Characterization of the Feeding Behavior of Three Erythroneura Species on Grapevine by Histological and DC-Electrical Penetration Graph Techniques

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-electrical 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 corresponding 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 grapevine 3

Damage on leaves quiescent and then immobilized by their abdomen with a Using a circular glass cage, leafhoppers were individually home-made vacuum. A 5-cm gold wire (20 lm diameter) restricted to feed on a zone (15 mm diameter) of the abax- was glued with water-based silver glue to the leafhopper’s ial face of an excised and undamaged leaf. A digital video- dorsum, and a copper electrode was inserted into the soil camera (Stringray F145C IRF; Allied Vision Technology, of the potted grapevine. Stadtroda, Germany) was fixed on a stereomicroscope Two Giga-4 DC-EPG systems (www.epgsystems.eu) (Leica M80; Leica Microsystems, Wetzlar, Germany) to were placed in a Faraday cage to record simultaneously record the effect of feeding by one leafhopper on the leaf the feeding behavior of eight independent leafhoppers from its adaxial face. To increase contrast, both faces of the on eight independent grapevines (one leafhopper per leaves were illuminated. From the video-recording, screen- plant). The Giga-4 output was connected to a DI-720 shots were taken at different times of feeding. This experi- analog-to-digital (AD) board (Dataq Instruments, ment was repeated 59. Akron, OH, USA), the substrate voltage was adjusted to To observe the effects of leafhopper feeding on leaf his- 0 V for each channel and the amplification on the Giga- tology, five undamaged leaves collected from healthy 4 was always set at 509. Waveforms were recorded using greenhouse-grown vines were used as control, and com- STYLET+ software (www.epgsystems.eu). All tested pared with five leaves fed upon by E. vitis and five by plants were 1 year old and were watered (soil was at sat- E. ziczac for 1 day, directly on the grapevine plants. Try- uration during the experiment) before use. Leafhoppers pan blue was used to stain and localize salivary sheaths in were placed on the abaxial face [i.e., their preferred feed- grapevine leaves. Control and damaged leaves were cut in ing site according to Robinson (1926) and Paxton samples of ca. 1 cm2 and fixed in 1:1 (vol/vol) ethanol:try- (1990)] of the fourth leaf from the apex of the grapevine pan blue solution (10 ml lactic acid, 10 g phenol, 10 ml and maintained in their natural position. Each leafhop- glycerol, 10 ml distilled water, 10 mg trypan blue). Leaf per was monitored for 4 h. EPG experiments were per- samples were boiled in the ethanol:trypan blue solution formed one species at a time, until 30 valid recordings for 1 min, and destained 1 h in chloral hydrate (5 g in were obtained per species. All the individuals that 2 ml). Samples were destained a second time for 16 h in escaped (by ungluing or leaving the plant) during the chloral hydrate, then placed in glycerol for conservation recording were discarded. All experiments were con- until making histological observations under a Leica M80 ducted at 23 °C, 60% r.h., and L16:D8 photoperiod. stereomicroscope and a light compound microscope (Leica DM LS; Leica Microsystems). EPG data analysis To correlate feeding behavior with histology and to Leafhopper feeding behaviors were individually analysed localize salivary sheaths in grapevine tissues, the feeding using the STYLET+ software, distinguishing the different behavior of leafhoppers was recorded by EPG (see below), waveforms according to their specific patterns. Calculation and interrupted during the different waveforms by remov- of 28 EPG parameters (e.g., number, frequency, and dura- ing the leafhopper. Leaf samples (ca. 25 mm2 around the tion of each waveform) was done with the EPG-Calc puncture site) were stained with trypan blue as previously open-access Software (Giordanengo, 2014) using the described. Tissues were then fixed in formalin:acetic acid: routine specifically developed to manage species with non- ethanol (2:1:17 by volume), dehydrated in a series of conventional characteristics and sequences of EPG wave- increasingly ethanol gradient baths, followed by paraffin forms. The general probing behavior was determined by embedding (Ruzin, 1999). Using a microtome, samples the number of probes performed by leafhoppers and total were cross-sectioned at 10 lm thickness. Cross sections duration of probing (Pr) vs. non-probing (NP) phases. were observed under a light compound microscope to Then, based on other studies and preliminary experiments, detect the occurrence of salivary sheaths stained in blue. we distinguished phases that reflect the duration of the When salivary sheaths were observed, samples were then feeding activities in non-vascular (i.e., stylet pathway and deparaffined, rehydrated, and stained using toluidine blue mesophyll ingestion) and vascular tissues. Number, mean, O (TBO). This experiment was repeated 59 for each and total duration of each feeding phase were calculated leafhopper species and each waveform, independent of the individually for each leafhopper and per species. Among EPG experiments describe below. the 30 leafhoppers tested per Erythroneura species, we noted the durations of the shortest and longest non-prob- Feeding behavior ing and probing phases. To compare the EPG parameters Unsexed adult leafhoppers were individually starved for of the three Erythroneura species, a Kruskal–Wallis test at 2.5 h in P100 cups (Solo Cup, Highland Park, IL, USA). a = 0.05 was performed using STATISTICA 10 software They were subjected at 20 °C for 40 s to make them (StatSoft, Tulsa, OK, USA). 4 Saguez et al.

