Aphidius colemani Behavior Changes Depending on Volatile Organic Compounds Emitted by Plants Infected with Viruses with Different Modes of Transmission.

Natural enemies are an additional component that may interact directly with the plant-virus-vector association, affecting viral dispersion. In our study, we conducted olfactometry assays to explore how single and mixed infections with CMV or/and CABYV modify the attractiveness of A. colemani to aphid-free and aphid-infested melon plants using two melon genotypes. Subsequently, we investigated the influence of CABYV-infected plants infested by A. gossypii on the parasitism rate and emergence of A. colemani in a dual-choice assay under greenhouse conditions. Our study demonstrates that males showed no preference for either infected or non-infected plants. Female parasitoids exhibit a preference for volatiles emitted by CMV and mixed-infected melon plants over clean air but not over mock-inoculated plants, suggesting a response influenced by plant genotype. Female parasitoid responses to CABYV and its interactions with aphids revealed a preference for mock-inoculated plants over CABYV-infected plants and a parasitism rate slightly higher (7.12%) on non-infected plants. Our study revealed that (1) parasitoids may reject olfactory cues from CABYV-infected plants, potentially interfering with the plant's "cry for help" response; (2) in the case of CMV, whether in single or mixed infections, non-infected plants are as attractive as infected ones to parasitoids. Our findings suggest that persistent viruses manipulate aphid parasitoid behavior to their advantage, promoting virus disease in melon crops.

Isolation of Extracellular Vesicles from Phloem Sap by Size Exclusion Chromatography.

Extracellular vesicles (EVs) are nanoparticles that are released by cells and participate in the transfer of information. It is now known that EVs from mammalian cells are involved in different physiological and pathophysiological processes (antigen presentation, tissue regeneration, cancer, inflammation, diabetes, etc.). In the past few years, several studies on plants have demonstrated that EVs are also key tools for plant intercellular and cross-kingdom communications, suggesting that these nanostructures may contribute to distinct aspects of plant physiology such as development, defense, reproduction, symbiotic relationships, etc. These findings are challenging the traditional view of signaling in plants. EVs are probably involved in the phloem's transport system, since this vascular tissue plays a crucial role in translocating nutrients, defensive compounds, and informational signals throughout the plant. The collection of phloem is experimentally challenging because sap is under high turgor pressure inside the sieve elements, which have a small diameter and are hidden within the plant organs. The goals of this work are to develop new protocols that allow us to detect EVs for the first time in the phloem of the plants, and to isolate these nanovesicles for in-depth analysis and characterization. Our protocols describe two distinct methods to collect the phloem sap from rice and melon. The first method (Basic Protocol 1) involves 'Aphid stylectomy by radiofrequency microcautery' using rice plants and the aphid Sitobion avenae. This is considered the least invasive method for collecting phloem sap. The second method, 'Stem incision', involves cutting the stem of melon plants for collecting the exuded sap. Phloem sap EVs are then isolated by size exclusion chromatography. The results obtained in this study represent the first report on typical EVs isolated from in vivo-collected phloem sap. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of EVs from phloem sap: Aphid stylectomy by radiofrequency microcautery Basic Protocol 2: Isolation of EVs from phloem sap: Stem incision method.

Kaolin particle films disrupt landing, settling behavior and feeding of Trioza erytrae on lemon plants.

BACKGROUND: The citrus greening disease or Huanglongbing (HLB) is the most devastating disease of citrus crops. Trioza erytreae is a vector of HLB. Since its introduction in Europe, the insect reached the northern region of Spain and the southern region of Portugal, threatening relevant citrus production areas. Limiting the spread of HLB vectors is mandatory to prevent this disease. In this work, we assessed the effect of kaolin, a white mineral clay, on the landing, settling behavior and feeding behavior of Trioza erytreae on lemon plants. RESULTS: After kaolin application, the number of plants on which the insect was found was significantly lower than on untreated plants in the laboratory and in the field. Moreover, there were significantly fewer T. erytreae and a shorter duration of phloem-related events on kaolin-treated than untreated plants. CONCLUSION: The use of kaolin could be a suitable and efficient tool for inclusion into integrated pest management programs or organic production to reduce populations of T. erytreae and subsequently limit the spread of HLB in citrus crops. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Flight performance and the factors affecting the flight behaviour of Philaenus spumarius the main vector of Xylella fastidiosa in Europe.

