Effect of methoxyphenols on grain aphid feeding behaviour

Methoxyphenols might be important in the resistance of cereals to aphids. Electrical penetration graph (EPG) recordings were used to determine the effect of caffeic, ferulic and sinapic acids, and scopoletin on the feeding behaviour of the grain aphidSitobion avenae (F.). Aphids on wheat seedlings treated systemically with these phenols showed reduced ingestion of phloem sap and salivation into sieve elements in most cases. The earlier pathway phases of probing were prolonged. Moreover increase in number of probes as well as reduction of total time of probing was observed. In addition, no O-demethylase activity was found in homogenates of aphids fed on moderately-resistant (phenolic rich) or susceptible (phenolic poor) wheat varieties. The significance of these results for understanding the resistance of cereals to aphids is discussed.     

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.     

Tissue localisation of lettuce resistance to the aphidNasonovia ribisnigri using electrical penetration graphs

Electrical penetration graphs (EPG's) ofNasonovia ribisnigri (Mosley) (Homoptera, Aphididae) on resistant and susceptible lettuce (Lactuca sativa, Compositae) showed a large reduction in the duration of the food uptake pattern (E2) on the resistant line. No differences in EPG's were observed before the phloem was reached. Therefore, resistance is believed to be located in the phloem vessel. Both mechanical blocking of the sieve element after puncturing and a difference in composition of the phloem sap are possible resistance factors. However, a chemical factor seems more likely because of the specificity of the resistance againstN. ribisnigri.     

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.     

Development and reproductive potential of the cereal aphid Sitobion avenae on resistant wheat lines (Triticum monococcum)

The effect of wheat resistance in lines of Triticum monococcum L., on the reproductive performances of the cereal aphid (Sitobion avenae F.) was investigated. Aphids were reared from birth to adult moult either on resistant or susceptible wheat lines, and transferred as apterae to both host genotypes. The influence of these transfers on the subsequent adult weight, gonad status and reproductive performances was evaluated. Aphids transferred from resistant to susceptible plants proved able to compensate for their poor nymphal growth, mainly through additional embryo growth and an increase in the number of matured embryos within the first 10 days of their adult life. Most aphids transferred from susceptible to resistant plants died within the first week following the transfer. Their most advanced embryos matured and were born, but subsequent embryo growth was quickly reduced. The reproductive strategies adopted by S. avenae when facing plant resistance, and the hypothesis of a resistant mechanism based on a poor nutritional state of the resistant plants are discussed.     

Locating a resistance mechanism to the cabbage aphid in two wild Brassicas

Feeding behaviour of the cabbage aphid,Brevicoryne brassicae, was monitored electronically on two resistantBrassica species,B. fruticulosa andB. spinescens, and compared with a susceptible controlB. oleracea var.capitata cv. Offenham Compacta. Aphids, monitored for 10 h on the under side of leaves, performed recognizable feeding behaviour on all species. Electrical Penetration Graphs (EPGs) of aphids on resistant and susceptible plants showed no difference in behaviour for aphids on resistantBrassica species compared to susceptible until stylets penetrated the phloem sieve elements when a large reduction in the duration of passive phloem uptake (E₂ pattern) onB. fruticulosa was indicated. Although feeding behaviour on 6 week-old plants ofB. spinescens was similar to the susceptible controls, behaviour on 10 week-old plants was similar to that recorded forB. fruticulosa. The mechanism of resistance is thought to be located in the sieve element as the normal sieve element salivation (E₁) signal was either quickly terminated by withdrawal of the stylets from the sieve element or continued as a disrupted E₂ pattern. Analysis of secondary plant compounds in the threeBrassica species only identified significant differences in the glucosinolate profile. No reproducible differences were detected in the concentration of phenolics or anthocyanins. The major glucosinolate component ofB. fruticulosa andB. spinescens was gluconapin rather than glucobrassicin and glucoiberin as found in the susceptible host plant. However, both pure glucosinolates and glucosinolate extracts from all three species did not reduce aphid survival on chemically-defined artificial diets. These results suggest that the mechanism of resistance may be a mechanical blocking of the sieve element or stylets rather than a difference in the secondary plant chemistry of glucosinolates and phenolics.     

Honeydew production and sap ingestion by the cereal aphids Rhopalosiphum padi and Metopolophium dirhodum on seedlings of resistant and susceptible wheat species

Measurements of the frequency and rate of honeydew excretion by Rhopalosiphum padi and Metopolophium dirhodum, feeding on seedlings of resistant and susceptible wheat (Triticum monococcum and T. aestivum) were used to estimate the quantity of sap ingested over 24 h. Ingestion was significantly reduced on the more resistant T. monococcum and for M. dirhodum this may be due in part to a reduction in the success of phloem location.     