by the length of leafhopper stylets. Then, upon feeding Results from one site on the leaf, the leafhoppers withdrew their Damage on the leaves stylets from the plant tissues and moved to another As the three Erythroneura species caused the same feeding site nearby. symptoms to grapevine leaves, no distinction was fur- Leaves stained with trypan blue revealed salivary ther done between them. Under natural or laboratory sheaths in the stipples (Figure 2A) localized in the tissues conditions, leaf parts injured by E. elegantula, E. vitis, and close to vessels (Figure 2C–F). A border of brownish and E. ziczac showed similar and characteristic stipples cells delimited the pigmented unfed areas (i.e., containing (i.e., contiguous depigmented zones that appeared as chloroplasts) and the stipples (Figure 2A). Salivary white spots on leaves) resulting from leafhoppers feed- sheaths presented a large diversity of shapes and localiza- ing on mesophyll cells. Video-recordings revealed that tions (Figure 2B–F). Leafhopper stylets penetrated the stylets rapidly moved under the epidermis (data not leaves obliquely or perpendicularly to the leaf surface. A shown). Cell content was removed rapidly in all direc- salivary ﬂange is produced at the probing site (Figure 2B, tions around the probing site, creating a stipple (de- D, and G) and salivary sheaths in leaf tissue were gener- prived of chloroplasts) in an area of few mm2 ally single and thin (Figure 2G) and some were ramiﬁed (Figure 1). The size of the affected area was restricted (Figure 2C).

Figure 1 Leaf depigmentation occurring during Erythroneura species feeding in palisade mesophyll. Adult leafhoppers were put on the abaxial face of a leaf and a series of pictures was taken at different times from the adaxial face of the leave. The arrow indicates the area where depigmentation occurred through time (time scale is in s).

A B

Figure 2 Close-up of damage caused by Erythroneura species on grapevine leaves stained by trypan blue. (A) White stipple (i.e., contiguous depigmented zones without chloroplasts) delimited by brownish cells (white arrow heads). Salivary sheaths (thin black arrows) appeared in blue. Thick black arrows indicate leaf veins. (B–G) Salivary sheaths. (B) Details of the red box (in A) showing oblique penetration of stylets in tissues, CEG salivary flange (sf) deposited around the penetration site (ps) in the leaf. (C–F) Types of salivary sheaths with single or ramified tracks in leaf tissues. Some punctures are close to vascular bundles (v). (G) Detail of a single track of salivary DF sheath close to stomata (s). Salivary flange (sf) is deposited at the penetration site (ps) in the leaf and the track is single and thin. Observations done under a stereomicroscope. Scale bars are in mm. Feeding behavior of Erythroneura species on grapevine 5

