Probing Behavior of Dichelops furcatus (F.) (Heteroptera: Pentatomidae) on Wheat Plants Characterized by Electropenetrography (EPG) and Histological Studies

Probing Behavior of Dichelops furcatus (F.) (Heteroptera: Pentatomidae) on Wheat Plants... The stink bug Dichelops furcatus (F.) (Heteroptera: Pentatomidae) has increased in abundance in recent years on the wheat, Triticum aestivum L., crop cultivated in the southern region of Brazil. To investigate the probing (stylet penetration) behaviors and nonprobing behaviors of D. furcatus on wheat plants, the electrical penetra- tion graph or electropenetrography (EPG) technique was applied. Nine EPG waveforms (types/subtypes) were identified and described on stem and on ear head of wheat plants, as follows: Z, Np, Df1a, Df1b, Df2, Df3a, Df3b, Df4a, and Df4b. For the waveforms Df1, Df2, Df3, and Df4, stylets were severed to determine, via histological studies, the location of the stylet tip and/or salivary sheath tip in plant tissue. Waveform Z was visually corre- lated with the bug standing still on the plant surface, whereas during Np the bug was walking. Df1a and Df1b represent initial stylet insertion, deep penetration of the stylets into the plant tissue, and secretion of salivary sheath. Df2 represents xylem sap ingestion on stem and on ear head. Waveforms Df3a and Df4a were related to the cell rupturing feeding strategy (laceration and maceration tactics) on stem and on ear head (seed endo- sperm), respectively. Waveforms Df3b and Df4b represent ingestion of cellular contents derived from cell rup- turing activities on stem and on ear head (seed endosperm), respectively. With this fundamental knowledge in hand, future studies can use EPG to develop novel pest management solutions. Key words: electronic feeding monitoring, feeding, histology, Triticum aestivum L, waveform identification The Neotropical stink bug Dichelops furcatus (F.), commonly called observed to cause damage on wheat plants cultivated in Brazil green-belly stink bug, occurs in different countries of South (Ferreira and Silveira 1991). In the United States (from Georgia America; in Brazil, it is recorded more often in the southern region north to Minnesota), several other pentatomids species are reported (Chiaradia et al. 2011). This stink bug is reported to feed on several on wheat, mainly species of the genus Euschistus [Euschistus servus plant species, including cultivated and non-cultivated plants (Say), Euschistus variolarius (Palisot de Beavois), and Euschistus (Smaniotto and Panizzi 2015). Among the cultivated plants, D. fur- ictericus (L.)], and Oebalus pugnax (F.) (Buntin and Greene 2004, catus has been observed as a pest of soybean, Glycine max (L.) Koch et al. 2016). Merrill, since the 1970s (Panizzi et al. 1977). D. furcatus feeds on both vegetative and reproductive stages, More recently, D. furcatus was observed to feed on heads of sun- causing significant damage to cultivated plants (Panizzi et al. 2016). flower, Helianthus annuus L. (Frota and Santos 2007), and on seed- Because of its recent occurrence in wheat fields and because of lack lings of corn, Zea mays (L.) causing injury that ended in seed yield of information on its feeding behavior, more studies are needed to lost (Roza-Gomes et al. 2011). Damage on this latter plant is similar better understand its feeding on wheat plants in different phenologi- to that of the congener Dichelops melacanthus (Dallas) (Avila and cal stages. To do so, the technique known as electrical penetration Panizzi 1995, Chocorosqui and Panizzi 2004), which also feeds on graph (EPG) or electropenetrography is useful; in this technique, any the reproductive structures of host plants. On wheat plants, piercing-sucking insect and a plant are made part of a simple electri- Triticum aestivum L., D. furcatus damages the crop in Southern cal circuit where a low current flows through a circuit, composed of Brazil causing seed yield reduction (Chocorosqui and Panizzi 2004, plant and feeding insect. Panizzi et al. 2016), where it has been observed to increase in abun- Both probing and nonprobing activities are captured by the EPG dance. Another pentatomid, Thyanta perditor (F.), has also been system and are presented as waveforms on computer screen V C The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 journals.permissions@oup.com by Ed 'DeepDyve' Gillespie user on 17 July 2018 2 Journal of Insect Science, 2017, Vol. 17, No. 2 (Tjallingii 1978, Walker 2000). For pentatomids, despite their eco- head-stage amplifiers (channels) were kept inside a Faraday cage. At nomic importance, only three species have had their feeding behav- the vegetative stage, the wheat seedlings were kept with their stems ior characterized using EPG [Edessa meditabunda (F.), and vertically positioned, whereas at the reproductive stage, the wheat Piezodorus guildinii (Westwood) on soybean plants (Lucini and plants containing a selected ear head were laid down along a plastic Panizzi 2016 and Lucini et al. 2016, respectively) and D. melacan- support (11  11 cm) and held in place using strips of Parafilm thus on maize seedlings (Lucini and Panizzi 2017)]. This is the (Pechiney Plastic Packaging, Menasha, WI). The plastic support was fourth study applying EPG technology to stink bugs, in this case, held horizontally by an alligator clip connected to a “helping hand” D. furcatus on wheat plants. It should yield valuable information holder. on its feeding on an economic host plant with potential to be used Waveforms caused by changes in electrical origins, i.e., on its chemical control targeting the action of systemic Resistance and electromotive force (emf) components, were cap- insecticides. tured and digitized at a sample rate of 100 Hz per channel using a The objectives of this study were, therefore, to 1) create a DI-710 (Dataq Instruments, Akron, OH) and recorded by using a waveform library characterizing the EPG waveforms produced by HP Pentium notebook with WinDaq Lite software (Dataq). Offset D. furcatus feeding on wheat plants during vegetative (stem) and and gain settings were adjusted for optimum view of the wave- reproductive (ear head) stages, 2) determine the biological mean- forms and to avoid rectifier fold-over to retain native waveform ings of each waveform recorded via electrical characteristics and polarity after rectification (Backus and Bennett 2009). To charac- histological studies; and 3) determine the feeding sites exploited by terize the waveforms, we considered the following characteristics: the bug on the vegetative and reproductive plant stages. shape, frequency, amplitude, and electrical origin (R and emf components). The frequency and amplitude were manually calcu- lated by expanding the x-axis to minimum compression and counting (see details of the methodology by Lucini and Panizzi Materials and Methods 2016). In addition, we used histological techniques to correlate Stink Bugs and Wheat Plants the waveforms and feeding sites, as done previously with three Adults of D. furcatus were field-collected at the Embrapa Wheat other species of pentatomids (Lucini and Panizzi 2016, 2017; Research Center during May to July 2016 located in Passo Fundo, Lucini et al. 2016). 0  0 RS, Brazil (28 15 S, 52 24 W). Stink bugs were weekly collected on wheat plants, taken to the laboratory and placed into rearing cages (25  20  20 cm), lined with filter paper to develop colonies. Experimental Design The cages were kept in a walk-in chamber at 256 1 C, 656 10% In the EPG experiment, we applied two different input impe- 7 9 RH, and a photoperiod of 14:10 (L:D) h. dance (Ri) levels, 10 and 10 Ohms, and a standardized voltage A standard food source was provided for the stink bugs, com- level of 50 mV of AC. Eighty insects were successfully recorded, posed of a mixture of green bean pods, Phaseolus vulgaris L., raw i.e., 40 stink bugs per Ri level (20 during vegetative stage and 20 shelled peanuts, Arachis hypogaea L., mature seeds of soybean, during reproductive stage at each Ri level). The stink bugs were Glycine max (L.) Merrill, and wheat seedlings; the mixture was monitored undisturbed for an 8-h access period, in a closed room replaced once per week. During this time, eggs were collected and with controlled conditions (256 2 C) and continuous light. placed inside of small plastic boxes (11  11  3.5 cm) to raise Representative waveforms excerpts at each Ri level were nymphs to obtain adults to be used in the experiments. assembled using Microsoft Power Point (Microsoft Corporation, Wheat seeds cv. BRS Parrudo (Embrapa) were seeded weekly in Redmond, WA). small (100 ml) and big (2 liters) pots kept in the greenhouse. Plants in stage 3 (V3: tillering stage) grown in small pots and plants in stage Histological Analysis to Determine the Stylet and/or Salivary Sheath 11.1 (R11.1: milk grain stage) (Large 1954) grown in big pots were Position in Plant Tissue separated and used in EPG recordings during vegetative and repro- Plant histology was conducted to determine the position of stylet ductive stages, respectively. Plants were grown using a standard oxy- and/or salivary sheath tip of D. furcatus on stem andonear soil prepared for greenhouse studies at Embrapa. head, which allowed us to correlate each feeding site with the different waveforms observed during EPG AC-DC recordings. EPG Recordings and Analysis of the Waveforms For that, a set of D. furcatus adult-females were recorded at Ri The feeding behavior of D. furcatus on wheat plants was monitored 10 Ohms and 50 mV AC signal. The EPG monitor was turned using a four-channel alternating current (AC)-direct current (DC) off every time a specific waveform was observed, and then the monitor (Backus and Bennett 2009; EPG Technologies, Inc., stylets were carefully cut using an entomological micro-scissors. Gainesville, FL). Before starting EPG recordings, adult-females (of After that, the plant tissues containing severed stylets were proc- approximately 2-wk old originated from eggs laid by field-collected essed to prepare semi-permanent slides according to Lucini and adults) were removed from the laboratory colony, placed in a small Panizzi (2016). plastic box (11  11  3.5 cm) lined with filter paper and starved For the stem histology, we used wheat plants in stage 8 (–8 - flag for ca.15 h before wiring. Then, the stink bugs were wired according leaf visible) (Large 1954), because the stem is more rigid allowing to the methodology described by Lucini and Panizzi (2016). This clean cuts of plant tissue (the feeding behavior and waveforms methodology consists in sanding the cuticle of the pronotum of bugs recorded in younger and older plants stages were the same). The to improve the wire attachment. position of the stylet tips and/or salivary sheath tips in stem and in After this procedure, stink bugs were individually connected to ear head structures were determined based on 10 samples for wave- an EPG amplifier and placed on stem wheat during vegetative stage form Df1a, 12 for Df2, 11 for Df3a, and 10 for Df4a. Digital images and on ear head during reproductive stage. To close the electrical were captured using Olympus BX50 (Shinjuku, Tokyo, Japan) circuit, the copper plant electrode (3-cm long) was inserted into the microscope coupled with a Sony DXC 107A video camera (Minato, soil of a pot containing each plant. Insects, plants, and the four Tokyo, Japan) linked to a computer. