Direct Current Electrical Penetration Graph Research Articles

DC‐EPG assisted comparison of European spittlebugs and sharpshooters feeding behaviour on grapevine

AbstractXylem‐feeding is apparently the only requirement making an insect a competent vector of the bacterium Xylella fastidiosa, an organism responsible for the devastation of the Southern Italian olive forest and nowadays considered one of the most feared threats to agriculture and landscape in Europe, including vineyards. Here, we used the direct current‐electrical penetration graph (DC‐EPG) technique to compare and describe the feeding behaviour on grapevine of four xylem‐feeding species considered candidate vectors of X. fastidiosa widespread in Europe, namely two spittlebugs (the meadow spittlebug Philaenus spumarius and the spittlebug Neophilaenus campestris) and two sharpshooter leafhoppers (the rhododendron leafhopper Graphocephala fennahi and the green leafhopper Cicadella viridis). We created a standard for the analysis of EPG waveforms recorded with a DC‐EPG device, describing feeding activities performed by these insects from stylet insertion into the plant to withdrawal. This standard, along with freely available software, has been developed to harmonize the calculation of feeding behavioural parameters in xylem‐feeders. The most relevant differences between the two vector taxa were the probing frequency and the dynamics of xylem ingestion. Sharpshooters tended to perform significantly more probes than spittlebugs. In contrast, the latter spent longer times in low‐frequency xylem ingestion, characterized by scattered contractions of the cibarial dilator muscle interspersed with periods of pump inactivity. Cicadella viridis was the species displaying the highest frequency of the electrical pattern found to be associated with X. fastidiosa inoculation in spittlebugs (Xe). Feeding behavioural data presented here represent an important step forward for deepening our knowledge of xylem‐sap feeding insects' interaction with both the host plants and the bacterium they transmit.

The probing behaviour of the planthopper Delphacodes kuscheli (Hemiptera: Delphacidae) on two alternating hosts, maize and oat

AbstractDelphacodes kuscheli is the most important natural vector of Mal de Río Cuarto virus (MRCV) in Argentina, a Fijivirus (Reoviridae) that causes important economic losses on maize production. Although this planthopper does not breed successfully on maize, virus transmission occurs when adults migrate from oat, a winter host and a reservoir of the virus, to maize. Probing behaviour on both hosts was recorded and analysed using the direct current electrical penetration graph (DC‐EPG) system. Eight main waveforms were identified during probing behaviour by D. kuscheli. These waveforms were interpreted with reference to that described for delphacids and were related to putative probing activities: np (non‐penetration or non‐probe); N1, N2 and N3 (stylet pathway: penetration, salivation, stylet movement and extracellular activities near the phloem region); N4‐a (sieve element salivation); N4‐b (phloem sap ingestion); N5 (xylem activity); and N6 (derailed stylet mechanics). The EPG variables showed that on maize, D. kuscheli exhibited longer periods of non‐probe and longer time from first probe to first phloem activity, with a higher percentage of probing spent in stylet pathway and xylem phase than on oat. Furthermore, on maize, fewer insects showed phloem phase activities (N4‐a and N4‐b) and sustained phloem ingestion than on oat, and the time on phloem ingestion was shorter than on oat. The constrained behaviour of D. kuscheli on maize suggests that plant resistance factors may be involved.

Characteristics of sugarcane white leaf phytoplasma transmission by the leafhopper Matsumuratettix hiroglyphicus

