TY - JOUR
AU1 - Cervantes, Felix A.
AU2 - Backus, Elaine A.
AU3 - Godfrey, Larry
AU4 - Akbar, Waseem
AU5 - Clark, Thomas L.
AB - Abstract Lygus lineolaris (Palisot de Beauvois) and Lygus hesperus Knight are among the most important pests affecting production of cotton in the United States. Lygus spp. use a cell rupture feeding strategy. However, the precise stylet-probing behaviors of adult Lygus spp. are not well understood or quantifiably related to cotton damage. The long-term goals of our research are to: 1) relate stylet probing to damage, and 2) quantitatively compare L. lineolaris feeding among resistant and susceptible host plants. The specific objectives of this study were to apply the latest technology in electropenetrography (EPG) to record adult, prereproductive Lygus spp. feeding, to identify and characterize all the waveforms, and to hypothesize their biological meanings. We used a third-generation AC–DC electropenetrograph to record nonprobing and probing waveforms of adult bugs on pinhead (<3 mm) cotton squares, and constructed a waveform library from the output. Recordings were obtained with both AC and DC applied signals and at different input impedances (10 6 –10 9 Ω). Three nonprobing waveforms were identified and visually correlated: Standing still (S), walking (W), and antennation (A). Probing waveforms were classified as: cell rupturing (CR), transition (T), and ingestion (I). T waveform is the first finding of an X wave for a nonsalivary sheath feeder in Hemiptera, implying that tasting/testing/acceptance behaviors can be performed by a cell rupture feeder. While waveform I is not performed in every probe, when performed, its appearance and structure were affected by applied signal and input impedance. Overall, time spent in nonprobing behaviors was longer than time spent on probing behaviors. Tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois) and Lygus hesperus Knight (Hemiptera: Miridae), are economically important pests of cotton in the mid-southern and western United Sates, respectively. Direct feeding damage by fifth instars and prereproductive adults is the most severe ( Cooper and Spurgeon 2012 , 2013 ). Cotton squares (floral buds) and bolls (developing fruits) abscise due to the damage to anthers and/or seeds produced by Lygus spp. feeding. While feeding of L. lineolaris has been studied previously, most of these studies were conducted with bioassays that rely on square abscission rates, within-plant distribution and preference, and age dependence. The specific behavioral mechanisms involved in L. lineolaris feeding are still relatively unknown. To elucidate these mechanisms, electropenetrography (EPG) provides the most rigorous and accurate means of studying the feeding behavior of piercing–sucking insects such as Lygus ( Van Helden and Tjallingii 2000 , Walker 2000 ). Information generated through EPG allows the identification of feeding mechanisms by histologically correlating waveforms with stylet locations and movement as well as salivary sheaths left in the plant tissue. This technique has been extensively used to study numerous hemipteran species such as aphids ( Miles et al. 1964 ; McLean and Kinsey 1965 , 1968 ) and leafhoppers ( Backus et al. 2009 ). However, phytophagous mirids produce no salivary sheath, instead they use a macerate-and-flush method ( Miles 1972 ; now termed tactic; Backus et al. 2005 ), in a feeding strategy recently renamed cell rupture feeding ( Backus et al. 2005a ). These insects use their stylets and watery saliva to lacerate a pocket of cells that is eventually flushed out with a copious flow of dilute saliva (maceration) and the contents ingested. The absence of a salivary sheath poses a limitation to the correlation of feeding behaviors observed in an EPG recording with histological sections, requiring other methods to define mirid behaviors. The only EPG report (to date) on Lygus feeding described the stylet-probing behaviors of L. hesperus third instars using a first-generation (AC Missouri-type) EPG monitor ( Cline and Backus 2002 ), and correlated waveforms via video-recording of body postures as well as feeding on artificial diets. The authors identified several probing and nonprobing behaviors of third instars on developing squares of cotton plants and leaves of other nonhost plants. With advancements in EPG technology, there was a need to examine a larger diversity of EPG waveforms, from prereproductive adults of both L. lineolaris and L. hesperus on cotton squares, and to compare them with those of the previously recorded third-instar nymphs. EPG recordings are produced by the flow of an electrical current passing through the plant and insect when the latter inserts its mouth parts (stylets) into electrified plant tissue. A tethered insect completes the electrical circuit and creates a plant–insect interface whose changes in voltage are observable as waveforms on a computer monitor. Voltage changes in the plant–insect interphase occur due to the effect of one of two components (also called electrical origins) on the flow of electrical current. They comprise electrical resistance (hereafter R component of the waveform) and biopotentials (hereafter electromotive force or emf component of the waveform; Tjallingii 1978 , 1985 ; Walker 2000 ). The R component represents electrical resistance to the passage of ionically charged fluids through the insect stylets. The emf component represents the changes in biologically generated voltages (biopotentials) that are independent of the nature and type of applied signal ( Backus and Bennett 2009 ). Some causes of biopotentials include charge separations due to plant cell membrane breakage by stylets and/or streaming potentials during rapidly alternating, constant-but-rhythmic fluid movement through thin capillaries like the stylet food and salivary canals (e.g., during ingestion; Walker 2000 ). Thus, identification of the electrical origin of a waveform facilitates the definition of its biological meaning. Clear identification of R and emf components is dependent on two main factors, the size of the insect being studied and the amplifier sensitivity (hereafter input resistance [Ri] [or more properly, impedance] on a scale of 10 6 to 10 13 Ω) of the EPG monitor. The proportion of each component detected or acquired in the outputted signal varies with Ri level, due to the R/emf responsiveness curve ( Backus and Bennett 2009 ). This curve demonstrates that, as the Ri level is decreased, detection of the emf component is de-emphasized and ultimately lost while the R component gains. In addition, EPG recordings using DC applied signal tend to emphasize emf, while those using AC signal tend to emphasize R. The most detailed and complete EPG output signal ideally should have a 50:50 R:emf balance. For small insects such as aphids, this balance is achieved at a Ri level of 10 9 Ω ( Tjallingii 1978 , 1985 ). However, as the size of the insect increases, the balance point shifts and Ri needs to decrease in order to reach the balance again, as is the case in large hemipterans such as sharpshooter leafhoppers ( Backus et al. 2015 ) and chinch bugs ( Backus et al. 2005b , Backus et al. 2013 ). The present study used a third-generation AC–DC EPG monitor ( Backus and Bennett 2009 ) that allows the researcher to obtain EPG recordings from both AC and DC applied signal, and to switch the level of Ri within and between recordings. Increases or decreases in the amplitude of the waveforms at different Ri levels would clearly identify the electrical origins of the waveforms based on the above-described sensitivities to R versus emf. Definitive identification of Lygus spp. feeding waveforms at different Ri levels will produce a waveform library. The objectives of this study were to compare, update, and expand upon the information in Cline and Backus(2002) , by identifying and electrically characterizing all EPG waveforms