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 3 Results make the histological sections, we also observed the presence of Df1b, because stems used to make the cuts were more developed General Overview of Dichelops furcatus Waveforms on Wheat and rigid (V8 stage). In general, both Df1a and Df1b were more Plants 7 9 clearly distinguished at Ri 10 Ohms (low Ri level) than at 10 The waveform coarse structure recorded for D. furcatus on wheat Ohms (high Ri level), thus supporting R as their primary electrical plants on vegetative and reproductive stages included nine different origin (Table 1). waveforms, which represented non-probing and probing activities. Df1a always preceded the waveform Df2 (see Ingestion Phase) Regarding the nomenclature, probing waveform types were denoted recorded on wheat stem (Figs. 1C and E and 2A)and wasalso often as Df [from Dichelops furcatus] plus a number and subtypes by an observed before the waveform Df3a (see Ingestion Phase) (Figs. 3B additional, lower-case letter, as used for other pentatomids (Lucini and 4B). Otherwise, on ear head, the waveforms Df2 and Df4a (see and Panizzi 2016, 2017; Lucini et al. 2016). below) were preceded by Df1a or Df1b. On stem and on ear head, the Two of those waveforms recorded represent nonprobing behav- waveforms Df1 (both subtypes) and Df2 were easily distinguished ior, namely, Z and Np, whereas probing waveforms were repre- from each other during recordings, and also between Ri levels (Figs. sented by another four waveforms, namely, Df1, Df2, Df3, and Df4. 1E and 2A). However, Df1 (both subtypes), Df3a (on stem), and All these waveforms, except Df2, were sub-divided into two differ- Df4a (on ear head) were occasionally not clearly distinguished from ent subtypes each, as follows: Df1a and Df1b, Df3a and Df3b, and one another, mainly when recorded at Ri 10 Ohms (Fig. 4B)(see Df4a and Df4b. The probing waveforms were grouped in two differ- Df3 and Df4). ent families: pathway (P) and ingestion (I). The family P consisted of two waveforms (Df1a and Df1b), and family I consisted of five waveforms (Df2, Df3a, Df3b, Df4a, and Df4b). In both plant struc- Ingestion Phase. Family I (Waveforms Df2, Df3, and Df4) tures evaluated (stem and ear head), waveforms presented similar The family I comprised five different waveforms, labeled Df2, Df3a, characteristics (electrical and appearance); therefore, all waveforms Df3b, Df4a, and Df4b. were grouped in the same table (Table 1). Waveform Df2. Df2 was always observed immediately after Nonprobing Waveforms. Represented by two waveforms, Z and waveform Df1a on stem (Fig. 1E), and Df1a (most frequently) and Np (Fig. 1A), nonprobing waveforms that were visually correlated Df1b on ear head (Fig. 2A) of wheat plants. The waveform Df2 was with their biological meanings. Waveform Z occurred when the bug composed of repetitive wave portions interspersed with peaks, in was standing still on the plant surface and was characterized by a general, downward oriented, which occurred at regular intervals very low amplitude without variation in appearance between Ri lev- over time (Figs. 1A, C, D, F and 2A and D, peaks and waves are els applied. Therefore, Z wave represented the baseline. Np was defined in Fig. 1C). observed when the bug was walking on the plant surface; it was 7 9 At both Ri levels (10 and 10 Ohms) and also on both plant characterized by irregular peaks with variable amplitude according structures evaluated, the appearance of waveform Df2 and its to Ri level applied. Results suggested a strong emf-component to Np peak orientation were similar, except the amplitudes varied wave although R was also present (Table 1). according to Ri level applied. Peak amplitude value was lower Probing Waveforms. Two main waveform families (P and I) 7 9 when applied Ri 10 Ohms (28%) compared to Ri 10 Ohms were described during feeding behavior of D. furcatus on stem and (60%) (Table 1). Regarding frequency, Df2 showed a regular on ear head of wheat plants, and they comprised seven different pattern with means of 3.1 Hz at both Ri levels. Thus, waveform types/subtypes, namely, Df1a, Df1b, Df2, Df3a, Df3b, Df4a, and Df2 presented a mixture of electrical components, with peaks Df4b. These will be described in the order in which they occurred originating from R-component, because they were clearly distin- during the feeding activities of D. furcatus on wheat plants. All guished at Ri 10 Ohms, as well as emf-component, because 7 9 probing waveforms, at both Ri levels (10 and 10 X), occurred peaks were still clearly distinguished and separated from wave below 0 V (i.e., were monophasic negative). portion at high Ri level (10 Ohms). Wave portions were more emphasized at Ri 10 Ohms; thus, waves were emf-dominated Pathway Phase. Family P (Waveform Df1) (compare Fig. 1C and F). Df1 occurred after nonprobing waveforms, Z or Np, and it was div- Waveform Df3. Waveform Df3 was divided into two subtypes, ided in two subtypes: Df1a and Df1b. Df1a was recorded in both Df3a and Df3b (Figs. 3A–E and 4A–F), which were recorded only plant structures evaluated (stem and ear head); it was characterized when D. furcatus fed on wheat stem. Df3a was often recorded after by irregular-peaks without a clear pattern (Figs. 1B and E and 2A– a short event of waveform Df1a (Figs. 3B and 4B); however, the sep- C), and almost always presented the largest amplitude value in the aration between them sometimes was not clear. Df3a was character- probe among all the waveforms registered. The subtype Df1b was ized by a generally stereotypical pattern with peaks oriented both recorded only on wheat ear head, and it was characterized by a upward and downward at both Ri levels (Figs. 3C and 4C). Df3a more distinct pattern (Fig. 2A–C) of random overlying/superim- showed a similar frequency at both Ri levels (means of 3.1 and 7 7 9 posed on low- to medium-amplitude waves (35 and 23% at Ri 10 3.0 Hz at Ri 10 and 10 Ohms, respectively). Nevertheless, Df3a and 10 Ohms, respectively), with a high and regular frequency, on was greatly variable in appearance (primarily amplitude) not only average ranging from 4.7 to 5.1 Hz between Ri levels (Table 1). across Ri levels, but also among individual bugs and even within the Df1b waveform always occurred after Df1a or alternating among same recording. As Ri level increased, the amplitude value decreased 7 9 events of Df1a (e.g., Df1a ! Df1b ! Df1a ! Df1b, and so on) (Fig. (57% at Ri 10 Ohms and 31% at Ri 10 Ohms) (Table 1). Thus, 2B and C). Df3a was R-dominated. The subtype Df1b was not always present, and it was visually Waveform Df3b was characterized as a regular wave of short correlated with protraction and retraction movements of the stylets event durations (in general, a few seconds) at both Ri levels, always in the ear head tissue, especially when the bug inserted its stylets in occurring interspersed with Df3a, as follows: Df3a ! Df3b ! Df3a the glume (outer layer) and in the lemma (layer below the glume ! Df3b ! and so on (Figs. 3D and E and 4D–F). Df3b was not that surrounds the seed). However, during recordings on stems to always clearly from Df3a within recordings, or it did not occur. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 4 Journal of Insect Science, 2017, Vol. 17, No. 2 Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Table 1. Summary of EPG AC-DC waveforms, their main electrical characteristics, and suggested biological meanings for each waveform recorded during probing and nonprobing activities of D. furcatus on stem and on ear head of wheat plants Phase Family Type or Plant structure Amplitude (%) Frequency (Hz) Electrical origin Suggested biological subtype observed meaning 7 9 7 9 Ri 10 Ri 10 Ri 10 Ri 10 Nonprobing – Z Stem/ear head Flat Flat – – – Standing still on the plant surface Np Stem/ear head Low-medium Medium-high Irregular Irregular Mostly emf/some R Walking on the plant surface Pathway P Df1a Stem/ear head 100 100 Irregular Irregular Mostly R Stylet penetration and salivary sheath secretion Df1b Ear head 35 (16–100) 23 (13–37) 5.1 (4.2–5.7) 4.7 (3.8–5.8) Mostly R Bug encountering a rigid cell layer requiring stylet protraction and retraction Ingestion I Df2 Stem/ear head 28 (11–53) 60 (29–100) 3.1 (2.6–3.9) 3.1 (2.4–4.2) Mixed; peak ¼ R/emf; Xylem sap ingestion wave ¼ emf-dominated Salivation/ I Df3a Stem 57 (37–90) 31 (19–52) 3.1 (2.8–3.5) 3.0 (2.9–3.2) Mostly R; emf is also Cell laceration, enzymatic ingestion present maceration of stem tissues and probably ingestion Ingestion I Df3b Stem 15 (7–19) 19 (15–22) 3.9 (3.5–4.2) 4.1 (4.0–4.2) Mostly emf-component Short ingestion of macer- ated stem tissues Salivation I Df4a Ear head 51 (32–79) 35 (18–55) Mostly irregular þ Mostly irregular þ Mostly R; emf is also Cell laceration, enzymatic burst regular peaks burst regular peaks present maceration of seed 3.5 Hz 3.8 Hz endosperm Ingestion I Df4b Ear head 20 (9–34) 27 (11–65) 3.5 (2.7–4.7) 4.0 (3.0–5.1) Mostly emf-component Ingestion of macerated seed endosperm Journal of Insect Science, 2017, Vol. 17, No. 2 5 Fig. 1. Waveforms recorded using EPG AC-DC from D. furcatus on stems of wheat plants during vegetative stage (–3 – tillering stage) at Ri 10 Ohms (A–C) and Ri 10 Ohms (D–F). (A) Compressed overview of a probing event (40 min), showing waveforms Df1a (pathway) and Df2 (xylem sap ingestion). (B) Expanded view of waveform Df1a. (C) Expanded view of waveform Df2 and definition of peak and wave portions. (D) Compressed overview of several waveform events (35 min), showing Df1a and Df2. (E) Expanded view of waveform Df1a and beginning of waveform Df2. (F) Expanded view of waveform Df2. (A has Windaq com- pression 400 [80 s/vertical div.], gain 16; B has compression 25 [5 s/vertical div.], gain 8; C has compression 3 [0.6 s/vertical div.], gain 32; D has compression 300 [60 s/vertical div.], gain 8; E has compression 50 [10 s/vertical div.], gain 8; F has compression 5 [1 s/vertical div.], gain 8). Arrowheads indicate beginning or end of a probe. 7 9 At both Ri levels, 10 and 10 Ohms, Df3b showed low-amplitude which were recorded only when D. furcatus fed on wheat ear head values (15 and 19%, respectively), as well as frequency values (3.9 (seed endosperm). Df4a was, in general, preceded by a short Df1b and 4.1 Hz, respectively) (Table 1). Df3b, in the same way as Df3a, wave, and sometimes by Df1a; however, in both cases, it was often was quite variable across Ri levels, among different bugs and also hard to separate the waveforms Df1a/Df1b and Df4a within the within the same bug recording. recording, especially at Ri 10 Ohms (Fig. 6A). Df4a showed irregu- Regarding the electrical origin of the waveform Df3, it presented larity in appearance, different from Df2 and even Df3a (which latter a mixture of electric components varying according to the wave sub- represents the same feeding strategy of the Df4a wave, but per- type. Subtype Df3a, in general, showed a decrease in amplitude formed in different tissues). Df4a presented a high-amplitude value 9 7 9 value when applied a Ri of 10 Ohms compared to a low Ri level when Ri of 10 Ohms was used (means of 51%) compared to 10 (10 Ohms), indicating a R-component. However, this subtype was Ohms (35%). Therefore, Df4a originated mostly from R- still clearly visible at 10 Ohms, indicating the emf-component, as component. well. Df3b was more emphasized at Ri 10 Ohms, thus emf is the In addition, Df4a was characterized not only by overlying main component (Table 1). irregular frequency, composed of peaks negative and positive ori- Waveform Df4. Waveform Df4 was also divided into two differ- ented at both Ri levels (Figs. 5A–C and 6A–C, Table 1)but also ent subtypes, namely, Df4a and Df4b (Figs. 5A–D and 6A–D), with underlying portions that were more regular (frequency Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 6 Journal of Insect Science, 2017, Vol. 17, No. 2 Fig. 2 Waveforms recorded using EPG AC-DC from D. furcatus on ear head of wheat plants during reproductive stage (R11.1: milk grain stage) at Ri 10 Ohms. (A) Compressed overview of several waveform events (50 min), showing Df1 (pathway) and Df2 (xylem sap ingestion). (B) Expanded view of both Df1 subtypes, Df1a and Df1b. (C) Details of the waveforms Df1a and Df1b. (D) Expanded view of waveform Df2. (A has Windaq compression 400 [80 s/vertical div.], gain 8;B has compression 10 [2 s/vertical div.], gain 8; C has compression 3 [0.6 s/vertical div.], gain 16; D has compression 3 [0.6 s/vertical div.], gain 16). Arrowheads indicate beginning or end of a probe. ranging from 3.5 to 3.8 Hz). Sometimes, within the same record- visible at 10 Ohms, indicating the emf-component, as well. Df4b ing, peaks were positive and negative oriented, and this inversion was more emphasized at Ri 10 Ohms, thus emf is the main com- was