Transmission of non-persistent plant viruses is related to aphid behaviour during superficial brief probes. A widely accepted hypothesis postulates that virus acquisition occurs during ingestion of plant cell contents, and inoculation during egestion or regurgitation of previously ingested sap. Although conceptually attractive, this ingestion-egestion hypothesis has not been clearly demonstrated. Furthermore, it overlooks the anatomy of the tips of the stylets (mouthparts) and, consequently, the potential role of salivation in the inoculation process. Here, we used the electrical penetration graph (EPG) technique to investigate aphid-stylet activities associated with uptake (acquisition) and release (inoculation) of two non-persistently transmitted viruses. Our results show that acquisition occurs primarily during the last sub-phase (II-3) of intracellular stylet punctures, whereas inoculation is achieved during the first sub-phase (II-1). An alternative mechanism to the ingestion-egestion hypothesis is proposed on the basis of our findings.

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 Bemisia tabaci were subjected to an acquisition access period of 72 h in Tomato yellow leaf curl virus (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 B. tabaci, 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 B. tabaci 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 B. tabaci. Our results show evidence that TYLCV directly manipulates the settling, probing and feeding behavior of its vector B. tabaci in a way that enhances virus transmission efficiency and spread. Furthermore, TYLCV-B. tabaci interactions are mutually beneficial to both the virus and its vector because B. tabaci feeds more efficiently after acquisition of TYLCV. This outcome has clear implications in the epidemiology and management of the TYLCV-B. tabaci complex.