Probing behavior of the corn leafhopper Dalbulus maidis on susceptible and resistant maize hybrids

The corn leafhopper Dalbulus maidis is the main vector of the pathogens that cause corn stunt, a major disease of maize in the Americas. As host resistance is an efficient tool to control diseases, the findings of a previous report showed that some corn hybrids are resistant to D . maidis . In this work, we assessed the probing behavior of D . maidis on susceptible and resistant corn hybrids using EPG (Electrical Penetration Graph) technology. Fifteen-day-old females were monitored for 20 hours, with access to hybrids DK390, DK670, DK79-10, and DK72-10. Hybrids DK390 and DK72-10 showed resistance to D . maidis in phloem, since insects feeding on these hybrids presented more salivation events in phloem without subsequent ingestion, which are seen as failed attempts to ingest. A reduction of the total duration of phloem ingestion was observed, and accordingly of the time spent by insects with access to these hybrids on xylem ingestion. The hybrid DK390 also had mesophyll resistance, seen as less probing time and a higher number of probes of short duration. These findings support and are consistent with previous research, providing useful information to characterize maize hybrids resistant to D . maidis , and so to corn stunt.

Effect of Soy Leaf Flavonoids on Pea Aphid Probing Behavior

Flavonoids detected in soybean Glycine max (L.) Merr. (Fabaceae) cause various alterations in the metabolism, behavior, and development of insect herbivores. The pea aphid Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae) poses potential threat to soybeans, but the effect of individual flavonoids on its feeding-associated behavior is relatively unknown. We monitored probing behavior (stylet penetration activities) of A. pisum on its preferred host plant, Pisum sativum L. untreated (control) and treated with 0.1% ethanolic solutions of flavonoids apigenin, daidzein, genistein, and kaempferol. We applied the electrical penetration graph (electropenetrography, EPG) technique, which visualizes the movements of aphid stylets within plant tissues. None of the applied flavonoids affected the propensity to probe the plants by A. pisum. However, apigenin enhanced the duration of probes in non-phloem tissues, which caused an increase in the frequency and duration of stylet mechanics derailment and xylem sap ingestion but limited the ingestion of phloem sap. Daidzein caused a delay in reaching phloem vessels and limited sap ingestion. Kaempferol caused a reduction in the frequency and duration of the phloem phase. Genistein did not affect aphid probing behavior. Our findings provide information for selective breeding programs of resistant plant cultivars to A. pisum.

Quercetin and Rutin as Modifiers of Aphid Probing Behavior

Rutin and its aglycone quercetin occur in the fruits, leaves, seeds, and grains of many plant species and are involved in plant herbivore interactions. We studied the effect of the exogenous application of rutin and quercetin on the probing behavior (= stylet penetration activities in plant tissues) of Acyrthosiphon pisum on Pisum sativum, Myzus persicae on Brassica rapa ssp. pekinensis, and Rhopalosiphum padi on Avena sativa using the electrical penetration graph technique (EPG = electropenetrography). The reaction of aphids to quercetin and rutin and the potency of the effect depended on aphid species, the flavonol, and flavonol concentration. Quercetin promoted probing activities of A. pisum within non-phloem and phloem tissues, which was demonstrated in the longer duration of probes and a trend toward longer duration of sap ingestion, respectively. M. persicae reached phloem in a shorter time on quercetin-treated B. rapa than on the control. Rutin caused a delay in reaching sieve elements by A. pisum and deterred probing activities of M. persicae within non-phloem tissues. Probing of R. padi was not affected by quercetin or rutin. The potency of behavioral effects increased as the applied concentrations of flavonols increased. The prospects of using quercetin and rutin in plant protection are discussed.