produced by prereproductive male and female adult L. lineolaris and L. hesperus while feeding on pin-head cotton squares (<3 mm). We also determined the electrical origins of the waveforms, created a waveform library, and then hypothesized about the biological meanings of the waveforms based on their electrical origins, further supporting and complementing the video and artificial diet correlations previously performed. The waveform library allows a researcher to judge the best monitor settings, and (in combination with a study on the effects of applied signal on behavior) choose a single setting (for future recordings) that will best represent the 50:50 R:emf ratio. The waveform library will facilitate further EPG studies on host plant resistance to Lygus spp. , insecticidal assays/bioassays to assess mode of action and appropriate dosage, repellant studies, and host plant resistance, among other potential management tactics. Materials and Methods Insects L. hesperus were field-collected from an organic alfalfa field at the University of California, Kearney Agricultural Research and Extension Center (Parlier, CA). Insects were placed inside a 0.30-m (12-in) collapsible plastic cage (“bug dorm,” BioQuip, Rancho Dominguez, CA) containing shredded paper, raw sunflower seeds and seven green bean pods for oviposition, and kept in a growth room at 27°C, 60% RH, and a photoperiod of 16:8 (L:D) h. After 2 d, green beans were transferred to a rearing cage and kept under the same environmental conditions until fifth-instar nymphs were observed. Insects were reared on organic green bean pods. For EPG recordings, individual fifth-instar nymphs were separated and placed in an individual cage with a green bean pod until they became adults. Only 2–4-d-old, prereproductive adults (both genders) were used in recordings. L. lineolaris egg packs were obtained from a rearing facility at the ARS-USDA facility in Stoneville, MS. Egg packs were then placed in rearing cages similar to the ones used for L. hesperus and kept under the same environmental conditions. Insects were reared on organic green bean pods and only 2–4-d-old, prereproductive adults (both genders) were used in recordings. Cotton Plants Cotton plants were grown from seed in a greenhouse using Sunshine mix #1 soil and fertilized with Jack’s LX 15:5:15 Ca-Mg (JR Peters INC, Allentown, PA). Plants were grown under a year-round photoperiod of 16:8 (L:D) h with a temperature ranging between 18 and 30°C. Cotton seeds cv. ‘Coker’ were obtained from Monsanto (St. Louis, MO). Cotton plants were grown until squares were observed. Pin-head squares (<3 mm) were used in all experiments. Insect Wiring and EPG Instrument Prereproductive adult Lygus spp. (2–4 d old) were removed from their cages and starved for 2 h before wiring. Insects were anesthetized with CO 2 for 30 s then immobilized by low suction under a stereomicroscope (Leica MZ12 5 , Leica Microsystems Ltd., Heerbrugg, Switzerland) during wiring. A 38.1 µm (in diameter; sold as 0.0015 in., Sigmund Cohn Corporation, Mt. Vernon, NY) gold wire (1–2 cm in length) was glued to the pronotum of the insect, using water-based silver glue (white household glue: water: silver flake [Inframat Advanced Materials LLC, Manchester, CT] 1:1:1 [vol: vol: wt]). Insects were then starved while dangling from the wire for 1 h before being placed on the cotton square and connected to the EPG monitor. A four-channel AC–DC EPG monitor ( Backus and Bennett 2009 ; EPG Equipment Co., Otterville, MO) was used to record nonprobing and probing behaviors of L. lineolari s and L. hesperus inside a Faraday cage. Four cotton plants were placed in a plastic pot tray inside the Faraday cage to electrically isolate them from the cage. A selected pin-head cotton square (<3 mm) from each plant was laid down along a Plexiglas stage (15 by 7 cm) after the bracts were carefully cut off to ensure direct access to the square. The cotton square was held in place using strips of Parafilm (Pechiney Plastic Packaging, Menasha, WI). The Plexiglas stage was held horizontally by an alligator clip connected to a “helping hand” holder (van Sickle Electronics, St. Louis, MO). Recordings were acquired with WinDaq Pro + acquisition software (DATAQ Instruments, Akron, OH) at a sample rate of 100 Hz per insect, and the signal was digitized using a WinDaq DI-720 analog-to-digital (A-D) board. The postrectification signal was analyzed for all recordings. However, the post- and prerectification signals were simultaneously recorded and checked to be sure that the offset function of the monitor was used properly, to avoid rectifier fold-over of the output signal and retain the native polarity of the waveform. The exact monitor gain for all the recordings was 6000× and WinDaq gain ranged from 4× to 64× (specified on each figure caption). Experimental Design Four insects were simultaneously EPG-recorded on pin-head cotton squares, one insect per plant, in a randomized complete block experiment with Ri levels 10 6 , 10 7 , 10 8 and 10 9 Ω used each day, with day as a block. Twenty insects per Ri level and type of applied signal, for a total of 160 insects (80 insects per experiment) were recorded. All channels were set at 50 mV applied signal voltage. The first experiment used AC applied signal for all channels; the second used DC (positive polarity) applied signal. Undisturbed, continuous waveforms were recorded for a 24-h access period. Representative waveform excerpts from all recordings for Ri level within experiment (applied signal type) were collected and assembled using Microsoft Excel (Microsoft Corporation, Redmond, WA). Waveform Naming Convention Waveforms were named according to terminological conventions previously established ( Backus 2000 ), where “probing” (synonymous with stylet penetration) includes all behaviors from start of stylet insertion into plant tissue until stylet withdrawal. “Nonprobing” represents all other behaviors that do not involve stylet penetration. Probing waveforms were named following a hierarchical scheme ( Almeida and Backus 2004 , Backus et al. 2005a ) modified for cell rupture feeders. Waveform phases were not used because they are most applicable to salivary sheath feeders whose stylets progress through various levels of plant tissue on the way to the vascular bundle. Waveform families describe coarse to medium-level details observed (WinDaq compressions of 11 to 100), showing consistent pattern and amplitude, labeled with capital letters. Waveform types are details and fine-structure of waveform families visible when resolution is increased (WinDaq compression of 2 to 5) and designated by the family’s capital letter(s) followed by a number, e.g., T1, T2. Quantification of L. lineolaris Behaviors Waveform categories were quantified based on stereotypical patterns. Frequencies (mean ± standard error [Hertz]) and relative amplitudes were calculated following the methodology described by van Helden and Tjallingii (2000) . One continuous, uninterrupted occurrence of a waveform type was considered a waveform event; each event’s duration was measured with WinDaq/Pro+ Waveform Browser software ( Serrano et al. 2000 ). The sum of all events of a certain waveform family or type was averaged per insect to give waveform duration per insect (WDI). Descriptive statistics of WDI and (mean) number of waveform events per insect (NWEI) were performed using the Backus 2.0 program ( Backus et al. 2007 , Ebert et al. 2015 ) for Statistical Analysis Software (SAS, Cary, NC). Percentage of time spend in probing and nonprobing behaviors, as well as percentage of probes with all each type/family of waveform events is presented. Results were analyzed within each experiment (type of voltage) but not statistically compared between them. Results General Overview of Lygus spp. Waveforms Nonprobing and Probing Behaviors The waveform coarse structure recorded for L. lineolaris and