The recent emergence of Xylella fastidiosa in Europe is a major threat to agriculture, including olive, almond and grape. Philaenus spumarius is the predominant vector of X. fastidiosa in Europe. Understanding vector movement is critical for developing effective control measures against bacterial spread. In this study, our goal was to set up a flight-mill protocol to assess P. spumarius flight potential and to analyse how different variables may affect its flight behaviour. We found that P. spumarius was able to fly ≈ 500 m in 30 min with a maximum single flight of 5.5 km in 5.4 h. Based on the observations, the flight potential of the females was higher in spring and autumn than in summer, and that of the males was highest in autumn. Moreover, we found that P. spumarius had a higher flight potential during the morning and the night than during the afternoon. Our results revealed that P. spumarius is likely to disperse much further than the established sizes of the infected and buffer zones designated by the EU. This knowledge on the flight potential of P. spumarius will be critical for improving management actions against P. spumarius and the spread of X. fastidiosa in Europe.

The role of plant labile carbohydrates and nitrogen on wheat-aphid relations.

Interactions between plants and herbivores are key drivers of evolution and ecosystem complexity. We investigated the role of plant labile carbohydrates and nitrogen on wheat-aphid relations in a 22 factorial combining [CO2] and nitrogen supply. We measured life history traits (assay 1) and feeding behaviour (assay 2) of bird-cherry oat aphid (Rhopalosiphum padi L.) and English grain aphid (Sitobion avenae F.) forced to feed on single leaf laminae, and reproduction of R. padi in a setting where insects moved freely along the plant (assay 3). Experimental setting impacted aphid traits. Where aphids were constrained to single leaf, high nitrogen reduced their fitness and discouraged phloem feeding. Where aphids could move throughout the plant, high nitrogen enhanced their reproduction. Aphid responses to the interaction between nitrogen and [CO2] varied with experimental setting. The number of R. padi adults varied tenfold with plant growing conditions and correlated negatively with molar concentration of sugars in stem (assay 3). This finding has two implications. First, the common interpretation that high nitrogen favours insect fitness because protein-rich animal bodies have to build from nitrogen-poor plant food needs expanding to account for the conspicuous association between low nitrogen and high concentration of labile carbohydrates in plant, which can cause osmotic stress in aphids. Second, the function of labile carbohydrates buffering grain growth needs expanding to account for the osmotic role of carbohydrates in plant resistance to aphids.

Changes in melon plant phytochemistry impair Aphis gossypii growth and weight under elevated CO2.

Elevated CO2 (eCO2) modifies plant primary and secondary metabolism that subsequently impacts herbivore insect performance due to changes in its nutritional requirements. This laboratory study evaluated interactions between Aphis gossypii Glover (Hemiptera: Aphididae) and melon (Cucumis melo L., Cucurbitaceae), previously acclimated two or six weeks to different CO2 levels, eCO2 (700 ppm) or ambient CO2 (400 ppm). Under eCO2, melon plants decreased nitrogen foliar concentration and increased carbon to nitrogen ratio, independently of acclimation period, significantly reducing the content of some amino acids (alanine, asparagine, glycine, isoleucine, lysine, serine, threonine, and valine) and increasing the carbohydrate (sucrose) content in melon leaves. The dilution in some essential amino acids for aphid nutrition could have aggravated the reduction in A. gossypii population growth reared on melon previously acclimated two weeks to eCO2, as well as the loss of aphid body mass from two successive generations of A. gossypii reared under eCO2 on plants previously acclimated two or six weeks to eCO2. The response to eCO2 of phloem feeders, such as aphids, is actually variable, but this study highlights a negative response of A. gossypii to this climate change driver. Potential implications on control of this pest in a global change scenario are discussed.

Feeding Behavior and Virus-Transmission Ability of Insect Vectors Exposed to Systemic Insecticides.

