1) Electropenetrography: EPG have shown that leafhoppers from the genus Erythroneura presented only few different phases of feeding behaviour. After a non-probing phase, leafhoppers started probing by puncturing the cuticle. Then, they penetrated the epidermis for few seconds before reaching the mesophyll cells (spongy and palissade mesophyll) (Fig. 3A). Feeding phases in mesophyll are characterized by recurrent and short drops (ca. every second), followed by a saw-toothed pattern that may correspond to the emptying of mesophyll cells. (Fig. 3B). Another waveform was associated with xylem ingestion as comparition with patterns observed in other insects. This waveform was characterized by sustained oscillations of the signal (Fig. 3C). No typical waveform associated with salivation or ingestion in phloem compartment was observed in Erythroneura species. The numbers of punctures and the mean times spent in each compartment by the 3 leafhopper species were determined. They varied depending on the species but, in general, Erythroneura leafhoppers spend more than 80% of their time feeding on mesophyll (Table 1). Julien SAGUEZ Julien SAGUEZ 1* 1* , Charles VINCENT , Charles VINCENT 1 , Chrystel OLIVIER , Chrystel OLIVIER 2 , Philippe GIORDANENGO , Philippe GIORDANENGO 3 Erythroneura spp. (Cicadellid) feeding behaviour on grapevine Erythroneura Erythroneura spp. ( spp. ( Cicadellid Cicadellid ) feeding ) feeding behaviour behaviour on grapevine on grapevine 1) Electropenetrography (EPG) Adult leafhoppers were inserted in an electrical circuit (Fig. 2). When leafhoppers penetrated plant tissues, we recorded variations of the electric signal that generated various waveforms, whose patterns are specific of each tissue. 2) Histology Leafhoppers were put on leaves to feed for one day. After leafhopper removing, leaves were stained with a blue trypan solution that specifically highlighted salivary sheaths of insects. Histological cross-sections of leaves were realized after planned interruptions of feeding bouts, during the different feeding phases observed by EPG. 2) Histology Staining with blue trypan allowed the localization of salivary sheaths in the depigmented areas of the leaves (Fig. 4A). Salivary sheaths were thin and short (Fig. 4B). Cross-sections grapevine leaves showed that these areas corresponded to the empty cells of mesophyll (Figs. 4C, 4D). Erythroneura species occasionally punctured in xylem vessels (Fig. 4E). (1) Saguez J., Olivier C., Lasnier J. and Vincent C. 2009 Phytoplasmas – leafhoppers – grapevines : an undesirable tritrophic interaction in Canada– Proceeding of the 2nd International Conference on Northern Viticulture, VitiNord 2009 Saint-Hyacinthe, Qc, Canada – 9-11 November 2009. Downloadable from: http://eduportfolio.org/6644 (2) Olivier C., Lowery T., Stobbs L., Vincent C., Galka B., Saguez J., Bittner L. , Johnson R., Rott M., Masters C. and Green M. 2009. First report of Aster Yellow phytoplasmas (‘Candidatus phytoplasma asteris’) in Canadian grapevines. Plant Disease. 93: 669. (3) Weintraub P. and Beanland L. 2006. Insect vectors of phytoplasmas. Annu. Rev. Entomol. 51: 91-111. (4) Giordanengo P. 2009. EPG-Calc 4.8, programme php pour calculer les paramètres EPG. Université de Picardie Jules Verne, Amiens, France. Downloadable from : http://www.u- picardie.fr/PCP/UTIL/epg.php The authors thank J. LASNIER (CoLab R&D) for his financial support and Pierre Lemoyne for his technical help. They also thank grapegrowers for the access to their vineyards. J. SAGUEZ was a post-doc of UQÀM. 1 Agriculture et Agroalimentaire Canada, Centre de Recherche et Développement en Horticulture, 430 Boulevard Gouin, Saint-Jean-sur-Richelieu Qc, J3B 3E6 CANADA * julien.saguez@agr.gc.ca 2 Agriculture et Agroalimentaire Canada, Centre