Electropenetrography (EPG): a Breakthrough Tool Unveiling Stink Bug (Pentatomidae) Feeding on Plants

In this article, we review and discuss the potential use of EPG (electropenetrography) as a powerful tool to unveil the feeding process of phytophagous stink bugs (pentatomids). These bugs are relatively big and vigorous, which presents a problem during wiring (i.e., attachment of the gold wire on the bug’s pronotum) for use in EPG. Once this challenge was overcome, using the sand paper-and-wire technique, several species have been studied using EPG, yielding waveforms that, coupled with histological studies, revealed the ingestion sites on different host plants. These sites include vascular tissues (xylem and phloem), parenchyma tissue, and seed endosperm. Stink bugs usually feed by secreting a gelling saliva to create a salivary sheath that surrounds the stylets and anchors/supports/lubricates them. However, using the cell rupture feeding strategy and the tactic of combined laceration (mechanical movements of the stylets) and maceration (action of chemical enzymes) breaks the plant cells enabling ingestion. The number of ingestion events and their duration is variable according to the feeding site. Waveforms generated have typical patterns according to the feeding site. Recent studies with several species of stink bugs have started to demonstrate the potential of EPG as a tool to unveil their feeding behavior. This may also be useful in the applied field of stink bug management, such as the development and screening of resistant genotypes and the action of chemical insecticides affecting their feeding and survivorship.     

Feeding Activity, Salivary Amylase Activity, and Superficial Damage to Soybean Seed by Adult Edessa meditabunda (F.) and Euschistus heros (F.) (Hemiptera: Pentatomidae)

Greenhouse and laboratory studies were conducted to evaluate feeding activity and superficial damage to soybean seed by the brown-winged stink bug, Edessa meditabunda (F.), and the Neotropical brown stink bug, Euschistus heros (F.). Soybean plants (cv. BRS 282), at R6 stage of development were used. Thirty pairs of each species were used individually for 48?h. Two daily observations (9:00?AM and 3:00?PM) were taken to record the number of bugs (feeding/resting) on plant parts. Harvested seeds imbibed in tetrazolium solution were photographed for measurement of the damaged surface. Adult E. meditabunda significantly preferred soybean stems (19.7 bugs) to pods (2.7). Feeding/resting was similar at 9:00?AM (mean number of 28.0 bugs) and 3:00?PM (24.3). Euschistus heros equally fed/stayed on stems (7.3 bugs) and pods (6.9), although most bugs (12.3) remained on the cage net; feeding/resting on all plant structures amounted to 13.7 bugs at 9:00?AM and 17.7 bugs at 3:00?PM. Amylase activity was greater for E. heros (41.61?±?0.89?U/mg) and almost none for E. meditabunda (2.35?±?0.14?U/mg). The superficial damage to seeds was significantly greater for E. meditabunda (22. 9?mm²) compared to E. heros (12.5?mm²). However, E. meditabunda caused less shrinkage of the seed tegument, while E. heros damage was deeper and seeds showed reduction in size.     

First Record of Edessa meditabunda (F.) on Lettuce in Mato Grosso State, Brazil

We report the first record of the stink bug Edessa meditabunda (F.) on lettuce Lactuca sativa in the organic vegetable garden ??Cheiro Verde?? in Tangará da Serra, State of Mato Grosso, Brazil (14°37??13?? S, 57°28??46?? W). During September, October, and November 2009, we collected 1,099 adults, 43 nymphs, and 29 egg masses. Feeding by this stink bug possibly caused early bolting.     

