TY - JOUR
AU - Rojas, Maria, G
AB - Abstract Probing behavior of Lygus lineolaris (Palisot de Beauvois) has previously been characterized with electropenetrography (EPG). Cell rupturing (CR) and ingestion (I) EPG waveforms were identified as the two main stylet-probing behaviors by adult L. lineolaris. However, characterization and identification of EPG waveforms are not complete until specific events of a particular waveform are correlated to insect probing. With the use of EPG, histology, microscopy, and chemical analysis, probing behavior of L. lineolaris on pin-head cotton squares was studied. Occurrences of waveforms CR and I were artificially terminated during the EPG recording. Histological samples of probed cotton squares were prepared and analyzed to correlate specific types and occurrences of feeding damage location and plant responses to insect feeding. Both CR and I occurred in the staminal column of the cotton square. Cell rupturing events elicited the production of dark-red deposits seen in histological staining that were demonstrated via chemical analysis to contain condensed tannins. We hypothesize that wounding and saliva secreted during CR triggered release of tannins, because tannin production was positively correlated with the number of probes with single CR events performed by L. lineolaris. Degraded plant tissue and tannins were removed from the staminal column during occurrence of waveform I. These results conclude the process of defining CR and I as probing waveforms performed by L. lineolaris on pin-head cotton squares. These biological definitions will now allow EPG to be used to quantitatively compare L. lineolaris feeding among different plant treatments, with the goal of improving pest management tactics against this pest. feeding behavior, input impedance, applied signal, stylet penetration, electrical penetration graph The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Heteroptera: Miridae), is a major pest of cotton in the mid-southern United States. When feeding on cotton, L. lineolaris prefers to feed on either meristematic tissue or developing reproductive organs, causing abscission of fruiting body, deformation of young fruit, necrosis, production of embryo-less or shriveled seeds, and reduction of vegetative growth (Strong 1970). Lygus bug feeding is one of the main causes of square abscission (Mauney and Henneberry 1984) by damaging the anther (Leigh et al. 1988), the staminal column (Williams and Tugwell 2000), and by causing various types of localized necrosis and irregular desiccation of pin-head squares (Leigh et al. 1988). Lygus lineolaris damage to cotton squares is caused by a cell rupture feeding strategy in which plant cells are slowly lacerated by intermittent or continuous probing of the stylets while highly enzymatic saliva is injected into the cells (termed maceration; Backus et al. 2005). This strategy results in the formation of a liquefied slurry of plant cell contents. The slurry is then ingested through the food canal in the stylets (Backus et al. 2005). With the use of electropenetrography (EPG), Cervantes et al. (2016) identified and electrically characterized the EPG waveforms that correspond to the behaviors performed during cell rupturing (waveform CR) and ingestion (waveform I), based on the electrical origins of their respective EPG waveforms. Preliminary histological correlation of both CR and I with stylet location and movement within the cotton square supported previous studies (Williams and Tugwell 2000) showing the absence of salivary sheaths by L. lineolaris (Cervantes et al. 2016). Absence of a salivary sheath poses limitations for the correlation of these two EPG waveforms with histological sections, an important step in the mandatory process of defining new waveforms. However, our preliminary studies also suggested certain changes in the plant anatomy as a consequence of Lygus feeding, also consistent with previous damage observations in the staminal column (Williams and Tugwell 2000). In the current study, we correlated the specific events of either the CR or I EPG waveforms performed by L. lineolaris on pin-head cotton squares and artificial diet with specific types and occurrences of feeding damage location and plant responses, as well as stylet movements. We also performed chemical analyses of cotton squares on which were performed waveform-correlated