L. hesperus included both nonprobing and probing waveforms. Nonprobing included three waveform families visually correlated with antennal tapping on the plant surface (A), walking (W), and standing still (S). Probing comprised another three waveform families: cell rupturing (CR), transition behaviors (T), and ingestion (I). Characteristics of the nonprobing and probing waveforms are summarized in Table 1 . Table 1. Summary of nonprobing and probing behaviors performed by L. lineolaris and L. hesperus on pin-head cotton squares Behavior . Family . Type . Repetition rate . Relative amplitude . Best seen at Ri levels . Suggested biological meaning . Origin . DC . AC . DC . AC . Nonprobing Standing still S Flat Flat Flat Flat 10 6 –10 9 Ω No movement or activity Antennation A Single spike Single spike High amplitude High amplitude 10 8 , 10 9 Ω Brief, repeated touches of antennal tips emf Walking W Irregular Irregular Low amplitude Low amplitude 10 8 , 10 7 Ω Walking R and emf Probing Cell rupturing CR Irregular Irregular 100% 100% 10 6 –10 9 Ω Stylet maceration and slow stylet moving R and emf Transition behaviors T 10 9 Ω Some R but mostly emf T1 4.6 ± 0.16 Hz 4 ± 0.21 Hz 8% 14% 10 7 –10 9 Ω Salivation and/or cibarial quivering R and emf T2 12.2 ± 0.53 Hz 15.71 ± 0.71 Hz 5% 2% 10 9 Ω Tasting precibarial valve fluttering emf T3 0.3% 1% 10 9 Ω Unknown emf Ingestion I 4 Hz 10 6 –10 9 Ω Sustained ingestion/cibarial pumping Some R but mostly emf Behavior . Family . Type . Repetition rate . Relative amplitude . Best seen at Ri levels . Suggested biological meaning . Origin . DC . AC . DC . AC . Nonprobing Standing still S Flat Flat Flat Flat 10 6 –10 9 Ω No movement or activity Antennation A Single spike Single spike High amplitude High amplitude 10 8 , 10 9 Ω Brief, repeated touches of antennal tips emf Walking W Irregular Irregular Low amplitude Low amplitude 10 8 , 10 7 Ω Walking R and emf Probing Cell rupturing CR Irregular Irregular 100% 100% 10 6 –10 9 Ω Stylet maceration and slow stylet moving R and emf Transition behaviors T 10 9 Ω Some R but mostly emf T1 4.6 ± 0.16 Hz 4 ± 0.21 Hz 8% 14% 10 7 –10 9 Ω Salivation and/or cibarial quivering R and emf T2 12.2 ± 0.53 Hz 15.71 ± 0.71 Hz 5% 2% 10 9 Ω Tasting precibarial valve fluttering emf T3 0.3% 1% 10 9 Ω Unknown emf Ingestion I 4 Hz 10 6 –10 9 Ω Sustained ingestion/cibarial pumping Some R but mostly emf Open in new tab Table 1. Summary of nonprobing and probing behaviors performed by L. lineolaris and L. hesperus on pin-head cotton squares Behavior . Family . Type . Repetition rate . Relative amplitude . Best seen at Ri levels . Suggested biological meaning . Origin . DC . AC . DC . AC . Nonprobing Standing still S Flat Flat Flat Flat 10 6 –10 9 Ω No movement or activity Antennation A Single spike Single spike High amplitude High amplitude 10 8 , 10 9 Ω Brief, repeated touches of antennal tips emf Walking W Irregular Irregular Low amplitude Low amplitude 10 8 , 10 7 Ω Walking R and emf Probing Cell rupturing CR Irregular Irregular 100% 100% 10 6 –10 9 Ω Stylet maceration and slow stylet moving R and emf Transition behaviors T 10 9 Ω Some R but mostly emf T1 4.6 ± 0.16 Hz 4 ± 0.21 Hz 8% 14% 10 7 –10 9 Ω Salivation and/or cibarial quivering R and emf T2 12.2 ± 0.53 Hz 15.71 ± 0.71 Hz 5% 2% 10 9 Ω Tasting precibarial valve fluttering emf T3 0.3% 1% 10 9 Ω Unknown emf Ingestion I 4 Hz 10 6 –10 9 Ω Sustained ingestion/cibarial pumping Some R but mostly emf Behavior . Family . Type . Repetition rate . Relative amplitude . Best seen at Ri levels . Suggested biological meaning . Origin . DC . AC . DC . AC . Nonprobing Standing still S Flat Flat Flat Flat 10 6 –10 9 Ω No movement or activity Antennation A Single spike Single spike High amplitude High amplitude 10 8 , 10 9 Ω Brief, repeated touches of antennal tips emf Walking W Irregular Irregular Low amplitude Low amplitude 10 8 , 10 7 Ω Walking R and emf Probing Cell rupturing CR Irregular Irregular 100% 100% 10 6 –10 9 Ω Stylet maceration and slow stylet moving R and emf Transition behaviors T 10 9 Ω Some R but mostly emf T1 4.6 ± 0.16 Hz 4 ± 0.21 Hz 8% 14% 10 7 –10 9 Ω Salivation and/or cibarial quivering R and emf T2 12.2 ± 0.53 Hz 15.71 ± 0.71 Hz 5% 2% 10 9 Ω Tasting precibarial valve fluttering emf T3 0.3% 1% 10 9 Ω Unknown emf Ingestion I 4 Hz 10 6 –10 9 Ω Sustained ingestion/cibarial pumping Some R but mostly emf Open in new tab Voltage at Baseline Voltage level at the baseline was negative at all input impedances, with AC applied signal being further from 0 V at higher Ri levels (−0.5 and −0.1 V at 10 9 and 10 8 Ω, respectively) and closer to 0 V at lower Ri levels (−0.01, −0.001 at 10 7 and 10 6 , respectively). With DC applied signal, baseline voltage levels remain negative at all input impedances with exception of 10 6 Ω when it became positive (−1, −0.1, and −0.009 V at 10 9 , 10 8 , and 10 7 Ω, respectively, and 0.002 V at 10 6 Ω) Waveform Polarities Appearance as well as polarity of waveforms changed with Ri level during recordings with DC applied voltage for both species. L. lineolaris probing waveforms at Ri 10 9 Ω were monophasic and positively positioned (i.e., with all waveforms above the baseline; hereafter termed monophasic positive) with upward-pointing (hereafter, positively oriented) peaks ( Fig. 1a ). At 10 8 Ω waveforms were biphasic (i.e., both above and below the baseline), but the bulk of the peaks were above baseline and positively oriented ( Fig. 1b ). At both 10 7 and 10 6 Ω waveforms were also biphasic. However, at 10 7 Ω most of the peaks were both positively and negatively (pointing downwards) oriented ( Fig. 1c ), while at 10 6 Ω the peaks of the probing waveforms were all oriented negatively. At all Ri levels, nonprobing waveforms were biphasic with peaks both positively and negatively oriented ( Fig. 1d ). Fig. 1. Open in new tabDownload slide Overview of EPG waveforms produced by L. lineolaris with DC applied signal on pin-head cotton squares (cv. Coker) using 50 mV at Ri 10 9 ( a ), 10 8 ( b ), 10 7 ( c ), and 10 6 ( d ) Ω. Monitor gain was set at 6,000× and Windaq gain 16×. Coarse structure of waveforms observed with Windaq compression 200 (50 s/vertical div). Boxed waveforms are enlarged in inset boxes. Arrowheads indicate beginning or end of a probe. Fig. 1. Open in new tabDownload slide Overview of EPG waveforms produced by L. lineolaris with DC applied signal on pin-head cotton squares (cv. Coker) using 50 mV at Ri 10 9 ( a ), 10 8 ( b ), 10 7 ( c ), and 10 6 ( d ) Ω. Monitor gain was set at 6,000× and Windaq gain 16×. Coarse structure of waveforms observed with Windaq compression 200 (50 s/vertical div). Boxed waveforms are enlarged in inset boxes. Arrowheads indicate beginning or end of a probe. With AC applied voltage, L. lineolaris waveforms were biphasic at all Ri levels ( Fig. 2 ). Probing always started with a downward spike below the baseline level but it became biphasic and positively oriented above the baseline by the end of the probe, especially at Ri 10 9 and 10 8 Ω ( Fig. 2a, b ). At lower Ri levels, 10 7 and 10 6 Ω ( Fig. 2c, d ), the bulk of the peaks remained negatively oriented below the baseline levels with sporadic low-amplitude, positively oriented peaks. Nonprobing waveforms were similar to those observed with DC applied signal, with biphasic peaks positively and negatively oriented above the baseline level. Fig. 2. Open in new tabDownload slide Overview of EPG waveforms produced by L. lineolaris with AC applied signal on pin-head cotton squares (cv. Coker) using 50 mV at Ri 10 9 ( a ), 10 8 ( b ), 10 7 ( c ), and 10 6 ( d ) Ω. Monitor gain was set at 6,000× and Windaq gain 16×. Coarse structure of waveforms observed with Windaq compression 200 (50 s/vertical div). Boxed waveforms are enlarged in inset boxes. Arrowheads indicate beginning or end of a probe. Fig. 2. Open in new tabDownload slide Overview of EPG waveforms produced by L. lineolaris with AC applied signal