Proton Nuclear Magnetic Resonance-Spectroscopic Discrimination of Wines Reflects Genetic Homology of Several Different Grape (V. vinifera L.) Cultivars.

BACKGROUND AND AIMS: Proton nuclear magnetic resonance spectroscopy coupled multivariate analysis (1H NMR-PCA/PLS-DA) is an important tool for the discrimination of wine products. Although 1H NMR has been shown to discriminate wines of different cultivars, a grape genetic component of the discrimination has been inferred only from discrimination of cultivars of undefined genetic homology and in the presence of many confounding environmental factors. We aimed to confirm the influence of grape genotypes in the absence of those factors. METHODS AND RESULTS: We applied 1H NMR-PCA/PLS-DA and hierarchical cluster analysis (HCA) to wines from five, variously genetically-related grapevine (V. vinifera) cultivars; all grown similarly on the same site and vinified similarly. We also compared the semi-quantitative profiles of the discriminant metabolites of each cultivar with previously reported chemical analyses. The cultivars were clearly distinguishable and there was a general correlation between their grouping and their genetic homology as revealed by recent genomic studies. Between cultivars, the relative amounts of several of the cultivar-related discriminant metabolites conformed closely with reported chemical analyses. CONCLUSIONS: Differences in grape-derived metabolites associated with genetic differences alone are a major source of 1H NMR-based discrimination of wines and 1H NMR has the capacity to discriminate between very closely related cultivars. SIGNIFICANCE OF THE STUDY: The study confirms that genetic variation among grape cultivars alone can account for the discrimination of wine by 1H NMR-PCA/PLS and indicates that 1H NMR spectra of wine of single grape cultivars may in future be used in tandem with hierarchical cluster analysis to elucidate genetic lineages and metabolomic relations of grapevine cultivars. In the absence of genetic information, for example, where predecessor varieties are no longer extant, this may be a particularly useful approach.

Activation of defence in sweet pepper, Capsicum annum, by cis-jasmone, and its impact on aphid and aphid parasitoid behaviour.

BACKGROUND: Two important pests of the sweet pepper, Capsicum annuum, are the peach potato aphid, Myzus persicae, and the glasshouse potato aphid, Aulacorthum solani. Current aphid control measures include the use of biological control agents, i.e., parasitic wasps, but with varying levels of success. One option to increase parasitoid efficiency is to activate plant defence. Therefore, sweet pepper plants were treated with the naturally occurring plant defence activator cis-jasmone, and its impact upon the behaviour and development of aphids and aphid parasitoids was investigated. RESULTS: Growth rate studies revealed that the intrinsic rate of population increase of A. solani and M. persicae on sweet pepper plants treated with cis-jasmone (cJSP) was not affected compared with untreated plants (UnSP), but the positive behavioural response of alate M. persicae towards the volatile organic compounds (VOCs) from UnSP was eliminated by cis-jasmone treatment 48 h previously (cJSP48). In addition, the aphid parasitoid Aphidius ervi preferred VOCs from cJSP48 compared with UnSP, and a significant increase in foraging time was also observed on cJSP. Analysis of VOCs collected from cJSP48 revealed differences compared with UnSP. CONCLUSION: There is evidence that treatment with cis-jasmone has the potential to improve protection of sweet pepper against insect pests.

Host acceptance by aphids: probing and larviposition behaviour of the bird cherry-oat aphid, Rhopalosiphum padi on host and non-host plants.

The probing and larviposition behaviour of the bird cherry-oat aphid, Rhopalosiphum padi on summer and winter host plants were investigated using electrical penetration graph (EPG) coupled with simultaneous video recording. In this way the precise probing history prior to parturition can be monitored and the location of possible reproductive stimulants identified. On the host plant, all gynoparae (autumn winged females that give birth to sexual females on bird cherry, Prunus padus, the primary host) and 55% of winged virginoparae (summer females which produce further virginoparae on barley, Hordeum vulgare, a secondary host) initiated larviposition before phloem contact. However, 90% of wingless virginoparae (on barley) contacted the phloem before first larviposition whilst 10% did not. Thus, phloem contact does not appear to be a pre-requisite for these aphid forms to initiate reproduction.