feeding, to correlate condensed tannins secreted as a plant response to salivation and wounding performed during CR. These results complete the process of defining the CR and I waveforms, so that future studies comparing L. lineolaris feeding via EPG can commence. Materials and Methods Insects Lygus lineolaris egg packs were obtained from a rearing facility at the ARS-USDA facility in Stoneville, MS. Egg packs were placed in a rearing cage (33 by 20 by 12 cm) containing shredded paper and a petri dish with a cotton pad soaked in water. Cages were kept in a walk-in incubator under 27 °C, 60–70% relative humidity (RH), and a photoperiod of 16:8 (L:D) h. After nymphs hatched, egg packs were removed from the cages and nymphs were reared on store-bought organic mature bean pods (Phaseolous vulgaris L.). Cages were dated to keep track of insect age. Dried beans were replaced by fresh beans on a need basis. For EPG recordings and correlation experiments, 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 this study, to ensure greatest likelihood of feeding. Cotton Plants Cotton plants were grown from seed in a greenhouse using Sunshine mix #1 soil (Sun Gro Horticulture, Agawam, MA) 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°C 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 Prereproductive adult L. lineolaris were removed from their cages and starved for 2 h before wiring. Insects were anesthetized with CO2 for 30 s, and then immobilized by low suction under a stereomicroscope (Leica MZ125, 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 Technologies Inc., Gainesville, FL) was used to record nonprobing and probing behaviors of L. lineolaris inside a Faraday cage (steel base, aluminum frame, copper mesh, 152 cm in length by 73 cm in width by 111 cm in height). Four cotton plants were placed individually on plastic trays to electrically isolate them from the bottom of 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. Recordings were conducted immediately after the bracts were cut, while the square was still attached to the plant. 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, 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 6,000× and WinDaq gain ranged from 4× to 64× (specified on each figure caption). Plant Tissue Histology Cotton squares were fixed in 6% (vol:vol) paraformaldehyde fixative in HEPES (N-(2-Hydroxyethyl)-piperazine-N’-(2-ethanesulfonic acid)) buffer. Samples were then washed with HEPES, dehydrated through a standard ethanol–isopropanol series, then infiltrated with Paraplast X-tra (St. Louis, MO) at 67°C (Berlyn and Miksche 1976). Paraffin blocks were serial sectioned at 10 μm using a rotary microtome (Microm HM355, Walldorf, Germany). Ribbons were mounted on slides and allowed to dry overnight on a slide warmer. All slides were dewaxed with 100% Citrisolv (Fisher Scientific, NJ), then stained with 0.5% (wt:vol) aqueous safranin and counterstained using 0.02% (wt:vol) ethanolic fast green (Berlyn and Miksche 1976). Slides were examined via bright field compound light microscopy with a Leica DM 5000B (Leica Microsystems, Jena, Germany), and imaged via a microscope-mounted Leica DMC 2900 digital camera coupled to a Dell Precision computer. Adobe Photoshop (Adobe Systems, San Jose, CA) was used to enhance image brightness and contrast. Tannin Extractions Condensed tannin extractions were performed using methods modified from Wallis et al. (2010). In brief, half of a cotton square per sample was placed in a 1.5-ml microcentrifuge tube and submerged in 100 µl of 70% acetone in water (Sigma-Aldrich, St. Louis, MO). These samples were then placed in a sonicating bath for 1 h at room temperature, and then the tissue was vacuum-infiltrated for 1 h at room temperature at −10 KPa. After vacuum infiltration, samples were placed at 4°C overnight. The supernatant was then removed, and washed with 50 µl of petroleum ether followed by 50 µl ethyl acetate (both from Sigma), with the top layer discarded after each wash. Samples were then left at room temperature overnight to evaporate excess organic solvent. Next, remaining sample extract was diluted 1:1 with water and moved to 2-ml microcentrifuge tubes. In total, 1.2 ml of n-butanol/acid reagent (a 95:5 solution of n-butanol and concentrated [12 N] HCl) was added per sample, followed by 20 µl of iron reagent [2% (wt/vol) ferric ammonium