Alfalfa leaf curl virus is efficiently acquired by its aphid vector Aphis craccivora but inefficiently transmitted

Alfalfa leaf curl virus (ALCV) is the first geminivirus for which aphid transmission was reported. Transmission by Aphis craccivora was determined previously to be highly specific and circulative. Using various complementary techniques, the transmission journey of ALCV was monitored from its uptake from infected plant tissues up to the head of its vector. ALCV was shown to be restricted to phloem tissues using fluorescence in situ hybridization (FISH) and electropenetrography (EPG) monitoring of virus acquisition. Furthermore, the virus is heterogeneously distributed in phloem tissues, as revealed by FISH and quantitative PCR of viral DNA acquired by EPG-monitored aphids. Despite the efficient ingestion of viral DNA, about 106 viral DNA copies per insect in a 15 h feeding period on ALCV-infected plants, the individual maximum transmission rate was 12 %. Transmission success was related to a critical viral accumulation, around 1.6×107 viral DNA copies per insect, a threshold that generally needed more than 48 h to be reached. Moreover, whereas the amount of acquired virus did not decrease over time in the whole aphid body, it declined in the haemolymph and heads. ALCV was not detected in progenies of viruliferous aphids and did not affect aphid fitness. Compared to geminiviruses transmitted by whiteflies or leafhoppers, or to luteoviruses transmitted by aphids, the transmission efficiency of ALCV by A. craccivora is low. This result is discussed in relation to the aphid vector of this geminivirus and the agroecological features of alfalfa, a hardy perennial host plant.


INTRODUCTION
Generally, plant viruses belonging to the same family are transmitted according to the same 29 transmission mode (1, 2). In particular, the viruses of the family Geminiviridae are all known 30 or suspected to be transmitted according to the circulative mode, although they are transmitted 31 by vectors belonging to different hemipteran groups. Thus, viruses of the genus Begomovirus 32 are transmitted by whiteflies (3), those of the genera Mastrevirus, Curtovirus,Becurtovirus,33 Turncurtovirus and probably Eragrovirus are transmitted by leafhoppers (4-6), those of the 34 genera Topocuvirus and Grablovirus are transmitted by treehoppers (7), and those of the genus 35 Capulavirus are transmitted by aphids (8)(9)(10). This means that geminiviruses were able to adapt 36 to different insect environment and particularly to counteract defense mechanisms and achieve 37 the complex virus-vector interactions associated with circulative transmission. Thereon, 38 compared to Luteoviridae or Nanoviridae, two families of viruses also transmitted in a 39 circulative manner but quasi exclusively by aphids (11), the family Geminiviridae stands in 40 striking contrast. With the exception of the recently discovered whitefly transmitted polerovirus 41 (12), it seems that members of Luteoviridae or Nanoviridae were not able to evolve all the 42 necessary interactions to be transmitted by other hemipteran groups, which altogether suggests 43 that the adaptation to other hemipteran groups is not straightforward.
44 Surprisingly, whilst diseases caused by leafhopper and whitefly transmitted geminiviruses were 45 reported more than 100 years ago (13-16), the aphid transmitted geminiviruses were discovered 46 less than 10 years ago (17). The circulative leafhopper transmission of maize streak virus 47 Preparation of fluorescent probes and labeling procedure 147 A fluorescent probe complementary to the CP gene of ALCV was prepared by random priming 148 with the BioPrime DNA labeling system (Invitrogen) and Alexa Fluor 568-labeled dUTP. The 149 template DNA was PCR amplified from the recombinant plasmid containing the ALCV 150 genome, using the following primer pair: CP_ALCV_620-F, 5'-GAA GAG GGC GAG AAC 151 GAC AG-3' and CP_ALCV_1025-R, 5'-GTG GTC TAT TTC AGC AGT TGC C -3'.

152
Ten µl of the probe was diluted in 290 µl of 20 mM Tris-HCl hybridization buffer (pH 8) 153 containing 0.9 M NaCl, 0.01% SDS and 30% formamide. The diluted probe was denatured 154 10 min at 100°C and rapidly cooled on ice for 15 min. In parallel, plant samples (petiole, leaf-155 discs or veins) were soaked 3 times 5 min in hybridization buffer. Plant samples were then 156 incubated overnight at 37°C in embryo dishes containing probe solutions and sealed with 157 parafilm membranes. After three washing steps of 5 min with hybridization buffer and two with 158 PBS, samples were mounted on microscope slides in Vectashield antifade mounting medium 159 containing 1.5 µg.ml -1 DAPI for staining nuclei. Observations were performed using a Zeiss 160 Confocal microscope and acquired in a stack mode.