The majority of plant viruses depend on Hemipteran vectors for their survival and spread. Effective management of these insect vectors is crucial to minimize the spread of vector-borne diseases, and to reduce crop damage. The aim of the present study was to evaluate the effect of various systemic insecticides on the feeding behavior of Bemisia tabaci and Myzus persicae, as well as their ability to interfere with the transmission of circulative viruses. The obtained results indicated that some systemic insecticides have antifeeding properties that disrupt virus transmission by their insect vectors. We found that some of the tested insecticides significantly reduced phloem contact and sap ingestion by aphids and whiteflies, activities that are closely linked to the transmission of phloem-limited viruses. These systemic insecticides may play an important role in reducing the primary and secondary spread of tomato yellow leaf curl virus (TYLCV) and turnip yellows virus (TuYV), transmitted by B. tabaci and M. persicae, respectively.

Aphid Resistance: An Overlooked Ecological Dimension of Nonstructural Carbohydrates in Cereals.

Nonstructural carbohydrates in cereals have been widely investigated from physiological, genetic, and breeding perspectives. Nonstructural carbohydrates may contribute to grain filling, but correlations with yield are inconsistent and sometimes negative. Here we ask if there are hidden functions of nonstructural carbohydrates, advance an ecological dimension to this question, and speculate that high concentration of nonstructural carbohydrates may challenge the osmotic homeostasis of aphids, thus providing a working hypothesis that connects nonstructural carbohydrates with aphid resistance in cereals. In the light of this proposition, the amount and concentration of nonstructural carbohydrates should be regarded as functionally different traits, with amount relevant to the carbon economy of the crop and concentration playing an osmotic role. We conclude with suggestions for experiments to test our hypothesis.

Feeding behavior, life history, and virus transmission ability of Bemisia tabaci Mediterranean species (Hemiptera: Aleyrodidae) under elevated CO2.

The continuous rise of CO2 concentrations in the atmosphere is reducing plant nutritional quality for herbivores and indirectly affects their performance. The whitefly (Bemisia tabaci, Gennadius) is a major worldwide pest of agricultural crops causing significant yield losses. This study investigated the plant-mediated indirect effects of elevated CO2 on the feeding behavior and life history of B. tabaci Mediterranean species. Eggplants were grown under elevated and ambient CO2 concentrations for 3 weeks after which plants were either used to monitor the feeding behavior of whiteflies using the Electrical Penetration Graph technique or to examine fecundity and fertility of whiteflies. Plant leaf carbon, nitrogen, phenols and protein contents were also analyzed for each treatment. Bemisia tabaci feeding on plants exposed to elevated CO2 showed a longer phloem ingestion and greater fertility compared to those exposed to ambient CO2 suggesting that B. tabaci is capable of compensating for the plant nutritional deficit. Additionally, this study looked at the transmission of the virus Tomato yellow leaf curl virus (Begomovirus) by B. tabaci exposing source and receptor tomato plants to ambient or elevated CO2 levels before or after virus transmission tests. Results indicate that B. tabaci transmitted the virus at the same rate independent of the CO2 levels and plant treatment. Therefore, we conclude that B. tabaci Mediterranean species prevails over the difficulties that changes in CO2 concentrations may cause and it is predicted that under future climate change conditions, B. tabaci would continue to be considered a serious threat for agriculture worldwide.

High levels of arbuscular mycorrhizal fungus colonization on Medicago truncatula reduces plant suitability as a host for pea aphids (Acyrthosiphon pisum).