Aphid pheromones.

Aphids are the main insect pests of agricultural crops in temperate regions causing major economic losses. Although broad-spectrum insecticides are available for control, alternative and more targeted methods are needed due to insecticide resistance and increasing environmental pressures. An alternative control method for aphids is to exploit their pheromones, which have been extensively studied in recent years. For example, aphids release alarm pheromones in response to natural enemy attack and these could be used to deter aphids from the crops. Sex pheromones have also been identified which could be used to interfere males locating conspecific females (oviparae), as well as for manipulating natural enemies. Several hypotheses relating to how species integrity is maintained via the aphid sex pheromone have been proposed. The composition and behavioral activity of these pheromones, and how their use could be implemented in integrated pest management systems to control aphids, is discussed.

Between plant and diurnal variation in quantities and ratios of volatile compounds emitted by Vicia faba plants.

Ratios of volatile phytochemicals potentially offer a means for insects to recognise their host-plant species. However, for this to occur ratios of volatiles would need to be sufficiently consistent between plants and over time to constitute a host-characteristic cue. In this context we collected headspace samples from Vicia faba plants to determine how consistent ratios of key volatile phytochemicals used in host location by one of its insect pests, the black bean aphid, Aphis fabae, were. These were (E)-2-hexenal, (Z)-3-hexen-1-ol, 1-hexanol, benzaldehyde, 6-methyl-5-hepten-2-one, octanal, (Z)-3-hexen-1-yl acetate, (R)-linalool, methyl salicylate, decanal, undecanal, (E)-caryophyllene, (E)-beta-farnesene, (S)-germacrene D, and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, which had previously been found to be electrophysiologically and behaviourally active to A. fabae. Although the quantities of volatiles produced by V. faba showed large between plant and diurnal variation, correlations between quantities of compounds indicated that the ratios of certain pairs of volatiles were very consistent. This suggests that there is a host-characteristic cue available to A. fabae in the form of ratios of volatiles.

Regulation of wing formation and adult development in an aphid host, Aphis fabae, by the parasitoid Aphidius colemani.

Nymphs of presumptive winged gynoparae of Aphis fabae (Hemiptera: Aphididae), were exposed to female parasitoids, Aphidius colemani (Hymenoptera: Aphidiidae) and stung once with the ovipositor. Wing development was inhibited and, when aphids were parasitised during the early stages, they did not reach the adult stage but mummies with rudimentary or no wingbuds are observed in the host's fourth-stadium. These and previous studies have suggested that wing development may be inhibited by factor(s) from the maternal parasitoid injected into the host at the time of oviposition. In an attempt to identify such factor(s), saline extracts of whole female parasitoids, abdomens, ovaries and venom glands were prepared. When a saline extract of venom glands was injected into late-second-stadium aphids, many develop to fourth-stadium nymphs with rudimentary wingbuds, indicating an effect on wing formation but also showed developmental arrest and often died when attempting to moult to the adult stage. It appears that host death may be related to physiological/biochemical interactions of parasitoid and host rather than just late stage parasitoid larvae ingesting the host's vital organs. Injections with extracts into later host stadia gave similar results with regard to development to the adult, although aphids injected in the late-fourth-stadium develop normally to the adult stage with no effect on wing formation. The results indicate that the earlier the injection before the final moult the greater the effect of the injected extract on preventing adult development. Extracts prepared from head+thorax do not affect aphid development and the results indicate that there is an active factor(s) - likely a protein - in the female parasitoid's venom that disrupts wing development and/or inhibits development to the adult stage. Surprisingly, injections of extracts from male parasitoids have similar effects but the location and function of such a factor(s) in males are unknown.

Autumn leaves seen through herbivore eyes.