Faba bean forisomes can function in defence against generalist aphids

Phloem sieve elements have shut‐off mechanisms that prevent loss of nutrient‐rich phloem sap when the phloem is damaged. Some phloem proteins such as the proteins that form forisomes in legume sieve elements are one such mechanism and in response to damage, they instantly form occlusions that stop the flow of sap. It has long been hypothesized that one function of phloem proteins is defence against phloem sap‐feeding insects such as aphids. This study provides the first experimental evidence that aphid feeding can induce phloem protein occlusion and that the aphid‐induced occlusions inhibit phloem sap ingestion. The great majority of phloem penetrations in Vicia faba by the generalist aphids Myzus persicae and Macrosiphum euphorbiae triggered forisome occlusion and the aphids eventually withdrew their stylets without ingesting phloem sap. This contrasts starkly with a previous study on the legume‐specialist aphid, Acyrthosiphon pisum, where penetration of faba bean sieve elements did not trigger forisome occlusion and the aphids readily ingested phloem sap. Next, forisome occlusion was demonstrated to be the cause of failed phloem ingestion attempts by M. persicae: when occlusion was inhibited by the calcium channel blocker lanthanum, M. persicae readily ingested faba bean phloem sap.     

Triticum monococcum lines with distinct metabolic phenotypes and phloem‐based partial resistance to the bird cherry–oat aphid Rhopalosiphum padi

Crop protection is an integral part of establishing food security, by protecting the yield potential of crops. Cereal aphids cause yield losses by direct damage and transmission of viruses. Some wild relatives of wheat show resistance to aphids but the mechanisms remain unresolved. In order to elucidate the location of the partial resistance to the bird cherry–oat aphid, Rhopalosiphum padi, in diploid wheat lines of Triticum monococcum, we conducted aphid performance studies using developmental bioassays and electrical penetration graphs, as well as metabolic profiling of partially resistant and susceptible lines. This demonstrated that the partial resistance is related to a delayed effect on the reproduction and development of R. padi. The observed partial resistance is phloem based and is shown by an increase in number of probes before the first phloem ingestion, a higher number and duration of salivation events without subsequent phloem feeding and a shorter time spent phloem feeding on plants with reduced susceptibility. Clear metabolic phenotypes separate partially resistant and susceptible lines, with the former having lower levels of the majority of primary metabolites, including total carbohydrates. A number of compounds were identified as being at different levels in the susceptible and partially resistant lines, with asparagine, octopamine and glycine betaine elevated in less susceptible lines without aphid infestation. In addition, two of those, asparagine and octopamine, as well as threonine, glutamine, succinate, trehalose, glycerol, guanosine and choline increased in response to infestation, accumulating in plant tissue localised close to aphid feeding after 24 h. There was no clear evidence of systemic plant response to aphid infestation.     

Probing behaviour of the green peach aphid Myzus persicae on resistant Prunus genotypes

Electrical penetration graphs of Myzus persicae (Sulzer) (Homoptera: Aphididae) feeding behaviour on four resistant and two susceptible genotypes of peach (Prunus persica L. Batsch) and related species showed that resistance was mainly linked to (i) reduced duration of phloem sap uptake, (ii) reduced percentage of pattern E1 (salivary secretion into sieve elements) followed by pattern E2 (sap ingestion) and (iii) increased number of shifts from E1 to E2 and back. These results suggest the unsuitability of phloem sap, and thus repetitive failures to initiate sustained ingestion. Extensive comparisons of the EPGs also revealed more specific trends. Aphids on the most susceptible cultivar GF305 produced significantly longer potential drops than on other peach genotypes. On the resistant Rubira, aphids generated more penetrations before the first E occurred, indicating the possible presence of a resistance factor before the phloem was reached. The clone P1908 of the wild species Prunus davidiana displayed traits of both susceptibility (less but longer probes) and resistance. In particular, aphids produced more E1, suggesting difficulties in preparing sieve elements before feeding. The aphid probing process could be correlated with aphid settling behaviour and bionomics, as previously reported, and gave evidence for the existence of different mechanisms underlying resistance in the tested genotypes against M. persicae.     