on pin-head cotton squares (cv. Coker) using 50 mV at Ri 10 9 ( a ), 10 8 ( b ), 10 7 ( c ), and 10 6 ( d ) Ω. Monitor gain was set at 6,000× and Windaq gain 16×. Coarse structure of waveforms observed with Windaq compression 200 (50 s/vertical div). Boxed waveforms are enlarged in inset boxes. Arrowheads indicate beginning or end of a probe. L. hesperus waveforms were biphasic at both 10 9 and 10 7 Ω with DC applied signal ( Fig. 3a, b ). At 10 9 Ω, most of the peaks from nonprobing and probing behaviors were positively oriented above the baseline ( Fig. 3a ). As with L. lineolaris , at the beginning of a probe at 10 7 Ω ( Fig. 3b ), the peaks of the waveforms were oriented negatively below the baseline level, but became positively oriented by the end of the probe. Nonprobing waveforms had most of their peaks positively oriented. Fig. 3. Open in new tabDownload slide Overview of EPG waveforms produced by L. hesperus with DC ( a , b ) and AC ( c , d ) applied signal on pin-head cotton squares (cv. Coker) using 50 mV at Ri 10 9 (a, c), and 10 7 (b, d) Ω. Monitor gain was set at 6,000× and Windaq gain 16×. Coarse structure of waveforms observed with Windaq compression 200 (50 s/vertical div). Boxed waveforms are enlarged in inset boxes. Arrowheads indicate beginning or end of a probe. Fig. 3. Open in new tabDownload slide Overview of EPG waveforms produced by L. hesperus with DC ( a , b ) and AC ( c , d ) applied signal on pin-head cotton squares (cv. Coker) using 50 mV at Ri 10 9 (a, c), and 10 7 (b, d) Ω. Monitor gain was set at 6,000× and Windaq gain 16×. Coarse structure of waveforms observed with Windaq compression 200 (50 s/vertical div). Boxed waveforms are enlarged in inset boxes. Arrowheads indicate beginning or end of a probe. For L. hesperus with AC applied voltage ( Fig. 3c, d ), waveforms at Ri 10 9 Ω were also biphasic with the bulk of peaks positively oriented above the baseline line ( Fig. 3c ). At Ri 10 7 Ω, nonprobing was biphasic with peaks oriented above and below the baseline. Probing started with negatively oriented peaks below the baseline but became biphasic with positive orientation by the time it reached ingestion ( Fig. 3d ). In summary, with the exception of L. lineolaris recordings using DC applied signal at Ri 10 9 (and sometimes also at 10 8 ) Ω, most probing waveforms from both species were biphasic with early-probe waveforms negatively oriented and positioned while later-probe (ingestion) waveforms were positively oriented and positioned. Biphasic polarity was accentuated by high Ri levels and reduced at low Ri levels, supporting that biphasic polarity is an emf component. Nonprobing Waveform Families During nonprobing, three waveform families were identified and characterized for both insect species; they were also visually correlated with their biological meanings. Family S Standing still while not probing (S) ( Fig. 4 ) was observed at all Ri levels and was recorded at or very close to the baseline level at 0 mV. S waveform did not show any visible change at different Ri levels or applied signal. Fig. 4. Open in new tabDownload slide Waveform A, visually correlated with dabbing of the antennae on the surface of the cotton square, the arrow shows a bout of several antennation spikes in rapid succession. Waveform W and Waveform S, visually correlated with the insect walking and standing still (respectively) on the cotton surface. Monitor gain was set at 6,000× and Windaq gain 32×. Medium structure of waveforms observed with Ri 10 9 Ω using 50 mV DC ( a ) and AC ( b ) applied signal. Windaq compression 10 (2 s/vertical div). Fig. 4. Open in new tabDownload slide Waveform A, visually correlated with dabbing of the antennae on the surface of the cotton square, the arrow shows a bout of several antennation spikes in rapid succession. Waveform W and Waveform S, visually correlated with the insect walking and standing still (respectively) on the cotton surface. Monitor gain was set at 6,000× and Windaq gain 32×. Medium structure of waveforms observed with Ri 10 9 Ω using 50 mV DC ( a ) and AC ( b ) applied signal. Windaq compression 10 (2 s/vertical div). Family A Antennation (A) ( Fig. 4 ) occurred when the insect performed single or repetitive light touches or taps of the antennal tips on the square or plant surface. A single antennation tap was visually correlated with a single high-amplitude spike that was always monophasic positive and above the baseline level ( Fig. 4 ). Rapid, repetitive antennal taps were observed as a bout of several single high-amplitude spikes ( Fig. 4 ). Waveform A was observed using both AC and DC applied signal. An emf origin of waveform A is supported by the lack of detection at lower Ri 10 6 and 10 7 Ω, using either applied signal type, suggesting that A does not have any R component. Perhaps a biopotential is generated when the antennal tip comes in contact with the plant surface. Family W Walking (W) was represented by an irregular pattern of frequent, low-amplitude biphasic peaks close to the baseline level ( Fig. 4 ). Like A, waveform W was observed with both types of applied signal voltage; however, amplitude was higher with AC applied signal ( Fig. 4a, b ) supporting some R component. W waveforms appeared similar across Ri levels but were especially visible at high Ri of 10 9 and 10 8 Ω, suggesting a strong emf component. Thus both R and emf components were present. Probing Waveform Families and Types There were also three main waveform families—CR, T, and I recorded during probing for both insect species. They will be described in the order in which they occur during a typical L. lineolaris or L. hesperus probe. Family CR CR always started with a high-amplitude peak (the highest in the probe) whose position and orientation was affected by Ri level and applied signal type. For example, with DC applied voltage, the initial peak was always monophasic positive at high Ri levels of 10 9 ( Fig. 5a ) and 10 8 Ω ( Fig. 5b ), and monophasic negative, negatively oriented, and originating below the baseline level when at low Ri levels 10 7 ( Fig. 5c ) and 10 6 Ω ( Fig. 5d ). In contrast, with AC applied signal, the initial CR peak of L. lineolaris always was monophasic negative at all Ri levels ( Fig. 6a, b ). For L. hesperus , with AC applied signal the initial CR peak was monophasic positive at Ri 10 9 Ω ( Fig. 6c ) but negative at 10 7 Ω ( Fig. 6d ). Fig. 5. Open in new tabDownload slide Medium structure of waveform CR for L. lineolaris recorded at Ri 10 9 ( a ), 10 8 ( b ), 10 7 ( c ), and 10 6 Ω ( d ) using DC applied signal and 50 mV. Monitor gain was set at 6,000× and Windaq gain 32×. Windaq compression 50 (large boxes) (10 s/vertical div) and 4 (small inset boxes) (0.8 s/vertical div). Arrows shows brief stylet withdrawal back to baseline followed by immediate stylet penetration. Fig. 5. Open in new tabDownload slide Medium structure of waveform CR for L. lineolaris recorded at Ri 10 9 ( a ), 10 8 ( b ), 10 7 ( c ), and 10 6 Ω ( d ) using DC applied signal and 50 mV. Monitor gain was set at 6,000× and Windaq gain 32×. Windaq compression 50 (large boxes) (10 s/vertical div) and 4 (small inset boxes) (0.8 s/vertical div). Arrows shows brief stylet withdrawal back to baseline followed by immediate stylet penetration. Fig. 6. Open in new tabDownload slide Medium structure of waveform CR for L. lineolaris ( a, b ) and L. hesperus ( c , d ) recorded at Ri 10 9 (a, c) and10 7 Ω (b, d) using AC applied signal and 50 mV. Monitor gain was set at 6,000× and Windaq gain 32×. Windaq compression 50 (large boxes) (10 s/vertical div) and 4 (small inset boxes) (0.8 s/vertical div). Fig. 6. Open in new tabDownload slide Medium structure of waveform CR for L. lineolaris ( a, b ) and L. hesperus ( c , d ) recorded at Ri 10 9 (a, c) and10 7 Ω (b, d) using AC applied signal and 50 mV. Monitor gain was set at 6,000× and Windaq gain 32×. Windaq compression 50 (large boxes) (10 