Why leaves of some trees turn red in autumn has puzzled biologists for decades, as just before leaf fall the pigments causing red coloration are newly synthesized. One idea to explain this apparently untimely investment is that red colour signals the tree's quality to herbivorous insects, particularly aphids. However, it is unclear whether red leaves are indeed less attractive to aphids than green leaves. Because aphids lack a red photoreceptor, it was conjectured that red leaves could even be indiscernable from green ones for these insects. Here we show, however, that the colour of autumnal tree leaves that appear red to humans are on average much less attractive to aphids than green leaves, whereas yellow leaves are much more attractive. We conclude that, while active avoidance of red leaves by aphids is unlikely, red coloration in autumn could still be a signal of the tree's quality, or alternatively serve to mask the over-attractive yellow that is unveiled when the green chlorophyll is recovered from senescing leaves. Our study shows that in sensory ecology, receiver physiology alone is not sufficient to reveal the whole picture. Instead, the combined analysis of behaviour and a large set of natural stimuli unexpectedly shows that animals lacking a red photoreceptor may be able to differentiate between red and green leaves.

Dolichodial: a new aphid sex pheromone component?

The sex pheromones of many aphid species from the subfamily Aphididae comprise a mixture of the iridoids (cyclopentanoids) (1R,4aS,7S,7aR)-nepetalactol and (4aS,7S,7aR)-nepetalactone. In this paper, we investigate whether other chemicals, in addition to nepetalactol and nepetalactone, are released from Dysaphis plantaginea (rosy apple aphid) oviparae as part of their sex pheromone. Four compounds present in an air entrainment sample collected from D. plantaginea oviparae feeding on apple (Malus silvestris c.v. Braburn) elicited electrophysiological responses from male D. plantaginea. Active peaks were tentatively identified by gas chromatography (GC) coupled with mass spectrometry, with identification confirmed by peak enhancement with authentic compounds on GC columns of different polarities. The electroantennography-active chemicals were (1R,4aS,7S,7aR)-nepetalactol, (4aS,7S,7aR)-nepetalactone, (1S,2R,3S)-dolichodial, and phenylacetonitrile. (1S,2R,3S)-Dolichodial elicited a behavioral response from male D. plantaginea and naïve-mated female parasitoids, Aphidius ervi. This is the first report of electrophysiological and behavioral responses from any aphid morph to (1S,2R,3S)-dolichodial. Whether or not (1S,2R,3S)-dolichodial is a third component of the aphid sex pheromone is discussed.

Shifting from clonal to sexual reproduction in aphids: physiological and developmental aspects.

Developmental biology is one of the fastest growing and fascinating research fields in life sciences. Among the wide range of embryonic development, a fundamental difference exists between organisms with sexual or asexual development. Aphids are unusual organisms which display alternative pathways of sexual and asexual development, the orientation of the pathway being determined by environmental conditions. These insects offer an adapted system in which to study developmental plasticity, because a side-by-side comparison of sexual and asexual development can be made in individuals with the same genotype. In this review, we describe the developmental mechanisms that have evolved in aphids for alternative sexual and asexual reproduction. In particular, we discuss how environmental cues orientate the reproductive mode of aphids from signal perception to endocrine regulation, and propose a comparative analysis of sexual and asexual gametogenesis and embryogenesis, which has been possible due to the development of molecular methods. As a result of the recent development of genomic resources in aphids, we expect these species will permit major advances in the study of the genomic basis underlying the choice of developmental fate and multiple reproduction strategies.

Identification of volatile compounds used in host location by the black bean aphid, Aphis fabae.