Probing behavior of Adelges laricis Vallot (Hemiptera: Adelgidae) on Larix decidua Mill: Description and analysis of EPG waveforms

Adelgidae are a sister group of Aphididae and Phylloxeridae within Hemiptera, Aphidoidea and occur exclusively on Pinaceae. The piercing-sucking mouthparts of Adelgidae are similar to those of aphids and it is believed that adelgids ingest sap from both the non-vascular and vascular (phloem) tissues. The aim of the present study was to identify and characterize the adelgid stylet activities during their penetration in plant tissues. The probing behavior of Adelges laricis Vallot (Hemiptera: Adelgidae) on European larch Larix decidua Mill. and sucrose diets was monitored using the DC-EPG (Electrical Penetration Graph technique = electropenetrography). The EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces, and associated with putative behavioral activities based on analogy with aphid activities. Waveform frequency, duration, and sequence were analysed as well. A. laricis generated EPG signals at two clearly distinct voltage levels positive and negative, suggesting extracellular and intracellular stylet penetration, respectively. The adelgid EPG patterns were ascribed to four behavioral phases, which were non-probing, pathway, phloem, and xylem phases. Non-probing referred to the position of the stylets outside the plant tissues. Pathway phase was represented by three waveform patterns that visualized extracellular stylet penetration in non-vascular tissues without potential drops (AC1), with serial short (1.2–1.5 s) potential drops (AC2), and with ‘aphid-like’ (5–10 s) potential drops (AC3). Phloem phase comprised three waveform patterns at intracellular level, which in all probability represented phloem salivation (AE1), and phloem sap passive (AE2) and active ingestion (AE3). AE3 was a newly described waveform, previously unreported from Hemiptera. Waveform AG represented the ingestion of xylem sap. The comparative analysis demonstrated that the gymnosperm-associated adelgids show certain similarities in probing behavior typical of aphids and phylloxerids on angiosperm plants. The present work is the first detailed analysis of specific adelgid stylet activities on gymnosperms.

Semipersistently Transmitted, Phloem Limited Plant Viruses Are Inoculated during the First Subphase of Intracellular Stylet Penetrations in Phloem Cells

The green peach aphid Myzus persicae Sulzer is the main vector of the semipersistently transmitted and phloem-limited Beet yellows virus (BYV, Closterovirus). Studies monitoring the M. persicae probing behavior by using the Electrical penetration graphs (EPG) technique revealed that inoculation of BYV occurs during unique brief intracellular punctures (phloem-pds) produced in companion and/or sieve element cells. Intracellular stylet punctures (or pds) are subdivided in three subphases (II-1, II-2 and II-3), which have been related to the delivery or uptake of non-phloem limited viruses transmitted in a non-persistent or semipersistent manner. As opposed to non-phloem limited viruses, the specific pd subphase(s) involved in the successful delivery of phloem limited viruses by aphids remain unknown. Therefore, we monitored the feeding process of BYV-carrying M. persicae individuals in sugar beet plants by the EPG technique and the feeding process was artificially terminated at each phloem-pd subphase. Results revealed that aphids that only performed the subphase II-1 of the phloem-pd transmitted BYV at similar efficiency than those allowed to perform subphase II-2 or the complete phloem-pd. This result suggests that BYV inoculation occurs during the first subphase of the phloem-pd. The specific transmission mechanisms involved in BYV delivery in phloem cells are discussed.

Relationships Among the Feeding Behaviors of a Mirid Bug on Cotton Leaves of Different Ages and Plant Biochemical Substances

Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae) tends to feed on young plant tissues. To explore the relationship between stylet probing behaviors of adult A. lucorum and conditions of cotton leaves, we conducted an experiment using electropenetrography (EPG). Behaviors were recorded on four cotton varieties, in relation to thickness and biochemical traits of differently-aged leaves. Cotton leaf age had a significant effect on the probing behavior of A. lucorum but cotton variety did not. One-day-old leaves of A. lucorum received the highest mean number of stylet probes (penetrations) per insect, and longest mean durations per insect of combined stylet probing or its components, cell rupture and ingestion behaviors. All of the leaf traits (thickness and biochemical substances) were similar among these four cotton varieties. Leaf thickness had a significantly negative effect on the same four variables above. Gossypol and tannin also had a negative impact on combined probing duration. Redundancy analysis showed that the four EPG variables were closely related to nutrient substances (amino acids, sugar, and water) while they had the opposite relationship with plant defense substances (gossypol and tannin). On cotton in the seedling stages, A. lucorum fed more readily on the youngest, thinnest leaves in our no-choice EPG experiments. Nutrients and chemical resistance substances determined the probing duration of A. lucorum. Our findings can contribute to better understanding of patterns of feeding and host consumption by A. lucorum, ultimately improving cotton resistance to A. lucorum.