AbstractThe leafhopper Matsumuratettix hiroglyphicus (Matsumura) (Hemiptera: Cicadellidae) is the most important vector of sugarcane white leaf (SCWL) phytoplasma that significantly affects the sugarcane crop in Asia. Here, we aimed to study the characteristics of SCWL phytoplasma transmission by M. hiroglyphicus. To this end, the stylet penetration activities performed during the acquisition access period (AAP) and inoculation access period (IAP) were investigated by the direct current electrical penetration graph technique and confirmed by quantitative polymerase chain reaction (qPCR). Additionally, the latent period (LP) of SCWL phytoplasma in the vector was determined by qPCR and localised by fluorescent in situ hybridisation. The results indicated that the acquisition of SCWL phytoplasma occurred during phloem ingestion (waveform D), whereas its inoculation was associated with salivation into the phloem sieve element (waveform C). The minimum AAP was 15 min and the minimum duration of phloem ingestion was 2.35 min. The minimum LP of SCWL phytoplasma in the vector was at least 14 days; then, SCWL phytoplasma moved to the salivary glands of the insect, enabling the transmission of the pathogen to the host plants. The minimum IAP for a successful transmission of SCWL phytoplasma to the host plants was 11–12 min, with a minimum duration of salivation into phloem of 1.35 min. The female vectors had higher SCWL phytoplasma copy numbers than the male vectors, and displayed faster AAP, IAP, and LP. Overall, our findings provide important information related to the feeding behaviour of M. hiroglyphicus and its effect on the transmission of SCWL phytoplasma.

Feeding behavior of Diaphorina citri and its transmission of ‘Candidatus Liberibacter asiaticus’ to citrus

AbstractHuanglongbing (HLB), also known as citrus greening, is currently the most destructive disease of citrus, responsible for huge economic losses in the world's major citrus production areas. The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits ‘Candidatus Liberibacter asiaticus’ (Clas), the pathogen responsible to cause HLB. Understanding of vector, pathogen, and host plant interactions is important for the management of this vector‐disease complex. We used the direct‐current electrical penetration graph (DC‐EPG) system to evaluate feeding behavior of Clas‐infected D. citri adults, and their potential to transmit the pathogen to healthy citrus, Citrus reticulata Blanco cv. Sunki (Rutaceae), following a 24‐h inoculation access period. Plants were tested for the presence of Clas by qPCR 6 months after inoculation. Findings suggest that inoculation was associated with salivation into the phloem sieve elements (waveform E1). The minimum feeding time for successful transmission by a single adult was 88.8 min, with a minimum E1 duration of 5.1 min. Regression analysis indicated a significant relationship between E1 duration and transmission efficiency. The adults successful in transmitting Clas to healthy citrus were able to penetrate and feed in the phloem much earlier than those which did not transmit. The minimum duration of E1 for a female was shorter than that of a male, but transmission was higher. However, durations of other EPG parameters were not significantly different between male and female. Feeding by single Clas‐infected D. citri adults on 6‐month‐old plants (Sunki) resulted in 23% HLB‐positive plants 6 months after inoculation. Multiple nymphs or adults could transmit the pathogen more efficiently than individual adults in the field, and further enhance the severity of the disease. Effective tactics are warranted to control D. citri and disrupt transmission of Clas.

Monitoring and characterization of DC electrical penetration graph waveforms of tea green leafhopper, Empoasca onukii, on tea plants

AbstractThe tea green leafhopper, Empoasca onukii (Homoptera: Cicadellidae), is a serious pest of “Yabukita”, the most popular tea cultivar in Japan. This study investigated its stylet‐probing behavior with a direct current (DC) electrical penetration graph (EPG) system. The EPG signals were classified into four distinct waveforms according to amplitude, frequency, voltage level and electrical origin. The waveforms were then characterized by fast Fourier transformation. The waveforms were correlated with distinct feeding behaviors: Np, non‐probing, when stylets were not inserted; Eo1, putative pathway and channel‐cutting phase, when stylets were inserted but not ingesting sap; Eo2, putative phloem phase, when the leafhoppers were probably ingesting from phloem; and Eo3, putative non‐phloem phase, when the leafhoppers were probably ingesting from mesophyll. Mean durations of waveforms showed that E. onukii likely ingested plant fluid mainly from phloem and partly from mesophyll. The description of DC EPG waveforms associated with feeding behaviors of this serious pest constitutes a fundamental step toward the understanding of the resistance mechanisms of the host plants against herbivorous insects.

Probing behavior of Empoasca vitis (Homoptera: Cicadellidae) on resistant and susceptible cultivars of tea plants.

