A Plant Virus Manipulates the Behavior of Its Whitefly Vector to Enhance Its Transmission Efficiency and Spread

<div><p>Plant viruses can produce direct and plant-mediated indirect effects on their insect vectors, modifying their life cycle, fitness and behavior. Viruses may benefit from such changes leading to enhanced transmission efficiency and spread. In our study, female adults of <i>Bemisia tabaci</i> were subjected to an acquisition access period of 72 h in <i>Tomato yellow leaf curl virus</i> (TYLCV)-infected and non-infected tomato plants to obtain viruliferous and non-viruliferous whiteflies, respectively. Insects that were exposed to virus-infected plants were checked by PCR to verify their viruliferous status. Results of the Ethovision video tracking bioassays indicated that TYLCV induced an arrestant behavior of <i>B. tabaci</i>, as viruliferous whitefly adults remained motionless for more time and moved slower than non-viruliferous whiteflies after their first contact with eggplant leaf discs. In fact, Electrical Penetration Graphs showed that TYLCV-viruliferous <i>B. tabaci</i> fed more often from phloem sieve elements and made a larger number of phloem contacts (increased number of E1, E2 and sustained E2 per insect, p<0.05) in eggplants than non-viruliferous whiteflies. Furthermore, the duration of the salivation phase in phloem sieve elements (E1) preceding sustained sap ingestion was longer in viruliferous than in non-viruliferous whiteflies (p<0.05). This particular probing behavior is known to significantly enhance the inoculation efficiency of TYLCV by <i>B. tabaci</i>. Our results show evidence that TYLCV directly manipulates the settling, probing and feeding behavior of its vector <i>B. tabaci</i> in a way that enhances virus transmission efficiency and spread. Furthermore, TYLCV-<i>B. tabaci</i> interactions are mutually beneficial to both the virus and its vector because <i>B. tabaci</i> feeds more efficiently after acquisition of TYLCV. This outcome has clear implications in the epidemiology and management of the TYLCV-<i>B. tabaci</i> complex.</p></div

Mean (± standard error) non-sequential EPG variable values (ranges in parenthesis) for the probing behavior of TYLCV-viruliferous and non-viruliferous <i>B. tabaci</i> adults on healthy eggplants during an eight-hour recording^a.

a<p><b>PPW</b>, proportion of individuals that produced the waveform type; <b>NWEI</b>, number of waveform events per insect; <b>WDI</b>, waveform duration (min) per insect; <b>WDE</b>, waveform duration (min) per event <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061543#pone.0061543-Backus1" target="_blank">[52]</a>. <b>Non-probe</b>, non-probe activity; <b>Probe</b>, probe activity. Waveforms: <b>C</b>, intercellular stylet pathway; <b>pd</b>, short intracellular punctures; <b>G</b>, xylem ingestion; <b>E</b> shows phloem-related activities: <b>E1</b>, correlates with salivation into phloem sieve elements <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061543#pone.0061543-Jiang2" target="_blank">[38]</a>; <b>E2</b>, regards as ingestion from phloem that is comparable to E2 of aphids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061543#pone.0061543-Prado1" target="_blank">[49]</a>; <b>sE2</b>: sustained E2 (longer than 10 minutes).</p>b<p>P-values according to a Chi-square 2×2 goodness of fit test or by a Fisher exact test when the expected values were lower than 5. Underline-type indicates significant differences (p≤0.05).</p>c<p>P-values according to a Student t-test¹ for Gaussian distribution variables and Mann Whitney U-test² for non-Gaussian distribution variables. Underline-type indicates significant differences (p≤0.05).</p>d<p>Potential drop (pd) duration is expressed in seconds.</p

Mean (± standard error) values for parameters of movement and displacement of TYLCV-viruliferous and non-viruliferous <i>B. tabaci</i> adult females on healthy eggplants discs during ten minutes of automatic tracking.

a<p>Statistical tests performed: T-Student test on Gaussian distribution parameter “Total distance moved” and “Frequency of movement”. Mann-Whitney non-parametric test on all other variables. Significant differences (p≤0.05) between both TYLCV-viruliferous and non-viruliferous <i>B.tabaci</i>.</p

EPGs recorded for <i>Bemisia tabaci</i>.

<p>(1) Probing and non-probing period; (2) waveform C, intercellular stylet pathway; (3) waveform pd, intracellular puncture; (4) waveform G, xylem ingestion; (5) waveform E1, salivation into phloem sieve elements; (6) waveform E2, ingestion from phloem sieve elements.</p

Mean (± standard error) sequential EPG variable values (ranges in parenthesis) for the probing behavior of non- viruliferous and viruliferous <i>B. tabaci</i> on healthy eggplants during an eight-hour recording^a.

a<p><b>PPW</b>, proportion of individuals that produced the waveform type; <b>NWEI</b>, number of waveform events per insect; <b>WDI</b>, waveform duration (min) per insect <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061543#pone.0061543-Backus1" target="_blank">[52]</a>. <b>Non-probe</b>, non-probe activity; <b>Probe</b>, probe activity. Waveforms: <b>C</b>, intercellular stylet pathway; <b>pd</b>, short intracellular punctures; <b>G</b>, xylem ingestion; <b>E</b> shows phloem-related activities: <b>E1</b>, correlates with salivation into phloem sieve elements <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061543#pone.0061543-Jiang2" target="_blank">[38]</a>; <b>E2</b>, regards as ingestion from phloem that is comparable to E2 of aphids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061543#pone.0061543-Prado1" target="_blank">[49]</a>; <b>sE2</b>: sustained E2 (longer than 10 minutes).</p>b<p>P-values according to a Chi-square 2×2 goodness of fit test or by a Fisher exact test when the expected values were lower than 5. Underline-type indicates significant differences (p≤0.05).</p>c<p>P-values according to a Student t-test¹ for Gaussian distribution variables and Mann Whitney U-test² for non-Gaussian distribution variables. Underline-type indicates significant differences (p<0.05).</p

EPG recording for <i>P</i>. <i>spumarius</i> on olive.

<p>Non probing (np), waveform C followed by Xc and Xi, with N interspersed, and flat waveform R. Below the general view, close-ups of the waveforms: a) C; b) Xc; c) Xi, high frequency; d) Xi, low frequency; e) R; f) N; g) transition Xi/R; h) drop similar to N during R.</p

Non-sequential and sequential EPG variables of adults of <i>P</i>. <i>spumarius</i> (males and females) on olive.

<p>Time is expressed in minutes.</p

Temporal development of stylets activity for female <i>P</i>. <i>spumarius</i> on olive.

<p>Temporal development of stylets activity for female <i>P</i>. <i>spumarius</i> on olive.</p

Micro-CT volume rendering reconstruction images and correlation waveform/stylets position.

<p>7) waveform R general view; 8) R, detailed view; 9) waveform N, lateral view including the <i>labium</i>; 10) waveform N. White arrows indicate the stylets tip.</p

Transition scheme of waveforms events for <i>P</i>. <i>spumarius</i> on olive cv Picual during the 8 hours EPG (likelihood of waveforms events).

<p>The values near the arrows correspond to the likelihood of a certain waveform being followed by another waveform type. Probabilities <2% are not shown. Waveform N was not represented in the diagram, because it occurs as brief interruption within waveforms Xc and Xi (and likely R).</p