Comparing undamaged (Figure 3) and leafhoppers-in- (Figures 6B–Dand7A–C), waveform G (Figures 6D and jured leaves (Figures 4 and 5), cross sections stained by 8), and waveform X (Figure 7D). trypan blue and TBO showed that Erythroneura species inserted their stylets perpendicularly or obliquely to the Non-probing waveform (NP). Non-probing waveform leaf surface (Figures 4 and 5) and ramified salivary sheaths (Figure 6A and B) had a baseline voltage of 0 V, the in spongy (Figure 4) and palisade (Figure 5) mesophyll. stylets being outside of the leaf. It occurred at the Palisade mesophyll cells damaged by Erythroneura species beginning of the recording when leafhoppers were put on were deprived of chloroplasts and their structure was dis- leaves. NP was also observed at various times during the organized (Figure 5) compared to undamaged leaves (Fig- recording. It corresponded to an interruption of feeding ure 3). The thinness of salivary sheaths did not allow the associated with movements of the leafhopper to find observation of salivary sheaths insertion in other plant tis- another site of puncture. During NP, several leafhoppers sues, notably in vessel bundles. cleaned their wings and tarsi or walked on the surface of the plant but these behaviors did not induce fluctuation Waveform characterization in voltage level. Waveform patterns of the three Erythroneura species feeding on Seyval Blanc leaves were similar (Figure 6A). Con- Waveform A. Once leafhopper stylets made contact with sequently, the characterization of waveforms will be the plant, this generated the first waveform before starting treated hereafter without species distinction. Similar the penetration in the leaf tissues. Penetration of leaf sequences of events were observed in the three species. cuticle and epidermis was characterized by a high, quick, After a leafhopper was put on the abaxial face of a leaf, a vertical, and positive increase of voltage that lasted few short (5–10 s) period of non-probing (NP) was observed. seconds, before voltage gradually or rapidly (Figure 6B) Then, stylets penetrated the leaf (i.e., probes or Pr), gener- decreased to baseline voltage. The amplitude of waveform ating a series of waveforms interspersed with non-probing A varied depending on several factors such as the phases. During probes, five different waveforms were iden- individuals, the substrate voltage, the electrode potential, tified, hereafter named non-probing waveform (NP) (Fig- and the resistance in the leafhopper-grapevine ure 6A and B), waveform A (Figure 6B), waveform C combination. Waveform A was consistently followed by a

Figure 3 Cross sections of an undamaged grapevine leaf stained by toluidine blue O. (A) Overview of a leaf section (as, air space). (B) Details showing the leaf structure and especially the palisade mesophyll composed of one or two layers of elongated cells tightly compacted and rich in chloroplasts, vascular parenchyma with vascular bundle, and the spongy mesophyll composed of rounded cells poor in chloroplasts and with large intracellular air spaces (as) to facilitate gas exchanges with stomata. Scale bars are in mm. 6 Saguez et al.

A A

Figure 4 Cross sections of grapevine leaf showing leafhopper stylet tracks and feeding in spongy mesophyll. (A) Overview of Figure 5 Cross sections of grapevine leaf showing Erythroneura the tracks in the leaf. (B) Detail of the red box shown in A. The leafhopper puncture in palisade mesophyll. (A) Overview of a ramiﬁed salivary sheaths (contour highlighted in red) were leaf section showing empty cells (ec) deprived of chloroplasts and stained by trypan blue. Leaf was stained with toluidine blue O. as, undamaged cells (uc) containing chloroplasts in palisade air space. Scale bars are in mm. mesophyll. (B) Detail of the red box shown in A. The ramiﬁed salivary sheaths are stained by trypan blue (contour highlighted in red). Intra- and intercellular punctures could be observed in sharp decrease in voltage and by the beginning of the spongy mesophyll and stylet tracks ending in the palisade mesophyll. Leaf was stained with TBO. Scale bars are in mm. waveform C (Figure 6B).

