The effect of cycloviolacin cyclotides on M. persicae probing and feeding behavior was investigated in vitro, using EPG (Mclean andKinsey, 1964; Tjallingii, 1978, 1985; Mayoral et al., 1996). In the present study, apterous female sornymphs of M.persicae were dorsally tethered on the abdomen with a gold wire (1.5 cm long, 20 Î¼m indiameter)and water-based conductive silver paint (EPG-Systems; Dil-lenburg 12,6703 CJ Wageningen, The Netherlands; www.epgsystems.eu).
Total time of non-probing was significantly longer and total time of probing was significantly shorter on cyO2 (100Î¼M),cyO13 (50Î¼M,100Î¼M) and cyO19 (50 Î¼M, 100 Î¼M) diets compared to the control sucrose diets.Although the number of probes was unaffected by the treatments,the mean probing time on cyO13 (100 Î¼M) diet was 10 times shorter(2.2 Â± 0.2min) to that on control sucrose diets (22.9 Â± 16.2 min; p=0.0007, Kruskal-Wallistest). The higher concentrations of cycloviolacin cyclotides prolonged aphid salivation into the diets and reduced aphid ingestion of the diets. The total time of salivation was the longeston for cyO3 (100Î¼M) and cyO13 (50Î¼M) diets (7 and 3.5 times longer than on sucrose diets, respectively). A similar trend was observed on cyO2 (100Î¼M), cyO3 (50Î¼M), cyO13 (100Î¼M) and cyO19 (100Î¼M) cycloviolacin-sucrose diets. The proportion of salivation in all aphid activities in the diets (d-E1/d-C+d-E1+d-G) was the highest after application of cyO2 (100Î¼M), cyO3 (100Î¼M), cyO13 (100Î¼M) and cyO19 (100Î¼M) cyclotide. The duration of total ingestion phase was shorter on cyO13 (100 Î¼M) and cyO19 (50 Î¼M) diets than on control diet (approximately 20 and 22 times shorter, respectively). A similar trend was observed on cyO3 (50Î¼M) and cyO19 (100Î¼M) cycloviolacin diets. The lowest proportion of ingestion during penetration of the diets (d-G/d-C+d-E1+d-G) was noteworthy on cyO13 (100Î¼M) and cyO19 (50Î¼M) (2% of all activities in the diets). ). Additionally, cyO13 (100Î¼M) and cyO19 (50Î¼M, 100Î¼M) cyclotides considerably reduced the number of probes with an ingestion phase (approximately six times for cyO13 and three times for both concentration of cyO19). The number of ingestion phases was 3.5 and four times lower on cyO19 (50Î¼M,100Î¼M, respectively) dietsthanon control diets. On cyO13 (100 Î¼M) diets, the number of ingestion phases tended to be lower, but without statistical significance. The proportion of aphids with sustained ingestion phase was lower on cyO2 (10Î¼M,100Î¼M), cyO3 (50Î¼M), cyO13 (100 Î¼M) and cyO19 (50 Î¼M) cyclotide-sucrose diets in comparison to control sucrose diets. Aphid sustained ingestion was completely inhibited after application of cyO13 (100Î¼Mâ€“no aphid showed d-G > 10min).