seven genera (Badnavirus, Caulimovirus, Closterovirus, Sequivirus, Trichovirus, Waikavirus and Crinivirus) that are transmitted by aphids or other homopterans (Hull, 2001 ; Powell et al., 1995
). The best-known semipersistent viruses are caulimoviruses and closteroviruses. The closterovirus Beet yellows virus (BYV) is found in the phloem (Esau et al., 1967
), while caulimoviruses are present in most plant tissues (Francki et al., 1985
; Kitajima et al., 1969
). Limburg et al. (1997)
found that the time threshold needed for acquisition of BYV was consistent with the mean time for aphids to reach the phloem. However, there is no experimental evidence showing whether caulimoviruses are acquired from epidermis, mesophyll or from vascular tissues.
A prerequisite for elucidation of the transmission mechanism of CaMV is a better characterization of the acquisition phase. In the present study, we investigated the probing and feeding behaviour of B. brassicae and M. persicae during the acquisition of CaMV using the EPG technique. The goal was to find out from which specific plant tissue(s) aphids usually acquire CaMV as well as to investigate aphid probing and feeding behavioural patterns associated with acquisition of the virus. We also compared the probing behaviour of the two main aphid vectors after short and long acquisition access periods to clarify the discrepancies seen in previous reports on the transmission mechanism of CaMV. To complement this study, we used immunoelectron microscopy to detect CaMV virions, P2 and P3 in the vascular tissue of infected plants.
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Methods |
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Non-viruliferous aphid clones of M. persicae and B. brassicae were reared under controlled conditions [(22/18 °C and 14/10 h (light/dark)] on Brassica rapa cv. Just Right. The clones of M. persicae and B. brassicae were started from single virginiparous females collected at Alcala de Henares and Villa del Prado (Madrid, Spain), respectively.
Fifteen-day-old seedlings of Brassica rapa cv. Just Right were used as test plants for all experiments because they show very clear symptoms when infected with CaMV. The seedlings were sprayed with Confidor (Bayer Hispania Industria) after the inoculation access period (1824 h), and transferred to the aphid-free growth chamber where they were checked regularly for symptoms during a 35 week period.
Transmission efficiency of CaMV by its main vectors.
The transmission efficiency of CaMV by M. persicae and B. brassicae was first tested under laboratory conditions. For this set of experiments, aphids were not connected to the EPG device. The transmission procedure was similar to the one described by Fereres et al. (1993) . Groups of 2530 aphids (young apterae adults) were placed inside plastic cages for a 1 h pre-acquisition period. Then, the last expanded leaf of a young infected turnip plant was detached from the plant and used for virus acquisition. Twenty aphids at a time were allowed to acquire the virus from the infected leaf. After a 5 min or 8 h acquisition access period (two different treatments), groups of five aphids were transferred to each turnip test plant for an 1824 h inoculation access period.
EPG recording set-up.
A gold wire (3 cm longx20 mm diameter) was attached to the dorsum of a young adult apterae aphid by immobilizing it with a vacuum-operated plate and touching the aphid with a small droplet of silver conducting paint (Pelco Colloidal Silver no. 16034, Ted Pella Inc., Reeding, CA, USA). The other end of the gold wire was attached to a copper wire (3 cm longx1 mm diameter) which was connected to one of the electrodes of the EPG system. A second electrode was connected to a copper post (0·2 cm diameterx10 cm long) which was inserted into the plant pot (Tjallingii, 1990 ).
EPG recordings were acquired at 100 Hz through a 4-channel Giga-99 DC-amplifier. This 1 giga-ohm input resistance DC-amplifier system has its own AD converter, which allows direct recording of the EPG signals onto the PC hard disk at the time that the EPG waveforms are displayed on the computer monitor. Data acquisition and screen display were controlled by Stylet 3.0 software and data analysis was performed with MacStylet v2.0 10 (Febvay et al., 1996
) software after data conversion.
EPG recording during acquisition of CaMV.