Behavioral and electrophysiological responses of winged Aphis fabae to volatiles of faba bean, Vicia faba (var. Sutton dwarf), plants were studied and semiochemicals used in host location were identified. In olfactometer bioassays, aphids spent significantly more time in the region of the olfactometer where V. faba volatiles from an intact plant were present than in control regions with clean air. This response also occurred when an air entrainment sample of a V. faba plant was used as the odor source. Coupled gas chromatography-electroantennography revealed the presence of 16 electrophysiologically active compounds in the air entrainment sample. Fifteen of these were identified as (Z)-3-hexen-1-ol, 1-hexanol, (E)-2-hexenal, benzaldehyde, 6-methyl-5-hepten-2-one, octanal, (Z)-3-hexen-1-yl acetate, (R)-(-)-linalool, methyl salicylate, decanal, undecanal, (E)-caryophyllene, (E)-beta-farnesene, (S)-(-)-germacrene D, and (E,E,)-4,8,12-trimethyl-1,3,7,11-tridecatetraene. An olfactometer response was observed to a 15-component synthetic blend that comprised all identified compounds at the same concentration and ratio as in the natural sample, with the aphids spending significantly more time in the treated regions of the olfactometer where volatiles were present than in the control regions. These data are discussed in the context of insect host location and crop protection.

Olfactory recognition of host plants in the absence of host-specific volatile compounds: Host location in the black bean aphid, Aphis fabae.

The black bean aphid, Aphis fabae, responds behaviorally to the odor of its host plant faba bean (Vicia faba) in olfactometer bioassays by spending more time in the treated than control regions. We have shown previously that a blend of fifteen volatile compounds emitted by V. faba elicits the same response as a headspace sample of an intact V. faba plant. Here we report that no single compound within this blend fully accounts for the behavioral response and that the responses to individual compounds are different when in the context of the blend. As none of the compounds are specific to the host, we have hypothesized that A. fabae responds preferentially to the blend of compounds when presented in a species-specific combination of volatiles or in ratios specific to V. faba. Future plans to test which of these two hypotheses pertains to host-seeking Aphis fabae are discussed.

Structure, ratios and patterns of release in the sex pheromone of an aphid, Dysaphis plantaginea.

Insect communication is primarily via chemicals. In Aphidinae aphids, the structure and ratio of iridoid (monoterpenoid) chemicals are known to be important components of the sex pheromone. However, for enhanced species specificity, it has been suggested that release of sex pheromone might be restricted to a narrow time period within the diel cycle. Here, we determine the structure, ratios and release patterns of iridoid chemicals produced by a serious global pest, the rosy apple aphid, Dysaphis plantaginea. Volatiles were collected from batches of oviparae (sexual females) and chemicals identified by gas chromatography, mass-spectrometry and microscale NMR spectroscopy. (1R,4aS,7S,7aR)-Nepetalactol and (4aS,7S,7aR)-nepetalactone were detected in a 3.7:1 ratio. To investigate timing of release, we constructed a sequential sampling device that allowed volatile chemicals to be captured hourly from 95 same-aged oviparae over 20 consecutive days. Release patterns of the two sex pheromone components show that D. plantaginea oviparae release high levels of the two components during photophase and low levels during scotophase. Release of the two components increased significantly during the first 3 h of photophase and thereafter remained at a high level until the onset of scotophase. The ratio of (1R,4aS,7S,7aR)-nepetalactol to (4aS,7S,7aR)-nepetalactone released did not change significantly between days two to 14 of the adult stadium, but from the 15th day onward there was a significant decrease in the relative amount of (1R,4aS,7S,7aR)-nepetalactol. Pheromone release was greatest on the eighth day of the adult stadium, with up to 8.4 ng of pheromone released per ovipara per hour. This is the first report on the full structural identification and ratios of volatile iridoid components collected from D. plantaginea oviparae and is also the most detailed temporal study on sex pheromone release from any aphid species. The lack of a temporally narrow and distinct period of very high sex pheromone release suggests that alternative mechanisms or factors for species recognition and isolation may be important. Findings are discussed broadly in relation to the biology of the aphid.

Antibiotics, primary symbionts and wing polyphenism in three aphid species.