s/vertical div) and 4 (small inset boxes) (0.8 s/vertical div). Following the initial peak, CR became a highly irregular waveform (i.e., without a clear pattern) that varied greatly in appearance not only across Ri levels and applied signals, but also among insects and even within the same individual insect. Under all circumstances, CR had long, meandering, and almost flat (i.e., very low-frequency) sections alternating in no uniform way with variable-frequency (sometimes even, high-frequency) sections of highly variable amplitude. There were no discernible patterns by Ri level, voltage type, or species ( Fig. 5 , inset boxes). The only exception was that CR peaks became higher and more frequent as Ri level decreased; as Ri level increased, the peaks shortened and became indiscernible from other parts of CR. Thus, the CR peaks had a strong R component. The CR waveform occurred in every probe regardless of duration. Family T In certain probes (but not in every probe), CR abruptly ended and the transition (T) waveform began. T always occurred after CR and always before (never after) an I waveform event (see next section). Unlike CR, T was a highly stereotypical and patterned waveform, occurring in repetitive episodes best seen in recordings of L. hesperus at Ri 10 9 Ω ( Fig. 7a, b ). Because Fig. 7a shows the clearest fine-structure of T, the three subdivisions of T (types T1, T2 and T3) will be described first for L. hesperus at Ri 10 9 Ω. The fine structure of T at other settings will be described by comparison with Fig. 7a . Fig. 7. Open in new tabDownload slide Fine structure of waveform type T recorded at Ri 10 9 Ω for L. hesperus using DC ( a ) and AC ( b ) applied signal and 50 mV, monitor gain was set at 6,000× and Windaq gain 64×; and for L. lineolaris using DC ( c ) and AC ( d ) applied signal and 50 mV, monitor gain was set at 6,000× and Windaq gain 64×. Windaq compression 4 (0.8 s/vertical div). Fig. 7. Open in new tabDownload slide Fine structure of waveform type T recorded at Ri 10 9 Ω for L. hesperus using DC ( a ) and AC ( b ) applied signal and 50 mV, monitor gain was set at 6,000× and Windaq gain 64×; and for L. lineolaris using DC ( c ) and AC ( d ) applied signal and 50 mV, monitor gain was set at 6,000× and Windaq gain 64×. Windaq compression 4 (0.8 s/vertical div). With DC voltage and 10 9 Ω ( Fig. 7a ), T1 was a medium amplitude (8%), highly regular waveform with a repetition rate of 4.6 ± 0.16 Hz and stereotypical duration of 3.46 ± 0.24 s. T1 abruptly changed into T2, which had a higher frequency than T1 (12.2 ± 0.53 Hz), much lower amplitude (5%), and shorter duration (1.56 ± 0.16 s). T2 abruptly ended at the onset of T3, which was a low-amplitude (0.3%), irregular, nearly flat waveform of very short duration (1.14 ± 0.04 s). Together, T1, T2, and T3 form an episode of T; a highly variable number of episodes can be performed in a probe, but never less than two. With 10 9 Ω but AC voltage ( Fig. 7b ), T fine structure changed. Amplitude of T1 increased (14%) but repetition rate remained similar (4 ± 0.21 Hz) with duration of 16.06 ± 2.86 s. T2 amplitude decreased (2%) with a repetition rate of 15.71 ± 0.71 Hz and duration of 6.27 ± 1.47 s. T3 remained somewhat flat with short amplitude (1%) and short duration (1.46 ± 0.29 s). For L. lineolaris , at Ri levels 10 9 Ω with DC voltage ( Fig. 7c ), the T waveform was less complex than for L. hesperus ( Fig. 7a ). T2 was completely missing and was replaced with a slightly longer duration of T1 (5.03 ± 0.82 s) of higher amplitude (50%), which nonetheless retained its signature frequency of 4–5 Hz. T3 had a shorter duration (0.50 ± 0.06 s) and amplitude of 8%. With AC voltage ( Fig. 7d ), all three types were visible. Amplitude of T1 increased to 18% with duration of 3.16 ± 0.82 s and repetition rate of 6.25 ± 0.28 Hz. T2 had a shorter amplitude (7%) with even shorter duration 0.88 ± 0.21 s and repetition rate of 13.12 ± 0.47 Hz. T3 again remained flat with short amplitude (0.2%) and short duration 0.23 ± 0.01 s. The fine structure of a T episode changed with species, Ri level, and applied signal voltage; however, within each Ri/voltage type, T episode appearance was highly stereotypical. When either Ri level or applied signal type was changed, fine structure of an episode of waveform T changed drastically for both Lygus spp. At Ri 10 8 Ω, T3 disappeared, similar to L. lineolaris at 10 9 Ω; at Ri 10 7 Ω with DC voltage, the T waveform was highly distorted with much of the regular pattern of T1 lost; T was completely lost at 10 6 Ω (data not shown). The fact that T was best seen at high Ri levels supports that the transition waveform is highly emf-dominated, as well as species-specific. In some probes, a short event of CR followed T before the recording proceeded to I; more often, T (when it occurred) immediately preceded I. Family I Waveform I is a high-frequency, very regular waveform composed of peaks and waves (that are, in turn, composed of multiple pointed or flat plateaus; Fig. 8 , peaks and waves defined in Fig. 8a ). The fairly stereotypical frequency was 4 Hz for L. lineolaris and L. hesperus . (NOTE: While peaks and waves generally were not considered waveform types per se , but rather fine structures of the commonly recorded I waveform, we have considered them types for sake of display in Table 1 ). Although frequency of I was moderately stereotypical, amplitude and peak orientation varied greatly with species, Ri level, and applied signal type. Fig. 8. Open in new tabDownload slide Fine structure of L. lineolaris waveform I recorded with DC (left) and AC (right) applied signal at Ri 10 9 ( a, e ), 10 8 ( b, f ), 10 7 ( c, g ), and 10 6 ( d, h ) Ω. Windaq compression 4 (0.8 s/vertical div). Each box represents a different insect engaged in sustained ingestion on its own cotton square. Windaq gain 64× (a, b, e, f) and 128× (c, d, g, h). Monitor gain set at 6,000×. Fig. 8. Open in new tabDownload slide Fine structure of L. lineolaris waveform I recorded with DC (left) and AC (right) applied signal at Ri 10 9 ( a, e ), 10 8 ( b, f ), 10 7 ( c, g ), and 10 6 ( d, h ) Ω. Windaq compression 4 (0.8 s/vertical div). Each box represents a different insect engaged in sustained ingestion on its own cotton square. Windaq gain 64× (a, b, e, f) and 128× (c, d, g, h). Monitor gain set at 6,000×. With DC applied signal, waveform I showed little variation for both L. lineolaris ( Fig. 8 ) and L. hesperus ( Fig. 9 ). For the L. lineolaris I, the main difference in appearance was the decrease in waveform amplitude as Ri decreased ( Fig. 8a, b, c, d ,). The tallest I waves were at 10 9 Ω and the shortest were at 10 6 Ω. Peaks were always negatively oriented, and also declined in amplitude slightly with decreasing Ri level. Fig. 9. Open in new tabDownload slide L. hesperus waveform I recorded with DC (left) and AC (right) applied signal at Ri 10 9 ( a , c ) and 10 7 ( b , d ) Ω. Fig. 9. Open in new tabDownload slide L. hesperus waveform I recorded with DC (left) and AC (right) applied signal at Ri 10 9 ( a , c ) and 10 7 ( b , d ) Ω. With AC applied signal, the fine structure of waveform I for L. lineolaris ( Fig. 8e, f, g, h ) showed greater differences among Ri levels. At Ri 10 9 Ω, the wave was regular with frequent, positive plateaus; peaks were barely taller than wave plateaus. At Ri 10 8 Ω, the main body of the wave was steady in voltage level and showed more regularity than at Ri 10 9 Ω, but the frequency of the positive peaks decreased while peak amplitude increased. At Ri 10 7 Ω, both amplitude and frequency of peaks decreased, and the peaks completely disappeared at Ri 10 6 Ω, leaving only the very short-amplitude wave. The finding that wave amplitude decreases with Ri level but does not completely disappear supports that the wave had both R and emf components. In contrast, the peaks were completely emf-dominated because they disappeared altogether at the