Feeding activities of the tea green leafhopper, Empoasca vitis (Gothe) (Homoptera: Cicadellidae), on resistant and susceptible cultivars of tea plants (Camellia sinensis L.) were recorded and analyzed using the direct current electrical penetration graph (EPG) system. Six distinct EPG waveforms characterizing the feeding behavior of the tea green leafhopper, categorized as waveforms A, C, E, S, F, and R, were obtained during the investigation. Duration of passive ingestion, possibly of phloem (E), was the longest among all the probing waveforms on susceptible cultivars, whereas durations of the salivation (S) waveform and stylet work waveform (F) became longer on resistant cultivars. The durations of waveforms S and F on the resistant cultivar Jiandecha were slightly longer than those on the less resistant cultivar Yunguidaye , and both were significantly longer than those on the susceptible cultivars Hangzhoudaye and Zhushan-1 . Waveform E was shorter on the resistant cultivar Jiandecha than on the less resistant cultivars Yunguidaye and was significantly shorter than on the susceptible cultivars ( Hangzhoudaye and Zhushan-1 ). It is suggested that E, S, and F are the important waveforms related to leafhopper feeding behavior and tea plant resistance. Based on the results, the resistance levels of tea cultivars against the tea leafhopper can be evaluated quickly by direct current EPG.

Effects of Soil-Applied Imidacloprid on Asian Citrus Psyllid (Hemiptera: Psyllidae) Feeding Behavior

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is one of the most important pests of citrus (Citrus spp.) because of its status as a vector of Candidatus Liberibacter asiaticus (Las), the bacterium associated with citrus greening disease. The use of insecticides for vector control is the primary method of managing the spread of this pathogen. Imidacloprid is an insecticide commonly applied to the root zone of young citrus trees to provide systemic protection from pests. The effects of imidacloprid on feeding behavior of D. citri have not been studied in much detail. The purpose of this study was to examine the effects of imidacloprid application on feeding behavior of D. citri and to determine whether use of this systemic insecticide could have any effect on pathogen transmission by D. citri. A direct current electrical penetration graph monitor was used to record D. citri feeding behaviors for 12-h periods on mature and young leaves of imidacloprid-treated and -untreated citrus seedlings. Overall, compared with untreated plants, the feeding behavior of D. citri was disrupted on imidacloprid-treated plants via reduction in the number of probes, as well as durations of average probes, initial stylet contact with phloem, phloem salivation, and phloem ingestion. The results of this study demonstrate that soil applications of imidacloprid can reduce the probability of citrus plants becoming inoculated with Las through a reduction in the number and duration of phloem salivation events by D. citri. Furthermore, Las acquisition from infected citrus is greatly reduced as a result of decreased phloem ingestion by D. citri on imidacloprid-treated plants.

Difference in Feeding Behaviors of Two Invasive Whiteflies on Host Plants with Different Suitability: Implication for Competitive Displacement

In China, Bemisia tabaci Q (commonly known as biotype Q) has rapidly displaced B (commonly known as biotype B) in the past 6 years. The mechanisms underlying such phenomenon have been studied extensively in recent years; however, we have not come to a definitive conclusion yet. In the present study, the differences in host suitability between B and Q whitefly adults to five host plants (cabbage, cotton, cucumber, poinsettia, and tomato) were evaluated based on their respective feeding behaviors using a direct-current electrical penetration graph (DC-EPG) system. Pair-wise comparisons of B. tabaci B and Q feeding on each of the five host plants clearly indicate that Q feeds better than B on tomato, cotton and poinsettia, while B feeds better than Q on cabbage and cucumber. The EPG parameters related to both phloem and non-phloem phases confirm that cabbage and cucumber are best suited to B, while tomato, cotton, and poinsettia are best suited to Q. Our present results support the contention that host suitability and adult feeding behavior contribute to the competitive displacement of biotype B by biotype Q. The discrepancy between field (previous studies) and laboratory results (this study), however, suggests that 1) whitefly displacement is apparently contributed by multiple factors; and 2) factor(s) other than the host plant suitability may play a vital role in dictating the whitefly biotypes in the field.