not due to rectifier fold-over (see Materials and Methods). ponent (Table 1). Furthermore, Df4a was variable in appearance between Ri levels, but also among bugs and within the recording of the same insect. In contrast, Df4b showed a highly regular pattern of short event Correlations of the Waveforms and Their Specific Feeding Sites via duration compared to Df4a, always occurring interspersed with Histological Analyses the latter (Figs. 5C and Dand 6C and D). Df4b presented a mean Df1. In the waveform Df1a recorded during the feeding activities of 7 9 amplitude value (20 and 27% at Ri 10 and 10 Ohms, respec- D. furcatus on wheat plant, the presence of a salivary sheath sur- tively) and a regular frequency, ranging from 3.5 to 4.0 Hz rounding the stylets was observed in all histological sections on stem between Ri levels (Table 1). (n¼ 10) (Fig. 7A). Furthermore, the tip of the salivary sheath and/or Df4 presented a mixture of electric components varying stylets terminated in cells of the parenchyma, confirming that Df1a according to the wave subtype. Df4a showed a decrease in ampli- represents pathway phase (insertion and penetration of the stylets 9 7 tude value when applied a Ri of 10 Ohms compared to a 10 deep into the plant tissue and secretion of the salivary sheath). Ohms, indicating a R-component. This subtype was still clearly However, there was an interesting difference about secretion of the Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 7 Fig. 3. Waveform Df3 recorded using EPG AC-DC from Dichelops furcatus on stems of wheat plants during vegetative stage (V3: tillering stage) at Ri 10 Ohms. (A) Compressed overview of a probing event (60 min) showing waveforms Df1a (pathway) and Df3 (laceration/maceration activities and ingestion). (B) Expanded view of waveform Df1a and beginning of waveform Df3a. (C) Expanded view of waveform Df3a. (D) Details of the waveforms Df3a and Df3b. (E) Expanded view of waveform Df3b. (A has Windaq compression 500 [100 s/vertical div.], gain 16; B has compression 30 [6 s/vertical div.], gain 16; C has com- pression 3 [0.6 s/vertical div.], gain 16; D has compression 20 [4 s/vertical div.], gain 8; E has compression 2 [0.4 s/vertical div.], gain 16). Arrowheads indicate beginning or end of a probe. salivary sheath during Df1a. When this waveform was recorded seed endosperm (n¼ 2), the salivary sheath was not completely before Df2 (n¼ 10, stem), the salivary sheath was completely formed (Fig. 7D–F). secreted (Fig. 7B), i.e., the full length of the stylets. However, when Df2. Cuts made during the waveform Df2 on stem showed that Df1a was recorded before Df3a on stem (n¼ 5), or before Df4a on in all histological sections (n¼ 10), the tip of the salivary sheath Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 8 Journal of Insect Science, 2017, Vol. 17, No. 2 Fig. 4. Waveform Df3 recorded using EPG AC-DC from Dichelops furcatus on stems of wheat plants during vegetative stage (V3: tillering stage) at Ri 10 Ohms. (A) Compressed overview of a probing event (50 min) showing waveforms Df1a (pathway) and Df3 (laceration/maceration activities and ingestion). (B) Expanded view of waveform Df1a and beginning of waveform Df3a. (C) Expanded view of waveform Df3a. (D) Details of the waveforms Df3a and Df3b. (E-F) Expanded views of waveform Df3b. (A has Windaq compression 400 [80 s/vertical div.], gain 16; B has compression 6 [1.2 s/vertical div.], gain 16; C has com- pression 3 [0.6 s/vertical div.], gain 32; D has compression 10 [2 s/vertical div.], gain 32; E and F have compression 2 [0.4 s/vertical div.], gain 32). Arrowheads indicate beginning or end of a probe. and/or stylets were positioned inside xylem cells (Fig. 7B). During not allow clarification of stylet tip positions, especially whether they Df2 in the wheat ear head, cuts made in the lemma layer (n¼ 2) were inside the xylem vessels or not. showed the stylets close to the xylem vessels (longitudinal position) Df3. During the Df3a wave (observed only in stems), the tip of in the plant tissue (Fig. 7C). However, the histological images did the stylets (n¼ 5) was always positioned in the parenchyma tissue Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 9 Fig. 5. Waveform Df4 recorded using EPG AC-DC from D. furcatus on ear head of wheat plants during reproductive stage (R11.1: milk grain stage) at Ri 10 Ohms. (A) Compressed overview of a probing event (140 min) showing waveforms Df1 (pathway) and Df4 (laceration/maceration activities and ingestion). (B) Expanded view of waveform Df4a. (C) Details of the waveforms Df4a and Df4b. (D) Expanded view of waveform Df4b. (A has Windaq compression 1,200 [240 s/ vertical div.], gain 8; B and D have compression 3 [0.6 s/vertical div.], gain 16; C has compression 30 [6 s/vertical div.], gain 8). Arrowheads indicate beginning or end of a probe. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 10 Journal of Insect Science, 2017, Vol. 17, No. 2 Fig. 6. Waveform Df4 recorded using EPG AC-DC from D. furcatus on ear head of wheat plants during reproductive stage (R11.1: milk grain stage) at Ri 10 Ohms. (A) Compressed overview of a probing event (60 min) showing waveforms Df1 (pathway) and Df4 (laceration/maceration activities and ingestion). (B) Expanded view of waveform Df4a. (C) Details of the waveforms Df4a and Df4b. (D) Expanded view of waveform Df4b. (A has Windaq compression 600 [120 s/vertical div.], gain 4; B has compression 3 [0.6 s/vertical div.], gain 16; C has compression 30 [6 s/vertical div.], gain 16; D has compression 3 [0.6 s/vertical div.], gain 32). Arrowheads indicate beginning or end of a probe. (Fig. 7D) and there was also an incomplete secretion of the salivary endosperm, and the formation of an incomplete salivary sheath sheath when this wave was recorded (Fig. 7E). (Fig. 7F, red arrow indicates the end of the salivary sheath). Df4. In the waveform Df4a (observed only in seed), the histologi- Moreover, cuts made of fresh stems, after recording of a Df3 cal sections (n¼ 2) showed the stylet tips positioned inside the seed wave event (n¼ 6), revealed a visibly damaged area (opaque region Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 11 Fig. 7. Cross sections of wheat stems (V8 stage: flag leaf visible) and ear head structures (R11.1 stage: milk grain stage) containing severed stylets and salivary sheath of the stink bug D. furcatus. (A) Salivary sheath ending in the parenchyma tissue during waveform Df1a on stem. (B) Salivary sheath and stylet tips ending in the xylem vessels during waveform Df2 on stem. (C) Stylet tips near of xylem vessels during waveform Df2 on ear head (lemma layer). (D) Stylet tips in paren- chyma tissue during waveform Df3a on stem. (E) Secretion of a flange (externally on the stem) and of an incomplete salivary sheath (internally in the tissue) dur- ing waveform Df3a on stem. (F) Stylet tips inside the seed endosperm during waveform Df4a and an incomplete salivary sheath secreted internally in the tissue (indicated by the red arrow in the detail box). Ep¼ stem epidermis, Ep lemma¼ lemma epidermis, Pa¼ parenchyma, Xy¼ xylem, Ph¼ phloem. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 12 Journal of Insect Science, 2017, Vol. 17, No. 2 Fig. 8. Cross sections of fresh stem (V8 stage: flag leaf visible) and fresh seed (R11.1 stage: milk grain stage) of wheat plants containing cut stylets of D. furcatus. (A) Section of a fresh wheat stem showing damaged area (opaque region surrounded by the red line) after recording a Df3 wave event on stem. (B) Immature wheat seed containing the flange (externally on lemma layer) and cut stylets during waveform Df4 on seed. (C) Section of a fresh seed showing the damaged area (opaque region) after recording a Df4 wave event on seed endosperm. surrounded by the red line, Fig. 8A). Similarly, cuts made in fresh using the AC-DC electropenetrography EPG technique. Seven differ- seeds during the waveform Df4a (n¼ 8) showed the stylets posi- ent waveforms related to its probing behavior on stem (vegetative tioned in the seed endosperm, and the secretion of flanges externally stage: V3) and ear head (reproductive sta–ge: R11.1) of wheat plant on protection layers of the seed (Fig. 8B). In addition, cuts made in were described. fresh seeds, after a Df4 wave event, showed a visually damaged area Some similarities with other stink bugs already studied via EPG in the seed endosperm (region of opaque white color, Fig. 8C). were observed during feeding of D. furcatus in both plant structures, as well as some interesting peculiarities. One of the most relevant similarities is the use of the same feeding strategy on stem and on ear head of wheat, in this case, the cell rupture feeding strategy Discussion [Backus et al. 2005–, previously known as laceration and flush strat- In this study, the probing behavior of the pentatomid D. furcatus, egy (Miles 1972)]. However, the waveforms recorded for each feed- found attacking the wheat crop in southern Brazil, was evaluated ing site, even using the same strategy, were completely different in Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 13 appearance and electrical characteristics, which demonstrates the correlated via histological analysis with activities in the xylem ves- complexity of the D. furcatus feeding process. In addition, when the sels, more specifically, sap ingestion. This wave was strongly similar same bug ingests from xylem vessels, it uses another feeding strat- in appearance to the Dm2 wave of D. melacanthus feeding on maize egy, known as salivary the sheath strategy, which was demonstrated stem (Lucini and Panizzi 2017), which was also correlated to xylem in the histological analyses. sap activities. In addition, both waves (Df2 and Dm2) share some Previous studies have shown that the same stink bug species may electrical characteristics, such as high-amplitude value at Ri 10 use only one of those strategies, such as E. meditabunda which uses Ohms, and a mixture of electrical origins for the peaks (R and emf) the salivary sheath when feeding on soybean stems (Lucini and and waves (emf). Panizzi 2016); or both strategies, as observed for P. guildinii on soy- In fact, all D. furcatus bugs evaluated in both plant structures bean plants (Lucini et al. 2016) and for D. melacanthus on maize ingested from the xylem vessels. Probably this is a strategy to avoid seedlings (Lucini and Panizzi 2017). Similarly, when feeding on dehydration and to maintain body water balance, as reported for wheat plants D. furcatus also uses both feeding strategies, switching other piercing-sucking insects such as aphids and psyllids (Spiller strategy according to the feeding site exploited. et al. 1990, Bonani et al. 2010, Pompon et al. 2010), and also for Pathway phase is represented by a single waveform, Df1, which other species of stink bug (Lucini and Panizzi 2016). In case of inges- was divided into two subtypes, Df1a and Df1b. Df1a was the first tion of water during feeding on wheat seed, this may also be a strat- waveform related to feeding of D. furcatus on the wheat plant, and egy for nutrient dilution, since the seed is highly concentrated in was observed on both plant structures (stem and ear head), whereas nutrients (Panizzi and Slansky 1985), as observed for P. guildinii Df1b was recorded only when bugs fed on wheat ear head. The sub- feeding on soybean seed endosperm (Lucini et al. 2016). type Df1a represents the insertion and deep penetration of the stylets During ingestion phase using cell rupture feeding on stem (wave- into the plant tissue and also the secretion of gelling saliva to form forms Df3a and Df3b), the bug (during the first subtype) makes the salivary sheath as observed in all the histological images from rapid, continuous, and deep protraction and retraction of the stylets stems of wheat during this waveform. In addition, the secretion of in the stem tissue. Such activity is reported in the literature to occur salivary flanges (external deposition of gelling saliva) was frequently during the activities of laceration (mechanical action of stylets) and observed on stem and also