Abstract. Plant penetration by Aphis fubue (Scopoli) was recorded by the electrical penetration graph (EPG) technique and followed by stylectomy during wave-forms that were suspected of indicating sieve element punctures. The severed stylets in the plant tissue were subsequently processed for transmission electron microscopy (TEM) and sectioned either transverse or longitudinal to the stylets. Two completely serially sectioned probes from the epidermis to the phloem were reconstructed.In one probe the stylet pathway went to a sieve element and showed many empty branches of salivary sheath material. Breaks in cell walls filled with sheath material demonstrated that the majority of cells bordering the track had been punctured, which supports earlier evidence from EPGs. All types of cells showed punctures and the highest number was found inside the vascular bundle. Very few cells died, which would appear to be important for virus transmission, and in others cellular reactions remained limited to some callose formation. The route of the stylets was intercellular and passed through the secondary wall material. The role of pectinase in intercellular penetration, and previous evidence for intracellular tracks are discussed. Most sieve elements had been punctured but only one was eventually accepted. Thus, reaching a sieve element in a host plant does not automatically imply its acceptance though the reason remains unclear. Gelation of phloem proteins was shown in the stylet canal.In a second probe, plant cytological and morphological correlations with the EPG were emphasized. Probes by other aphid-plant combinations showed great similarity.
Successful phloem feeding requires overcoming a number of phloem-related plant properties and reactions. The most important hurdle is formed by the phloem wound responses, such as coagulating proteins in the phloem sieve elements of the plant and in the capillary food canal in the insect's mouth parts, i.e. the stylets. It seems that in order to prevent protein clogging inside a sieve element, ejection of watery saliva plays an important role. This ejection is detected in the electrical penetration graph (EPG) as E1 salivation and always precedes phloem sap ingestion. During this feeding from sieve elements, another regular and concurrent salivation also occurs, the watery E2 salivation. This E2 saliva is added to the ingested sap and, it probably prevents phloem proteins from clogging inside the capillary food canal. Whatever the biochemical mode of action of the inhibition of protein coagulation might be, in some plants aphids do not seem to be able to prevent clogging, which may explain the resistance to aphids in these plants. The relevance of this hypothesis is demonstrated by new experimental results and is related to new EPG results from plants with phloem-located resistance.
A simple amplifier is used for electronic recording of aphid penetration behaviour. Several different patterns of electrical signal are described, some of which can be correlated with different aspects of feeding behaviour. RÉSUMÉ ENREGISTRER ÉLECTRONIQUEMENT LE COMPORTEMENT DE LA PÉNÉTRATION CHEZ LES PUCERONS Le comportement de la pénétration chez les pucerons (Homoptera) est enregistré électroniquement par un amplificateur simple. Cette méthode fut introduite par McLean & Kinsey (1964). Les ondes différentes du signal électrique sont descrites à nouveau et classifiées de A à F. Des expériences avec isotopes radio‐actives ont mis en évidence une corrélation significative d'ingestion avec l'onde D ainsi qu'avec A, B en C ensemble. Il apparaît que la sécretion de la salive est seulement en corrélation avec les ondes A, B et C ensemble et non avec D. Cependent en même temps que l'onde D, l'onde E apparait et il a été mis en évidence par expériences électrophysiologiques que cette dernière represente l'activité de la pompe à salive. Il semble que la salive radio‐active produite pendant D est ingerée directement après excrétion. Des observations visuelles démontrent une corrélation de l'onde B avec la formation fourreau salivaire. Aucune recherche n'a encore été faite sur les relations entre les ondes A, C et F et le comportement.
Aphids, which constitute one of the most important groups of agricultural pests, ingest nutrients from sieve tubes, the photoassimilate transport conduits in plants. Aphids are able to successfully puncture sieve tubes with their piercing mouthparts (stylets) and ingest phloem sap without eliciting the sieve tubes' normal occlusion response to injury. Occlusion mechanisms are calciumtriggered and may be prevented by chemical constituents in aphid saliva injected into sieve tubes before and during feeding. We recorded aphid feeding behavior with the electrical penetration graph (EPG) technique and then experimentally induced sieve tube plugging. Initiation of sieve tube occlusion caused a change in aphid behavior from phloem sap ingestion to secretion of watery saliva. Direct proof of ''unplugging'' properties of aphid saliva was provided by the effect of aphid saliva on forisomes. Forisomes are proteinaceous inclusions in sieve tubes of legumes that show calcium-regulated changes in conformation between a contracted state (below calcium threshold) that does not occlude the sieve tubes and a dispersed state (above calcium threshold) that occludes the sieve tubes. We demonstrated in vitro that aphid saliva induces dispersed forisomes to revert back to the nonplugging contracted state. Labeling Western-blotted saliva proteins with 45 Ca 2؉ or ruthenium red inferred the presence of calcium-binding domains. These results demonstrate that aphid saliva has the ability to prevent sieve tube plugging by molecular interactions between salivary proteins and calcium. This provides aphids with access to a continuous flow of phloem sap and is a critical adaptation instrumental in the evolutionary success of aphids.calcium-binding ͉ plant-aphid interaction ͉ plugging ͉ saliva proteins ͉ sieve element
Transmission of non-persistent plant viruses is related to aphid behaviour during superficial brief probes. A widely accepted hypothesis postulates that virus acquisition occurs during ingestion of plant cell contents, and inoculation during egestion or regurgitation of previously ingested sap. Although conceptually attractive, this ingestionegestion hypothesis has not been clearly demonstrated. Furthermore, it overlooks the anatomy of the tips of the stylets (mouthparts) and, consequently, the potential role of salivation in the inoculation process. Here, we used the electrical penetration graph (EPG) technique to investigate aphid-stylet activities associated with uptake (acquisition) and release (inoculation) of two nonpersistently transmitted viruses. Our results show that acquisition occurs primarily during the last sub-phase (II-3) of intracellular stylet punctures, whereas inoculation is achieved during the first sub-phase (II-1). An alternative mechanism to the ingestion-egestion hypothesis is proposed on the basis of our findings.The transmission of non-persistent plant viruses is unique to aphids (Homoptera : Aphididae) because they exhibit specific and characteristic activities during brief (a few seconds or minutes) and superficial probes, involved in host plant recognition (Pollard, 1973). Two different hypotheses have been proposed to explain the mechanism of transmission. The first, so-called stylet-borne hypothesis (Kennedy et al., 1962),
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