Impacts of transgenic Bt cotton on the stylet penetration behaviors of Bemisia tabaci biotype B: Evidence from laboratory experiments

Abstract Stylet penetration behaviors of Bemisia tabaci biotype B on two transgenic cotton lines “GK12” and “GK19” expressing Bt toxic protein Cry1A (Bt cotton) and a non‐Bt conventional cotton line “Simian‐3” (CK cotton) were recorded with the direct current electrical penetration graph (DC‐EPG) technique. Our results suggested that EPG waveform patterns, types and characteristics [non‐probe (NP), pathway (C), potential drops (pd) and phloem phase (E(pd))] of Bemisia tabaci biotype B were very similar on the three cotton lines. There were no obvious differences of pathway variables among whiteflies on the three cotton lines. Some phloem variables related to E(pd)1 differed. Duration of 1st E(pd)1 and mean duration of E(pd)1 on both GK12 and GK19 were significantly shorter than that on CK cotton (P < 0.05). Fewer whiteflies on GK have long E(pd)1. Other phloem variables including total duration of E(pd) summed, mean E(pd) duration and percentage of whiteflies reaching the phloem phase were similar among the three cotton lines.

Mi-mediated aphid resistance in tomato: tissue localization and impact on the feeding behavior of two potato aphid clones with differing levels of virulence

The Mi-1.2 gene in tomato, Solanum lycopersicum L. (Solanaceae), confers resistance against several herbivores, including the potato aphid, Macrosiphum euphorbiae (Thomas) (Hemiptera: Sternorrhyncha: Aphididae) and the sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Sternorrhyncha: Aleyrodidae). Previous studies on the tissue localization of resistance have given varying results; whitefly resistance was attributed to factors localized in the mesophyll or epidermis, whereas aphid resistance was attributed to factors localized in the phloem. Our study utilizes the direct current electrical penetration graph (DC-EPG) technique to compare aphid feeding behavior on resistant (Mi-1.2+) and susceptible (Mi-1.2-) tomato plants. This study also compares the impact of resistance on the feeding behavior of two aphid clones that vary in their virulence, or their ability to survive and reproduce on resistant plants. Previous work had shown that the avirulent WU11 clone is almost completely inhibited by resistance, whereas the semi-virulent WU12 clone can colonize resistant hosts. Here, DC-EPG analysis shows that both aphid clones take longer to initiate cell sampling and to establish a confirmed sieve element phase on resistant plants than on susceptible hosts, and have shorter ingestion periods on resistant plants. However, the magnitude of these deterrent effects is far less for the semi-virulent clone than for the avirulent aphids. In particular, the WU12 clone is less sensitive to factors that limit sieve element ingestion, showing shorter non-probe duration and rapidly establishing sustained phloem ingestion on resistant plants when compared to the WU11 clone. We conclude that, in addition to previously described factors in the phloem that inhibit ingestion, Mi-mediated aphid resistance also involves factors (possibly in the mesophyll and/or epidermis) that delay initiation of phloem salivation, and that act in the intercellular spaces to deter the first cell sampling. Furthermore, the relative effectiveness of these components of resistance differs among insect populations.

Stylet penetration behaviors of the cotton aphid Aphis gossypii on transgenic Bt cotton