Waveform C. After waveform A, the voltage level went before the resumption of a typical pattern of waveform C down and waveform C generally presented a typical saw- (Figure 7D). The voltage level may slightly increase and toothed pattern (Figure 6C) characterized by repeated decrease during the occurrence of this waveform X. brief drops that presented a frequency of 1–4Hz.Each drop was followed by several consecutive spikes in the Waveform G. Waveform G was consistently preceded opposite direction and had a lower amplitude and followed by waveform C and was composed of regular (Figure 6C). The amplitudes and shapes of these spikes oscillations (Figures 6D and 8). Between waveforms C and varied during C phases. Occasionally, the first drop had G, the voltage level gradually increased before reaching a smaller amplitude and the following upward spikes had more-or-less constant level with sustained oscillations. higher amplitude (black arrows in Figure 7A–C) Waveform G varied with a frequency of about 6.5–10 Hz compared with the typical saw-thooted profile (Figures 6D and 8A). The pattern of waveform G varied (Figure 6C). Some spikes had variable hill-shapes (black occasionally, showing reduced amplitude and less triangles in Figure 7B and C). pronounced spikes (Figure 8B–D). In most cases, at the end of waveform G, voltage level decreased under the Waveform X. When produced, waveform X always baseline and waveform C immediately followed. However, occurred during waveform C (Figure 7D). After a period in some cases, the voltage level of waveform G quickly of saw-toothed pattern, the voltage stabilized and became decreased and was followed by a secondary waveform with very flat. Peaks, drops, spikes, or oscillations were a pattern similar to waveform G with smaller amplitude occasionally observed and voltage gradually increased and higher frequency, as shown in Figure 6D between 8 Feeding behavior of Erythroneura species on grapevine 7

Figure 6 Typical EPG patterns of Erythroneura species. (A) Overview of 1 h of feeding behavior recording, showing the different waveforms, i.e., non-probing (NP) and waveforms A, C, and G. (B–D) Detail (10 s) of waveforms A, C, and G, respectively. Arrow head indicates the secondary waveform (smaller amplitude and higher frequency) that occasionally occurs at the end of waveform G. and 9.2 s (see the black arrow head). This secondary Non-probing vs. probing phases. The total duration of NP waveform occurred occasionally and, when observed, had phases represented 14–18% of the total time of recording a duration that largely varied independent of the duration and did not statistically differ between the three of the previous part of waveform G (range: few seconds to Erythroneura species. The mean duration of a NP phase 3min). was 2–3 min (range: 1 s to several hours). The probing phases (Pr) represented >80% of the time spent by Occurrence and duration of EPG phases leafhopper feeding on the various grapevine tissues and Although the waveforms had similar patterns and grouped together A, C, G, and X phases. The first probe sequence, their frequency and duration varied, depend- usually occurred in the first 2 min after placing a ing on the species but also on the individuals. Using leafhopper on a grapevine leaf (Table 1). Its duration Giordanengo (2014) the feeding behavior of E. elegan- varied from 11 to 20 min. tula, E. vitis,andE. ziczac was categorized in five EPG phases: (1) non-probing (NP), (2) probing (Pr), (3) C Cphases. The duration of waveform A was generally phase, corresponding to the combination of waveforms ≤5 s (data not shown). That is why we included it with A and C, (4) G phase associated with waveform G, and the duration of C phases. The shortest C phases lasted (5) X phase associated with waveform X. The parame- 0.2–0.3 s and occurred between a waveform G and a ters calculated are presented in Table 1. The Kruskal– NP phase, before leafhoppers changed feeding site. In Wallis test detected no significant difference among the contrast, the longest C phases lasted up to several three species. hours (Table 1). 8 Saguez et al.