Induced resistance by Myzus persicae in the peach cultivar `Rubira'

The effect of a previous infestation by the green peach aphid Myzus persicae (Sulzer) on the settling behaviour and reproduction of the same aphid species was investigated in the resistant peach cultivar Rubira, and compared with that observed in the susceptible control cultivar GF305. A previous infestation of 48 h triggered induced resistance in Rubira. There were significantly fewer aphids settling on preinfested than on uninfested plants, indicating an increased rejection of Rubira as a host plant. The level of induced resistance in preinfested plants was positively related to the duration of the first infestation. In GF305, previous infestation had no detrimental effect on aphid settlement and even slightly enhanced larviposition by adult females. The aphid probing behaviour after a 48-h preinfestation was also monitored for 8 h with the electrical peneration graph (EPG) technique. On preinfested GF305, most EPG parameters indicated an enhanced host plant acceptance. On preinfested GF305, aphids produced less sieve element salivation and more continuous sap ingestion than on uninfested GF305, indicating that the previous aphids provoked changes in plant properties beneficial to the test aphids. In Rubira, a major induced factor of resistance was thought to be expressed in the sieve element as phloem sap ingestion was 4-fold shorter on preinfested than on uninfested plants. The time taken by the aphid stylets to reach a sieve element was also significantly increased on preinfested Rubira, suggesting the induction of resistance factors outside the phloem. The originality of the Rubira/M. persicae interaction is discussed in the perspective of a better understanding of plant induced responses to aphids.     

Sieve element occlusion provides resistance against Aphis gossypii in TGR-1551 melons

Feeding behavior and plant response to feeding were studied for the aphid Aphis gossypii Glover on susceptible and resistant melons(cv.Iroquois and TGR-1551,respectively).Average phloem phase bout duration on TGR-1551 was<7% of the duration on Iroquois.Sixty-seven percent of aphids on TGR-1551 never produced a phloem phase that attained ingestion(EPG waveform E2)in contrast to only 7% of aphids on Iroquois.Average bout duration of waveform E2(scored as zero if phloem phase did not attain E2)on TGR-1551 was<3% of the duration on Iroquois.Conversely,average bout duration of EPG waveform El(sieve element salivation)was 2.8 times greater on TGR-1551 than on Iroquois.In a second experiment,liquid nitrogen was used to rapidly cryofix leaves and aphids within a few minutes after the aphids penetrated a sieve element.Phloem near the penetration site was then examined by confocal laser scanning microscopy.Ninety-six percent of penetrated sieve elements were occluded by protein in TGR-1551 in contrast to only 28% in Iroquois.Usually in TGR-1551,occlusion was also observed in nearby nonpenetrated sieve elements.Next,a calcium channel blocker,trivalent lanthanum,was used to prevent phloem occlusion in TGR-1551,and A.gossypii feeding behavior and the plants phloem response were compared between lanthanum-treated and control TGR-1551.Lanthanum treatment eliminated the sieve element protein occlusion response and the aphids readily ingested phloem sap from treated plants.This study provides strong evidence that phloem occlusion is a mechanism for resistance against A.gossypii in TGR-1551.     

The resistance of lettuce (Lactuca sativa L.) toNasonovia ribisnigri: Bionomics ofN. ribisnigri on near isogenic lettuce lines

The intrinsic rate of increase (r_m-values), mean relative growth rates and mortality ofNasonovia ribisnigri (Mosley) (Homoptera, Aphididae) on different lines of lettuce (Lactuca sativa L.) were determined. Near isogenic resistant and susceptible lines, plus their ancestors, were used. Bionomics ofN. ribisnigri on the resistant lines (NrNr) with the dominantNasonovia resistance gene (Nr-gene) differed clearly from the susceptible lines (nrnr). Mortality was high, no larvae reached adulthood, and no reproduction nor honeydew production was seen on the resistant lines. Transfer of aphids to susceptible plants after a period of 2 days on the resistant lines showed no signs of intoxication of aphids. Apparently there is no feeding on the resistant lines. It is not clear whether the aphids can not reach the phloem or do not accept it on the resistant line.     