This study sheds light on a poorly understood area in insect-plant-microbe interactions, focusing on aphid probing and feeding behavior on plants with varying levels of arbuscular mycorrhizal (AM) fungus root colonization. It investigates a commonly occurring interaction of three species: pea aphid Acyrthosiphon pisum, barrel medic Medicago truncatula, and the AM fungus Rhizophagus irregularis, examining whether aphid-feeding behavior changes when insects feed on plants at different levels of AM fungus colonization (42% and 84% root length colonized). Aphid probing and feeding behavior was monitored throughout 8 h of recording using the electrical penetration graph (EPG) technique, also, foliar nutrient content and plant growth were measured. Summarizing, aphids took longer to reach their 1st sustained phloem ingestion on the 84% AM plants than on the 42% AM plants or on controls. Less aphids showed phloem ingestion on the 84% AM plants relative to the 42% AM plants. Shoots of the 84% AM plants had higher percent carbon (43.7%) relative to controls (40.5%), and the 84% AM plants had reduced percent nitrogen (5.3%) relative to the 42% AM plants (6%). In conclusion, EPG and foliar nutrient data support the hypothesis that modifications in plant anatomy (e.g., thicker leaves), and poor food quality (reduced nitrogen) in the 84% AM plants contribute to reduced aphid success in locating phloem and ultimately to differences in phloem sap ingestion. This work suggests that M. truncatula plants benefit from AM symbiosis not only because of increased nutrient uptake but also because of reduced susceptibility to aphids.

Barley yellow dwarf virus Can Be Inoculated During Brief Intracellular Punctures in Phloem Cells Before the Sieve Element Continuous Salivation Phase.

The distinguished intracellular stylet puncture called phloem-pd (potential drop [pd]) produced by Myzus persicae has been associated with the transmission of the semipersistently transmitted, phloem-limited Beet yellows virus (BYV, Closterovirus). However, the production of intracellular punctures in phloem cells (phloem-pd) by other aphid species and their role in the transmission of persistently transmitted, phloem-limited viruses are still unknown. Previous studies revealed that inoculation of the persistently transmitted, phloem-limited Barley yellow dwarf virus (BYDV, Luteovirus) is associated mainly with the sieve element continuous salivation phase (E1 waveform). However, the role of brief intracellular punctures that occur before the E1 phase in the inoculation of BYDV by aphids is unknown. We aimed to investigate whether the bird cherry-oat aphid Rhopalosiphum padi (Hemiptera: Aphididae) produced a stereotypical phloem-pd and to study its role in the inoculation of BYDV. The feeding behavior of viruliferous R. padi individuals in barley (Hordeum vulgare) was monitored via the electrical penetration graph (EPG) technique. The feeding process was artificially terminated after the observation of specific EPG waveforms: standard-pds, phloem-pd, and E1. Analysis of the EPG recordings revealed the production of a phloem-pd pattern by R. padi, in addition to a short, distinct E1-like pattern (short-E1), both resulting in successful inoculation of BYDV. Also, the transmission efficiency of BYDV was directly proportional to the time spent by aphids in intracellular salivation in phloem cells. Finally, we discussed the main differences between the inoculation process of semipersistent and persistently transmitted phloem-limited viruses by aphids.

Sulfoxaflor and Natural Pyrethrin with Piperonyl Butoxide Are Effective Alternatives to Neonicotinoids against Juveniles of Philaenus spumarius, the European Vector of Xylella fastidiosa.

The threat imposed by the bacterial pathogen Xylella fastidiosa to crops of utter importance to European agriculture such as olive, stone fruit and grapevine calls for immediate research against the meadow spittlebug, Philaenus spumarius (L.), the main European vector. Management tools should consider reducing juveniles of vector populations growing on weeds or cover crops during spring as nymphs have limited movement and do not contribute to disease spread. We examined a wide range of insecticides with different modes of action against P. spumarius nymphs in laboratory and semi-field glasshouse conditions. Pyrethroids (delthamethrin and λ-cyhalothrin) and natural pyrethrin (Pirecris®) + piperonyl butoxide (PBO) efficacy surpassed 86% after 24 h of exposure, without significant differences in the PBO amount tested. The inclusion of PBO caused a 3-fold increase in the mortality of P. spumarius nymphs compared to pyrethrin alone. Sulfoxaflor (Closer®) exhibited similar efficacy at 48 and 72 h but it was slow acting and mortality only reached 60% at 24 h. The LC90 was 34 ppm at 72 h. Pymetrozine, spirotetramat, azadirachtin and kaolin were not effective against nymphs (mortality <33%) although in azadirachtin-treated plants, mortality had a 3-fold increase from 24 to 72 h. Our results will help decision-making policy bodies to set up a sustainable integrated pest management of P. spumarius in areas where X. fastidiosa becomes a problem.