Figure 7 Waveforms C and X. (A) Five seconds of recording of characteristic waveforms C followed by 5 s of a waveform C with several positive-voltage spikes (black arrows). (B) Waveform C with several spikes (black arrows) and some hill-shape patterns (black triangles). (C) Waveform C with characteristic pattern, spikes (black arrows), hilly patterns (black triangles), and without spikes (white triangles). (D) Waveform X that occurred during waveform C pattern.

Xphases. X phases were recorded in only ca. 34% of the punctures. Although each G phase had a mean duration of three leafhopper species (Table 1) and no significant ca. 2 min, the first G phases were generally longer than differences were observed among the species (E. elegantula subsequent ones and lasted 5–6 min (maximum 45 min). vs. E. vitis: v2 = 0.318, d.f. = 1, P = 0.57; E. elegantula vs. The shortest duration of G phases were ≤2 s, whereas E. ziczac: v2 = 1.832, d.f. = 1, P = 0.18; E. vitis vs. longest G phases varied from 24 to 46 min. E. ziczac: v2 = 0.635, d.f. = 1, P = 0.43). Although we could not associate this waveform with a specific feeding Discussion behavior, X phases were not baseline and we considered them as parts of the probes and not as non-probing Damage and histological observations phases. The total duration of these X phases was <35 min Our results indicated that, feeding on grapevine leaves, and their mean duration ranged from 7 to 15 min. These Erythroneura species emptied leaf cells, as observed during phases randomly appeared during C phases and their the video recordings, inducing the appearance of stipples minimum duration was ca. 1.5–2minandmaylast confined to the cells punctured by the stylets. Damaged 40 min to 1 h. areas did not present necrotic symptoms, and adjacent cells appeared undamaged. The white appearance of stip- Gphases. The first G phase generally occurred few ples is due to the ingestion of mesophyll cell content by seconds or minutes after the beginning of the first probe leafhoppers resulting in emptied cells containing air, nota- but sometimes occurred after one or two short exploring bly in the upper palisade mesophyll cells. These results Feeding behavior of Erythroneura species on grapevine 9

Figure 8 Waveform G. (A) Typical waveform G with high voltage amplitude. (B) Waveform G with small voltage amplitude. (C, D) Variations of the shape of the waveform G. Boxes show details of the waveforms. confirmed previous histological studies conducted on Consequently, we deducted that Erythroneura species pos- leafhoppers belonging to the Typhlocybinae, including the sess characteristics of both ‘lacerate and flush feeders’ and tribe Erythroneurini (Smith, 1926; Smith & Poos, 1931; ‘lance-and-ingest feeders’. Pollard, 1968). Our results also demonstrated that Erythroneura species EPG waveforms characterization inserted their stylets perpendicularly or obliquely to the The nomenclature of waveforms recorded with DC-EPG leaf surface and formed tenuous salivary sheaths. Based on was first established for aphids and is related to stylet path- our video recordings, we observed that Erythroneura spe- ways in epidermis and parenchyma (waveforms A, B, C, cies quickly emptied the mesophyll cells by rapid stylet pd, and F), xylem ingestion (G), and phloem phases (E1 protraction and retraction movements and by deeply pen- and E2). However, leafhoppers feed on plants with differ- etrating plant tissues, suggesting that Erythroneura species ent strategies than aphids and whiteflies. In leafhoppers, can be classified as a ‘lacerate and flush feeder’ (Smith & waveform nomenclature established by several authors Poos, 1931; Backus et al., 1988, 2005). However, ‘lacerate greatly varies depending on the species studied (Kawabe & and flush feeders’ typically do not produce salivary McLean, 1980; Hunter & Backus, 1989; Buduca et al., sheaths. In contrast, we demonstrated that Erythroneura 1996; Lett et al., 2001; Stafford & Walker, 2009; Trezbicki species produce thin and incomplete salivary sheaths et al., 2012). (pseudosheaths) characteristic of the ‘lance-and-ingest’ The first EPG study on mesophyll feeders was done by feeders (Backus et al., 2005). However, ‘lance-and-ingest’ Marion-Poll et al. (1987) on Zynginidia scutellaris (Her- leafhoppers specifically feed on vascular tissues. rich-Sch€affer), a leafhopper pest of maize. They suggested 10 Saguez et al.