Behavioral Comparisons of Ingestion and Excretion by Selected Species of Pentatomids: Evidence of Feeding on Different Food Sources Supports Pest Status

Laboratory studies were conducted on food ingestion and excretion by selected species of pentatomids on different food sources to support their pest status. We compared the frequency and time of feeding on vegetative (stem) and reproductive (seed) structures of soybean, Glycine max (L.) Merrill and of maize, Zea mays L. by Piezodorus guildinii (Westwood), Dichelops melacanthus (Dallas), and Edessa meditabunda (F.); in addition, the amount of excreta (feces) produced were compared for D. melacanthus feeding on seed of soybean and stem of maize seedling. The feeding behavior of E. meditabunda and P. guildinii on soybean, and of D. melacanthus on maize was recorded using the electropenetrography (EPG) technique. Excretion was estimated using water sensitive paper recording number and area of fecal drops. Results indicated that E. meditabunda on soybean stem repeated events of ingestion (both xylem and phloem sap) over four times per bug during the 8 h of recording for ca. 53 min per event. Dichelops melacanthus on maize seedling repeated each ingestion event over three times per bug for ca. 24 min per event. Piezodorus guildinii feeding on soybean stem repeated each ingestion 1.2 times per bug for ca. 40 min per event; on seed endosperm, it fed for a longer time, ca. 80 min per event, each event repeated only 0.5 times per bug. Number of excretory drops was higher (9.9 drops per bug) when D. melacanthus fed on maize seedling than on soybean seed (1.4 drops per bug). A larger amount of saliva/regurgitate liquid food was expelled when bugs fed on the former than on the later food.     

Plant penetration activities by the flatid planthopper Metcalfa pruinosa (Hemiptera: Fulgoroidea): an electrical penetration graph‐histology analysis

The citrus flatid planthopper, Metcalfa pruinosa Say (Hemiptera: Flatidae), is a very polyphagous native insect in North America and currently a serious pest in Europe and South Korea. To understand the feeding behaviour of M. pruinosa, stylet penetration behaviour of M. pruinosa was investigated with an electrical penetration graph (EPG) system. This study reports seven EPG waveforms related to M. pruinosa feeding behaviour: np (no stylet penetration), Mp1 (initiation of stylet penetration), Mp2 (stylet movement and salivation), Mp4 (phloem feeding), Mp4‐H (honeydew excretion), Mp5 (xylem feeding) and Mp6 (unknown). To determine respective feeding behaviour related to the Mp4 and Mp5 waveforms, stylets were cut with a laser beam, and the location of the stylet tip within plant tissue was examined. We found plant sap was exuded from the severed stylets only when the Mp4 waveform was observed, suggesting phloem sap ingestion. The stylet tip was located in the xylem region, indicating xylem‐feeding activity, when the Mp5 waveform was observed. The analysis of 24 different EPG parameters suggests that M. pruinosa stylets reached the vascular bundle of a plant within ca. 5 min and spend ca. 70% of the time feeding on xylem and phloem feeding. This is the first study that reports seven distinctive EPG waveforms with respect to the feeding behaviour of M. pruinosa which could help determine host specificity and host plant susceptibility.     

Spatial distribution and sampling plan of the phytophagous stink bug complex in different soybean production systems

Phytophagous stink bugs are major soybean pests, and knowledge of spatial distribution models of the pest in the crop is fundamental to establishing an appropriate sequential sampling plan, and thus, allowing the correct utilization of control strategies. This work aimed to study the spatial distribution of phytophagous stink bugs in soybean grown in different cropping systems and determine a sequential sampling plan. The experiment was conducted in Maracaju, MS, Brazil, during the agricultural year of 2012/2013. Soybean cultivars BRS 284 and SYN 1163 RR were placed in an experimental area comprising six fields (two soybean cultivars × three cropping systems). Sampling was performed weekly, using a beat cloth per plot and counting the number of stink bugs found, virtually throughout the soybean reproductive period. Concerning the statistical analyses, we used the dispersion indexes (variance‐to‐mean ratio, Morisita's index, exponent k of the negative binomial and Green's coefficient) and probabilistic methods of frequency adjustment (negative binomial and Poisson). Adult and nymph phytophagous stink bugs showed aggregate disposition in the field regardless of the cropping system, and their numbers were adjusted to the negative binomial probability distribution. There was no difference in the behaviour of adult and nymphs considering the tested cultivars. We elaborated a practical sequential sampling plan for phytophagous stink bug complexes, considering crops intended for the production of grains and seeds.     