Phloem specific aphid resistance in Cucumis melo line AR 5: effects on feeding behaviour and performance of Aphis gossypii

The feeding behaviour, excretion rate, and life history traits of the cotton-melon aphid, Aphis gossypii (Glover) (Homoptera, Aphididae), were measured on a resistant melon, Cucumis melo L., breeding line, AR 5. The site of resistance detection by the aphids was determined using the electrical penetration graph (EPG) technique. EPG recordings showed that resistance is expressed within the host plant, rather than on its surface, because the time to first stylet penetration was not significantly different between AR 5 and the closely related susceptible breeding line, PMR 5. EPG patterns associated with stylet pathway activities of the aphids were not significantly different between the resistant and susceptible lines. Significant behavioural differences were observed only after stylets contacted phloem sieve elements. On AR 5, the duration of salivation after sieve element puncture (waveform E1) was significantly longer, and the number of aphids showing phloem sap ingestion (waveform E2) was significantly reduced. We conclude that the resistance mechanism producing the effects seen in this study acts within the phloem sieve elements. Monitoring of excretion rates on the two genotypes showed that aphid feeding was delayed and greatly reduced on the resistant genotype. Comparisons of aphid life history traits and population development between host plant genotypes showed that the effects of resistance act throughout aphid development and are highly effective at slowing down population increase.     

Feeding by sugarcane aphid,Melanaphis sacchari,on sugarcane cultivars with differential susceptibility and potential mechanism of resistance

Feeding behavior of Melanaphis sacchari Zehntner (Hemiptera: Aphididae) was studied on sugarcane, Saccharum spp. (Poaceae), cultivars HoCP 91‐555 (resistant), LCP 85‐384 (moderately resistant), and L 97‐128 (susceptible) using the electrical penetration graph (EPG) technique. Constitutive concentrations of total phenolics and available carbohydrates, water potential at the whole‐leaf tissue level, and free amino acids (FAAs) in phloem sap extracts, and in honeydew produced by aphids fed on L 97‐128 and HoCP 91‐555 were determined. Cultivar did not influence time for M. sacchari to access phloem sieve elements. Total time in sieve elements was ca. two‐fold greater on L 97‐128 than on HoCP 91‐555, whereas it did not differ from LCP 85‐384 in either cultivar. The mean duration of individual events associated with phloem sap ingestion was ca. 50% shorter on both HoCP 91‐555 and LCP 85‐384 than on L 97‐128. Although cultivar effects were not detected for levels of total phenolics, available carbohydrates, and water potential, two free essential amino acids, histidine and arginine, were absent from phloem sap in HoCP 91‐555. Two free essential amino acids, leucine and isoleucine, and two free non‐essential amino acids, tyrosine and proline, were absent from honeydew of aphids fed on HoCP 91‐555. These results suggest that despite apparent biosynthesis of some FAAs, the absence of important FAAs in the phloem sap of HoCP 91‐555 and the inability of M. sacchari and its endosymbionts (e.g., Buchnera) to derive specific free essential and non‐essential amino acids from other ingested molecules, possibly along with other unidentified factors, underlie the pest's decreased phloem sap ingestion and consequently reduced growth potential on HoCP 91‐555.     

Role of phloem sap quality and exudation characteristics on performance of pea aphid grown on lucerne genotypes

The phloem sap of two lucerne (Medicago sativa L.) genotypes inducing differences in pea aphid,Acyrthosiphon pisum Harris (Homoptera: Aphididae) performances (weight, fecundity, survival), was collected by stylectomy (radio frequency microcautery). Sugars and amino acids were assayed. No significant difference in their concentrations could be established between genotypes. It is thus unlikely that a substantial part of the varietal resistance of these lucerne genotypes to pea aphid could be related to the content in sugars or amino acids. The same conclusion was drawn for the sugar/amino acid ratio. The amino acid profiles were globally similar. They nevertheless allowed a discrimination between the two genotypes on a few amino acids (alanine, leucine, isoleucine, arginine, ornithine) only. Moreover, the degree of resistance can be related neither to GABA concentration, nor to that of sulfur amino acids, because these constituants were either absent, or in trace amounts, whatever the lucerne genotype taken into account. Stylectomy parameters revealed some interesting differences in the sap exudation of the two genotypes. Average volume of exudation by stylet and average duration of exudation were respective 2.8 and 2.3 times higher in the susceptible genotype. These results suggest a reduced flow of phloem sap, after puncturing of the sieve-tubes by the aphid stylets on the resistant genotype; this could result in a greater difficulty in phloem sap ingestion by aphids settled on the resistant genotype and thus in suboptimal performances.     