Table 1 Mean ( SEM) electrical penetration graph (EPG) phases and parameters measured during a 4 h-recording session for three Erythroneura leafhopper species deposited on Seyval Blanc grapevine cultivar

Kruskal–Wallis EPG phases and parameters E. elegantula E. vitis E. ziczac H P d.f. Non-probing (NP) No. NP 21.3 2.1 15.5 1.2 19.7 2.2 5.1072 0.078 87 Mean duration of NP (s) 138.9 16.1 154.1 26.2 178.6 37.5 0.0593 0.97 87 Total duration of NP (s) 2632.3 293.0 2085.6 255.5 2569.6 313.8 1.3541 0.50 87 Minimum duration of NP (s) 4.7 1.0 5.1 0.7 6.8 1.1 0.4856 0.78 87 Maximum duration of NP (s) 1270.0 212.9 790.4 114.3 1037.7 242.6 2.6322 0.27 87 Probes (Pr) No. Pr 21.9 2.0 16.3 1.2 20.5 2.2 5.3257 0.070 87 Mean duration of Pr (s) 775.7 139.2 862.5 69.2 746.3 87.7 5.6587 0.059 87 Total duration of Pr (s) 11628.9 298.6 12135.9 264.5 11632.0 310.4 1.5203 0.47 87 Time to first Pr (s) 43.4 15.1 87.9 55.8 47.4 20.4 3.3456 0.19 87 Duration of first Pr (s) 1225.9 342.1 700.9 108.8 801.1 147.1 0.1566 0.92 87 C phase (C) No. C 45.8 3.3 35.4 1.9 41.0 3.2 5.2931 0.071 87 Mean duration of C (s) 236.3 21.3 288.9 19.1 260.3 20.5 5.4591 0.065 87 Total duration of C (s) 9192.8 359.7 9524.1 412.9 9393.9 369.7 0.3185 0.86 87 Minimum duration of C (s) 1.8 0.3 2.7 0.4 1.8 0.3 3.5231 0.17 87 Maximum duration of C (s) 1302.6 165.9 1368.6 120.0 1577.0 290.0 0.9062 0.64 87 Gphase(G) No.G 25.4 2.2 19.5 1.3 20.8 1.5 4.3603 0.11 87 Mean duration of G (s) 111.2 13.9 138.9 19.7 135.4 26.9 0.3980 0.82 87 Total duration of G (s) 2354.6 220.6 2386.8 264.6 2121.8 217.2 0.8528 0.65 87 Time to first G from first Pr (s) 48.7 10.9 271.4 108.5 70.3 27.2 4.9113 0.086 87 No. Pr before first G1 1.1 0.1 1.4 0.2 1.1 0.0 3.0136 0.22 87 Duration of first G (s) 359.3 72.6 278.7 62.0 344.5 89.8 1.9342 0.38 87 Minimum duration of G (s) 8.5 2.0 10.2 2.0 8.1 2.2 1.9752 0.37 87 Maximum duration of G (s) 638.1 91.0 573.6 71.1 630.6 111.9 0.1349 0.93 87 Undefined phase (X) No. X 2.3 1.1 0.7 0.2 1.8 0.6 1.7015 0.43 28 Mean duration of X (s) 475.8 151.9 879.6 184.2 446.8 78.0 2.7989 0.25 28 Total duration of X (s) 1870.1 371.4 1947.0 561.3 2064.6 572.0 1.6061 0.45 28 Minimum duration of X (s) 309.4 149.0 710.7 196.1 200.5 42.3 4.1417 0.13 28 Maximum duration of X (s) 878.6 254.9 1193.9 237.6 1147.5 368.8 1.7205 0.42 28 n2 81013