sulphate dodecahydrate salt (FeNH4(SO4)2 * 12H2O) diluted in 2 M HCl] (all reagents from Sigma). Additional negative controls were prepared, which used 100% water in lieu of the sample. Resultant solutions were vortexed and then placed on a heat block at 95 °C for 1 h. After the samples cooled back to room temperature, 100 µl of the final solution were placed into wells of a microplate. Sample optical densities (ODs) at 550 nm were then recorded with blanks subtracted by an Epoch (Biotek, Winooski, VT) plate reader using the provided Gen5 (ver 2.0, Biotek) software. Final reported ODs were adjusted by original sample weights. Correlation of Probing Behaviors Originally, we attempted to correlate CR and I with stylet position in the cotton square. However, this proved to be difficult owing to the lack of a salivary sheath and clear marks that would indicate the location of the stylets inside the square. Instead, histological imaging of cotton squares consistently revealed the presence of deposits that stained dark red after a probe consisting of a single CR event (hereafter called a CR event) or several probes each consisting of a single CR event. The dark red-stained deposits appeared to decrease in area or disappeared with ingestion (I). Thus, two experiments were conducted to identify the nature of these dark-red deposits and their relationship with probing behaviors. A third experiment using artificial diet was conducted to correlate the ingestion (I) waveform. Experiment 1 To correlate the occurrence of EPG-probing waveforms to damage location in pin-head cotton squares, EPG recordings were artificially interrupted after the waveform of interest was observed. Probing waveforms were cell rupturing (CR) and ingestion (I; Cervantes et al. 2016). Recordings were interrupted after: 1) a single CR event, 2) several CR events (4–5; typically in separate probes), or 3) a single probe consisting of a single CR event directly followed by a single I event (about 20 min of I, within the same probe; n = 8 per treatment), for a total of 24 insects on squares. After the artificial termination of a probe, the entire plant was transferred to a fume hood where the pin-head cotton square was cut from the plant and immediately placed in 6% (vol:vol) paraformaldehyde fixative. The areas of dark red-stained patches on a representative histological section of each cotton square were measured using Image Pro Plus software (Media Cybernetics, Inc., MD). Statistical comparisons of red-stained areas among feeding treatments were performed by using mixed model analysis of variance (PROC GLIMMIX, SAS Institute 2001) followed by least significant difference test for pairwise comparisons. Experiment 2 To correlate the occurrence of CR events with red-stained areas and tannin concentration, four insects were simultaneously EPG-recorded on pin-head cotton squares for 1 h. In total, 12 recordings were performed. After the recordings ended, squares on which insects performed ingestion (i.e., CR plus I waveforms; n = 2) and did not perform any behavior (n = 3) were discarded. The seven remaining cotton squares, wherein insects performed from one to several CR events without I, were separated from the cotton plant and cut in half. One half of the square was fixed and prepared for histological sectioning. The other half was freeze-dried for measurement of the concentration of tannins in the square, via chemical analysis. Seven additional cotton squares that were not fed to L. lineolaris (although bracts were cut off) were prepared similarly as control tissue. Comparisons of red-stained areas and tannin concentration in CR-squares with control squares was performed by using mixed model analysis of variance (PROC GLIMMIX, SAS Institute 2001) followed by least significant difference test for pairwise comparisons. Total probing duration and number of probes obtained from EPG recordings were correlated with red-stained area and tannin concentration using a regression analysis in Excel (Microsoft Excel 2013). Hereafter, we considered the terms “probe” and “event” as synonymous, because L. lineolaris typically performs only one event of either CR or I in each stylet probe. Experiment 3 Eight prereproductive adult L. lineolaris were EPG-recorded and simultaneously videotaped while feeding on artificial diet (Cline and Backus 2002), containing particles of red Chinese stick