The role of sinigrin in host plant recognition by aphids during initial plant penetration

Plant penetration behaviour (probing) of the cabbage aphid, Brevicoryne brassicae, and the pea aphid, Acyrthosiphon pisum, was studied on excised leaves of broad beans, Vicia faba, kept in water or in a 1% aqueous solution of sinigrin. Using the DC EPG (Electrical Penetration Graph) technique it was shown that the cabbage aphid on sinigrin-untreated bean leaves showed numerous short probes into epidermis and mesophyll. None of these aphids showed either phloem salivation or ingestion waveforms on untreated leaves. In contrast, on sinigrin-treated bean leaves, 35% of the probing time was spent on phloem sap ingestion (E2) and almost all aphids reached phloem vessels and started feeding. The duration of phloem salivation before phloem ingestion and the mean duration of phloem ingestion periods were similar on a host and a sinigrin-treated non-host plant. However, the total probing time by B. brassicae was 10% longer, the total phloem sap ingestion time was twice as long, and the time to the first phloem phase within a probe was three times shorter on the host plant compared to sinigrin-treated broad beans. Acyrthosiphon pisum also responded to the addition of sinigrin to broad beans, but in this case sinigrin acted as a deterrent. On sinigrin-treated leaves, A. pisum terminated probes before ingestion from phloem vessels, and none of these aphids showed phloem salivation and ingestion on treated leaves. Glucosinolates were detected in the mesophyll cells of the brassicaceous plant, Sinapis alba. Based on this finding and in addition to the foregoing EPG analysis of aphid probing on these plants and broad beans, our hypothesis is that aphids may recognise their host plants as soon as they probe the mesophyll tissue and before they start ingestion from phloem vessels.     

Interactions of Russian wheat aphid, a hymenopterous parasitoid and resistant and susceptible slender wheatgrasses

Interactions among three trophic levels of resistant and susceptible slenderwheat grasses, Elymus trachycaulum (Link) Goule ex Shinners ex. H.F. Lewis, Russian wheat aphid, Diuraphis noxia (Mordvilko), and a hymenopterous parasitoid were studied in the laboratory and greenhouse. These relationships were compared with a commercial susceptible wheat Triticum aestivum L. variety. Aphids reared on the resistant entries showed significantly lower weights and numbers. Significant reduction of parasitoid mummy weight and adult size was positively correlated with the effects on the aphids. Resistant entries also induced a longer prereproductive period for both the aphids and parasitoids. Numbers of aphids and aphid damage were significantly modified by the addition of parasitoids. Parasitism was higher on plants that did not have leaf rolling. These findings may indicate that antibiosis resistance studied here is not the most desirable because it decreases natural enemy vitality.     

Xylem ingestion by winged aphids

When aphids and their host plant are incorporated in a DC electrical circuit, phloem and xylem ingestion register as separate waveforms of the electrical penetration graph (EPG) signal. Aphids are primarily phloem feeders; xylem ingestion is seldom reported but can be induced experimentally by fasting the insects in desiccating conditions. In experiments with the black bean aphid, Aphis fabae Scop., young winged (alate) and unwinged (apterous) virginoparous adults were collected from their natal host plants (broad bean, Vicia faba L.) and allowed 3-h continuous EPG-recorded access to V. faba seedlings. Several aphids (47% of both morphs) showed ingestion from phloem sieve elements. Alate aphids also showed frequent xylem ingestion (60% of individuals), but no apterous aphids exhibited this activity. The EPG technique involves attachment of a fine gold wire electrode to each insect, a process that may affect normal behaviour at the plant surface. However, when the technique was modified to monitor the stylet activities of freely-settled aphids, high levels of xylem ingestion by alates were also recorded. The results suggest that the developmental physiology of winged aphids somehow predisposes them to xylem ingestion, possibly as a result of dehydration during the teneral period. Alate aphids may reduce their weight by fasting before take-off, giving aerodynamic benefits, but making rehydration, via xylem uptake, a priority following plant contact.     

Salivation into sieve elements in relation to plant chemistry: the case of the aphid Sitobion fragariae and the wheat, Triticum aestivum

Extended sieve element salivation (E1 waveform in the electrical penetration graph) is a characteristic activity during early sieve element punctures, particularly in resistant plants. In order to explore a chemically-mediated mechanism of resistance associated with sieve element salivation, we compared the pattern of feeding behaviour of the aphid, Sitobion fragariae (Walker), on two cultivars of the wheat Triticum aestivum L., with different concentrations of hydroxamic acids (Hx). During 24 h of electronic monitoring, aphids dedicated over 50% of the total time to phloem ingestion from the sieve elements. Total time allocated to E1 in the experiment, time to first E1 within the experiment, time allocated to E1 before a sustained phloem ingestion (E2) and the contribution of sieve element salivation to the phloem phase (E1/[E1+E2]) were significantly higher in the high-Hx cultivar. The increased salivation in plants with higher contents of Hx suggests the existence, at least in this system, of a chemically-mediated sieve element constraint.