on ear head of wheat. The subtype Df1b maceration (enzymatic –action: digestive enzymes secreted in watery probably represents the bug encounter of a rigid cell layer requiring saliva) (Miles 1972, 1987), which are tactics of the cell rupture strat- stylet protraction and retraction. egy (Backus et al. 2005). This behavior was also noted for other Similar to the present study, other EPG studies (Lucini and sucking insects, such as the leafhoppers Empoasca fabae (Harris), Panizzi 2016, 2017; Lucini et al. 2016) with other species of penta- Empoasca kraemeri (Ross & Moore) (Calderon and Backus 1992), tomids showed that pathway phase was correlated, via histological and Empoasca vitis Go ¨ the (Jin et al. 2012), as well as stink bugs, studies, with salivary sheath secretion. It can be completely or Lygus hesperus Knight (Cline and Backus 2002), P. guildinii (Lucini incompletely formed, depending on the feeding strategy used by the et al. 2016), and D. melacanthus (Lucini and Panizzi 2017). In the bug. Complete secretion occurs when the bug uses the salivary last species, in one of the waveforms recorded on maize seedlings sheath strategy to ingest from vessels of the vascular tissues: xylem (called Dm3a), the bug moved its stylets in the stem tissue similar to and phloem (Bonani et al. 2010; Backus et al. 2013; Pearson et al. that observed herein for the waveform Df3a of D. furcatus on wheat 2014; Lucini and Panizzi 2016, 2017; Lucini et al. 2016; Seo et al. stem. Moreover, during Df3a, there was the secretion of an incom- 2016); incomplete secretion occurs during the cell rupture feeding plete salivary sheath, as observed for D. melacanthus using the cell strategy (Miles 1972, Backus et al. 2005). rupture strategy (Lucini and Panizzi 2017). Likewise, it was observed in the histological images of D. furca- In the subtype Df3b, it was observed that the stylets remained tus on wheat stem that during feeding activities in xylem vessels, the motionless inside the plant tissue for a short period of time, and salivary sheath was completely formed and followed the pathway then, the stylets moved rapidly and vigorously in and out again the stylets to the vessels. However, during the cell rupture activities (wave Df3a). Therefore, Df3b represents the ingestion of degraded on stem and on seed, a partial salivary sheath was observed to be cellular contents (composed of cells from parenchyma and possibly formed. In the histological images of these plant structures during vascular vessels) via laceration and maceration activities, as cell rupturing, a salivary sheath surrounding the stylet was observed described during the Dm3b wave of D. melacanthus (Lucini and only at the beginning of its insertion point in the plant tissue, similar Panizzi 2017). However, the waveform Df3b was not clearly distin- to D. melacanthus on stem of maize seedlings (Lucini and Panizzi guished and/or observed in all recordings in which the Df3a wave 2017). was recorded. Thus, we believe that during Df3a, the ingestion proc- We believe that Df1b represents the stylet work to overcome ess may be occurring simultaneously with the laceration and macer- some physical barrier that makes the penetration of the stylets into ation activities of the stem cells, as described for D. melacanthus the plant tissue quite difficult. The structures that make up the during the Dm3a wave (Lucini and Panizzi 2017). wheat spikelet are rigid and are the only layers responsible for pro- In addition to the similarities in the behavior of the two stink tecting the seed. bugs D. melacanthus and D. furcatus, the subtypes Dm3a/Df3a and A waveform very similar to Df1b, namely, Pg1d, was described Dm3b/Df3b showed others similarities in appearance and in electri- for P. guildinii fed on different parts of the soybean pod. Pg1d was cal characteristics. In the histological images made during the Df3a, correlated with the bug forcing the stylets downward and subse- it was not possible to observe a damaged area or cells destruction in quently retracting them upward. This indicates the presence of a the insertion region of the stylets. However, in fresh sections of the rigid cell layer (which was observed in histological analyses of soy- wheat stem was possible to clearly observe a damaged area of opa- bean pods) that was difficult to penetrate with the stylets, requiring que white coloration. stylet laceration to reach the seed (Lucini et al. 2016). During the ingestion phase using cell rupture feeding in the During the ingestion phase, one of the waveforms recorded was wheat ear head (seed endosperm: waveforms Df4a and Df4b), Df2, which was recorded when the bug fed on stem and on any D. furcatus made a continuous and deep movement of the stylets in structure of the ear head (palea, glume, lemma, or awn). Df2 was the wheat seed endosperm as reported during the waveform Pg3a Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 14 Journal of Insect Science, 2017, Vol. 17, No. 2 of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings. for P. guildinii feeding on soybean seed endosperm (Lucini et al. Entomol. Exp. Appl. 134: 35–49. 2016). Therefore, the Df4a wave represents the dissolution of endo- Buntin, G. D., and J. K. Greene. 2004. Abundance and species composition sperm cells via laceration (mechanical action) and maceration (enzy- of stink bugs (Heteroptera: Pentatomidae) in Georgia winter wheat. matic action) for future ingestion. During this waveform, a salivary J. Entomol. Sci. 39: 287–290. sheath was observed surrounding the stylets only at the beginning of Calderon, J. D., and E. A. Backus. 1992. Comparison of the probing behaviors the insertion point in the seed endosperm, i.e., the salivary sheath of Empoasca fabae and E. kraemeri (Homoptera: Cicadellidae) on resistant was incompletely secreted inside the tissue, which was observed in and susceptible cultivars of common beans. J. Econ. Entomol. 85: 88–99. the histological sections. Chiaradia, L. A., A. Rebonatto, M. A. Smaniotto, M. R. F. Davila, and C. N. In cuts made of fresh wheat seeds, shortly after the recording of Nesi. 2011. Arthropods associated with soybean crops. Rev. Ci^ enc. the Df4 wave, we observed a damaged area in the seed endosperm Agrovet. 10: 29–36. Chocorosqui, V. R., and A. R. Panizzi. 2004. Impact of cultivation systems on caused by the activities of laceration, maceration and ingestion; this Dichelops melacanthus (Dallas) (Heteroptera: Pentatomidae) populations is similar to what was reported for P. guildinii during the waveform and damage and its chemical control on wheat. Neotrop. Entomol. 33: Pg3 recorded on soybean seed endosperm (Lucini et al. 2016). 487–492. During the subtype Df4b, the stylets of D. furcatus were motion- Cline, A. R., and E. A. Backus. 2002. Correlations among AC electronic moni- less inside the seed tissue for a short period of time (which represents toring waveforms, body postures, and stylet penetration behaviors of the ingestion period); after that, they moved their stylets again to Lygehaviorrus (Hemiptera: Miridae). Environ. Entomol. 31: 538–549. break more cells (Df4a wave). This activity was continually Ferreira, E., and P. M. Silveira. 1991. Dano de Thyanta perditor (Hemiptera: repeated; however, the bug spent most of the time in laceration and Pentatomidae) em trigo (Triticum aestivum L.). An. Soc. Entomol. Bras. 20: maceration activities to prepare the food for latter ingestion, which 165–171. usually took a short time. Frota, R. T., and R. S. S. Santos. 2007. Pentatomidae bugs associated with sunflower crops in the northwest of Rio Grande do Sul state and the action In conclusion, our results demonstrated that D. furcatus feeding of Euschistus heros (Fabricius, 1794) (Hemiptera: Pentatomidae) on sun- on wheat plants during vegetative (stem) and reproductive (ear flower seeds. Biotemas. 20: 65–71. head) stages use two different feeding strategies. When in xylem ves- Jin, S., Z. M. Chen, E. A. Backus, X. L. Sun, and B. Xiao. 2012. sels (ingestion of xylem sap) on wheat stem (and probably on ear Characterization of EPG waveforms for the tea green leafhopper, Empoasca head although not histological demonstrated in this work), D. furca- vitis Go ¨ the (Hemiptera: Cicadellidae), on tea plants and their correlation tus used the salivary sheath strategy, with its complete salivary with stylet activities. J. Insect Physiol. 58: 1235–1244. sheath secretion. In contrast, when on seed endosperm and again on Koch, R. L., A. Rich, and T. Pahs. 2016. Statewide and season-long surveys stem, the bug used the cell rupture strategy as well, using the lacera- for Pentatomidae (Hemiptera: Heteroptera) of Minnesota wheat. Ann. tion and maceration tactics to break cell pockets for ingesting. At Entomol. Soc. Am. 109: 396–404. this time, a salivary sheath was also observed in the plant tissue, Large, E. C. 1954. Growth stages in cereals. Illustration of the Feekes scale. Plant Pathol. 3: 128–129. however, not completely formed. These results demonstrate the Lucini, T., and A. R. Panizzi. 2016. Waveform characterization of the soybean plasticity of this insect’s feeding behavior, which involve a switch in stem feeder Edessa meditabunda: overcoming the challenge of wiring penta- strategy according to the feeding site exploited. tomids for EPG. Entomol. Exp. Appl. 158: 118–132. Lucini, T., and A. R. Panizzi. 2017. Feeding behavior of the stink bug Dichelops melacanthus (Heteroptera: Pentatomidae) on maize seedlings: an Acknowledgments EPG analysis at multiple input impedances and histology correlation. Ann. This study was partially supported by a National Council of Research and Entomol. Soc. Am. 110: in press. Doi.org/10.1093/aesa/saw070. Technology of Brazil grant 471517/2012-7 to A.R.P. and by a scholarship Lucini, T., A. R. Panizzi, and E. A. Backus. 2016. Characterization of an EPG from CAPES (Ministry of Education) of Brazil to T.L. in order to obtain the waveform library for redbanded stink bug, Piezodorus guildinii (Hemiptera: Doctor of Science (Entomology) degree at the Federal University of Paranaat Pentatomidae), on soybean plants. Ann. Entomol. Soc. Am. 109: 198–210. Curitiba, Brazil. We also thank the Embrapa Unit at Passo Fundo, RS, for Miles, P. W. 1972. The saliva of Hemiptera. Adv. Insect Physiol. 9: 183–125. support. Appreciation is also extended to Elaine Backus and Paula Mitchell Miles, P. W. 1987. Plant-sucking bugs can remove the content of cells without for reviewing and earlier draft of the article. Approved by the Publication mechanical damage. Experientia. 43: 937–939. Committee of the Embrapa National Wheat Research Center, Passo Fundo, Panizzi, A. R., and F. Slansky, Jr. 1985. Legume host impact on performance RS, Brazil, under number 5408/2016. of adult Piezodorus guildinii (Westwood) (Hemiptera: Pentatomidae). Environ. Entomol. 14: 237–242. Panizzi, A. R., S. Corr^ea, D. L. Gazzoni, E. B. Oliveira, G. G. Newman, and S. References Cited G. Turnipseed. 1977. Insects of soybean in Brazil. Bol. Tec., n. 1. Embrapa, Avila, C. J., and A. R. Panizzi. 1995. Occurrence and damage by Dichelops Londrina. (Neodichelops) melacanthus (Dallas) (Heteroptera: Pentatomidae) on corn. Panizzi, A. R., T. Agostinetto, T. Lucini, and P. R. V. S. Pereira. 2016. Effect An. Soc. Entomol. 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Koenders, and W. F. Tjallingii. 1990. Xylem ingestion by aph–s – a the study of homopteran feeding behavior. Thomas Say Publications in strategy for maintaining water balance. Entomol. Exp. Appl. 55: 101–104. Entomology, Entomological Society of America, Lanham, MD. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Insect Science Oxford University Press

Probing Behavior of Dichelops furcatus (F.) (Heteroptera: Pentatomidae) on Wheat Plants Characterized by Electropenetrography (EPG) and Histological Studies

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Abstract