To investigate the behavioural events associated with acquisition of CaMV, aphids were first connected to the EPG device and then placed on a CaMV-infected source plant. Aphid probing was artificially interrupted by removing the aphid from the infected plant with a paintbrush just after the following events: (a) aphids were allowed to make a single intracellular puncture (=potential drop, pd) during the first probe. Aphids that were unable to make an intracellular puncture within 3 min of the beginning of the first probe were discarded; (b) aphids were allowed a 5 min acquisition access period starting after the beginning of the first probe; (c) aphids were allowed a 30 min acquisition access period starting after the beginning of the first probe; (d) aphids were allowed to carry out a committed phloem ingestion phase (E2 15 min). Aphids that were unable to reach the phloem within 3 h were discarded. To reduce the time to reach the phloem, aphids were placed on the abaxial surface of the last expanded leaf.
Groups of starved (for 1 h before virus acquisition) and non-starved aphids were used for the studies described under treatments (a) and (b). Table 1 shows the specific EPG variables that were calculated and analysed for each of the treatments described above.
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Statistical analysis.
Transmission rate, calculated as a percentage, was compared among the different treatment groups using a 2 analysis (Abacus Concepts, 1989
) or by Fishers Exact test (SAS Institute, 1996
) when the expected values were lower than 5. The formula of Gibbs & Gower (1960)
was used to calculate the probability of transmission by a single aphid when groups of aphids were used to determine transmission efficiency.
The behavioural variables obtained by EPG recording from aphids that transmitted CaMV were compared with those from aphids that did not transmit by means of a MannWhitney U test, because these variables followed a non-Gaussian distribution. These comparisons allowed us to correlate specific aphid behavioural events with their ability to acquire CaMV.
The 17 behavioural variables calculated for treatment d (Table 1) were introduced into a stepwise-forward logistic regression model (Afifi & Clark, 1990
), using SPSS computer software (SPSS, 2001
), to determine which were really critical for acquisition of CaMV. All EPG data obtained for transmitters and non-transmitters of both aphid species under treatment d (51 recordings) were pooled. The probability function used was of the type P(C)=eu/1+eu, where P(C) represents the probability of acquisition of CaMV. The function u=bo+b1f1(X1)+b2f2(X2)+...7+bnfn(Xn) includes the 16 behavioural variables that determine virus acquisition according to the model. The model also calculates the constant values (bi) and includes the transformations (fi) necessary to achieve normality of the variables. The usefulness of the stepwise logistic regression method is to determine which of the variables considered in the experiment are most appropriate to estimate the probability for virus acquisition.
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Results |
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There were significant differences (P<0·05) in the probing behaviour between the two aphid species during the 5 min acquisition access period. We found that the total probing time (251·6±10·37 s vs 110·1±11·18 s, P<0·05), the probing time until the first pd was produced (52·1±6·98 s vs 14·2±3·05 s, P<0·05), the duration of intracellular punctures (17·3±1·49 s vs 11·4±0·81 s, P<0·05) and the time from the last pd to the end of the probe (63·7±8·83 s vs 23·9±4·86 s, P<0·05) was significantly longer for B brassicae than for M. persicae. Also, the number of intracellular punctures was larger for B. brassicae than for M. persicae (2·8±0·24 vs 2·2±0·17, P<0·05). In spite of the apparent behavioural differences observed between the two aphid species, there were no significant differences in their ability to transmit CaMV after a 5 min acquisition access time.
EPG variables that best explained the acquisition of non-persistent viruses during a 5 min acquisition access period were irrelevant for the acquisition of CaMV (Table 4). The time from the beginning of the probe until the first pd was produced (1stC1stpd) was the only EPG variable that showed significant differences between the group of B. brassicae that was able to transmit and the one that failed to transmit CaMV. Aphids that transmitted CaMV produced the first pd faster than aphids that failed to transmit the virus. In the case of M. persicae, aphids that were able to acquire CaMV spent less time probing than the ones that did not transmit the virus (Table 4
).
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Discussion |
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Chalfant & Chapman (1962) suggested that M. persicae transmitted CaMV in a non-persistent manner, but our results agree with those reported by Markham et al. (1987)
that show a semipersistent type of virusvector relationship. We found out that CaMV was preferentially acquired from the phloem by M. persicae and that pre-acquisition starvation and long acquisition access periods (8 h) did not change the rate of virus transmission. Furthermore, analysis of the data recorded during aphid probing and feeding activities revealed that EPG variables that best explained acquisition of non-persistently transmitted viruses (Collar et al., 1997
) are irrelevant for CaMV acquisition. However, in the case of B. brassicae, the individuals that transmitted CaMV during a 5 min acquisition access period produced the first pd faster than those that failed to transmit the virus. This result suggests that acquisition of P2 and P3virion complexes during short probes is more efficient after a superficial (epidermal) than after deeper (mesophyll) intracellular stylet punctures.