lowest Ri level. For L. hesperus , the type of applied signal, at all Ri levels but especially at Ri 10 9 Ω, greatly affected the appearance of the I waveform. With the DC applied signal, the constant frequency and amplitude of the wave indicated strong emf component ( Fig. 9a, b ). This regularity was almost completely lost with AC applied signal, revealing the underlying R component waveform because AC tends to accentuate R. The arching form of the 10 9 Ω AC waveform suggests that the arches seen in the DC recording are the R component, with the peak-and-wave emf component superimposed on top of the R component ( Fig. 9a, c ). Thus, for reasons unknown, L. hesperus I waveform has a stronger R component than does the I waveform of L. lineolaris . Quantification of Behaviors Waveform duration per insect (WDI) and number of waveform events per insect (NWEI) were calculated ( Table 2 ). With both AC and DC applied signal, the event with the longest WDI was standing still (S). With AC applied signal, ingestion (I) had the second longest duration followed by cell rupturing (CR). With DC signal, CR was slightly longer than I. Walking (W) and antennation (A) had the shortest waveform duration per insect. Overall, percentage of time spent on non-probing behaviors was longer than on probing behaviors ( Table 2 ). NWEI was also larger for two out of three nonprobing events (W and S), and also larger than probing behaviors (CR and I). Moreover, only about 12 and 15% of the time (AC and DC voltage, respectively), did the insects performed probes that included all three of the probing waveforms ( Table 2 ). Table 2. Calculated waveform duration per insect (WDI) (mean ± SEM), percentage of total recording time spent in nonprobing and probing behaviors, number of waveform events per insect (NWEI) (mean ± SEM), and percentage of probes with all three waveform types included Waveform . WDI (s) . Time spent in behavior (%) . NWEI . Probes with all probing events (%) . DC . S 12603 ± 579.18 79.5 34.38 ± 2.29 W 1356.44 ± 102.53 40.98 ± 2.62 A 40.97 ± 7.31 15.61 ± 1.47 CR 1967.9 ± 519.74 20.4 9.53 ± 1.81 15.8 I 1639.57 ± 297.47 1.8 ± 0.21 AC S 13064 ± 837.79 69.9 39.29 ± 5.04 W 2238.22 ± 386.69 41.62 ± 5.25 A 3.71 ± 2.49 2.75 ± 0.71 CR 2265.19 ± 592.72 30.1 14.9 ± 2.49 12.7 I 4320.29 ± 830.89 2.18 ± 0.22 Waveform . WDI (s) . Time spent in behavior (%) . NWEI . Probes with all probing events (%) . DC . S 12603 ± 579.18 79.5 34.38 ± 2.29 W 1356.44 ± 102.53 40.98 ± 2.62 A 40.97 ± 7.31 15.61 ± 1.47 CR 1967.9 ± 519.74 20.4 9.53 ± 1.81 15.8 I 1639.57 ± 297.47 1.8 ± 0.21 AC S 13064 ± 837.79 69.9 39.29 ± 5.04 W 2238.22 ± 386.69 41.62 ± 5.25 A 3.71 ± 2.49 2.75 ± 0.71 CR 2265.19 ± 592.72 30.1 14.9 ± 2.49 12.7 I 4320.29 ± 830.89 2.18 ± 0.22 Data from recordings with DC and AC voltage for L. lineolaris across all input impedances and 50 mV applied signal. Open in new tab Table 2. Calculated waveform duration per insect (WDI) (mean ± SEM), percentage of total recording time spent in nonprobing and probing behaviors, number of waveform events per insect (NWEI) (mean ± SEM), and percentage of probes with all three waveform types included Waveform . WDI (s) . Time spent in behavior (%) . NWEI . Probes with all probing events (%) . DC . S 12603 ± 579.18 79.5 34.38 ± 2.29 W 1356.44 ± 102.53 40.98 ± 2.62 A 40.97 ± 7.31 15.61 ± 1.47 CR 1967.9 ± 519.74 20.4 9.53 ± 1.81 15.8 I 1639.57 ± 297.47 1.8 ± 0.21 AC S 13064 ± 837.79 69.9 39.29 ± 5.04 W 2238.22 ± 386.69 41.62 ± 5.25 A 3.71 ± 2.49 2.75 ± 0.71 CR 2265.19 ± 592.72 30.1 14.9 ± 2.49 12.7 I 4320.29 ± 830.89 2.18 ± 0.22 Waveform . WDI (s) . Time spent in behavior (%) . NWEI . Probes with all probing events (%) . DC . S 12603 ± 579.18 79.5 34.38 ± 2.29 W 1356.44 ± 102.53 40.98 ± 2.62 A 40.97 ± 7.31 15.61 ± 1.47 CR 1967.9 ± 519.74 20.4 9.53 ± 1.81 15.8 I 1639.57 ± 297.47 1.8 ± 0.21 AC S 13064 ± 837.79 69.9 39.29 ± 5.04 W 2238.22 ± 386.69 41.62 ± 5.25 A 3.71 ± 2.49 2.75 ± 0.71 CR 2265.19 ± 592.72 30.1 14.9 ± 2.49 12.7 I 4320.29 ± 830.89 2.18 ± 0.22 Data from recordings with DC and AC voltage for L. lineolaris across all input impedances and 50 mV applied signal. Open in new tab Discussion This study is the first to characterize and describe EPG waveforms of prereproductive adults of two Lygus species. Like all mirids, Lygus spp. feed very differently than sternorrhynchans (e.g., aphids) and auchenorrhynchans (e.g., leafhoppers) because they do not form salivary sheaths as part of their stylet-probing behaviors; therefore, their stylets do not follow a specific pathway revealed by the sheath. Instead, Lygus spp. perform a macerating or lacerating behavior as part of a cell rupture strategy ( Backus et al. 2005b , Backus et al. 2007 ), previously named macerate-and-flush by Miles (1972) . These insects use their stylets and watery saliva to lacerate and macerate cell contents that are eventually flushed out with additional flow of dilute saliva combined with ingestion. The absence of a salivary sheath poses a limitation for the correlation of feeding behaviors observed on an EPG recording with histological sections. In order to compensate for absence of a salivary sheath and difficulty in identifying correlated landmarks in plant tissue (histological sections of probed cotton squares) for the EPG waveforms produced by Lygus feeding, we comprehensively determined the electrical origin of these waveforms by constructing a waveform library using different Ri (input impedance) levels and applied signal types. This is only possible due to the previous EPG studies that have been conducted on multiple hemipteran species that have provided strong theoretical background on the electrical components and origin of EPG waveforms (R and emf components; Tjallingii 1978 , 1985 ; Walker 2000 ; Backus and Bennett 2009 ; Backus et al. 2013 ). With the advent of the new AC–DC electropenetrograph with selectable Ri levels, construction of waveform libraries ( Backus et al. 2013 , Pearson et al. 2014 , Lucini et al. 2016 ) has expanded the depth and versatility of electrical origin studies. We herein present hypotheses for the biological meanings of Lygus spp. waveforms, based both on electrical origins and on comparison with appearances of waveforms from other species whose meanings are known. Known or Hypothesized Biological Meanings of Waveforms Nonprobing Waveforms Walking and standing-still waveforms have been detected via EPG with other insect species ( Youn et al. 2011 ). However, to our knowledge, the present work is the first report of a non-probing waveform (A) visually correlated with tapping of the insect antennae on the plant surface. This addition to the nonprobing waveforms will make EPG a useful actigraph of general Lygus spp. behavior on the plant surface, in addition to measuring stylet probing waveforms. This will be valuable to future studies of Lygus spp. behavior, because these insects spend less than a quarter of their time on a cotton square in stylet probing behaviors. Probing Waveforms: CR The Lygus spp. CR waveform bears almost no resemblance to early-probe waveforms from salivary sheath feeders. On the other hand, when adult CR waveform was recorded using Ri levels of 10 6 and 10 7 Ω and AC applied signal, it was strikingly similar to the B waveform of third-instar L. lineolaris nymphs recorded with an AC monitor ( Cline and Backus 2002 ). In both that older paper and our low-Ri recordings, B/CR had high peaks dominated by R component, implying they represented salivation. Loss of regularity and definition in our Lygus spp. CR waveform at higher Ri levels means there was little emf in CR. We hypothesize the loss of emf is caused by secretion of highly enzymatic watery saliva that rapidly dissolves cellular contents (maceration) ( Shackel et al. 2005 ), thus depriving the electrical insect-plant interface of much of the cellular structure that contributes to waveform fine structure in other insect recordings. We