1Number of initiated or completed probes before the first G phase. 2Number of leafhoppers (out of 30) for which undefined phases were observed in their EPG profile. The Pearson’s v2 test indicates no significant difference between the numbers of leafhoppers that realized this phase (P>0.05). that waveforms generated by Z. scutellaris were associated suggests that the high increase in voltage and the follow- with penetration, salivation, and ingestion. In our study, ing peaks that occur at the beginning of waveform A may waveforms generated by the three Erythroneura species correspond to waveforms A in aphids and could reflect presented typical and distinct patterns that are easily rec- epidermis penetration and production of salivary flange ognizable and also associated with penetration, salivation, and sheath material secretion by leafhoppers. This and ingestion. The transition from one waveform to hypothesis could not be confirmed by correlating the another was clear, being typically associated with an EPG waveform with histological observation because the increase or a decrease of voltage. duration of waveform A was too brief and, in many cases, Waveform A is characteristic of the beginning of stylet salivary sheaths were not observed. During the brusque penetration in the leaf. Sheath material deposition (i.e., interrupting of the feeding behavior, salivary sheaths salivary flange) was previously described in Typhlocybi- could have been torn out or destroyed during or after nae: a salivary sheath develops around the stylets imme- removal of the stylets. However, in other cross sections, diately after contact with the leaf cuticle (Pollard, 1968). salivary sheaths inserted in the epidermis were observed, The duration of waveform A was short (few seconds) suggesting intracellular penetrations. Stylet insertion before the stylets penetrate deeper into other tissues. This never occurred in the stomata, as also mentioned by Feeding behavior of Erythroneura species on grapevine 11