Our work shows that aphids were able to acquire CaMV after just a single intracellular puncture, which was produced within the first minute after the beginning of the probe. Van Hoof (1958) showed that M. persicae needs at least 2 min to cross the epidermis of PVY-infected potato plants. Therefore, as opposed to other semipersistent viruses, we confirmed that CaMV is acquired from superficial plant tissues, including the epidermis. The rate of CaMV acquisition did not increase with increasing number of intracellular punctures (Fig. 1
). However, the total duration of intracellular punctures was one of the variables selected by the stepwise logistic regression model to explain the acquisition of CaMV. This analysis shows that the virus is acquired more frequently when the total duration of intracellular stylet activities is long. The length of intracellular stylet punctures has been related to the volume of sap ingested by an aphid (Collar et al., 1997
; Powell et al., 1995
). It seems logical that aphids ingesting larger volumes of superficial cell contents are more likely to acquire CaMV electron-lucent inclusion bodies and therefore, are more competent for subsequent acquisition of CaMV virions.
The model of Drucker et al. (2002) for sequential acquisition of CaMV by aphids from infected cells has two steps that are consistent with the findings reported in the present work. The model proposes that an aphid stylet pierces the plasmalemma of an infected mesophyll cell and ingests part of the cell contents, possibly including an electron-lucent inclusion body. Immediately after acquisition, P3 is liberated and ingested while P2, perhaps together with a few P3virion complexes, attaches to the aphid stylet (or foregut) cuticle. Our results demonstrating that acquisition of CaMV after a single intracellular stylet puncture in superficial tissues is a rare but possible event supports the proposed model. The model further suggests that after the initial stylet punctures in infected cells, the aphid is P2-loaded and thus transmission competent, ready to acquire more P3virion complexes during subsequent feeding. The fact that we here describe a significant increase of CaMV acquisition during the phase of committed phloem ingestion is also consistent with the proposed model. Indeed, while P2 could not be detected in the phloem sieve lumen, P3virion complexes were very frequently observed. We propose that aphids mainly loaded with free P2 acquired during multiple intracellular stylet punctures of epidermal and mesophyll cells can act as a sieve for trapping CaMV virions during committed phloem ingestion. Beside further suggesting that the sequential acquisition of P2 and P3virion complexes is predominant during CaMV transmission, our electron microscopic data represent the first indication that the CaMV operates long-distance movement in the vascular tissues of infected host plants in the form of viral particles complexed with P3.
Work by Limburg et al. (1997) suggests that the closterovirus Beet yellows virus (BYV), another semipersistent virus, is acquired from the sieve elements by Aphis fabae Scopoli during phloem ingestion. However, this work could not exclude the possibility that the virus is also acquired from non-phloem tissues because the electronic device they used (an AC-amplifed monitor) could not detect intracellular stylet punctures during stylet pathway activities (Reese et al., 2000
). They were only able to correlate the average time needed to reach the sieve elements (20 min) with the minimum time needed for BYV acquisition. Therefore, it is not possible to conclude that closteroviruses are acquired exclusively from the phloem. However, closteroviruses seem to be phloem-restricted and no helper proteins have been formally proposed (Hull, 2001
), suggesting that their transmission strategy is different from the one described for CaMV. This fact opens the debate that semipersistent viruses do not share uniform transmission properties and another classification such as the one proposed by Pirone & Blanc (1996)
should be used when referring to viruses transmitted in a non-circulative manner.
The exact location of cuticle receptors in the mouthparts of vectors of semipersistently transmitted viruses remains unclear. Some authors have shown that semipersistently transmitted viruses are found mainly in the foregut of aphid vectors (Murant et al., 1976 ) or leafhoppers (Ammar & Nault, 1991
; Childress & Harris, 1989
). López-Abella et al. (1988)
suggested that semipersistent viruses may differ from non-persistently transmitted viruses in the tenacity with which virions are carried in a transmissible state in the foreguts of aphids, or that perhaps there are two binding sites, one in the aphid stylet and another in the foregut. More studies are necessary to identify the specific retention sites of CaMV virions in a transmissible form within the aphid feeding apparatus.
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Acknowledgments |
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References |
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Received 13 May 2002;
accepted 26 July 2002.