161
EPG system 162 We used the electropenetrography (EPG) technique (27,28) to investigate which specific stylet 163 penetration activities of A. craccivora individuals were associated with the transmission of the 164 virus. By connecting an insect and its host plant to an electrical circuit, EPG allows variations 165 in biopotentials and electrical resistance to be recorded (referred to as electrical waveforms) 166 and related to different feeding activity patterns (29,30). In particular, localization of the aphid 167 stylets in the phloem is characterized by two typical waveforms, E1 and E2. The E1 waveform 168 occurring few seconds after the stylet penetration in the sieve element has been related to 169 salivation and described as the essential phase for inoculation of persistently or semi-170 persistently transmitted plant viruses by aphids (31), whiteflies (32), or leafhoppers (33). The 171 E1 waveform is generally -but not necessarily -followed by a short transition period and typical 172 E2 waveforms. The latter has been related to passive sap ingestion and concurrent secretion of 173 watery saliva (31). An assumption that is commonly accepted is that phloem-restricted viruses 174 transmitted by homopterans are acquired only during the ingestion phase (i.e. waveform E2).
probing and ingestion activities of virus-free newly emerged A. craccivora adults on leaves of 179 virus-infected broad bean plants. The electrical circuits containing insects, plants, and 180 electrodes were placed in a Faraday cage to isolate them from electromagnetic interference. The 181 electrical signals between the electrodes were converted into digital signals via the Di710-UL 182 (DATAQ, Akron, OH, USA) analog-to-digital board. The digital signals were visualized and 183 recorded on a computer using Probe 3.5 software (EPG Systems, The Netherlands). Recordings 184 were made under constant temperature (23±1°C).

185
A gold wire (Ø 18.5 µm, 2-3 cm long) was fixed to the thorax cuticle of the backside of the 186 insects using a drop of silver glue (EPG Systems, The Netherlands). During this procedure, 187 insects were held stationary at the tip of a plastic pipe in which a slight suction was applied.

188
The other end of the gold wire was pasted to a copper electrode with the silver glue. This 189 electrode (5 cm long, 2 mm in diameter) was rammed into the soil beside the plant whereas the 190 insect was placed onto a leaf. Before each beginning of recordings, individuals were given a 191 fasting period of 30 minutes. Eight individuals were recorded in parallel. The voltage source 192 was tuned as described by Tjallingii (35), so that the amplifier output signal was between +5  195 Signals were analyzed using the software Stylet+a (EPG Systems, The Netherlands).

196
Individuals of many aphid species have been monitored by EPG over the past 50 years, 197 including A. craccivora (e.g. 36, 37), allowing an unambiguous interpretation of our own 198 recordings, in particular the waveforms correlated with the stylet location in the phloem (E1 199 and E2). Young apterous adult aphids (⋍2 days old) were given access individually to upper leaves of 203 ALCV-infected broad bean plants and their feeding behavior was monitored by EPG-recording.

204
In experiment 1, 24 individuals were stopped at the end of their first phloem salivation phase 205 (E1) (modality 1), whereas 30 individuals were monitored up to 4 hours, a period that was 206 expected to contain almost one phloem-feeding phase (E2) (modality 2). Indeed, preliminary 207 analysis revealed that most aphids reached phloem in less than one hour. After the monitoring, aphids were stored individually at -20°C. Total DNA was extracted from each aphid and viral 209 DNA was quantified by qPCR as described above. Negative controls consisted of three adult 210 aphids of the same batch that were given access to upper leaves of healthy broad bean plants. 211 In experiment 2, 26 individuals were monitored for 4 hours. For each individual, we calculated 212 the variable WDi -Waveform Duration by Insect, i.e. sum of durations of all its events of one 213 waveform type made by each individual insect that produced that waveform -related to E2 after IAP and PCR-tested for ALCV detection.

225
In experiment 4, the transmission rate of ALCV was determined with aphid batches of various 226 sizes in eight independent transmission tests (Suppl Table 1). The batch size were of 1, 5, 10, 227 20, 30, 40, or 100 individuals depending on the test. The average transmission rate determined 228 for each batch size was used to plot a curve showing the expected transmission rate as a function 229 of the number of individuals per test plants. The theoretical transmission rate TR is defined as 230 TR = 1-(1-pi) n in which pi is the probability that at least one aphid is infective in a population, 231 1-pi the probability for an aphid not to be infective, and n the batch size.