Pectin Methylesterases Modulate Plant Homogalacturonan Status in Defenses against the Aphid Myzus persicae.

Because they suck phloem sap and act as vectors for phytopathogenic viruses, aphids pose a threat to crop yields worldwide. Pectic homogalacturonan (HG) has been described as a defensive element for plants during infections with phytopathogens. However, its role during aphid infestation remains unexplored. Using immunofluorescence assays and biochemical approaches, the HG methylesterification status and associated modifying enzymes during the early stage of Arabidopsis (Arabidopsis thaliana) infestation with the green peach aphid (Myzus persicae) were analyzed. Additionally, the influence of pectin methylesterase (PME) activity on aphid settling and feeding behavior was evaluated by free choice assays and the Electrical Penetration Graph technique, respectively. Our results revealed that HG status and HG-modifying enzymes are significantly altered during the early stage of the plant-aphid interaction. Aphid infestation induced a significant increase in total PME activity and methanol emissions, concomitant with a decrease in the degree of HG methylesterification. Conversely, inhibition of PME activity led to a significant decrease in the settling and feeding preference of aphids. Furthermore, we demonstrate that the PME inhibitor AtPMEI13 has a defensive role during aphid infestation, since pmei13 mutants are significantly more susceptible to M. persicae in terms of settling preference, phloem access, and phloem sap drainage.

EPG combined with micro-CT and video recording reveals new insights on the feeding behavior of Philaenus spumarius.

The meadow spittlebug Philaenus spumarius plays a key role in the transmission of the bacterium Xylella fastidiosa to olive in Apulia (South Italy). Currently, available data on P. spumarius feeding behavior is limited, and a real-time observation of the different steps involved in stylet insertion, exploratory probes, and ingestion, has never been carried out. Therefore, we performed an EPG-assisted characterization of P. spumarius female feeding behavior on olive, in order to detect and analyze the main EPG waveforms describing their amplitude, frequency, voltage level, and electrical origin of the traces during stylet penetration in plant tissues. Thereafter, each of the main waveforms was correlated with specific biological activities, through video recording and analysis of excretion by adults and excretion/secretion by nymphs. Furthermore, the specific stylet tips position within the plant tissues during each of the waveforms observed was assessed by microcomputer tomography (micro-CT). Additional EPG-recordings were carried out with males of P. spumarius on olive, in order to assess possible sex-related differences. P. spumarius feeding behavior can be described by five main distinct waveforms: C (pathway), Xc (xylem contact/pre-ingestion), Xi (xylem sap ingestion), R (resting), N (interruption within xylem phase). Compared to males, females require shorter time to begin the first probe, and their Xi phase is significantly longer. Furthermore, considering the single waveform events, males on olive exhibit longer np and R compared to females.

Ultrastructure of compatible and incompatible interactions in phloem sieve elements during the stylet penetration by cotton aphids in melon.

Resistance of the melon line TGR-1551 to the aphid Aphis gossypii is based on preventing aphids from ingesting phloem sap. In electrical penetration graphs (EPGs), this resistance has been characterized with A. gossypii showing unusually long phloem salivation periods (waveform E1) mostly followed by pathway activities (waveform C) or if followed by phloem ingestion (waveform E2), ingestion was not sustained for more than 10 min. Stylectomy with aphids on susceptible and resistant plants was performed during EPG recording while the stylet tips were phloem inserted. This was followed by dissection of the penetrated leaf section, plant tissue fixation, resin embedding, and ultrathin sectioning for transmission electron microscopic observation in order to study the resistance mechanism in the TGR. The most obvious aspect appeared to be the coagulation of phloem proteins inside the stylet canals and the punctured sieve elements. Stylets of 5 aphids per genotype were amputated during sieve element (SE) salivation (E1) and SE ingestion (E2). Cross-sections of stylet bundles in susceptible melon plants showed that the contents of the stylet canals were totally clear and also, no coagulated phloem proteins occurred in their punctured sieve elements. In contrast, electron-dense coagulations were found in both locations in the resistant plants. Due to calcium binding, aphid saliva has been hypothesized to play an essential role in preventing/suppressing such coagulations that cause occlusion of sieves plate and in the food canal of the aphid's stylets. Doubts about this role of E1 salivation are discussed on the basis of our results.