also suspect this seldom-recorded (with EPG) maceration behavior explains the extreme variation in appearance of CR at different Ri levels. We chose to name our early-probe waveform after this cell rupturing behavior, because there is probably no “pathway” per se that Lygus spp. stylets follow to a sought-after cell for ingestion. There are other peculiarities (compared with waveforms of salivary sheath feeders) of our Lygus spp. CR waveforms also. When recorded at Ri of 10 9 Ω, most early-probe/pathway waveforms of other insects are negatively oriented (waveform peaks point downward from an otherwise neutral or positive baseline; termed monophasic negative) regardless of AC or DC applied signal. Negative-going signals are considered indicative of intracellular stylet penetration ( Backus and Bennett 2009 ). At lower Ri levels, especially 10 6 and 10 7 Ω, waveform polarity of typical pathway in sheath feeders inverts and become monophasic positive ( Backus and Bennett 2009 , Backus et al. 2013 ). Yet, surprisingly and for the first time published, Lygus spp. CR waveforms do the reverse; they are monophasic positive at 10 9 Ω and monophasic negative at all lower Ri levels, regardless of applied signal type (AC or DC). We suspect that the above reversal of CR polarity relative to Ri level again may be due to the extreme maceration of cellular structure/contents caused by Lygus spp. salivation. The insect certainly is inserting its mouthparts intracellularly, interior to the plant cells, causing breakage of cell walls and membranes. Without a salivary sheath to protect the plant cells, the electrolyte concentration may change, with breakdown of charge separation between interior and exterior of the cells, thus negatively charging the area surrounding the mouthparts. This negative charge may be so strong that it is even detectable at low Ri levels. However, why the voltage level of recordings at 10 9 Ω would become positive is unknown. Regardless of the cause, the reversal of polarity by Ri level is a cautionary tale for EPG research, i.e., that one cannot always assume intercellular versus extracellular stylet position from signal voltage level alone. Probing Waveforms: T The transition waveform (T) was observed mainly at high input impedances. It is worth noting that this waveform was not seen by Cline and Backus (2000) in their study of third-instar nymphs. This is probably because T waveform was not seen at 10 6 Ω in the present study (the same Ri level as the AC monitor used in the earlier study). Interestingly, a T- like waveform has been observed when immature instars are recorded at higher Ri levels using the AC-DC monitor (F.A.C., unpublished data). When T occurred, it marked the end of a CR event and may or may not have been directly followed by ingestion (I). Sometimes, a short event of CR followed T before onset of I. Nonetheless, T did not follow I, only preceded it. The structure and detail of the T waveform observed at high input impedance (10 9 Ω) suggest a strong emf origin for this waveform. This electrical origin is further supported by the loss in clarity and structure at lower input impedances and complete disappearance at Ri 10 6 Ω. Waveform types T2 and T3 become less visible at 10 7 Ω. In contrast, the R component of T1 is maintained as the impedance at 10 7 Ω; it seems to completely mask T2 and decrease the clarity of T3. The additional clarity of all three T types observed with DC applied signal (compared with AC) recordings at high input impedances provides additional support that emf is the dominant electrical origin of T, with some R also found in T1. This is because DC applied signal emphasizes the detection of emf components while AC applied signal emphasizes the R component. The highly stereotypically repeating pattern of the T waveform resembles an X-wave in appearance. X-waves have previously been reported for other hemipterans that form salivary sheaths ( Wayadande and Nault 1993 , Backus et al. 2009 , Backus et al. 2013 , Lucini and Panizzi 2016 ) and are 100% correlated with initial stylet contact, testing, tasting, overcoming vascular cell defenses (e.g., P proteins in phloem, cavitation in xylem) via salivation ( Walker and Medina-Ortega 2012 ), and final acceptance or rejection of the preferred ingestion tissue. Thus, based on the electrical origin of the T waveform types and their similarity with X-waves previously described for other hemipteran species, especially sharpshooter leafhoppers ( Backus 2016 ) we hypothesized specific biological processes that may be represented by T1, T2, and T3 ( Table 1 ). We justify these comparisons, despite the differences in foregut anatomy of leafhoppers (with acetabular [wineglass-shaped] precibarium and cibarium) versus heteropterans (with tubular precibarium and cibarium) because in both cases there is a flap-like, pressure-sensitive valve located medially in the precibarium (E.A.B, unpublished data) and pumping of the cibarium is performed similarly ( Goodchild 1966 ). Thus, the acts of tasting and swallowing probably are performed very similarly in leafhoppers and heteropterans. At present, the components of the sharpshooter X wave are the most thoroughly defined, biologically, of any hemipteran X wave ( Backus 2016 ), so they provide the best comparison available. The intermediate amplitude and frequency of T1 resembles that of waveform C1 in the X-wave of sharpshooters ( Backus et al. 2009 , Backus 2016 ) in which the rapid rise and fall of voltage is probably caused by a rapid rise and immediate drop of the cibarial diaphragm, taking up fluid insufficient for swallowing but enough for tasting. The rapid drop of the cibarial diaphragm would propel the fluid back into the plant. The degree of uplift of the diaphragm represents changes in the R component of the waveform, explaining why T1 is the portion of the T waveform that is maintained as input impedance decreases. We therefore propose the hypothesis that T1 represents an initial fluid-uptake/egestion behavior accompanied possibly by salivation. T2 (intermediate amplitude, very high frequency) shows similarity to the sharpshooter waveform B1s (another component of the X wave), which is thought to represent fluttering of the precibarial valve and/or quivering of the cibarial diaphragm ( Backus 2016 ). Actions of these valves and pumps are thought to push fluids brought into the precibarium back and forth across the precibarial chemosensilla ( Backus and McLean 1982 , 1983 , 1985 ; Backus 1988 ). The appearance of T2 suggests a rhythmic behavior in which streaming potentials might be created (emf component) and are observed at high input impedances. We therefore hypothesize that T2 represents tasting of internal fluids of the plant that have previously been exposed to the saliva ejected during T1. T3 resembles the B1w waveform of sharpshooters, also a component of the sharpshooter X wave. It was previously correlated with secretion of saliva ( Backus et al. 2009 ), therefore we propose that T3 may represents secretion of without co-occurrence of other behaviors described above. Backus et al. (2009) state that a true X-wave represents stylet penetration of the preferred ingestion cell type for a vascular-specializing species, either phloem sieve elements or xylem tracheary elements. Unlike the hemipteran species for which X-waves have been previously described, L. lineolaris and L. hesperus are not thought to make salivary sheaths that end in a vascular cell, but rather engage in an array of feeding behaviors that cause cell rupturing and degradation of cell content without following a specific pathway ( Miles 1972 ). To our knowledge, this paper is the first report of an X wave-like waveform for a cell rupture-feeding, cimicomorphan heteropteran. It seems likely that the maceration of cellular contents triggered by salivation during the CR waveform would require some time to reach an optimum