EPG technique as a tool to reveal host plant acceptance by xylem sap‐feeding insects

The discovery of a new exotic insect herbivore triggers responses from biosecurity agencies, one of which is the decision of whether or not to attempt eradication. Rapid determination of the host range of the new invader is necessary, but when sap‐sucking insects are first collected from plants, the lack of visible signs of feeding damage makes it difficult to determine their host status. We investigated the Electrical Penetration Graph (EPG‐DC) technique as tool to assess host range of a xylem sap‐feeding invader, using Carystoterpa fingens (Hemiptera: Aphrophoridae), a New Zealand endemic xylem feeder, as a model insect. Real‐time probing and feeding events over a 12‐h recording period were compared for adult C. fingens on 18 plant species. Hebe azure, a known host, was designated the ‘reference plant species’ against which events on all other plants were statistically compared. EPG waveforms were categorized on their amplitude, frequency, voltage and electrical origin, and six parameters (time taken to first probe, time to first xylem ingestion from first probe, total probing time, number of xylem‐ingesting events, duration of the longest xylem‐ingesting event and total xylem ingestion time) were measured. The total xylem ingestion period (i.e. the actual feeding period) on each plant species expressed as a percentage of total probing time was considered the best parameter for comparing the host status of plants with H. azure. Although the EPG data overestimated the actual host range of C. fingens, we consider that they provided a reasonable first guide to the potential host status of the unknown plants, and so might usefully be used to rapidly assess whether a plant from which a new invader was collected was a host, or whether the association was merely incidental.     

Effect of resistant and susceptible soybean cultivars on the attraction of egg parasitoids under field conditions

The association of resistant crop varieties and biological pest control has great potential for pest management, as resistant cultivars may affect herbivore population dynamics by interfering with their biology and search behaviour for host plants. In addition, resistant varieties can also affect the efficiency of natural enemies. The aims of this work were to evaluate the influence of the soybean cultivars Dowling (resistant), IAC 100 (resistant) and Silvânia (susceptible) under field conditions on: (i) the abundance of parasitoids in the Platygastridae family and the stink bug Euschistus heros and (ii) the parasitism of stink bug eggs by these natural enemies. There was no difference between cultivars in stink bug distribution in the field. The parasitoids Telenomus podisi, Trissolcus teretis, Tr. urichi and Tr. brochymenae emerged from the sentinel eggs of E. heros, and T. podisi was the most abundant species observed during the reproductive stage R6. The cultivars Dowling and IAC 100 attracted a higher number of Platygastridae parasitoids, T. podisi and Tr. teretis, and the abundance of these parasitoids during the period from R1 to R7 might have being sufficient to control the stink bug population. These results suggest that the cultivars Dowling and IAC 100 have important attributes that should be taken into account in the implementation and development of new cultivars in breeding programmes that aim to obtain plants more resistant to stink bugs and more attractive to natural enemies.     

Field biology of Edessa rufomarginata (Hemiptera: Pentatomidae): phenology, behavior, and patterns of host plant use

Pentatomids may cause direct and indirect damage to important crop plants. Biological and ecological features of phytophagous stink bugs in natural environments, however, remain poorly documented. Here, we provide an ecological account of Edessa rufomarginata De Geer on Caryocar brasiliense (Caryocaraceae) in the Brazilian savanna. The phenology of E. rufomarginata matched that of its host plant, with immatures developing in the wet season simultaneously with the production of vegetative and reproductive plant tissue. Females do not exhibit parental care and lay eggs more frequently on larger plants. Oviposition frequency, however, does not differ between plants with and without flowers/fruits. Nymphs and adults usually feed on stem parts and more rarely on flower buds and fruits. First- and second-instar nymphs remain aggregated, but disperse as third-instar nymphs. Adults and nymphs were more abundant on mature stems of C. brasiliense compared with other plant locations. Ants visiting the plant to search for extrafloral nectar occasionally tap the abdomen of E. rufomarginata nymphs with their antennae to obtain honeydew. This is the first record of trophobiotic interactions between Edessa stinkbugs and ants, and one of the few for heteropterans. The interaction of the stink bug with other natural enemies, such as predaceous Heniartes (Reduviidae), was also observed. Given the pest status of Edessa species for crop plants, additional field studies on host plants, interaction with ants, and natural enemies in native habitats are needed for an effective management of these stink bugs in tropical agricultural systems.     