ink (Oriental Art Supply, Huntington Beach, CA), for 6 h. Briefly, the artificial diet contained 6% sucrose, 4% agarose, and 2.5% ink particles (wt:vol). Stylets were observed at 50× under a videomicrography set up, making sure that stylets were in focus at all times. We attempted to correlate and make notes of stylet movement, flow of ink particles, ingestion, salivation, or fluid egestion with specific EPG-probing waveforms. Results Experiment 1 Cotton squares exposed to a single or multiple CR events consistently showed dark-red stained areas in the staminal column (Fig. 1A, B; dark red is black in the gray-scale images). When cotton squares were exposed to an I event, dark-red stained areas disappeared and clear amorphous spaces were observed (Fig. 1C). Red area from a single CR event and from multiple CR events was significantly larger than the red area seen on squares exposed to CR + I event in one probe (Fig. 2). No significant difference in red area was observed between cotton squares exposed to a single CR or multiple CR events (F = 60.57, df = 2, 21, P < 0.0001; Fig. 2). Fig. 1. Open in new tabDownload slide Histological correlations of CR (cell rupturing) and I (ingestion) waveforms of L. lineolaris. Each part shows three paraffin cross sections from the same area of probed cotton square (different cotton squares), below the correlated EPG recording. (A) Small amount of red stain (depicted as black in these images) from a single, CR-containing probe. (B) Larger amounts of red stain (black) from multiple, contiguous CR-containing probes. (C) Relative loss of red stain (black) after a long probe with multiple, shallow CR events, followed by a long, deeper I event. I, expanded in insects. Fig. 1. Open in new tabDownload slide Histological correlations of CR (cell rupturing) and I (ingestion) waveforms of L. lineolaris. Each part shows three paraffin cross sections from the same area of probed cotton square (different cotton squares), below the correlated EPG recording. (A) Small amount of red stain (depicted as black in these images) from a single, CR-containing probe. (B) Larger amounts of red stain (black) from multiple, contiguous CR-containing probes. (C) Relative loss of red stain (black) after a long probe with multiple, shallow CR events, followed by a long, deeper I event. I, expanded in insects. Fig. 2. Open in new tabDownload slide Red-stained (black) area (μm2; Mean ± SEM) in the staminal column of pin-head cotton squares exposed to a single CR, multiple CR events, or I by L. lineolaris during an EPG recording. Similar letters above bars indicate no significant difference between treatments. Fig. 2. Open in new tabDownload slide Red-stained (black) area (μm2; Mean ± SEM) in the staminal column of pin-head cotton squares exposed to a single CR, multiple CR events, or I by L. lineolaris during an EPG recording. Similar letters above bars indicate no significant difference between treatments. Experiment 2 Red-stained area was significantly larger in cotton squares exposed to CR events performed by L. lineolaris than on control squares (F = 11.74, df = 1, 12, P = 0.005; Fig. 3A). Tannin concentration was also higher in cotton squares exposed to CR events than in control squares (F = 5.02, df = 1, 12, P = 0.04; Fig. 3B). Tannin concentration was significantly correlated with the red-stained area in squares exposed to CR events (F = 14.49, df = 1, 5, P = 0.01; Fig. 4A) but not so in control squares (F = 0.93, df = 1, 5, P = 0.37; Fig. 4B). Both red-stained area (Fig. 4C) and tannin concentration (Fig. 4D) were significantly correlated with the number of CR-only probes performed by L. lineolaris (F = 8.84, df = 1, 5, P = 0.03; F = 11.93, df = 1, 5, P = 0.01, respectively). Fig. 3. Open in new tabDownload slide Area of red stains (A) [Mean ± SEM] in cotton squares exposed to CR events by L. lineolaris and control squares. Relative condensed-tannin concentration (B) [Mean ± SEM] in cotton squares exposed to CR events by L. lineolaris and control squares. Letters above bars indicate significant difference between treatments. Fig. 3. Open in new tabDownload slide Area of red stains (A) [Mean ± SEM] in cotton squares exposed to CR events by L. lineolaris and control squares. Relative condensed-tannin concentration (B) [Mean ± SEM] in cotton squares exposed to CR events by L. lineolaris and control squares. Letters above