232
Kinetics of ALCV accumulation in aphids (experiment 5) 233 Young apterous adult aphids were allowed to feed on ALCV infected broad bean plants for 2h, 234 6h, 15h 24h, 48h and 82h. Individuals were collected at each time point. Their total DNA was 235 extracted in pools of five individuals, and the amount of ALCV DNA was quantified by qPCR.

236
The same test was performed with AAPs of 2h, 4h, 6h, 19h, 24h, 48h, 72h and 96h except that 237 DNA was extracted from individual aphids. Some of the individuals that were not shifted to 238 infected plants for an AAP were collected and tested as negative controls (time point 0h).
Eight independent transmission tests were carried out with 5 or 10 apterous adult aphids per 241 test plant. The transmission rate was determined with 19 or 20 test plants per AAP duration.

242
AAP duration ranged between 1 and 120h and IAP duration was of 5 days.  Minimum latency and inoculation period (experiment 8) 257 In the first set of four transmission tests (tests 1-4), five to ten young apterous adult aphids were 258 given a 15-hours AAP on ALCV infected broad bean plants and then shifted to test plants for 259 various IAP durations between 3h and 48h. Similarly, five to ten aphids were given AAPs of In a first test, aphids were reared on ALCV infected broad bean plants for 2 weeks. Apterous bean seedlings for a four-hour laying, and removed thereafter. At day 4 (D4), nymphs were 290 removed except one. From day 6 (D6), individuals were daily observed to detect the age at 291 which each individual lay its first nymphs (i.e. to determine d, the pre-reproductive time). Then, 292 the number of nymphs produced during a period of d days was counted for each individual.

293
When nymphs retained for laying turned out to be alate, they were excluded from the test.

294
Likewise, the countings that were performed with adults that were not found at the end of the 295 experiment, or that produced very few or no nymphs were excluded from the statistical analysis 296 to avoid interpretation bias. Due to this selection, 6 viruliferous and 9 non-viruliferous 297 individuals were excluded from the analysis. The rm value was estimated for each individual for 298 comparison of mean differences, but the rm values presented in the results are the group values, 299 and standard errors were calculated using the bootstrap technique (42).

302
To interpret the results from experiments 2, 5 and 6, we applied loess (acronym for locally 303 weighted regression) smoothing to fit a curve through points in each scatterplot (44) labelling co-localized with cell nuclei and was detected in relatively few cells, with a maximum 326 of 3 labeled cells per cross-section. Specific labelling was restricted to areas located between 327 xylem and sclerenchyma, the location of phloem (Fig. 1a,b). ALCV specific labelling was 328 detected also in phloem of vascular tissues pulled from broad bean leaf lamina (Fig. 1c). The  (Fig. 2d). Indeed, there is no correlation between E2 cumulative time and the amount of virus 358 ingested by the aphid (Kendall's rank correlation thau, τ = 0.0027). For example, two aphids 359 for which the E2 waveform was recorded for about one hour, acquired each as many or more 360 viral DNA copies (0.5x10 6 ) than aphids for which E2 recording lasted more than 2h30. On the 361 other hand, one aphid for which E2 recording lasted almost two hours in phase E2 has acquired 362 very little viral DNA. Nevertheless, it is noteworthy that individuals that fed more than 107 min were all detected qPCR positive with more than 10 5 copies of viral DNA, suggesting that in spite of heterogeneous distribution, the virus is apparently accessible at any sites of the phloem 365 network. 366 This result suggests that phloem-feeding durations and viral DNA accumulation may be 367 positively correlated with longer AAP durations. This prediction was validated by monitoring 368 viral DNA accumulation in individuals that were given access to ALCV infected plants for 369 durations ranging between 2 and 96 hours (Fig. 3). Aphids accumulate a large amount of virus 370 in the first 15-19 hours of acquisition. During this initial period, viral load per insect increased 371 sharply up to 4-7x10 5 copies. Thereafter, the viral increase was lower with only 10 times 372 increase up to a plateau of about 2 to 7x10 6 copies that were reached at 48 hours. Interestingly, 373 aphids individuals that were given a 2-hour AAP were not all PCR-positive in spite of the fact 374 that they were tested in pools of 10 individuals (4 of the 12 pools that were given a 2-hour AAP 375 were negative). This result is consistent with the heterogeneous virus acquisition observed with 376 the EPG-monitored individuals that were given 4 hour AAPs (Fig. 2d).