level of liquefaction prior to ingestion. We propose that the Lygus spp. X wave functions primarily for tasting and testing the plant tissues for this optimum level of liquefaction; if adequate, then ingestion can ensue. If not yet adequate, more CR behavior could be performed prior to ingestion. This would explain why T sometimes does not immediately precede I, as occurs with other X waves. In support of our hypothesis, T never followed an I event within a probe. Because the common ancestor of all hemipterans is thought to have been a cell rupture feeder ( Goodchild 1966 ), it is possible that this rather simple (both in waveform appearance and in tasting and testing function) X wave of cimicomorphans is the evolutionary ancestor to the X wave of salivary sheath feeders. Probing Waveforms: I The Lygus spp. I waveform in our study is fundamentally patterned as a repetitive, peak-and-wave structure, apparently caused by relatively equal representation of both emf and R components. This peak-and-wave structure has been correlated (in other heteropterans) with ingestion (i.e., uptake of fluid into the functional foregut that is then swallowed; Backus et al. 2013 , Lucini and Panizzi 2016 , Lucini et al. 2016 ). The I waveform of adult Lygus spp. is similar to (at least, in voltage level and sequence during a probe) the C1 and C2 waveforms of L. lineolaris nymphs recorded using an AC monitor and correlated with ingestion via body posture, inward flow of carbon particles in artificial diet recordings, plus comparison with then-recorded ingestion waveforms ( Cline and Backus 2002 ). (However, C1 and C2 [like all ingestion waveforms recorded with that earlier AC monitor] did not have clear peak-and-wave fine structure, probably due to artifactual loss of emf component by low input impedance (Ri) level of 10 6 Ω AC, as well as rectifier fold-over [ Backus and Bennett 2009 ]). Therefore, we propose that the renamed I waveform represents ingestion by adult Lygus spp. Despite the unity in appearance of the peak-and-wave, there also was interesting variability in fine-structure details of I across Ri levels and applied signal type, especially in L. hesperus recordings. For L. lineolaris , the amplitude of the wave decreased with decreasing Ri level, for both AC and DC applied signal. Thus, a tiny wave was still visible even at Ri 10 6 Ω, using either AC or DC applied signals. This supports both R and emf as electrical origins. Sometimes, an underlying arch-like structure was superimposed on the overlying wave structure, especially evident when the peaks were negative-going, as with DC applied signal. The cause of this underlying arch is apparent when considering the unusual appearance (compared with that of L. lineolaris ) of the L. hesperus I waveform using AC applied signal. The peak-and-wave structure was completely abolished and replaced by a less regular waveform with intermittent, downward spikes of unknown mechanism. At 10 9 Ω, repeating arches were seen. At 10 7 Ω, irregular, downward peaks dominated. We propose that the underlying arch-like structure in both Lygus spp. I waveform is composed of this highly R-dominated motif. For both species, there was more emf in peaks than R, because peaks were slightly larger at 10 6 Ω for DC applied signal than for AC signal. In addition, peaks from AC recordings became shorter and less frequent as Ri level decreased. Interestingly, peaks in recordings from DC applied signal were negative-going at all Ri levels, while those from AC applied signal were positive-going at higher Ri levels but negative-going at lower Ri levels. (Unlike in past AC monitor recordings like Cline and Backus [2002] , this difference cannot be due rectifier fold-over for the AC applied signal, because we scrupulously used the offset function to remove fold-over; we compared prerectification and postrectification output signals to assure that they were identical and signal polarity was always native.) The highest peaks were at 10 8 Ω with AC signal. Perhaps a clue to this enigma is seen at 10 7 Ω, where peaks were biphasic, with both positive- and negative-going parts. We suggest that there are at least two mechanisms that generate peaks, one that is R-dominant (generating positive-going peaks) and one that is emf-dominant (generating negative-going peaks). In some unknown manner, the two mechanisms are slightly offset temporally, so that some portions cancel each other out at certain Ri levels. The mechanism of the wave, for both Lygus spp., seems similar to those of other species. Wave emf probably represented alternating-direction streaming potentials that are rhythmically produced and destroyed during cibarial pumping and swallowing ( Dugravot et al. 2008 ) of macerated plant tissue. That said, frequency generally was not affected because it is caused by cibarial pumping ( Dugravot et al. 2008 ). Lifting and lowering of the cibarial diaphragm during pumping probably also caused a small, rhythmic R component. For the mechanism of the peak, the R component may be caused by higher uplift of the cibarial diaphragm (leading to enlargement of the cibarial space), every few pumps, as has been shown for sharpshooters ( Dugravot et al. 2008 ). Alternatively, there could be spurts of saliva produced on occasion during ingestion. Quantification of Behaviors With DC applied signal, tarnished bugs spent about 20% of their time in probing behaviors and of this about 17% of their time in ingestion; thus, ingestion represented <3% of the recording time of each insect. However, our recordings with AC signal showed probing time increased to ∼30% of the recording time, but ingestion still remained short. Cline and Backus (2002) also observed that third instar nymphs of L. hesperus spent only about 30% of their time in probing behaviors. Moreover, the number of probes with ingestion ranged only from 1 to 2 for each insect. These observations are consistent with the feeding strategy used by adult Lygus spp. in our study and matched the findings of Cline and Backus (2002) . Several test probes (lone CR events), when saliva is probably injected into the plant tissue, occurred; these were followed by several periods of walking or waiting (presumably for saliva to degrade the cell contents) before ingestion. CR probably encompasses several stylet activities occurring simultaneously in the intracellular space, salivation, and stylet movement through the plant tissue but with little or no ingestion taking place. In conclusion, this study has generated for the first time an EPG waveform library for prereproductive adults of L. linolaris and L. hesperus , laying the groundwork for future EPG quantitative studies. Also, for the first time an X-wave like waveform (T) has been found and reported for a nonsalivary sheath feeder. Finding of the T waveform might be helpful in increasing our understanding of the feeding behavior of Lygus spp. and aid in the improvement of management of Lygus spp. populations through host plant resistance and other control methods. 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USDA is an equal opportunity provider and employer. Published by Oxford University Press on behalf of Entomological Society of America 2016. This work is written by US Government employees and is in the public domain in the United States. Published by Oxford University Press on behalf of Entomological Society of America 2016. This work is written by US Government employees and is in the public domain in the United States.
TI -  Characterization of an EPG Waveform Library for Adult Lygus lineolaris and Lygus hesperus (Hemiptera: Miridae) Feeding on Cotton Squares 
JF - Annals of the Entomological Society of America
DO - 10.1093/aesa/saw039
DA - 2016-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/characterization-of-an-epg-waveform-library-for-adult-lygus-lineolaris-cjL9TOByGJ
SP - 684
EP - 697
VL - 109
IS - 5
DP - DeepDyve
ER -