The possible role of the primary Buchnera symbionts in wing polyphenism is examined in three aphid species. Presumptive winged aphids were fed on antibiotic-treated beans to destroy these symbionts. As previously reported, this leads to inhibited growth and low/zero fecundity. When such treatment is applied to the short-day-induced gynoparae (the winged autumn migrant) of the black bean aphid, Aphis fabae, it also causes many insects to develop as wingless or winged/wingless intermediate adult forms (apterisation). However, whilst antibiotic treatment of crowd-induced, long-day winged forms of the pea aphid, Acyrthosiphon pisum (a green and a pink clone) and the vetch aphid, Megoura viciae has similar effects on size and fecundity, it does not affect wing development. Food deprivation also promotes apterisation in A. fabae gynoparae but not in the crowd-induced winged morphs of the other two species. Thus, it appears that apterisation in A. fabae is not a direct effect of antibiotic treatment or a novel role for symbionts but is most likely related to impaired nutrition induced by the loss of the symbiont population.

Interactions between host-plant volatiles and the sex pheromones of the bird cherry-oat aphid, Rhopalosiphum padi and the damson-hop aphid, Phorodon humuli.

The bird cherry-oat aphid, Rhopalosiphum padi (L.), and the damson-hop aphid, Phorodon humuli (Schrank), migrate at the same time of year and colonize closely related Prunus spp. as primary hosts, but utilize (1R,4aS,7S,7aR)-nepetalactol and (1RS,4aR,7S,7aS)-nepetalactol, respectively, as sex pheromones. Interactions between these sex pheromones and benzaldehyde and methyl salicylate, plant volatiles common to primary hosts of both species, were investigated to assess whether they confer reproductive isolation between these species. Female autumn migrants (gynoparae) and males of these two species were caught in the field with water traps baited with their respective sex pheromones. Rhopalosiphum padi gynoparae and males also responded positively to benzaldehyde. Release of either benzaldehyde or methyl salicylate with the conspecific sex pheromone increased catches of both species of aphid. However, releasing both plant volatiles with the sex pheromone of R. padi increased catches of gynoparae and males, but reduced those with the sex pheromone of P. humuli. These results support the hypothesis that specific plant volatiles synergize responses of autumn migrating aphids to their sex pheromone. Because these interactions are species-specific, they may be important in allowing males to discriminate between conspecific sexual females (oviparae) and those of other aphid species.

Host plant selection by aphids: behavioral, evolutionary, and applied perspectives.

As phloem feeders and major vectors of plant viruses, aphids are important pests of agricultural and horticultural crops worldwide. The processes of aphid settling and reproduction on plants therefore have a direct economic impact, and a better understanding of these events may lead to improved management strategies. Aphids are also important model organisms in the analysis of population differentiation and speciation in animals, and new ideas on plant utilization influence our understanding of the mechanisms generating biological diversity. Recent research suggests that the dominant cues controlling plant preference and initiation of reproduction are detected early during the stylet penetration process, well before the nutrient supply (phloem) is contacted. Aphids regularly puncture cells along the stylet pathway and ingest cytosolic samples, and the cues stimulating settling and parturition likely are metabolites present in peripheral (nonvascular) plant cells. We discuss these findings and their implications for aphid evolution and management.

Electroantennogram responses of the three migratory forms of the damson-hop aphid, Phorodon humuli, to aphid pheromones and plant volatiles.