The stink bug Dichelops furcatus (F.) (Heteroptera: Pentatomidae) has increased in abundance in recent years on the wheat, Triticum aestivum L., crop cultivated in the southern region of Brazil. To investigate the probing (stylet penetration) behaviors and nonprobing behaviors of D. furcatus on wheat plants, the electrical penetra- tion graph or electropenetrography (EPG) technique was applied. Nine EPG waveforms (types/subtypes) were identified and described on stem and on ear head of wheat plants, as follows: Z, Np, Df1a, Df1b, Df2, Df3a, Df3b, Df4a, and Df4b. For the waveforms Df1, Df2, Df3, and Df4, stylets were severed to determine, via histological studies, the location of the stylet tip and/or salivary sheath tip in plant tissue. Waveform Z was visually corre- lated with the bug standing still on the plant surface, whereas during Np the bug was walking. Df1a and Df1b represent initial stylet insertion, deep penetration of the stylets into the plant tissue, and secretion of salivary sheath. Df2 represents xylem sap ingestion on stem and on ear head. Waveforms Df3a and Df4a were related to the cell rupturing feeding strategy (laceration and maceration tactics) on stem and on ear head (seed endo- sperm), respectively. Waveforms Df3b and Df4b represent ingestion of cellular contents derived from cell rup- turing activities on stem and on ear head (seed endosperm), respectively. With this fundamental knowledge in hand, future studies can use EPG to develop novel pest management solutions. Key words: electronic feeding monitoring, feeding, histology, Triticum aestivum L, waveform identification The Neotropical stink bug Dichelops furcatus (F.), commonly called observed to cause damage on wheat plants cultivated in Brazil green-belly stink bug, occurs in different countries of South (Ferreira and Silveira 1991). In the United States (from Georgia America; in Brazil, it is recorded more often in the southern region north to Minnesota), several other pentatomids species are reported (Chiaradia et al. 2011). This stink bug is reported to feed on several on wheat, mainly species of the genus Euschistus [Euschistus servus plant species, including cultivated and non-cultivated plants (Say), Euschistus variolarius (Palisot de Beavois), and Euschistus (Smaniotto and Panizzi 2015). Among the cultivated plants, D. fur- ictericus (L.)], and Oebalus pugnax (F.) (Buntin and Greene 2004, catus has been observed as a pest of soybean, Glycine max (L.) Koch et al. 2016). Merrill, since the 1970s (Panizzi et al. 1977). D. furcatus feeds on both vegetative and reproductive stages, More recently, D. furcatus was observed to feed on heads of sun- causing significant damage to cultivated plants (Panizzi et al. 2016). flower, Helianthus annuus L. (Frota and Santos 2007), and on seed- Because of its recent occurrence in wheat fields and because of lack lings of corn, Zea mays (L.) causing injury that ended in seed yield of information on its feeding behavior, more studies are needed to lost (Roza-Gomes et al. 2011). Damage on this latter plant is similar better understand its feeding on wheat plants in different phenologi- to that of the congener Dichelops melacanthus (Dallas) (Avila and cal stages. To do so, the technique known as electrical penetration Panizzi 1995, Chocorosqui and Panizzi 2004), which also feeds on graph (EPG) or electropenetrography is useful; in this technique, any the reproductive structures of host plants. On wheat plants, piercing-sucking insect and a plant are made part of a simple electri- Triticum aestivum L., D. furcatus damages the crop in Southern cal circuit where a low current flows through a circuit, composed of Brazil causing seed yield reduction (Chocorosqui and Panizzi 2004, plant and feeding insect. Panizzi et al. 2016), where it has been observed to increase in abun- Both probing and nonprobing activities are captured by the EPG dance. Another pentatomid, Thyanta perditor (F.), has also been system and are presented as waveforms on computer screen V C The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 journals.permissions@oup.com by Ed 'DeepDyve' Gillespie user on 17 July 2018 2 Journal of Insect Science, 2017, Vol. 17, No. 2 (Tjallingii 1978, Walker 2000). For pentatomids, despite their eco- head-stage amplifiers (channels) were kept inside a Faraday cage. At nomic importance, only three species have had their feeding behav- the vegetative stage, the wheat seedlings were kept with their stems ior characterized using EPG [Edessa meditabunda (F.), and vertically positioned, whereas at the reproductive stage, the wheat Piezodorus guildinii (Westwood) on soybean plants (Lucini and plants containing a selected ear head were laid down along a plastic Panizzi 2016 and Lucini et al. 2016, respectively) and D. melacan- support (11  11 cm) and held in place using strips of Parafilm thus on maize seedlings (Lucini and Panizzi 2017)]. This is the (Pechiney Plastic Packaging, Menasha, WI). The plastic support was fourth study applying EPG technology to stink bugs, in this case, held horizontally by an alligator clip connected to a “helping hand” D. furcatus on wheat plants. It should yield valuable information holder. on its feeding on an economic host plant with potential to be used Waveforms caused by changes in electrical origins, i.e., on its chemical control targeting the action of systemic Resistance and electromotive force (emf) components, were cap- insecticides. tured and digitized at a sample rate of 100 Hz per channel using a The objectives of this study were, therefore, to 1) create a DI-710 (Dataq Instruments, Akron, OH) and recorded by using a waveform library characterizing the EPG waveforms produced by HP Pentium notebook with WinDaq Lite software (Dataq). Offset D. furcatus feeding on wheat plants during vegetative (stem) and and gain settings were adjusted for optimum view of the wave- reproductive (ear head) stages, 2) determine the biological mean- forms and to avoid rectifier fold-over to retain native waveform ings of each waveform recorded via electrical characteristics and polarity after rectification (Backus and Bennett 2009). To charac- histological studies; and 3) determine the feeding sites exploited by terize the waveforms, we considered the following characteristics: the bug on the vegetative and reproductive plant stages. shape, frequency, amplitude, and electrical origin (R and emf components). The frequency and amplitude were manually calcu- lated by expanding the x-axis to minimum compression and counting (see details of the methodology by Lucini and Panizzi Materials and Methods 2016). In addition, we used histological techniques to correlate Stink Bugs and Wheat Plants the waveforms and feeding sites, as done previously with three Adults of D. furcatus were field-collected at the Embrapa Wheat other species of pentatomids (Lucini and Panizzi 2016, 2017; Research Center during May to July 2016 located in Passo Fundo, Lucini et al. 2016). 0  0 RS, Brazil (28 15 S, 52 24 W). Stink bugs were weekly collected on wheat plants, taken to the laboratory and placed into rearing cages (25  20  20 cm), lined with filter paper to develop colonies. Experimental Design The cages were kept in a walk-in chamber at 256 1 C, 656 10% In the EPG experiment, we applied two different input impe- 7 9 RH, and a photoperiod of 14:10 (L:D) h. dance (Ri) levels, 10 and 10 Ohms, and a standardized voltage A standard food source was provided for the stink bugs, com- level of 50 mV of AC. Eighty insects were successfully recorded, posed of a mixture of green bean pods, Phaseolus vulgaris L., raw i.e., 40 stink bugs per Ri level (20 during vegetative stage and 20 shelled peanuts, Arachis hypogaea L., mature seeds of soybean, during reproductive stage at each Ri level). The stink bugs were Glycine max (L.) Merrill, and wheat seedlings; the mixture was monitored undisturbed for an 8-h access period, in a closed room replaced once per week. During this time, eggs were collected and with controlled conditions (256 2 C) and continuous light. placed inside of small plastic boxes (11  11  3.5 cm) to raise Representative waveforms excerpts at each Ri level were nymphs to obtain adults to be used in the experiments. assembled using Microsoft Power Point (Microsoft Corporation, Wheat seeds cv. BRS Parrudo (Embrapa) were seeded weekly in Redmond, WA). small (100 ml) and big (2 liters) pots kept in the greenhouse. Plants in stage 3 (V3: tillering stage) grown in small pots and plants in stage Histological Analysis to Determine the Stylet and/or Salivary Sheath 11.1 (R11.1: milk grain stage) (Large 1954) grown in big pots were Position in Plant Tissue separated and used in EPG recordings during vegetative and repro- Plant histology was conducted to determine the position of stylet ductive stages, respectively. Plants were grown using a standard oxy- and/or salivary sheath tip of D. furcatus on stem andonear soil prepared for greenhouse studies at Embrapa. head, which allowed us to correlate each feeding site with the different waveforms observed during EPG AC-DC recordings. EPG Recordings and Analysis of the Waveforms For that, a set of D. furcatus adult-females were recorded at Ri The feeding behavior of D. furcatus on wheat plants was monitored 10 Ohms and 50 mV AC signal. The EPG monitor was turned using a four-channel alternating current (AC)-direct current (DC) off every time a specific waveform was observed, and then the monitor (Backus and Bennett 2009; EPG Technologies, Inc., stylets were carefully cut using an entomological micro-scissors. Gainesville, FL). Before starting EPG recordings, adult-females (of After that, the plant tissues containing severed stylets were proc- approximately 2-wk old originated from eggs laid by field-collected essed to prepare semi-permanent slides according to Lucini and adults) were removed from the laboratory colony, placed in a small Panizzi (2016). plastic box (11  11  3.5 cm) lined with filter paper and starved For the stem histology, we used wheat plants in stage 8 (–8 - flag for ca.15 h before wiring. Then, the stink bugs were wired according leaf visible) (Large 1954), because the stem is more rigid allowing to the methodology described by Lucini and Panizzi (2016). This clean cuts of plant tissue (the feeding behavior and waveforms methodology consists in sanding the cuticle of the pronotum of bugs recorded in younger and older plants stages were the same). The to improve the wire attachment. position of the stylet tips and/or salivary sheath tips in stem and in After this procedure, stink bugs were individually connected to ear head structures were determined based on 10 samples for wave- an EPG amplifier and placed on stem wheat during vegetative stage form Df1a, 12 for Df2, 11 for Df3a, and 10 for Df4a. Digital images and on ear head during reproductive stage. To close the electrical were captured using Olympus BX50 (Shinjuku, Tokyo, Japan) circuit, the copper plant electrode (3-cm long) was inserted into the microscope coupled with a Sony DXC 107A video camera (Minato, soil of a pot containing each plant. Insects, plants, and the four Tokyo, Japan) linked to a computer. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 3 Results make the histological sections, we also observed the presence of Df1b, because stems used to make the cuts were more developed General Overview of Dichelops furcatus Waveforms on Wheat and rigid (V8 stage). In general, both Df1a and Df1b were more Plants 7 9 clearly distinguished at Ri 10 Ohms (low Ri level) than at 10 The waveform coarse structure recorded for D. furcatus on wheat Ohms (high Ri level), thus supporting R as their primary electrical plants on vegetative and reproductive stages included nine different origin (Table 1). waveforms, which represented non-probing and probing activities. Df1a always preceded the waveform Df2 (see Ingestion Phase) Regarding the nomenclature, probing waveform types were denoted recorded on wheat stem (Figs. 1C and E and 2A)and wasalso often as Df [from Dichelops furcatus] plus a number and subtypes by an observed before the waveform Df3a (see Ingestion Phase) (Figs. 3B additional, lower-case letter, as used for other pentatomids (Lucini and 4B). Otherwise, on ear head, the waveforms Df2 and Df4a (see and Panizzi 2016, 2017; Lucini et al. 2016). below) were preceded by Df1a or Df1b. On stem and on ear head, the Two of those waveforms recorded represent nonprobing behav- waveforms Df1 (both subtypes) and Df2 were easily distinguished ior, namely, Z and Np, whereas probing waveforms were repre- from each other during recordings, and also between Ri levels (Figs. sented by another four waveforms, namely, Df1, Df2, Df3, and Df4. 