Stylet penetration activities of the whitefly Bemisia tabaci associated with inoculation of the crinivirus Tomato chlorosis virus.

Bemisiatabaci is an important vector of numerous plant viruses, including the emergent semi-persistently transmitted crinivirus Tomato chlorosis virus (ToCV). Its vector feeding behaviour is complex, with important implications for virus transmission, epidemiology and control. Thus, the objective of this study was to investigate the role of the stylet penetration activities of B. tabaci in the inoculation of ToCV in tomatoes by using the electrical penetration graph (EPG) technique. EPG recordings were classified into six categories depending on the waveforms observed. The results showed that ToCV inoculation is mainly associated with stylet activities in phloem sieve elements (E1 waveform), as there was a significant increase in the rate of transmission when whiteflies performed waveform E1. The precise stylet activities - either salivation or egestion - associated with virion release, presumably from retention sites in the foregut, need further investigation.

Electrical penetration graph technique as a tool to monitor the early stages of aphid resistance to insecticides.

BACKGROUND: Sulfoxaflor, a new insecticide from the sulfoximine chemical family, and imidacloprid, a widely used neonicotinoid insecticide, were tested to assess the susceptibility and feeding behaviour of two populations of Myzus persicae: Mp61, which exhibited target-site R81T resistance to neonicotinoids, and Mp1989, a laboratory clone maintained since 1989 as a susceptible reference. RESULTS: The imidacloprid LC50 value for Mp61 was 16 times higher than for Mp1989, showing a moderate level of resistance. Sulfoxaflor LC50 values for Mp61 and Mp1989 were much closer. The probing behaviour, as assessed by electrical penetration graphs (EPGs), of both populations was clearly altered by sulfoxaflor, which reduced the ability of aphids to find and feed from the phloem. The feeding behaviour of the susceptible Mp1989 population was much more severely affected than the moderately resistant Mp61 population on imidacloprid-treated plants. PCR assays of both aphid populations followed by DNA sequencing identified differences between populations in the point mutation in the ß-subunit of the nicotinic acetylcholine receptor linked to the resistant gene against the neonicotinoid insecticide. CONCLUSIONS: Sulfoxaflor provoked feeding cessation more rapidly than imidacloprid in both aphid populations. Sharp differences in feeding behaviour were detected between the susceptible and the moderately resistant neonicotinoid-resistant aphid populations. The EPG technique can be used as a useful tool to give new insights into the functional effects of new chemical compounds and for early detection of low to moderate levels of resistance of sap-feeding insects to insecticides. The potential of this technique was validated by molecular analysis of the R81T mutation target site.

Resistance to Cucurbit aphid-borne yellows virus in Melon Accession TGR-1551.

The genetic control of resistance to Cucurbit aphid-borne yellows virus (CABYV; genus Polerovirus, family Luteoviridae) in the TGR-1551 melon accession was studied through agroinoculation of a genetic family obtained from the cross between this accession and the susceptible Spanish cultivar 'Bola de Oro'. Segregation analyses were consistent with the hypothesis that one dominant gene and at least two more modifier genes confer resistance; one of these additional genes is likely present in the susceptible parent 'Bola de Oro'. Local and systemic accumulation of the virus was analyzed in a time course experiment, showing that TGR-1551 resistance was expressed systemically as a significant reduction of virus accumulation compared with susceptible controls, but not locally in agroinoculated cotyledons. In aphid transmission experiments, CABYV inoculation by aphids was significantly reduced in TGR-1551 plants, although the virus was acquired at a similar rate from TGR-1551 as from susceptible plants. Results of feeding behavior studies using the DC electrical penetration graph technique suggested that viruliferous aphids can salivate and feed from the phloem of TGR-1551 plants and that the observed reduction in virus transmission efficiency is not related to reduced salivation by Aphis gossypii in phloem sieve elements. Since the virus is able to accumulate to normal levels in agroinoculated tissues, our results suggest that resistance of TGR-1551 plants to CABYV is related to impairment of virus movement or translocation after it reaches the phloem sieve elements.