bars indicate significant difference between treatments. Fig. 4. Open in new tabDownload slide Relationship between red-stained area and relative condensed-tannin concentration in pin-head cotton squares exposed to CR events by L. lineolaris (A) and in control squares not exposed to L. lineolaris (B). Relationship between number of CR-only containing probes with red-stained area (C) and relative condensed-tannin concentration (D). Fig. 4. Open in new tabDownload slide Relationship between red-stained area and relative condensed-tannin concentration in pin-head cotton squares exposed to CR events by L. lineolaris (A) and in control squares not exposed to L. lineolaris (B). Relationship between number of CR-only containing probes with red-stained area (C) and relative condensed-tannin concentration (D). Experiment 3 Two probing waveforms were observed during diet recordings, cell rupturing (CR) and ingestion (I) (Fig. 5A). Cell rupturing always started with an initial monophasic negative peak followed by irregular and regular peaks of high amplitude (87% of the highest peak voltage). Peaks with regular frequency had a repetition rate of 5 ± 0.42 Hz that decreased to 2.5 ± 0.18 Hz toward the end of the CR waveform (Fig. 5B). Ingestion waveform (when it occurred) always followed CR with high amplitude (71%) and a stereotypical frequency of 4 Hz (Fig. 5C). These waveform characteristics from insects feeding on artificial diet are similar to those seen when insects fed on plants. During the CR waveform on the artificial diet, the stylets perforated the parafilm layer and penetrated into the diet with small deposits of visible, clear saliva extruded from the stylet tips; extension of the stylets into the diet was slow-moving. Slow retraction and subsequent reinsertion of the stylets without separating the labium from the diet surface was observed during this waveform; however, these behaviors did not coincide with specific peaks of spikes owing to the irregularity of the CR waveform. During I waveform, particles of Chinese stick ink moved toward the stylets and, after some time in I, a hollow space formed in the diet. These observations show that the contents of the semiviscous diet were being consumed; thus I represent ingestion. Fig. 5. Open in new tabDownload slide Overview of EPG waveforms produced by L. lineolaris with AC applied signal on artificial diet using 2 mV and Ri 107 Ω. Monitor gain was set at 6000× and Windaq gain at 4×. Coarse structure of waveforms observed with Windaq compression 100 (20 s/vertical div) (A). Boxed waveforms are enlarged in inset boxes, CR (cell rupturing) (B), I (ingestion) (C) Windaq compression 4 (0.8 s/vertical div) and Windaq gain at 4×. Fig. 5. Open in new tabDownload slide Overview of EPG waveforms produced by L. lineolaris with AC applied signal on artificial diet using 2 mV and Ri 107 Ω. Monitor gain was set at 6000× and Windaq gain at 4×. Coarse structure of waveforms observed with Windaq compression 100 (20 s/vertical div) (A). Boxed waveforms are enlarged in inset boxes, CR (cell rupturing) (B), I (ingestion) (C) Windaq compression 4 (0.8 s/vertical div) and Windaq gain at 4×. Discussion Implications of EPG Results Probing behavior of Lygus spp. has previously been studied (Sevacherian 1974, Cline and Backus 2002, Cooper and Spurgeon 2012, Cervantes et al. 2016). However, this is the first study that directly correlates individual EPG waveforms of prereproductive adult Lygus lineolaris to direct feeding damage on pin-head cotton squares, providing new information on the effects of specific EPG-probing waveforms and the plant responses elicited by such behaviors. Cell rupturing (CR) and ingestion (I) form the bulk of EPG-probing waveforms produced by L. lineolaris feeding on pin-head cotton squares (Cervantes et al. 2016). These waveforms comprise the main behaviors of the macerate-and flush-feeding tactic within the cell rupture feeding strategy (Backus et al. 2005) used by L. lineolaris. With this feeding tactic, cells are thought to be slowly lacerated by intermittent or continuous probing of the stylets while highly enzymatic saliva (Strong and Kruitwagen 1968, Laurema et al. 1985, Agblor et al. 1994, Shackel et al. 2005, Frati et al. 2006, Celorio-Mancera et al. 2009) is injected into the cells. This tactic results in the formation of a slurry of plant cell contents. The slurry is