Naito (1977) in the green rice leafhopper, Nephotettix those observed during xylem sap ingestion in various cincticeps Uhler. hemipteran pests such as aphids (Prado & Tjallingii, Single salivary sheath tracks were often thin and difficult 1994), whiteflies (Lei et al., 1999), planthoppers (Buduca to observe in their full length. Consequently, we allowed et al., 1996), leafhoppers (Lett et al., 2001; Stafford & leafhoppers to feed for several seconds or minutes before Walker, 2009), and psyllids (Bonani et al., 2010; Civolani interrupting the feeding behaviour. As a result, ramified et al., 2011). Although this pattern indicates an active tracks were observed in spongy mesophyll, showing that ingestion of fluid, our study does not allow to conclude leafhopper stylets penetrated spongy mesophyll intercellu- that waveforms G observed in Erythroneura species corre- larly. Numerous intracellular punctures were observed in sponded to feeding activities in xylem vessels. However, cells of the spongy and palisade mesophyll, suggesting that we observed two kinds of waveforms G with short (few mesophyll is the main source of food. This statement is seconds) or long durations (several minutes to hours) supported by the emptying of the mesophyll cells observed (data not shown) with a mean duration of ca. 600–650 s. by video-recording and the occurrence of empty cells in Long waveforms G were the most abundant (data not histological preparations. Furthermore, in aphids and shown), can reach ca. 2400 s, and generally occurred at the whiteflies, low-voltage levels represent intracellular posi- beginning of the recording or after a long period of non- tions of the stylets. In Erythroneura species waveform C probing. During the long waveforms G, no stipples also had low-voltage level, reinforcing our statement. The appeared on video-recordings suggesting that leafhoppers comparison of number of probes and the duration of C were not feeding on mesophyll. Consequently, although phases also support the hypothesis that waveform C is Erythroneura species are mainly mesophyll feeders, punc- associated with mesophyll feeding. Waveform C was com- tures in xylem may allow them to quickly find water and posed of different patterns. The first leaf tissue encoun- contribute to the regulation of their osmotic potential as tered by leafhopper stylets under the lower epidermis is previously observed in other piercing-sucking insects spongy mesophyll, whose structure differs from the pal- (Pompon et al., 2010, 2011). Consequently, long wave- isade mesophyll. Spongy mesophyll is composed of few forms G (ca. 270–350 s) observed at the beginning of the layers of rounded cells poor in chlorophyll with thin cell EPG recordings may reflect rehydration after starvation. walls. These cells are not compacted and there are large This is supported by the fact that subsequent G phases intercellular air spaces to facilitate gas exchanges. In con- recorded after short non-probing phases were shorter or trast, palisade mesophyll is composed of one or few layers not necessarily observed. Besides, some salivary sheaths of elongated cells tightly compacted and rich in chloro- were observed closed the leaf veins even if we have no evi- plasts. The difference of cell structure and their organisa- dence that stylets reach the vascular bundles due to the tion generate different membrane potentials depending on production of pseudosheaths by Erythroneura species. the tissues. EPG waveforms mainly reflect the electromo- In contrast, short waveforms G (<120 s) have also been tive forces and resistance components associated with pen- observed (data not shown) and could be associated with etration, salivation, and ingestion of plant fluids. mesophyll feeding as previously observed on Circulifer Consequently, puncturing and sucking palisade or spongy tenellus (Baker), and suggested by Stafford & Walker mesophyll induced different EPG signals, explaining the (2009). In C. tenellus, waveform G was produced both in variability in patterns of waveform C. For example, when xylem and in mesophyll and short waveforms G were asso- stylets were located in the spongy mesophyll, we presumed ciated with mesophyll ingestion whereas long waveforms they contact intercellular air spaces that may influence the G were associated with xylem ingestions. The variations of electric potential recorded in this tissue. Salivation and waveform G shapes, also observed in C. tenellus (Stafford ingestion of palisade or spongy mesophyll cells may also & Walker, 2009) were generally observed at the end of G have generated different waveform C patterns due to the phases and before waveform C. Unfortunately, at this differences of cell structure and chemical composition. point of the study, we could not explain these variations of The spikes and large peaks observed during waveform C waveform G shapes, even using histological observations had an elevation of voltage and shapes similar to waveform because Erythroneura species produced thin and tenuous A and could correspond to saliva injections in plant tissues salivary sheaths often undetectable in leaf cross sections, as observed in other piercing-sucking insects. especially in xylem vessels. Although Erythroneura species are generally described In our model grapevine – Erythroneura species, our as mesophyll feeders, our EPG profiles showed different results also revealed important variation of voltage waveforms, suggesting different feeding activities (punc- between the various waveforms (e.g., transition between tures, salivation, or ingestion) in different grapevine tis- waveforms A and C or alternating increases or decreases sues. Waveform G presented a typical pattern similar to between waveforms C and G). The phenomena causing 12 Saguez et al. this important variation of voltage needs to be clarified in paper. Julien Saguez was a postdoc fellow of the Universite further studies. du Quebec aMontreal, Canada. In summary, our study showed that the three Ery- throneura species induced the same histological damage References on grapevine leaves. They also had the same feeding behavior and generated the same waveforms patterns. 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Annals of associated with activities in phloem (this indicating that the Entomological Society of America 85: 437–444. they probably do not feed in phloem sap or are accidental Backus EA, Hunter WB & Arne CN (1988) Technique for stain- phloem feeders), we detected few phytoplasma-positive ing leafhopper (Homoptera: Cicadellidae) salivary sheaths and individuals collected in vineyards (Olivier et al., 2014). eggs within unsectioned plant tissue. Journal of Economic Riedle-Bauer et al. (2006) also reported the occurrence of Entomology 81: 1819–1823. Bois Noir-phytoplasma in mesophyll-feeders or Backus EA, Serrano MS & Ranger CM (2005) Mechanisms of Typhlocibinae in grapevine that are not usually known as hopperburn: an overview of insect taxonomy, behavior, and – Bois Noir vectors. In contrast, Macrosteles species and Em- physiology. 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