DOI: 10.1111/eea.12353 Characterization of the feeding behavior of three Erythroneura species on grapevine by histological and DC-electrical penetration graph techniques Julien Saguez1*, Pierre Lemoyne1, Philippe Giordanengo2,3,ChrystelOlivier4, Jacques Lasnier5,YvesMauffette6 & Charles Vincent1 1Agriculture et Agroalimentaire Canada, 430 Boulevard Gouin, Saint-Jean-sur-Richelieu, Quebec J3B 3E6, Canada, 2Universite de Picardie Jules Verne, 33 Rue St Leu, 80039 Amiens Cedex, France, 3Institut Sophia Agrobiotech, UMR 1355 INRA/Universite Nice Sophia Antipolis/7254 CNRS, 400 route des Chappes, 06903 Sophia Antipolis Cedex, France, 4Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, Saskatchewan S7N 0X2, Canada, 5Co-Lab R&D div. Ag-Cord, 655 Rue Delorme, Granby, Quebec J2J 2H4, Canada, and 6UniversiteduQuebec a Montreal, 141 Rue du President-Kennedy, Montreal, Quebec H2X 3Y5, Canada Accepted: 22 July 2015 Key words: mesophyll-feeder, piercing-sucking insect, plant tissues, salivary sheath, stylet penetration, Vitis, xylem, Auchenorrhyncha, Hemiptera, Cicadellidae, Vitaceae, DC-EPG Abstract Feeding behavior of three leafhopper species – Erythroneura vitis (Harris), Erythroneura ziczac (Walsh), and Erythroneura elegantula (Say) (Hemiptera: Cicadellidae) – reared on grapevine, Vitis vinifera L. cv. ‘Seyval blanc’ (Vitaceae), was investigated using histological techniques and DC-electri- cal penetration graphs (DC-EPG). Histological studies revealed that the Erythroneura species induced white stipples on the leaves and that these leafhoppers produced thin salivary sheaths in grapevine leaf tissues. The DC-EPG system allowed the characterization of five waveforms associated with stylet penetration and feeding in leaf tissues. These waveforms were characteristic of feeding phases corre- sponding to epidermis penetration pathway, salivation, and ingestion. We calculated 28 parameters (e.g., number of probes, duration of phases, and time spent in the various tissues) to describe and compare the feeding behavior of the Erythroneura species. We conclude that the three Erythroneura species are mainly mesophyll feeders but may probably also feed in other tissues such as xylem. symptoms (Christensen et al., 2005), including yellowing Introduction or reddening of foliage, rolling of leaves, flower sterility, Leafhoppers can induce serious economic losses to proliferation of shoots, fruit abortion, decline, and vineyards (Bostanian et al., 2012; Olivier et al., 2012) as eventually grapevine death. Specific interactions devel- they vector plant pathogens such as viruses (Putman, oped between hemipteran pests (i.e., piercing-sucking 1941; Nielson, 1968; Nault & Ammar, 1989; Mesfin insects) and their host plants have been studied by et al., 1995), bacteria (Purcell et al., 1979), and phyto- histological techniques (Smith & Poos, 1931; Baber & plasmas (Weintraub & Beanland, 2006; Olivier et al., Robinson, 1951; DeLong, 1971; Gunthardt€ & Wanner, 2012). Phytoplasmas are obligate pathogens that live and 1981) to describe stylet location and feeding activities in reproduce in the phloem of their host plants and in the plants. organs of their insect vectors. In grapevines (Vitis spp., The contact of stylets with plant tissues generates varia- Vitaceae), phytoplasmas induce Grapevine Yellow (GY), tion of electrical resistance and voltage (named wave- a destructive disease occurring in most grape-growing forms) that reflect the various feeding phases associated regions worldwide. GY-infected plants show an array of with tissue penetration, salivation, or ingestion of plant fluids (McLean & Kinsey, 1964; Tjallingii, 1978, 1985; Backus & Bennett, 1992; Backus et al., 2009). Developed *Correspondence: Julien Saguez, Agriculture et Agroalimentaire to investigate aphid-feeding behavior (McLean & Kinsey, Canada, 430 Boulevard Gouin, Saint-Jean-sur-Richelieu, Quebec J3B 1964; Tjallingii, 1978, 1985), electrical penetration graph 3E6, Canada. E-mail: [email protected] (EPG) was adapted to other piercing–sucking insects such © 2015 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 1–14, 2015 1 2 Saguez et al. as whiteflies (Janssen et al., 1989; Johnson & Walker, rence of phytoplasmas in Erythroneura species in 1999; Lei et al., 1999; Jiang et al., 2000; Lett et al., 2001; Canadian vineyards, based on PCR tests, was unexpected Liu et al., 2012), psyllids (Ullman & McLean, 1988; (Olivier et al., 2014). Bonani et al., 2010; Civolani et al., 2011), mealybugs Erythroneura species are important economical pests (Calatayud et al., 1994; Huang et al., 2012), thrips (Thysa- in North America due to their abundance and their noptera) (Harrewijn et al., 1996; Kindt et al., 2003, 2006), extensive damage caused in vineyards (Bostanian et al., and phylloxera (Phylloxeridae) (Kingston et al., 2007). 