then ingested through the food canal in the stylets (Backus et al. 2005). Cell laceration and saliva-induced maceration have also been observed in third-instar nymphs of Lygus hesperus Knight (Cline and Backus 2002), and correlated with EPG waveforms. The authors identified their waveforms A and B as representing initial stylet penetration, during which salivation and stylet movement through the plant tissue occur. The waveform CR performed by L. lineolaris and observed in our study resembles a combination of waveforms A and B of L. hesperus. Cline and Backus (2002) also identified a less frequent waveform, F that occurred when stylets were being retracted and moved in a different direction. Our diet study showed that during CR, spikes of medium amplitude (similar to those of waveform F) were frequent and occurred while stylets moved through the diet. Waveform I also showed similarities in appearance to the C2 ingestion waveform observed in Cline and Backus (2002). However, those authors did not observe movement of ink particles in artificial diet toward or away from the insect stylets during this waveform. In our study, ink particles moved toward the stylets during I, leaving an empty space. It is possible that our adult insects are stronger than Cline and Backus’s third-instar nymphs used in their (2002) study, and that our use of a semiviscous artificial diet did not impose a limitation to adult Lygus on the diet uptake. Thus, the A, B, F, and C2 waveform correlations for L. hesperus from the Cline and Backus (2002) paper also apply to our CR waveform for L. lineolaris, and further reinforce our interpretation that CR represents maceration behaviors consisting of slow stylet movements and secretion of enzymatic watery saliva. We did not choose to subdivide our CR waveform as did Cline and Backus (2002), for ease of measurement for future quantitative studies. Unlike sternorrhynchans and auchenorrhyncans, which form salivary sheaths upon probing on the plant and leaving behind a clear landmark that can be identified and observed through histology and microscopy, cell-rupturing feeders such as L. lineolaris do not leave marks on the plant tissue that clearly indicate stylet location or position in the plant tissue. Thus, EPG waveform correlation for these species can be particularly challenging. Evidence of direct feeding damage in our histologically sectioned cotton squares could, however, be observed as anatomical changes of the plant tissue, i.e., dark spots colored deep orange-red with safranin-fast green staining protocol. Dark- red areas, originally thought to be artifacts of the staining procedure, were not seen with the same frequency and amount in control sections, and were chemically confirmed in this study to be deposits of tannins probably produced as a plant defense response. Occurrence of these dark-red areas was consistent with previous observations by Williams and Tugwell (2000), who indicated that most of the damage by Lygus feeding on cotton squares happened in the staminal column. A single CR event performed by L. lineolaris is capable of eliciting tannin production by the cotton plant in the pin-head square. During a CR event, cell wounding is inflicted, and tannin production as plant defense response seems to increase with amount of wounding and salivation. Our work showed that the more wounding and salivation (more CR events), the more tannin concentration was observed on pin-head cotton squares. Tannins are phenolic compounds produced by plants as a response to insect damage, therefore playing a role in pest resistance (Lege et al. 1995). Tannins in general have detrimental effects on development and growth of insects as well as acting as feeding deterrents to many insects. They cause protein precipitation by covalent or hydrogen bonding of the protein. The proteins bind to reduce nutrient absorption and cause midgut lesions (Barbehenn and Constable 2011). Tannins also chelate metal ions, thereby reducing their bioavailability. Tannins have negative effects on growth and development of phytophagous insects. For example, tannins have been shown to inhibit the growth of young larvae of cotton bollworm, tobacco budworm, and pink bollworm (Barbehenn and Constable 2011). That said, our study showed that L. lineolaris was not deterred from ingesting from cotton squares that produced condensed tannins in response