de Recherche de Saskatoon, 107 Place des Sciences, Saskatoon SK, S7N 0X2 CANADA 3 Université de Picardie Jules Verne, UFR des Sciences, Biologie des Plantes et Contrôle des Insectes Ravageurs, 33 Rue St Leu, 80 039 Amiens Cedex 1, FRANCE Introduction Materials and Methods Results and Discussion Conclusion References Acknowledgments © 2011 Entomology 2011 – ESA 59 th Annual Meeting – November 13-16, Reno, Nevada, USA E) Figure 1: Leafhopper species collected in Canadian vineyards. A) Erythroneura vitis B) Erythroneura tricincta C) Erythroneura comes D) Macrosteles quadrilineatus E) Erythroneura ziczac A) B) C) D) E) Leafhoppers (Cicadellidae) are piercing-sucking insects that feed on several plant species including grapevine. Leafhoppers suck plant tissues and fluids such as xylem or phloem saps. Between 2006 and 2008, ca. 110 leafhopper species were collected in vineyards from British Columbia, Ontario and Quebec (1). The most abundant species was Macrosteles quadrilineatus and Erythroneura specimens represented the most important genus (Fig. 1). Using PCR methods, these species, collected in Canadian vineyards, were detected positive for Aster Yellow phytoplasmas (2). Phytoplasmas are mainly acquired and transmitted during the feeding process of leafhoppers (3). In order to understand the mechanisms of phytoplasma acquisition and transmission, the feeding behaviour of Erythroneura elegantula, E. vitis and E. ziczac was investigated by electropenetrography (EPG). We also realized cross-sections of grapevine leaves to identify histological damage caused by leafhoppers during the feeding process. amplification 50x EPG signal (waveforms) Waveform analysis Statistical analysis EPG-Calc (4) ground + - R Figure 2: Electropenetrography system 9330 9331 9332 9333 9334 9335 933 -5 -4 -3 -2 -1 0 1 2 3 4 5 Mesophyll 6960 6961 6962 6963 6964 6965 696 -5 -4 -3 -2 -1 0 1 2 3 4 5 6963 6964 6965 6966 6967 6968 Xylem 8518 8519 8520 8521 8522 8523 8524 8525 8526 8527 8528 -5 -4 -3 -2 -1 0 1 2 3 4 5 4 3 2 1 A) B) C) Figure 3: Typical waveforms recorded by EPG. A) Beginning of food intake. (1) Non-probing; (2) Cuticle penetration; (3) Puncture in epidermis; (4) Puncture in mesophyll. B) Feeding in mesophyll. C) Feeding in xylem. Red arrows indicate depolarisations (probes in cells). Parameters E. ziczac E. vitis E. elegantula Number of tested individals 10 10 10 Total duration of non-probing (sec) 2984 2307 2734 Number of non-probing phases 20.5 17.1 22.7 Total duration of feeding (sec) 11416 12093 11666 Total time in mesophyll (sec) 8970 9078 8541 Number of phases in mesophyll 40 34 52 Mean time in mesophyll (sec) 273 268 179 Total time in xylem (sec) 2446 3015 3125 Number of phases in xylem 20 18 36 Mean time in xylem (sec) 126 150 84 Table 1: Erythroneura feeding behaviour in plant compartments. Leafhoppers from the genus Erythroneura preferentially fed on mesophyll of vines. However, phytoplasmas are mainly found in phloem sap. Because Erythroneura species do not feed in phloem and because these species were detected phytoplasma-positive, our results raise the question of phytoplasma acquisition by Erythroneura in mesophyll, a compartment known to occasionally contain phytoplasmas. A) B) E) Xylem vessels Salivary sheath Salivary sheaths Points of entry in plant tissues C) Upper epidermis Palissade mesophyll Spongy mesophyll Inner epidermis Xylem vessel Point of entry in plant tissues D) Empty cell Undamaged cell Figure 4: A) Depigmented areas containing salivary sheaths. B) Close-up of depigmented area showing the salivary sheaths. C) Transversal cross section of an undamaged grapevine leaf. D) Transversal cross-section of a damaged grapevine leaf, showing salivary sheath and damage in mesophyll. E) Punctures done in or close to xylem vessels.