A high accumulation of ALCV is necessary for efficient transmission but not always
The accumulation dynamics of ALCV in aphids revealed that viral DNA content reaches more 380 than 10 6 viral DNA copies per insect within two days of AAP (Fig. 3) determine their ability to transmit the acquired virus (Fig. 4a). The viral amounts assessed by 385 qPCR in the 34 individuals collected alive at the end of the 5-day IAP were consistent with the 386 threshold hypothesis. Indeed the four individuals that were able to transmit ALCV to their test 387 plant were among the eight individuals that exhibited the highest virus content, i.e above 388 1.6 x 10 7 viral DNA copies. Aphids whose viral amount was below this threshold failed to 389 transmit ALCV. The 4 non-transmitters whose viral content was above the threshold suggest 390 that, although necessary, accumulating virus above a threshold is not always sufficient to 391 transmission. Other parameters, presently unknown, might also determine the efficiency of viral 392 inoculation.
durations of AAP and IAP, -3 and 5 days respectively. Using pi⋍11.8%, theoretical 395 transmission rates (TR) were estimated as a function of the number of individuals used per test 396 plant according to the formula TR = 1-(1-pi) n (Fig 4b, red curve). Surprisingly, the transmission 397 rates derived from transmission tests performed with different numbers of individuals per test 398 plants (1,5,10,20,30,40;Suppl Table 1), were all below the expected rates (Fig. 4b). The To determine the minimum AAP duration that is needed to reach an optimal transmission rate, 411 5 or 10 individuals per test plants were used as defined above. Figure 5 shows that up to 48h, 412 the transmission rate is a quasi-linear function of acquisition time, reaching a maximum of 413 about 50 to 60%. Increasing acquisition time beyond 48h does not increase the transmission 414 rate, even up to 5 days. It is noteworthy that the maximum transmission rate is not reached 415 following a 24 hour AAP although the most intensive virus accumulation was achieved in less 416 than 24h (Fig. 3). These results are consistent with the threshold hypothesis. Indeed, it seems 417 that the moderate virus accumulation that occurs after the initial intensive virus accumulation 418 (19h) is critical for an optimal transmission rate. Unexpectedly, in some tests, low transmission 419 rates were observed although the experimental conditions were the same.  The decrease of viral content in the hemolymph and the head compartment was thought to limit 442 the amount of virus that is potentially released from the insect through salivary glands, and 443 hence, the transmission rate. To test the effect of IAP duration on the transmission rate, we used 444 a 48-or 72-hour AAP which, according to previous tests produced viruliferous insects that 445 exhibit optimal infectivity (Fig. 5). With these experimental conditions, it was expected that the 446 virus accumulation would not be a limiting factor for virus inoculation. However, in spite of 447 AAP durations compatible with high virus accumulation, the inoculation was successful only 448 with an IAP duration of at least 24 hours (Table 1). Additional transmission tests were 449 performed to investigate the minimum latency period. To do this we had to use individuals that 450 were given an AAP that was long enough to produce infective aphids (Fig. 5) and short enough 451 to limit the risk of exceeding the potential minimum time of latency during AAP. Using 15 452 hours AAPs (Table 1)

476
Surprisingly aphid-transmitted geminiviruses were detected less than 10 years ago, whilst 477 leafhopper and whitefly transmitted geminiviruses were discovered more than 100 years ago 478 (3,5,6,9,17). We hypothesized that the delayed discovery of aphid-transmitted geminiviruses 479 is due to their low dissemination rate within cultivated plants compared to that of non-aphid The non-correlation between the amount of acquired ALCV and the durations of AAP observed 516 during a 4-hour AAP fades with longer AAPs. Thus, we hypothesize that, due to the generalized 517 contamination of the sieve tube network, the ALCV content of individual aphids, irrespective 518 of their puncture spot, increase steadily during the first 24 hours of AAP, the accumulation 519 phase exhibiting the highest increase. Such a correlation between duration of AAP and virus 520 amount in the vector was also detected with other geminiviruses and is a common feature of 521 non-propagative transmission (50).