to the CR events preceding an I event. Moreover, ingestion removed tannins that had accumulated in the cotton square. Although we did not test survival or effect on new generations, feeding deterrence was not observed. The lack of feeding deterrence suggests that some sort of avoidance or detoxification mechanism such as extraoral digestion may be taking place prior to ingestion. It should be noted that other feeding deterrence compounds, such as the polyphenolics, were not measured here, and it was possible that these too were avoided or detoxified by L. lineolaris. Lygus lineolaris saliva contains several digestive enzymes and several studies have reported genes encoding proteinases (Zhu et al. 2003), polygalacturanases (PG; Strong and Kruitwagen 1968, Laurema et al. 1985, Agblor et al. 1994, Agusti and Cohen 2000, Frati et al. 2006, Wright et al. 2006, Allen and Mertens 2008, Celorio-Mancera et al. 2009), and ribonucleases (Allen and Walker 2012). Most of the enzymes secreted in Lygus saliva exhibit pectin-degrading activity that is responsible for feeding damage (Celorio-Mancera et al. 2009). Our histological sections showed presence of amorphous clear spaces in the cotton square that were overlapped by the presence of the dark-red tannin deposits. These clear spaces are probably caused by deposition and accumulation of L. lineolaris saliva, loosening cell walls and degrading cytoplasm, followed by ingestion of the degraded material. Phenolases are the primary salivary enzymes that detoxify tannins in host plants. Such enzymes have, to our knowledge, only been reported in two Lygus species. Phenolase activity has been observed in gland extracts of Lygus rugulipennis Poppius and L. hesperus (Strong and Kruitwagen 1968, Laurema et al. 1985). Our results suggest that tannin production in cotton squares is a direct response to the amount of wounding and salivation inflicted by L. lineolaris, yet the insect is capable of overcoming the plant defense to complete its feeding. Although we cannot say whether tannins are detoxified after ingestion by L. lineolaris, if so, then it is possible that L. lineolaris saliva may also contain phenolase activity. This could be an interesting direction for future research. A more rigorous study of the enzymatic profile of L. lineolaris saliva should be conducted to identify salivary enzymes with phenolase activity. The use of newer scientific equipment has made possible these types of more in-depth studies. With the use of mass spectrometry, three new salivary enzymes were identified in L. hesperus that target plant-defense compounds (laccase, glucose dehydrogenase, and xanthine dehydrogenase; Cooper et al. 2013). Moreover, the genetic expression of polygalacturonase in L. lineolaris has also been identified (Walker and Allen 2010), opening new avenues of research to explore the use of gene expression disruption and gene knockdown as potential control tactics. The results of this study have direct implications for our understanding of the interactions between L. lineolaris and cotton as a feeding host. Lygus lineolaris is capable of overcoming cotton plant defenses, with no feeding deterrence observed in this study. If specific gene regulation is taking place to produce salivary enzymes that aid in the detoxification of the plant slurry created during Lygus feeding, and the transcriptional profiles of these genes is studied, new options such as RNA interference could be explored to knockdown expression of such genes. Acknowledgments We sincerely thank Jackie Van De Viere, Nancy Goodell and Ernesto Duran, ARS Parlier, for helping to rear plants and insects for the study. Funding for this project was provided by Monsanto Co. to USDA-ARS (Backus). References Cited Agblor A. , Henderson H. M. , Madrid F. J . 1994 . Characterization of alpha-mylase and polygalacturonase from Lygus spp. (Heteroptera: Miridae) . Food Res. Int . 27 : 321 – 326 . 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TI - Correlation of Electropenetrography Waveforms From Lygus lineolaris (Hemiptera: Miridae) Feeding on Cotton Squares With Chemical Evidence of Inducible Tannins
JO - Journal of Economic Entomology
DO - 10.1093/jee/tox198
DA - 2017-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/correlation-of-electropenetrography-waveforms-from-lygus-lineolaris-eeLCq7Puc3
SP - 2068
VL - 110
IS - 5
DP - DeepDyve
ER -