A non-persistently transmitted-virus induces a pull-push strategy in its aphid vector to optimize transmission and spread.

Plant viruses are known to modify the behaviour of their insect vectors, both directly and indirectly, generally adapting to each type of virus-vector relationship in a way that enhances transmission efficiency. Here, we report results of three different studies showing how a virus transmitted in a non-persistent (NP) manner (Cucumber mosaic virus; CMV, Cucumovirus) can induce changes in its host plant, cucumber (Cucumis sativus cv. Marumba) that modifies the behaviour of its aphid vector (Aphis gossypii Glover; Hemiptera: Aphididae) in a way that enhances virus transmission and spread non-viruliferous aphids changed their alighting, settling and probing behaviour activities over time when exposed to CMV-infected and mock-inoculated cucumber plants. Aphids exhibited no preference to migrate from CMV-infected to mock-inoculated plants at short time intervals (1, 10 and 30 min after release), but showed a clear shift in preference to migrate from CMV-infected to mock-inoculated plants 60 min after release. Our free-choice preference assays showed that A. gossypii alates preferred CMV-infected over mock-inoculated plants at an early stage (30 min), but this behaviour was reverted at a later stage and aphids preferred to settle and reproduce on mock-inoculated plants. The electrical penetration graph (EPG) technique revealed a sharp change in aphid probing behaviour over time when exposed to CMV-infected plants. At the beginning (first 15 min) aphid vectors dramatically increased the number of short superficial probes and intracellular punctures when exposed to CMV-infected plants. At a later stage (second hour of recording) aphids diminished their feeding on CMV-infected plants as indicated by much less time spent in phloem salivation and ingestion (E1 and E2). This particular probing behaviour including an early increase in the number of short superficial probes and intracellular punctures followed by a phloem feeding deterrence is known to enhance the transmission efficiency of viruses transmitted in a NP manner. We conclude that CMV induces specific changes in a plant host that modify the alighting, settling and probing behaviour of its main vector A. gossypii, leading to optimum transmission and spread of the virus. Our findings should be considered when modelling the spread of viruses transmitted in a NP manner.

Comparative analysis of Solanum stoloniferum responses to probing by the green peach aphid Myzus persicae and the potato aphid Macrosiphum euphorbiae.

Plants protect themselves against aphid attacks by species-specific defense mechanisms. Previously, we have shown that Solanum stoloniferum Schlechtd has resistance factors to Myzus persicae Sulzer (Homoptera: Aphididae) at the epidermal/mesophyll level that are not effective against Macrosiphum euphorbiae Thomas (Homoptera: Aphididae). Here, we compare the nymphal mortality, the pre-reproductive development time, and the probing behavior of M. persicae and M. euphorbiae on S. stoloniferum and Solanum tuberosum L. Furthermore, we analyze the changes in gene expression in S. stoloniferum 96 hours post infestation by either aphid species. Although the M. euphorbiae probing behavior shows that aphids encounter more probing constrains on phloem activities-longer probing and salivation time- on S. stoloniferum than on S. tuberosum, the aphids succeeded in reaching a sustained ingestion of phloem sap on both plants. Probing by M. persicae on S. stoloniferum plants resulted in limited feeding only. Survival of M. euphorbiae and M. persicae was affected on young leaves, but not on senescent leaves of S. stoloniferum. Infestation by M. euphorbiae changed the expression of more genes than M. persicae did. At the systemic level both aphids elicited a weak response. Infestation of S. stoloniferum plants with a large number of M. persicae induced morphological changes in the leaves, leading to the development of pustules that were caused by disrupted vascular parenchyma and surrounding tissue. In contrast, an infestation by M. euphorbiae had no morphological effects. Both plant species can be regarded as good host for M. euphorbiae, whereas only S. tuberosum is a good host for M. persicae and S. stoloniferum is not. Infestation of S. stoloniferum by M. persicae or M. euphorbiae changed the expression of a set of plant genes specific for each of the aphids as well as a set of common genes.