AbstractStylet penetration behaviors of cotton aphids Aphis gossypii Glover on a transgenic cotton line “GK‐12” expressing Bt toxic protein of Cry1A (Bt cotton) and a non‐Bt conventional cotton line “Simian‐3” (CK cotton) were recorded with the direct current electrical penetration graph (DC‐EPG) technique. Cotton aphids reared on Bt cotton (abbreviated as Bt‐aphids) and its parental non‐Bt control line (CK‐aphids) for more than 20 generations each, were used for recordings on two cotton lines. Among 47 selected parameters reflecting the activities of aphid stylets within plant tissues, there were eight parameters of CK‐aphids showing significant differences between the performances of CK‐aphids on Bt cotton and CK cotton, while for Bt‐aphids, all the parameters were statistically equal between the performances on the two cotton lines. All parameters with significant differences indicated that CK‐aphids could penetrate into Bt cotton more easily, but the phloem saps of Bt cotton were not as good as those of regular cotton for CK‐aphids. Based on the present results, we concluded that there were some factors in Bt cotton affecting penetration behaviors of CK‐aphids, but it just took several generations for CK‐aphids to completely adapt Bt cotton, and Bt‐aphids could feed on two cotton lines without difficulty.

Probing behavior of the tea green leafhopper on different tea plant cultivars

The probing behaviors of the tea green leafhopper, Empoasca vitis (Gothe), on 9 tea cultivars were studied using video-text Direct Current-Electrical Penetration Graph, i.e., DC-EPG. The following 7 types of waveforms produced by the leafhopper stylet probing were determined: A, stylet pathway formation; S, salivation when stylets pierce into and stay in phloems; C, active ingestion before stylets reach phloems; E and F, passive ingestion in phloems; R, the insect resting with its stylet inserted into the leaf tissue and NP without probing. The 9 tested tea cultivars were categorized into 3 groups by the cluster analysis according to the number of probes per insect, waveform durations, or duration per probe of various waveforms on different tea cultivars. Waveforms S, E and F correlated to the main feeding activity of the leafhopper and may provide valuable information on predicting the resis-tance level of the tea plants to the leafhopper. The resistance level of the 9 tea cultivars to the leafhopper was ranked based on the durations of waveforms S, E and F, as well as the duration per probe including various waveforms. The ranking order of the resis-tance was: Longjingchangye > Hangdan > Zhenghedabaicha > Qianmei 601 > Hongyafoshuo > Zhongcha 102 > Zhongcha 302 > Longjing 43 > Anjibaicha, which corresponded to the resistance level determined by the population density (infestation) of the leaf-hopper on the 9 tea cultivars in the tea fields. Our study suggests that this simple and convenient DC-EPG technique might have great potential as a reliable tool to predict the resistance of tea cultivars to the tea leafhopper.

Effects of treatments for electrical penetration graph recordings on behaviour and biology of Aphis craccivora (Aphididae)

Abstract. Two laboratory studies were conducted to investigate effects of treatments for direct “current electrical penetration graph (DC‐EPG) recordings or ‘tether effect’, on behaviour and reproductive performance of cowpea aphid Aphis craccivora Koch. The experiments constituted a control study in application of DC‐EPGs to analyse cowpea aphid feeding behaviour and host plant resistance mechanisms. Resistant (ICV‐12) and susceptible (ICV‐1) cultivars of cowpea Vigna unguiculata (L.) Walp were used. EPG treatments included two groups of aphids: tethered aphids that were exposed to DC electricity via an attachment of a thin, flexible gold wire on their dorsum using a droplet of adhesive silver paint, and ‘free’ (untethered) aphids with a dorsal spot of silver paint only. EPGs of the tethered aphids were recorded continuously for c.! h, whereas from the ‘free’ aphids recordings were done only for brief periods of 2–5 min, by temporarily contacting a gold wire to the spot of silver paint. Waveform signals generated from resistance fluctuations and electromotive forces, and representing aphid stylet penetration behaviour were recorded. A separate experiment was conducted to investigate effects of EPG treatments on aphid survivorship and population growth. Overall, EPG treatments did not significantly affect aphid stylet penetration behaviour or life‐table parameters. However, effects of crop cultivar on those characteristics were significant. Waveform E2, which denotes aphid ingestion in phloem sieve elements, and non‐penetration behaviour were important indicators of aphid resistance in ICV‐12. Also, apart from the number of aphid generations, other life‐table parameters were useful indicators of ICV‐12 resistance. Thus, DC‐EPGs provided a reliable technique for studying aphid stylet behaviour, and investigation of aphid resistance in cowpeas.