1E and 2A). However, Df1 (both subtypes), Df3a (on stem), and All these waveforms, except Df2, were sub-divided into two differ- Df4a (on ear head) were occasionally not clearly distinguished from ent subtypes each, as follows: Df1a and Df1b, Df3a and Df3b, and one another, mainly when recorded at Ri 10 Ohms (Fig. 4B)(see Df4a and Df4b. The probing waveforms were grouped in two differ- Df3 and Df4). ent families: pathway (P) and ingestion (I). The family P consisted of two waveforms (Df1a and Df1b), and family I consisted of five waveforms (Df2, Df3a, Df3b, Df4a, and Df4b). In both plant struc- Ingestion Phase. Family I (Waveforms Df2, Df3, and Df4) tures evaluated (stem and ear head), waveforms presented similar The family I comprised five different waveforms, labeled Df2, Df3a, characteristics (electrical and appearance); therefore, all waveforms Df3b, Df4a, and Df4b. were grouped in the same table (Table 1). Waveform Df2. Df2 was always observed immediately after Nonprobing Waveforms. Represented by two waveforms, Z and waveform Df1a on stem (Fig. 1E), and Df1a (most frequently) and Np (Fig. 1A), nonprobing waveforms that were visually correlated Df1b on ear head (Fig. 2A) of wheat plants. The waveform Df2 was with their biological meanings. Waveform Z occurred when the bug composed of repetitive wave portions interspersed with peaks, in was standing still on the plant surface and was characterized by a general, downward oriented, which occurred at regular intervals very low amplitude without variation in appearance between Ri lev- over time (Figs. 1A, C, D, F and 2A and D, peaks and waves are els applied. Therefore, Z wave represented the baseline. Np was defined in Fig. 1C). observed when the bug was walking on the plant surface; it was 7 9 At both Ri levels (10 and 10 Ohms) and also on both plant characterized by irregular peaks with variable amplitude according structures evaluated, the appearance of waveform Df2 and its to Ri level applied. Results suggested a strong emf-component to Np peak orientation were similar, except the amplitudes varied wave although R was also present (Table 1). according to Ri level applied. Peak amplitude value was lower Probing Waveforms. Two main waveform families (P and I) 7 9 when applied Ri 10 Ohms (28%) compared to Ri 10 Ohms were described during feeding behavior of D. furcatus on stem and (60%) (Table 1). Regarding frequency, Df2 showed a regular on ear head of wheat plants, and they comprised seven different pattern with means of 3.1 Hz at both Ri levels. Thus, waveform types/subtypes, namely, Df1a, Df1b, Df2, Df3a, Df3b, Df4a, and Df2 presented a mixture of electrical components, with peaks Df4b. These will be described in the order in which they occurred originating from R-component, because they were clearly distin- during the feeding activities of D. furcatus on wheat plants. All guished at Ri 10 Ohms, as well as emf-component, because 7 9 probing waveforms, at both Ri levels (10 and 10 X), occurred peaks were still clearly distinguished and separated from wave below 0 V (i.e., were monophasic negative). portion at high Ri level (10 Ohms). Wave portions were more emphasized at Ri 10 Ohms; thus, waves were emf-dominated Pathway Phase. Family P (Waveform Df1) (compare Fig. 1C and F). Df1 occurred after nonprobing waveforms, Z or Np, and it was div- Waveform Df3. Waveform Df3 was divided into two subtypes, ided in two subtypes: Df1a and Df1b. Df1a was recorded in both Df3a and Df3b (Figs. 3A–E and 4A–F), which were recorded only plant structures evaluated (stem and ear head); it was characterized when D. furcatus fed on wheat stem. Df3a was often recorded after by irregular-peaks without a clear pattern (Figs. 1B and E and 2A– a short event of waveform Df1a (Figs. 3B and 4B); however, the sep- C), and almost always presented the largest amplitude value in the aration between them sometimes was not clear. Df3a was character- probe among all the waveforms registered. The subtype Df1b was ized by a generally stereotypical pattern with peaks oriented both recorded only on wheat ear head, and it was characterized by a upward and downward at both Ri levels (Figs. 3C and 4C). Df3a more distinct pattern (Fig. 2A–C) of random overlying/superim- showed a similar frequency at both Ri levels (means of 3.1 and 7 7 9 posed on low- to medium-amplitude waves (35 and 23% at Ri 10 3.0 Hz at Ri 10 and 10 Ohms, respectively). Nevertheless, Df3a and 10 Ohms, respectively), with a high and regular frequency, on was greatly variable in appearance (primarily amplitude) not only average ranging from 4.7 to 5.1 Hz between Ri levels (Table 1). across Ri levels, but also among individual bugs and even within the Df1b waveform always occurred after Df1a or alternating among same recording. As Ri level increased, the amplitude value decreased 7 9 events of Df1a (e.g., Df1a ! Df1b ! Df1a ! Df1b, and so on) (Fig. (57% at Ri 10 Ohms and 31% at Ri 10 Ohms) (Table 1). Thus, 2B and C). Df3a was R-dominated. The subtype Df1b was not always present, and it was visually Waveform Df3b was characterized as a regular wave of short correlated with protraction and retraction movements of the stylets event durations (in general, a few seconds) at both Ri levels, always in the ear head tissue, especially when the bug inserted its stylets in occurring interspersed with Df3a, as follows: Df3a ! Df3b ! Df3a the glume (outer layer) and in the lemma (layer below the glume ! Df3b ! and so on (Figs. 3D and E and 4D–F). Df3b was not that surrounds the seed). However, during recordings on stems to always clearly from Df3a within recordings, or it did not occur. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 4 Journal of Insect Science, 2017, Vol. 17, No. 2 Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Table 1. Summary of EPG AC-DC waveforms, their main electrical characteristics, and suggested biological meanings for each waveform recorded during probing and nonprobing activities of D. furcatus on stem and on ear head of wheat plants Phase Family Type or Plant structure Amplitude (%) Frequency (Hz) Electrical origin Suggested biological subtype observed meaning 7 9 7 9 Ri 10 Ri 10 Ri 10 Ri 10 Nonprobing – Z Stem/ear head Flat Flat – – – Standing still on the plant surface Np Stem/ear head Low-medium Medium-high Irregular Irregular Mostly emf/some R Walking on the plant surface Pathway P Df1a Stem/ear head 100 100 Irregular Irregular Mostly R Stylet penetration and salivary sheath secretion Df1b Ear head 35 (16–100) 23 (13–37) 5.1 (4.2–5.7) 4.7 (3.8–5.8) Mostly R Bug encountering a rigid cell layer requiring stylet protraction and retraction Ingestion I Df2 Stem/ear head 28 (11–53) 60 (29–100) 3.1 (2.6–3.9) 3.1 (2.4–4.2) Mixed; peak ¼ R/emf; Xylem sap ingestion wave ¼ emf-dominated Salivation/ I Df3a Stem 57 (37–90) 31 (19–52) 3.1 (2.8–3.5) 3.0 (2.9–3.2) Mostly R; emf is also Cell laceration, enzymatic ingestion present maceration of stem tissues and probably ingestion Ingestion I Df3b Stem 15 (7–19) 19 (15–22) 3.9 (3.5–4.2) 4.1 (4.0–4.2) Mostly emf-component Short ingestion of macer- ated stem tissues Salivation I Df4a Ear head 51 (32–79) 35 (18–55) Mostly irregular þ Mostly irregular þ Mostly R; emf is also Cell laceration, enzymatic burst regular peaks burst regular peaks present maceration of seed 3.5 Hz 3.8 Hz endosperm Ingestion I Df4b Ear head 20 (9–34) 27 (11–65) 3.5 (2.7–4.7) 4.0 (3.0–5.1) Mostly emf-component Ingestion of macerated seed endosperm Journal of Insect Science, 2017, Vol. 17, No. 2 5 Fig. 1. Waveforms recorded using EPG AC-DC from D. furcatus on stems of wheat plants during vegetative stage (–3 – tillering stage) at Ri 10 Ohms (A–C) and Ri 10 Ohms (D–F). (A) Compressed overview of a probing event (40 min), showing waveforms Df1a (pathway) and Df2 (xylem sap ingestion). (B) Expanded view of waveform Df1a. (C) Expanded view of waveform Df2 and definition of peak and wave portions. (D) Compressed overview of several waveform events (35 min), showing Df1a and Df2. (E) Expanded view of waveform Df1a and beginning of waveform Df2. (F) Expanded view of waveform Df2. (A has Windaq com- pression 400 [80 s/vertical div.], gain 16; B has compression 25 [5 s/vertical div.], gain 8; C has compression 3 [0.6 s/vertical div.], gain 32; D has compression 300 [60 s/vertical div.], gain 8; E has compression 50 [10 s/vertical div.], gain 8; F has compression 5 [1 s/vertical div.], gain 8). Arrowheads indicate beginning or end of a probe. 7 9 At both Ri levels, 10 and 10 Ohms, Df3b showed low-amplitude which were recorded only when D. furcatus fed on wheat ear head values (15 and 19%, respectively), as well as frequency values (3.9 (seed endosperm). Df4a was, in general, preceded by a short Df1b and 4.1 Hz, respectively) (Table 1). Df3b, in the same way as Df3a, wave, and sometimes by Df1a; however, in both cases, it was often was quite variable across Ri levels, among different bugs and also hard to separate the waveforms Df1a/Df1b and Df4a within the within the same bug recording. recording, especially at Ri 10 Ohms (Fig. 6A). Df4a showed irregu- Regarding the electrical origin of the waveform Df3, it presented larity in appearance, different from Df2 and even Df3a (which latter a mixture of electric components varying according to the wave sub- represents the same feeding strategy of the Df4a wave, but per- type. Subtype Df3a, in general, showed a decrease in amplitude formed in different tissues). Df4a presented a high-amplitude value 9 7 9 value when applied a Ri of 10 Ohms compared to a low Ri level when Ri of 10 Ohms was used (means of 51%) compared to 10 (10 Ohms), indicating a R-component. However, this subtype was Ohms (35%). Therefore, Df4a originated mostly from R- still clearly visible at 10 Ohms, indicating the emf-component, as component. well. Df3b was more emphasized at Ri 10 Ohms, thus emf is the In addition, Df4a was characterized not only by overlying main component (Table 1). irregular frequency, composed of peaks negative and positive ori- Waveform Df4. Waveform Df4 was also divided into two differ- ented at both Ri levels (Figs. 5A–C and 6A–C, Table 1)but also ent subtypes, namely, Df4a and Df4b (Figs. 5A–D and 6A–D), with underlying portions that were more regular (frequency Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 6 Journal of Insect Science, 2017, Vol. 17, No. 2 Fig. 2 Waveforms recorded using EPG AC-DC from D. furcatus on ear head of wheat plants during reproductive stage (R11.1: milk grain stage) at Ri 10 Ohms. (A) Compressed overview of several waveform events (50 min), showing Df1 (pathway) and Df2 (xylem sap ingestion). (B) Expanded view of both Df1 subtypes, Df1a and Df1b. (C) Details of the waveforms Df1a and Df1b. (D) Expanded view of waveform Df2. (A has Windaq compression 400 [80 s/vertical div.], gain 8;B has compression 10 [2 s/vertical div.], gain 8; C has compression 3 [0.6 s/vertical div.], gain 16; D has compression 3 [0.6 s/vertical div.], gain 16). Arrowheads indicate beginning or end of a probe. ranging from 3.5 to 3.8 Hz). Sometimes, within the same record- visible at 10 Ohms, indicating the emf-component, as well. Df4b ing, peaks were positive and negative oriented, and this inversion was more emphasized at Ri 10 Ohms, thus emf is the main com- was not due to rectifier fold-over (see Materials and Methods). ponent (Table 1). Furthermore, Df4a was variable in appearance between Ri levels, but also among bugs and within the recording of the same insect. In contrast, Df4b showed a highly regular pattern of short event Correlations of the Waveforms and Their Specific Feeding Sites via duration compared to Df4a, always occurring interspersed with Histological Analyses the latter (Figs. 5C and Dand 6C and