522
From 15 hours AAP, the number of copies of viral DNA per individual reaches 10 6 , and was in 523 the range of 10 6 to 10 7 between 2 and 4 days AAP. While these amounts are slightly higher than 524 that of MSV in its leafhopper vector following a 6-day AAP (< 10 6 DNA copies)(51), they were 525 lower than those of tomato yellow leaf curl virus and watermelon chlorotic stunt virus in their 526 whitefly vector following AAPs of 5 or 6 days (10 8 DNA copies or more) (52, 53) 527

Transmission success depends on a high viral amount threshold in aphids 528
Previous transmission studies with geminiviruses showed that the infectivity of viruliferous 529 vectors was correlated with AAP durations (e.g., 54). These results suggest that insect 530 infectivity depends on its virus content but to our knowledge, a threshold of virus concentration 531 beyond which an infection is possible was to our knowledge never defined. Here, by checking 532 the virus concentrations in individual viruliferous aphids each tested for infectivity, we came 533 up with a virus concentration threshold below which insects are not infective. As the 534 quantification was done after the 5-day IAP, the threshold of 1.6 x 10 7 copies of viral DNA 535 corresponds to the persistent virus that is most likely internalized and therefore relevant for 536 infectivity.

537
Although necessary for infectivity, reaching this concentration threshold is not sufficient, 538 because 50% of the individuals that reached the threshold were not infective during the 5-day 539 IAP. As aphids are genetically highly homogeneous due to their clonal multiplication, the 540 contrasted infectivities may be associated with stochastic phenomena, like the site of 541 inoculation, a particular feeding behavior, particular physiological conditions influencing the 542 virus distribution along the transmission route. Moreover, the influence of endosymbionts 543 cannot be excluded from individuals that may potentially exhibit contrasting infection status 544 (55).
following increasing AAP durations can be interpreted according to a critical threshold of 547 ALCV DNA content which is around 10 6 viral DNA copies per individual (Fig. 3). Indeed, the 548 maximum infection rate, around 50%, was reached only from 48 hours AAP (Fig. 5), which 549 according to the accumulation dynamics of ALCV in A. craccivora (Fig. 3) is the time needed 550 for a majority of individuals to reach viral contents that are higher than 10 6 copies. Thus, 551 although the ALCV content of 24-hour AAP individuals was only slightly lower than that of 552 48-hour AAP individuals, the differential transmission rate is of about 2. These results suggest 553 that the ALCV content threshold that was found to be necessary for individual aphid 554 transmission cannot be achieved collectively, by the addition of individual contributions. This 555 hypothesis was supported by the low transmission rates obtained with groups of aphids ranging 556 from 5 to 40 individuals. Indeed, they were all below the expected (theoretical) transmission 557 rate calculated from the observed individual transmission rate (12%) (Fig. 4b). Indeed, if this 558 threshold could have been collectively achieved the transmission probability would have been 559 higher than the rate estimated from the probability of having at least one individual per group.

560
The low infectivity of aphids is associated with the low persistence of ALCV in hemolymph 561 and head.

562
The highest individual transmission rate of ALCV by its vector A. craccivora was 12%, an The low transmission rate of ALCV by A. craccivora in comparison with that of other 596 geminiviruses is consistent with the hypothesis that the delayed discovery of capulaviruses may 597 be ascribed to their low dissemination to and within cultivated plants. Unlike nanoviruses and 598 luteoviruses which evolved efficient transmission with aphids, it seems that geminiviruses did 599 not. Thus, it is thought that capulaviruses adapted to aphid transmission only in host 600 environments where high transmission efficiency is not critical for survival. Alfalfa meets this 601 criteria. Indeed, it is a hardy perennial plant that exhibit a high tolerance to non-biotic stresses 602 like drought and extreme temperatures. This tolerance is expected to have at least two beneficial 603 effects on ALCV fitness. Firstly the window of opportunity to be carried to a new plant is 604 extremely wide and may extend over several years. Secondly, as alfalfa is hardier than other 605 plants, it is relatively more conducive to insect feeding under biotic stresses and therefore may 606 attract aphid vectors. The hardy perennial feature also applies to E. caput-medusae, the host of 607 EcmLV, and probably also to P. lanceolata the host of PlLV. The french bean Severe leaf curl 608 virus would seem to be an exception as it is the only capulavirus isolated from an annual host. below 2% (Akram, personal communication). Finally, according to preliminary results, ALCV 638 The authors declare that there are no conflicts of interest.     transmission rate (pi) was deduced from formula , TR = 1-(1-pi) n , knowing TR and n.