2003; Lowery & Judd, 2007; Saguez et al., 2014). Deci- EPG has also been used to study the feeding behavior of phering their feeding behavior may help to elucidate Auchenorrhynca, including treehoppers, planthoppers, whereandhowtheycanacquirephytoplasmasand sharpshooters – economically important pests known to whether they could be competent vectors of phytoplas- vector Pierce’s disease on grapevines (Backus et al., 2009; mas. Our objectives were to study the damage and to Krugner & Backus, 2014) –,andleafhoppers. characterize the feeding behavior of three abundant Ery- Whereas feeding on plants, leafhoppers can acquire throneura species in Canadian vineyards with both histo- and transmit pathogens (viruses, bacteria, and phytoplas- logical and EPG techniques. mas) that cause economically important plant diseases. However, their feeding behavior is still poorly docu- Materials and methods mented. One of the reasons is that their feeding behavior is more diverse than that of other piercing-sucking Plant material insects, notably because, depending on their subfamilies, Uninfected potted greenhouse-grown vines (Vitis vinifera leafhoppers may feed on various plant tissues (Nielson, L. cv. ‘Seyval Blanc’) were established in 2007 from healthy 1968; Backus et al., 2005). The feeding behaviour of sev- vine shoots collected in a commercial vineyard of Dun- eral Typhlocibinae was investigated by EPG (Marion-Poll ham, Quebec (45°110N, 72°860W). To reduce variability et al., 1987; Hunter & Backus, 1989; Backus et al., 2005; between tested plants, one potted greenhouse-grown vine Jin et al., 2012; Miao et al., 2014) because leafhopper was selected and micropropagated to obtain genetically species belonging to this subfamily cause various symp- identical plants. Briefly, internodes (2–4 cm) of Seyval toms and diseases on plants (e.g., stipples, hopperburn, Blanc were excised, thoroughly washed for 1 h in distilled and phytoplasma diseases). water at 4 °C, dipped in 75% (vol/vol) ethanol for 1 min In Canada, ca. 100 leafhopper species have been found and 30 min in 1.5% (vol/vol) sodium hypochlorite, then in vineyards (Bostanian et al., 2003; Saguez et al., 2014), washed 39 in sterile distilled water. Explants were cut off the most abundant genera being Erythroneura, Macrosteles, to exclude damaged tissues and then grown in Murashige and Empoasca (Saguez et al., 2014). GY phytoplasmas also and Skoog medium pH 5.7 (Sigma-Aldrich, St-Louis, have been detected in Canadian grapevines and leafhop- MO, USA) without growing hormones. After 6 weeks, we pers (Olivier et al., 2009, 2014). Among the 37 leafhopper obtained 42 viable plants that were potted and transferred species detected to be phytoplasma-positive in Canadian to the greenhouse, where they were kept under the same vineyards, some are known to be GY carriers (i.e., phyto- growing conditions until their use. All experiments were plasma detected in leafhoppers but vectorship ability not done at 23 °C, 60% r.h., and L16:D8 photoperiod and 30 formally demonstrated) or vectors (i.e., transmission of plants were used for EPG tests (each plant was used at least phytoplasmas formally demonstrated) (Olivier et al., 19 per leafhopper species). 2014). The difference between carriers and vectors may be related to feeding behavior. For example, the phytoplasma Insect rearing vector Macrosteles quadrilineatus (Forbes) preferentially Erythroneura vitis (Harris), Erythroneura elegantula (Say), feed on phloem but can also feed on other plant tissues and Erythroneura ziczac (Walsh) colonies were each initi- (Smith, 1926; Putman, 1941; Hunter & Backus, 1989; ated from 10 individuals collected in commercial vine- 0 0 Backus et al., 2005). The phytoplasma carriers Empoasca yards located in Dunham (45°11 N, 72°86 W), Quebec, fabae (Harris) and Erythroneura species (Olivier et al., Canada, in summer 2008. All colonies were maintained in 2014) belong to the Typhlocibinae subfamily (Hemiptera: environmental chambers on potted ‘Seyval Blanc’ grapevi- Cicadellidae) that are considered as mesophyll feeders nes and reared at 23 °C, 60% r.h., and L16:D8 photope- (Naito, 1977). However, E. fabae is not a strict mesophyll riod (Saguez & Vincent, 2011). Nymphs were reared on feeder and can feed in phloem (Hunter & Backus, 1989). Seyval Blanc leaves placed on Petri dishes containing 0.7% In contrast, the feeding behaviour of Erythroneura species agar (wt/vol), until age-synchronized adults emerged is poorly documented and no report they feed in other tis- (Saguez & Vincent, 2011). All experiments were conducted sues than mesophyll is available. Consequently, the occur- with 5-day-old adults. Feeding behavior of Erythroneura species on