D). Df4b presented a mean Df1. In the waveform Df1a recorded during the feeding activities of 7 9 amplitude value (20 and 27% at Ri 10 and 10 Ohms, respec- D. furcatus on wheat plant, the presence of a salivary sheath sur- tively) and a regular frequency, ranging from 3.5 to 4.0 Hz rounding the stylets was observed in all histological sections on stem between Ri levels (Table 1). (n¼ 10) (Fig. 7A). Furthermore, the tip of the salivary sheath and/or Df4 presented a mixture of electric components varying stylets terminated in cells of the parenchyma, confirming that Df1a according to the wave subtype. Df4a showed a decrease in ampli- represents pathway phase (insertion and penetration of the stylets 9 7 tude value when applied a Ri of 10 Ohms compared to a 10 deep into the plant tissue and secretion of the salivary sheath). Ohms, indicating a R-component. This subtype was still clearly However, there was an interesting difference about secretion of the Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 7 Fig. 3. Waveform Df3 recorded using EPG AC-DC from Dichelops furcatus on stems of wheat plants during vegetative stage (V3: tillering stage) at Ri 10 Ohms. (A) Compressed overview of a probing event (60 min) showing waveforms Df1a (pathway) and Df3 (laceration/maceration activities and ingestion). (B) Expanded view of waveform Df1a and beginning of waveform Df3a. (C) Expanded view of waveform Df3a. (D) Details of the waveforms Df3a and Df3b. (E) Expanded view of waveform Df3b. (A has Windaq compression 500 [100 s/vertical div.], gain 16; B has compression 30 [6 s/vertical div.], gain 16; C has com- pression 3 [0.6 s/vertical div.], gain 16; D has compression 20 [4 s/vertical div.], gain 8; E has compression 2 [0.4 s/vertical div.], gain 16). Arrowheads indicate beginning or end of a probe. salivary sheath during Df1a. When this waveform was recorded seed endosperm (n¼ 2), the salivary sheath was not completely before Df2 (n¼ 10, stem), the salivary sheath was completely formed (Fig. 7D–F). secreted (Fig. 7B), i.e., the full length of the stylets. However, when Df2. Cuts made during the waveform Df2 on stem showed that Df1a was recorded before Df3a on stem (n¼ 5), or before Df4a on in all histological sections (n¼ 10), the tip of the salivary sheath Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 8 Journal of Insect Science, 2017, Vol. 17, No. 2 Fig. 4. Waveform Df3 recorded using EPG AC-DC from Dichelops furcatus on stems of wheat plants during vegetative stage (V3: tillering stage) at Ri 10 Ohms. (A) Compressed overview of a probing event (50 min) showing waveforms Df1a (pathway) and Df3 (laceration/maceration activities and ingestion). (B) Expanded view of waveform Df1a and beginning of waveform Df3a. (C) Expanded view of waveform Df3a. (D) Details of the waveforms Df3a and Df3b. (E-F) Expanded views of waveform Df3b. (A has Windaq compression 400 [80 s/vertical div.], gain 16; B has compression 6 [1.2 s/vertical div.], gain 16; C has com- pression 3 [0.6 s/vertical div.], gain 32; D has compression 10 [2 s/vertical div.], gain 32; E and F have compression 2 [0.4 s/vertical div.], gain 32). Arrowheads indicate beginning or end of a probe. and/or stylets were positioned inside xylem cells (Fig. 7B). During not allow clarification of stylet tip positions, especially whether they Df2 in the wheat ear head, cuts made in the lemma layer (n¼ 2) were inside the xylem vessels or not. showed the stylets close to the xylem vessels (longitudinal position) Df3. During the Df3a wave (observed only in stems), the tip of in the plant tissue (Fig. 7C). However, the histological images did the stylets (n¼ 5) was always positioned in the parenchyma tissue Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 9 Fig. 5. Waveform Df4 recorded using EPG AC-DC from D. furcatus on ear head of wheat plants during reproductive stage (R11.1: milk grain stage) at Ri 10 Ohms. (A) Compressed overview of a probing event (140 min) showing waveforms Df1 (pathway) and Df4 (laceration/maceration activities and ingestion). (B) Expanded view of waveform Df4a. (C) Details of the waveforms Df4a and Df4b. (D) Expanded view of waveform Df4b. (A has Windaq compression 1,200 [240 s/ vertical div.], gain 8; B and D have compression 3 [0.6 s/vertical div.], gain 16; C has compression 30 [6 s/vertical div.], gain 8). Arrowheads indicate beginning or end of a probe. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 10 Journal of Insect Science, 2017, Vol. 17, No. 2 Fig. 6. Waveform Df4 recorded using EPG AC-DC from D. furcatus on ear head of wheat plants during reproductive stage (R11.1: milk grain stage) at Ri 10 Ohms. (A) Compressed overview of a probing event (60 min) showing waveforms Df1 (pathway) and Df4 (laceration/maceration activities and ingestion). (B) Expanded view of waveform Df4a. (C) Details of the waveforms Df4a and Df4b. (D) Expanded view of waveform Df4b. (A has Windaq compression 600 [120 s/vertical div.], gain 4; B has compression 3 [0.6 s/vertical div.], gain 16; C has compression 30 [6 s/vertical div.], gain 16; D has compression 3 [0.6 s/vertical div.], gain 32). Arrowheads indicate beginning or end of a probe. (Fig. 7D) and there was also an incomplete secretion of the salivary endosperm, and the formation of an incomplete salivary sheath sheath when this wave was recorded (Fig. 7E). (Fig. 7F, red arrow indicates the end of the salivary sheath). Df4. In the waveform Df4a (observed only in seed), the histologi- Moreover, cuts made of fresh stems, after recording of a Df3 cal sections (n¼ 2) showed the stylet tips positioned inside the seed wave event (n¼ 6), revealed a visibly damaged area (opaque region Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 11 Fig. 7. Cross sections of wheat stems (V8 stage: flag leaf visible) and ear head structures (R11.1 stage: milk grain stage) containing severed stylets and salivary sheath of the stink bug D. furcatus. (A) Salivary sheath ending in the parenchyma tissue during waveform Df1a on stem. (B) Salivary sheath and stylet tips ending in the xylem vessels during waveform Df2 on stem. (C) Stylet tips near of xylem vessels during waveform Df2 on ear head (lemma layer). (D) Stylet tips in paren- chyma tissue during waveform Df3a on stem. (E) Secretion of a flange (externally on the stem) and of an incomplete salivary sheath (internally in the tissue) dur- ing waveform Df3a on stem. (F) Stylet tips inside the seed endosperm during waveform Df4a and an incomplete salivary sheath secreted internally in the tissue (indicated by the red arrow in the detail box). Ep¼ stem epidermis, Ep lemma¼ lemma epidermis, Pa¼ parenchyma, Xy¼ xylem, Ph¼ phloem. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 12 Journal of Insect Science, 2017, Vol. 17, No. 2 Fig. 8. Cross sections of fresh stem (V8 stage: flag leaf visible) and fresh seed (R11.1 stage: milk grain stage) of wheat plants containing cut stylets of D. furcatus. (A) Section of a fresh wheat stem showing damaged area (opaque region surrounded by the red line) after recording a Df3 wave event on stem. (B) Immature wheat seed containing the flange (externally on lemma layer) and cut stylets during waveform Df4 on seed. (C) Section of a fresh seed showing the damaged area (opaque region) after recording a Df4 wave event on seed endosperm. surrounded by the red line, Fig. 8A). Similarly, cuts made in fresh using the AC-DC electropenetrography EPG technique. Seven differ- seeds during the waveform Df4a (n¼ 8) showed the stylets posi- ent waveforms related to its probing behavior on stem (vegetative tioned in the seed endosperm, and the secretion of flanges externally stage: V3) and ear head (reproductive sta–ge: R11.1) of wheat plant on protection layers of the seed (Fig. 8B). In addition, cuts made in were described. fresh seeds, after a Df4 wave event, showed a visually damaged area Some similarities with other stink bugs already studied via EPG in the seed endosperm (region of opaque white color, Fig. 8C). were observed during feeding of D. furcatus in both plant structures, as well as some interesting peculiarities. One of the most relevant similarities is the use of the same feeding strategy on stem and on ear head of wheat, in this case, the cell rupture feeding strategy Discussion [Backus et al. 2005–, previously known as laceration and flush strat- In this study, the probing behavior of the pentatomid D. furcatus, egy (Miles 1972)]. However, the waveforms recorded for each feed- found attacking the wheat crop in southern Brazil, was evaluated ing site, even using the same strategy, were completely different in Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 13 appearance and electrical characteristics, which demonstrates the correlated via histological analysis with activities in the xylem ves- complexity of the D. furcatus feeding process. In addition, when the sels, more specifically, sap ingestion. This wave was strongly similar same bug ingests from xylem vessels, it uses another feeding strat- in appearance to the Dm2 wave of D. melacanthus feeding on maize egy, known as salivary the sheath strategy, which was demonstrated stem (Lucini and Panizzi 2017), which was also correlated to xylem in the histological analyses. sap activities. In addition, both waves (Df2 and Dm2) share some Previous studies have shown that the same stink bug species may electrical characteristics, such as high-amplitude value at Ri 10 use only one of those strategies, such as E. meditabunda which uses Ohms, and a mixture of electrical origins for the peaks (R and emf) the salivary sheath when feeding on soybean stems (Lucini and and waves (emf). Panizzi 2016); or both strategies, as observed for P. guildinii on soy- In fact, all D. furcatus bugs evaluated in both plant structures bean plants (Lucini et al. 2016) and for D. melacanthus on maize ingested from the xylem vessels. Probably this is a strategy to avoid seedlings (Lucini and Panizzi 2017). Similarly, when feeding on dehydration and to maintain body water balance, as reported for wheat plants D. furcatus also uses both feeding strategies, switching other piercing-sucking insects such as aphids and psyllids (Spiller strategy according to the feeding site exploited. et al. 1990, Bonani et al. 2010, Pompon et al. 2010), and also for Pathway phase is represented by a single waveform, Df1, which other species of stink bug (Lucini and Panizzi 2016). In case of inges- was divided into two subtypes, Df1a and Df1b. Df1a was the first tion of water during feeding on wheat seed, this may also be a strat- waveform related to feeding of D. furcatus on the wheat plant, and egy for nutrient dilution, since the seed is highly concentrated in was observed on both plant structures (stem and ear head), whereas nutrients (Panizzi and Slansky 1985), as observed for P. guildinii Df1b was recorded only when bugs fed on wheat ear head. The sub- feeding on soybean seed endosperm (Lucini et al. 2016). type Df1a represents the insertion and deep penetration of the stylets During ingestion phase using cell rupture feeding on stem (wave- into the plant tissue and also the secretion of gelling saliva to form forms Df3a and Df3b), the bug (during the first subtype) makes the salivary sheath as observed in all the histological images from rapid, continuous, and deep protraction and retraction of the stylets stems of wheat during this waveform. In addition, the secretion of in the stem tissue. Such activity is reported in the literature to occur salivary flanges (external deposition of gelling saliva) was frequently during the activities of laceration (mechanical action of stylets) and observed on stem and also on ear head of wheat. The subtype Df1b maceration (enzymatic –action: digestive enzymes secreted in watery probably represents the bug encounter of a rigid cell layer requiring saliva) (Miles 1972, 1987), which are tactics of the cell rupture strat- stylet protraction and retraction. egy (Backus et al. 2005). This behavior was also noted for other Similar to the present study, other EPG studies (Lucini and sucking insects, such as the leafhoppers Empoasca