荔枝蝽取食行为的研究

在越冬前和产卵期,荔枝蝽Tessaratoma papillosa Drury在寄主植物-荔枝树的花枝、嫩枝和老枝上都有取食行为。但在这两个不同时期, 其取食行为有显著差异。在越冬前,只有50%～70%的成虫在各类枝条上取食;而处于产卵期的荔枝蝽成虫,在各类枝条上,100%积极取食,且其取食前时间显著短于越冬前在同类枝条上的取食前时间。同一时期在不同枝叶上,荔枝蝽的取食行为也有显著差异。被置于嫩枝叶和花枝上的成虫取食前时间明显短于被置于老枝叶上者;在有选择的情况下,两个时期的成虫都明显地选择在花枝和嫩枝上取食。经分析测定：在不同生长时期,荔枝树的花枝和嫩枝的含水量与含氮量均较老枝叶中的高,可溶性糖含量的变化较大;组织结构也存在明显差异。     

Identification of Stink Bugs Using an Electronic Nose

Stink bugs are recognized as pests of several economically important crops, including cotton, soybean and a variety of tree fruits. The Cyranose 320 was used for the classified investigation of stink bug. Stink bugs including males and females of the southern green stink bugs, Nezara viridula, were collected from crop fields around College Station, TX. Results show that the released chemicals and chemical intensity are both critical factors, which determine the rate that the Cyranose 320 correctly identified the stink bugs. The Cyranose 320 shows significant potential in identifying stink bugs, and can classify stink bug samples by species and gender.     

Aphid activities during sieve element punctures

Aphid salivation in sieve elements and phloem sap ingestion were linked to waveforms in the Electrical Penetration Graph (EPG). Non-viruliferousRhopalosiphum padi (L.) (Hemiptera, Aphididae) on barley yellow dwarf virus (BYDV) infected wheat could acquire the virus, which was used as an indication for phloem sap ingestion, whereas virus inoculation by viruliferous aphids on healthy plants was associated with salivation in sieve elements or other phloem cells. Probing was monitored and the waveforms recorded were related to ELISA results of test plants. The EPG patterns A, B, and C are indicative of the stylet pathway phase, whereas patterns E1 and E2 reflect the phloem (sieve element) phase with an unknown activity (E1) or with ingestion and concurrent salivation (E2). Aphids showing pathway and E1 rarely acquired virus, suggesting that little or no phloem sap ingestion can occur during these patterns, whereas those showing additionally pattern E2 did so substantially, indicating phloem sap ingestion. The main pattern related to virus inoculation was E1, although some aphids were able to inoculate plants during pathway. Pattern E1 clearly reflects the most important salivation into sieve elements. Pattern E2 had no clear contribution to virus inoculation, supporting the present hypothesis that during this pattern the saliva is mixed with the phloem sap in the single canal at the stylet tips and ingested immediately, without reaching the plant tissue. Sustained sap ingestion did not affect virus inoculation. So, BYDV inoculation mainly occurs during the first period of a sieve element puncture which is always formed by E1. Implications on persistent virus transmission are discussed.     