Behavioral effects induced by organic insecticides can be exploited for a sustainable control of the Orange Spiny Whitefly Aleurocanthus spiniferus

The Orange Spiny Whitefly (OSW) Aleurocanthus spiniferus (Hemiptera: Aleyrodidae) represents a new serious threat to Citrus spp., grapevine and ornamental plants in the whole Mediterranean area. Such threat urgently calls for the development of a sustainable control strategy, including insecticides compatible with biological control, and applicable also in organic citrus farming that represent an essential part of Mediterranean agricultural economy. Therefore, we evaluated the toxicity and the effects on host searching, oviposition, and probing and feeding behavior exerted on OSW by organic insecticides supposed to have limited side effects on environment and ecosystem services, i.e. sweet orange essential oil (EO), extract of Clitoria ternatea (CT), mineral oil, pyrethrin and azadirachtin. Despite none of the compounds caused a significant mortality of any of the OSW instars, we observed interesting effects on whitefly behavior: (i) EO and pyrethrin showed a relevant repellent effect, with impairment of both adults landing and oviposition on treated plants; (ii) CT and pyrethrin strongly affected probing behavior. Here, in the light of our findings, we discuss possible OSW sustainable control strategies and further research perspectives.

Effect of Naringenin and Its Derivatives on the Probing Behavior of Myzus persicae (Sulz.)

Substances that alter insect behavior have attracted a lot of attention as potential crop protection agents. Naringenin (5,7,4′-trihydroxyflavanone) is a naturally occurring bioactive flavanone. We evaluated the influence of naringenin on aphid activities during individual phases of probing and feeding and the effect of structural modifications of naringenin on its activity towards aphids. We monitored the probing behavior of Myzus persicae (Sulz.) (Hemiptera: Aphididae) using the Electrical Penetration Graph (EPG) technique. The chemical modifications were the substitution of hydrogen atoms with methyl, ethyl or pentyl groups and the replacement of the carbonyl group in naringenin and its derivatives with an oxime moiety. Depending on the substituents, the activity of naringenin-derived compounds varied in potency and mode of action. Naringenin was an attractant of moderate activity, which enhanced sap ingestion. The naringenin derivative with two methyl groups—7,4′-di-O-methylnaringenin—was a deterrent, which hindered aphid probing in non-phloem tissues. Naringenin oxime derivatives with methyl substituents—7,4′-di-O-methylnaringenin oxime, 7-O-methylnaringenin oxime, and 5,7,4′-tri-O-methylnaringenin oxime—and the derivative with a pentyl substituent—7-O-pentylnaringenin oxime—were strong attractants which stimulated aphid probing in non-phloem tissues and the ingestion of phloem sap.

Drench Application of Systemic Insecticides Disrupts Probing Behavior of Diaphorina citri (Hemiptera: Liviidae) and Inoculation of Candidatus Liberibacter asiaticus

Candidatus Liberibacter asiaticus (CLas) is a phloem-limited bacterium that is associated with the Huanglongbing (HLB) disease of citrus and transmitted by the psyllid, Diaphorina citri. There are no curative methods to control HLB and the prevention of new infections is essential for HLB management. Therefore, the objective of our study was to determine the effects of systemic insecticides, such as the neonicotinoids imidacloprid, thiamethoxam, and a mixture of thiamethoxam and chlorantraniliprole (diamide) on the probing behavior of CLas-infected D. citri and their effect on CLas transmission. The electrical penetration graph (EPG-DC) technique was used to monitor the stylet penetration activities of CLas-infected D. citri on sweet orange [Citrus sinensis (L.) Osbeck] ‘Valencia’ treated with systemic insecticides. Systemic insecticides disrupted the probing behavior of CLas-infected D. citri, in a way that affected CLas transmission efficiency, particularly by negatively affecting the stylet activities related to the phloem phase. All insecticides reduced (by 57–73%) the proportion of psyllids that exhibited sustainable phloem ingestion (waveform E2 > 10 min), with significant differences observed on plants treated with thiamethoxam and thiamethoxam + chlorantraniliprole. The transmission rate of CLas with high inoculum pressure (five CLas-infected D. citri per plant and a seven-day inoculation access period) to untreated control plants was 93%. In contrast, CLas transmission was reduced to 38.8% when test plants were protected by systemic insecticides. Our results indicated that all insecticides tested presented a potential to reduce CLas inoculation by an average of 59%; therefore, these insecticides can be used to reduce the spread of HLB.