fabae (Harris), Panizzi 2016, 2017; Lucini et al. 2016) with other species of penta- Empoasca kraemeri (Ross & Moore) (Calderon and Backus 1992), tomids showed that pathway phase was correlated, via histological and Empoasca vitis Go ¨ the (Jin et al. 2012), as well as stink bugs, studies, with salivary sheath secretion. It can be completely or Lygus hesperus Knight (Cline and Backus 2002), P. guildinii (Lucini incompletely formed, depending on the feeding strategy used by the et al. 2016), and D. melacanthus (Lucini and Panizzi 2017). In the bug. Complete secretion occurs when the bug uses the salivary last species, in one of the waveforms recorded on maize seedlings sheath strategy to ingest from vessels of the vascular tissues: xylem (called Dm3a), the bug moved its stylets in the stem tissue similar to and phloem (Bonani et al. 2010; Backus et al. 2013; Pearson et al. that observed herein for the waveform Df3a of D. furcatus on wheat 2014; Lucini and Panizzi 2016, 2017; Lucini et al. 2016; Seo et al. stem. Moreover, during Df3a, there was the secretion of an incom- 2016); incomplete secretion occurs during the cell rupture feeding plete salivary sheath, as observed for D. melacanthus using the cell strategy (Miles 1972, Backus et al. 2005). rupture strategy (Lucini and Panizzi 2017). Likewise, it was observed in the histological images of D. furca- In the subtype Df3b, it was observed that the stylets remained tus on wheat stem that during feeding activities in xylem vessels, the motionless inside the plant tissue for a short period of time, and salivary sheath was completely formed and followed the pathway then, the stylets moved rapidly and vigorously in and out again the stylets to the vessels. However, during the cell rupture activities (wave Df3a). Therefore, Df3b represents the ingestion of degraded on stem and on seed, a partial salivary sheath was observed to be cellular contents (composed of cells from parenchyma and possibly formed. In the histological images of these plant structures during vascular vessels) via laceration and maceration activities, as cell rupturing, a salivary sheath surrounding the stylet was observed described during the Dm3b wave of D. melacanthus (Lucini and only at the beginning of its insertion point in the plant tissue, similar Panizzi 2017). However, the waveform Df3b was not clearly distin- to D. melacanthus on stem of maize seedlings (Lucini and Panizzi guished and/or observed in all recordings in which the Df3a wave 2017). was recorded. Thus, we believe that during Df3a, the ingestion proc- We believe that Df1b represents the stylet work to overcome ess may be occurring simultaneously with the laceration and macer- some physical barrier that makes the penetration of the stylets into ation activities of the stem cells, as described for D. melacanthus the plant tissue quite difficult. The structures that make up the during the Dm3a wave (Lucini and Panizzi 2017). wheat spikelet are rigid and are the only layers responsible for pro- In addition to the similarities in the behavior of the two stink tecting the seed. bugs D. melacanthus and D. furcatus, the subtypes Dm3a/Df3a and A waveform very similar to Df1b, namely, Pg1d, was described Dm3b/Df3b showed others similarities in appearance and in electri- for P. guildinii fed on different parts of the soybean pod. Pg1d was cal characteristics. In the histological images made during the Df3a, correlated with the bug forcing the stylets downward and subse- it was not possible to observe a damaged area or cells destruction in quently retracting them upward. This indicates the presence of a the insertion region of the stylets. However, in fresh sections of the rigid cell layer (which was observed in histological analyses of soy- wheat stem was possible to clearly observe a damaged area of opa- bean pods) that was difficult to penetrate with the stylets, requiring que white coloration. stylet laceration to reach the seed (Lucini et al. 2016). During the ingestion phase using cell rupture feeding in the During the ingestion phase, one of the waveforms recorded was wheat ear head (seed endosperm: waveforms Df4a and Df4b), Df2, which was recorded when the bug fed on stem and on any D. furcatus made a continuous and deep movement of the stylets in structure of the ear head (palea, glume, lemma, or awn). Df2 was the wheat seed endosperm as reported during the waveform Pg3a Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018 14 Journal of Insect Science, 2017, Vol. 17, No. 2 of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings. for P. guildinii feeding on soybean seed endosperm (Lucini et al. Entomol. Exp. Appl. 134: 35–49. 2016). Therefore, the Df4a wave represents the dissolution of endo- Buntin, G. D., and J. K. Greene. 2004. Abundance and species composition sperm cells via laceration (mechanical action) and maceration (enzy- of stink bugs (Heteroptera: Pentatomidae) in Georgia winter wheat. matic action) for future ingestion. During this waveform, a salivary J. Entomol. Sci. 39: 287–290. sheath was observed surrounding the stylets only at the beginning of Calderon, J. D., and E. A. Backus. 1992. Comparison of the probing behaviors the insertion point in the seed endosperm, i.e., the salivary sheath of Empoasca fabae and E. kraemeri (Homoptera: Cicadellidae) on resistant was incompletely secreted inside the tissue, which was observed in and susceptible cultivars of common beans. J. Econ. Entomol. 85: 88–99. the histological sections. Chiaradia, L. A., A. Rebonatto, M. A. Smaniotto, M. R. F. Davila, and C. N. In cuts made of fresh wheat seeds, shortly after the recording of Nesi. 2011. Arthropods associated with soybean crops. Rev. Ci^ enc. the Df4 wave, we observed a damaged area in the seed endosperm Agrovet. 10: 29–36. Chocorosqui, V. R., and A. R. Panizzi. 2004. Impact of cultivation systems on caused by the activities of laceration, maceration and ingestion; this Dichelops melacanthus (Dallas) (Heteroptera: Pentatomidae) populations is similar to what was reported for P. guildinii during the waveform and damage and its chemical control on wheat. Neotrop. Entomol. 33: Pg3 recorded on soybean seed endosperm (Lucini et al. 2016). 487–492. During the subtype Df4b, the stylets of D. furcatus were motion- Cline, A. R., and E. A. Backus. 2002. Correlations among AC electronic moni- less inside the seed tissue for a short period of time (which represents toring waveforms, body postures, and stylet penetration behaviors of the ingestion period); after that, they moved their stylets again to Lygehaviorrus (Hemiptera: Miridae). Environ. Entomol. 31: 538–549. break more cells (Df4a wave). This activity was continually Ferreira, E., and P. M. Silveira. 1991. Dano de Thyanta perditor (Hemiptera: repeated; however, the bug spent most of the time in laceration and Pentatomidae) em trigo (Triticum aestivum L.). An. Soc. Entomol. Bras. 20: maceration activities to prepare the food for latter ingestion, which 165–171. usually took a short time. Frota, R. T., and R. S. S. Santos. 2007. Pentatomidae bugs associated with sunflower crops in the northwest of Rio Grande do Sul state and the action In conclusion, our results demonstrated that D. furcatus feeding of Euschistus heros (Fabricius, 1794) (Hemiptera: Pentatomidae) on sun- on wheat plants during vegetative (stem) and reproductive (ear flower seeds. Biotemas. 20: 65–71. head) stages use two different feeding strategies. When in xylem ves- Jin, S., Z. M. Chen, E. A. Backus, X. L. Sun, and B. Xiao. 2012. sels (ingestion of xylem sap) on wheat stem (and probably on ear Characterization of EPG waveforms for the tea green leafhopper, Empoasca head although not histological demonstrated in this work), D. furca- vitis Go ¨ the (Hemiptera: Cicadellidae), on tea plants and their correlation tus used the salivary sheath strategy, with its complete salivary with stylet activities. J. Insect Physiol. 58: 1235–1244. sheath secretion. In contrast, when on seed endosperm and again on Koch, R. L., A. Rich, and T. Pahs. 2016. Statewide and season-long surveys stem, the bug used the cell rupture strategy as well, using the lacera- for Pentatomidae (Hemiptera: Heteroptera) of Minnesota wheat. Ann. tion and maceration tactics to break cell pockets for ingesting. At Entomol. Soc. Am. 109: 396–404. this time, a salivary sheath was also observed in the plant tissue, Large, E. C. 1954. Growth stages in cereals. Illustration of the Feekes scale. Plant Pathol. 3: 128–129. however, not completely formed. These results demonstrate the Lucini, T., and A. R. Panizzi. 2016. Waveform characterization of the soybean plasticity of this insect’s feeding behavior, which involve a switch in stem feeder Edessa meditabunda: overcoming the challenge of wiring penta- strategy according to the feeding site exploited. tomids for EPG. Entomol. Exp. Appl. 158: 118–132. Lucini, T., and A. R. Panizzi. 2017. Feeding behavior of the stink bug Dichelops melacanthus (Heteroptera: Pentatomidae) on maize seedlings: an Acknowledgments EPG analysis at multiple input impedances and histology correlation. Ann. This study was partially supported by a National Council of Research and Entomol. Soc. Am. 110: in press. Doi.org/10.1093/aesa/saw070. Technology of Brazil grant 471517/2012-7 to A.R.P. and by a scholarship Lucini, T., A. R. Panizzi, and E. A. Backus. 2016. Characterization of an EPG from CAPES (Ministry of Education) of Brazil to T.L. in order to obtain the waveform library for redbanded stink bug, Piezodorus guildinii (Hemiptera: Doctor of Science (Entomology) degree at the Federal University of Paranaat Pentatomidae), on soybean plants. Ann. Entomol. Soc. Am. 109: 198–210. Curitiba, Brazil. We also thank the Embrapa Unit at Passo Fundo, RS, for Miles, P. W. 1972. The saliva of Hemiptera. Adv. Insect Physiol. 9: 183–125. support. Appreciation is also extended to Elaine Backus and Paula Mitchell Miles, P. W. 1987. Plant-sucking bugs can remove the content of cells without for reviewing and earlier draft of the article. Approved by the Publication mechanical damage. Experientia. 43: 937–939. Committee of the Embrapa National Wheat Research Center, Passo Fundo, Panizzi, A. R., and F. Slansky, Jr. 1985. Legume host impact on performance RS, Brazil, under number 5408/2016. of adult Piezodorus guildinii (Westwood) (Hemiptera: Pentatomidae). Environ. Entomol. 14: 237–242. Panizzi, A. R., S. Corr^ea, D. L. Gazzoni, E. B. Oliveira, G. G. Newman, and S. References Cited G. Turnipseed. 1977. Insects of soybean in Brazil. Bol. Tec., n. 1. Embrapa, Avila, C. J., and A. R. Panizzi. 1995. Occurrence and damage by Dichelops Londrina. (Neodichelops) melacanthus (Dallas) (Heteroptera: Pentatomidae) on corn. Panizzi, A. R., T. Agostinetto, T. Lucini, and P. R. V. S. Pereira. 2016. Effect An. Soc. Entomol. Bras. 24: 193–194. of green-belly stink bug, Dichelops furcatus (F.) on wheat yield and develop- Backus, E. A., and W. H. Bennett. 2009. The AC-DC Correlation Monitor: ment. Crop Prot. 79: 20–25. New EPG design with flexible input resistors to detect both R and emf com- Pearson, C. C., A. Backus, H. J. Shugart, and J. E. Munyaneza. 2014. ponents for any piercing-sucking hemipteran. J. Insect Physiol. 55: Characterization and correlation of EPG waveforms of Bactericera cocker- 869–884. elli (Hemiptera: Triozidae): Variability in waveform appearance in relation Backus, E. A., M. Rangasamy, M. Stamm, H. J. McAuslane, and R. Cherry. to applied signal. Ann. Entomol. Soc. Am. 107: 650–666. 2013. Waveform library for chinch bugs (Hemiptera: Heteroptera: Pompon, J., D. Quiring, P. Giordanengo, and Y. Pelletier. 2010. 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Koenders, and W. F. Tjallingii. 1990. Xylem ingestion by aph–s – a the study of homopteran feeding behavior. Thomas Say Publications in strategy for maintaining water balance. Entomol. Exp. Appl. 55: 101–104. Entomology, Entomological Society of America, Lanham, MD. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/65/3775837 by Ed 'DeepDyve' Gillespie user on 17 July 2018

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