Attractiveness of conspecific stink bugs to adult stink bug-baited traps in soybean fields

The attractiveness of live adult stink bugs used as baits in traps in soybean fields, Milyang, Korea, to conspecific stink bugs was evaluated. Both sexes of bean bug, Riptortus pedestris Fabricius (Hemiptera: Alididae), and one-banded stink bug, Piezodorus hybneri Gmelin (Hemiptera: Pentatomidae), were attracted to conspecific male adults-baited traps. Likewise, both sexes of brown-marmorated stink bug, Halyomorpha halys Stål (Hemiptera: Pentatomidae), and sole bug, Dolycoris baccarum L. (Hemiptera: Pentatomidae), were attracted to traps baited with conspecific male stink bugs. However, in Nezara antennata Scott (Hemiptera: Pentatomidae), both male and female used as baits in traps were attractive to conspecific adults. Accordingly, these results suggest that the only male adults of H. halys and D. baccarum and both sexes of N. antennata are attractive to conspecific male stink bugs.     

cis-Jasmone indirect action on egg parasitoids (Hymenoptera: Scelionidae) and its application in biological control of soybean stink bugs (Hemiptera: Pentatomidae)

In many plants, the secondary metabolite cis-jasmone activates the metabolic pathway that produces volatile organic compounds attractive to natural enemies and, sometimes, repellent to herbivores. Previous studies indicate that the feeding damage caused by the herbivore Euschistus heros or the exogenous application of cis-jasmone in soybean plants induces the release of herbivore-induced plant volatiles (HIPVs) with a similar chemical profile and these compounds can attract the stink bug egg parasitoid Telenomus podisi (Scelionidae). Herein we tested in field conditions the effect of exogenous application of cis-jasmone in soybean plants on the parasitoid and stink bug community and on stink bug egg parasitism. In two areas, one within a soybean and another within a Crotalaria matrix, we randomly distributed 2 m² plots, with soybean plants induced (treatment, n = 5) or not induced by cis-jasmone (control, n = 5) in the field. We sampled the parasitoid community weekly with yellow sticky traps (n = 3/plot) and monitored parasitism with sentinel eggs of E. heros (n = 150/plot). We also monitored the population of stink bugs weekly, by sampling each plot with shake-cloth technique. The abundance of Scelionidae was highest overall and also in treated plots during the first four weeks in the area with a soybean matrix, but decreased thereafter. The richness of parasitoid families was similar between treatment and control plots in the area with a soybean matrix, but higher in control plots in the area with a Crotalaria matrix. Evenness was higher in control plots in the area with soybean matrix, whereas the reverse occurred in the area with a Crotalaria matrix. Results suggest that treatment with cis-jasmone effectively attracted and enhanced the population of scelionid parasitoids, but had no effect on the occurrence and intensity of parasitism and in the number of stink bugs.     

Biological control of soybean stink bugs by inoculative releases of Trissolcus basalis

Field studies of inoculative releases of Trissolcus basalis (Wollaston) (Hymenoptera: Scelionidae) in early-maturing soybean, used as a trap crop, were conducted during four consecutive seasons to evaluate the parasitoid's potential as an IPM tactic for use against stink bug (Pentatomidae) species. Fifteen thousand adults of the parasitoid were released per hectare in the trap crop when the first stink bugs were detected in the experimental area. The stink bug population density was reduced by an average of 54% in the trap crop and by 58% in the main crop. The inoculative releases produced a reduction and delay in the stink bug population peak, mainly represented by Nezara viridula (L.), Piezodorus guildinii (Westwood) and Euschistus heros (Fabr.), which were held below economic threshold levels during the most critical stages of stink bug attack on soybean (pod and seed fill, R3–R6). As a result, seed quality was better in areas where T. basalis was released, demonstrating the efficacy of inoculative releases of this egg-parasitoid which could be an important component of the soybean IPM program in Brazil.     

An analysis of the feeding behavior of three stages of Toxoptera citricida by DC electrical penetration graph waveforms

With the purpose of studying the feeding behavior of the brown citrus aphid pest, Toxoptera citricida (Kirkaldy) (Hemiptera: Aphididae), we compared stylet probing behaviors of third and fourth instars and adults on Citrus unshiu Marc (Rutaceae) seedlings using the electrical penetration graph (EPG) technique. EPG waveforms exhibited the full suite of stylet behaviors – stylet pathway, intracellular stylet puncture, phloem salivation (E1), sieve ingestion (E2), and xylem sap ingestion activities, plus the non‐penetration (Np) waveform. Before the phloem phase, the number of probes was significantly higher for third‐instar nymphs than for adults. Overall duration of Np events by adults was significantly lower than the duration of third and fourth instars. The number of short probes of the fourth instars was significantly higher than that of the adults. In the phloem phase, adults made more frequent and longer E1 events than the third and fourth instars. Third instars made more frequent but shorter E2 events, whereas adults made fewer but longer events. These results showed adults gained nutrients by increasing feeding time during phloem ingestion. Thus, the probability of phloem‐associated virus acquisition and transmission of T. citricida was higher in adults than in nymphs.     

Characterization of electrical penetration graphs of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings

Detailed information on probing behavior of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is critical for understanding the transmission process of phloem‐limited bacteria (Candidatus Liberibacter spp.) associated with citrus ‘huanglongbing’ by this vector. In this study, we investigated stylet penetration activities of D. citri on seedlings of Citrus sinensis (L.) Osbeck cv. Pêra (Rutaceae) by using the electrical penetration graph (EPG‐DC system) technique. EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces during stylet penetration into plant tissues. The main waveforms were correlated with histological observations of salivary sheath termini in plant tissues, to determine the putative location of stylet tips. The behavioral activities were also inferred based on waveform similarities in relation to other Sternorrhyncha, particularly aphids and whiteflies. In addition, we correlated the occurrence of specific waveforms with the acquisition of the phloem‐limited bacterium Ca. Liberibacter asiaticus by D. citri. The occurrence of a G‐like xylem sap ingestion waveform in starved and unstarved psyllids was also compared. By analyzing 8‐h EPGs of adult females, five waveforms were described: (C) salivary sheath secretion and other stylet pathway activities; (D) first contact with phloem (distinct from other waveforms reported for Sternorrhyncha); (E1) putative salivation in phloem sieve tubes; (E2) phloem sap ingestion; and (G) probably xylem sap ingestion. Diaphorina citri initiates a probe with stylet pathway through epidermis and parenchyma (C). Interestingly, no potential drops were observed during the stylet pathway phase, as are usually recorded in aphids and other Sternorrhyncha. Once in C, D. citri shows a higher propensity to return to non‐probing than to start a phloem or xylem phase. Several probes are usually observed before the phloem phase; waveform D is observed upon phloem contact, always immediately followed by E1. After E1, D. citri either returns to pathway activity (C) or starts phloem sap ingestion, which was the longest activity observed.     

Adjacent Habitat Influence on Stink Bug (Hemiptera: Pentatomidae) Densities and the Associated Damage at Field Corn and Soybean Edges

The local dispersal of polyphagous, mobile insects within agricultural systems impacts pest management. In the mid-Atlantic region of the United States, stink bugs, especially the invasive Halyomorpha halys (Stål 1855), contribute to economic losses across a range of cropping systems. Here, we characterized the density of stink bugs along the field edges of field corn and soybean at different study sites. Specifically, we examined the influence of adjacent managed and natural habitats on the density of stink bugs in corn and soybean fields at different distances along transects from the field edge. We also quantified damage to corn grain, and to soybean pods and seeds, and measured yield in relation to the observed stink bug densities at different distances from field edge. Highest density of stink bugs was limited to the edge of both corn and soybean fields. Fields adjacent to wooded, crop and building habitats harbored higher densities of stink bugs than those adjacent to open habitats. Damage to corn kernels and to soybean pods and seeds increased with stink bug density in plots and was highest at the field edges. Stink bug density was also negatively associated with yield per plant in soybean. The spatial pattern of stink bugs in both corn and soybeans, with significant edge effects, suggests the use of pest management strategies for crop placement in the landscape, as well as spatially targeted pest suppression within fields.