Electroantennogram (EAG) responses were recorded from alate fundatrigeniae (spring migrants), gynoparae (the winged female form that produces sexual females) and males, the three migratory forms of the damson-hop aphid, Phorodon humuli (Schrank). EAG responses of gynoparae and males showed typical dose response characteristics to (E)-2-hexenal, (-)-R-carvone, hexanenitrile and (1RS,4aR,7S,7aS)-nepetalactol, the sex pheromone of this species. The 34 plant volatiles elicited broadly similar EAG response profiles in the three migratory forms. Green leaf volatiles produced large responses in all forms; however, the relative order of responsiveness varied. EAG responses to isomers of the monoterpene carvone differed between forms, with males being most, and spring migrants least, responsive. The hop-plant volatile and aphid alarm pheromone, (E)-beta-farnesene, evoked similar EAG responses in all forms. By contrast, males were most responsive to the three sex pheromone components, (-)-(4aS,7S,7aR)-nepetalactol, (+)-(4aS,7S,7aR)-nepetalactone and (1RS,4aR,7S,7aS)-nepetalactol. Males were no more responsive to their own sex pheromone, (1RS,4aR,7S,7aS)-nepetalactol, than to the other aphid sex pheromone components tested. Spring migrants and gynoparae also responded to the three sex pheromone components. This study indicates that migratory forms of P. humuli detect a wide range of volatile compounds, and that they are equally well-adapted for the detection of volatiles associated with host and non-host plants and with other species of aphid.

Electrophysiological characterisation of olfactory sensilla in the black bean aphid, Aphis fabae.

Electrophysiological responses of three different olfactory sensilla (proximal primary rhinaria (PPR), distal primary rhinaria (DPR) and secondary rhinaria (SR)) to the sex pheromone components, nepetalactol, nepetalactone, and a plant volatile, (E)-2-hexenal, were investigated in four different morphs of the black bean aphid, Aphis fabae Scopoli. The DC responses recorded directly from the antennal sensilla and termed electrosensillograms (ESGs) were generally much larger (up to 14 mV) than electroantennogram (EAG) responses (up to 2.5 mV). Characteristic morph-specific response profiles to these compounds were observed in each type of rhinarium and response waveforms were different between (E)-2-hexenal and the sex pheromone components. (E)-2-Hexenal elicited the largest responses at PPR, while nepetalactol and nepetalactone elicited the largest responses at SR in gynoparae and males. Nepetalactol and nepetalactone also showed significant activities on DPR and PPR in all morphs. In contrast, (E)-2-hexenal had almost no activity on SR. However, almost all of the SR investigated in males and gynoparae were sensitive to both nepetalactol and nepetalactone. A small sub-set of male SR responded mainly to nepetalactol. SR of winged virginoparae did not respond to the sex pheromone compounds. Paraffin oil (the solvent control) also elicited significant responses at PPR in virginoparae but not in other morphs. In a further experiment, SR of winged virginoparae showed no response to 30 other plant volatile compounds or the alarm pheromone component, (E)-beta-farnesene. Nepetalactol and nepetalactone had similar dose-response profiles in the SR of gynoparae. The results indicate that SR in males and gynoparae are highly specialised to detect sex pheromone compounds, while the DPR and PPR are relatively broadly tuned to both plant volatiles and sex pheromone components. The presence of SR in winged virginoparae that are not responsive to sex pheromone components, alarm pheromone, or any of the plant volatile compounds tested may indicate a possible role of these sensilla to detect, as yet, unknown compound(s) with a high specificity. The present study also suggests that PPR may play a role in detecting paraffin-related compounds such as cuticular hydrocarbons of plants.

Reproductive response of generalist and specialist aphid morphs with the same genotype to plant secondary compounds and amino acids.

Many studies have paid particular attention to the role of either secondary plant compounds or amino acids as determinants of host-plant range in phytophagous insects. Here we examine the relative importance of both of these classes of compound in host acceptance by generalist and specialist morphs of the black bean aphid, Aphis fabae, that are morphologically similar and genetically identical. Eleven secondary plant compounds and six amino acids with known biological activity in aphids are presented to insects within an artificial membrane system as single compounds, mixtures of same-class compounds and combined mixtures of the two classes of compound. It is found that 1) when specific single secondary compounds and amino acids are presented to generalist and specialist morphs of A. fabae, differential responses are exclusively consistent with plant-use strategy for amino acids but not for secondary compounds, 2) neither secondary compound nor amino acid mixtures give reproductive responses entirely consistent with plant range, but the response to secondary compounds is broadly consistent with plant range whereas the response to amino acids is not, 3) when secondary compounds and amino acid mixtures are combined, the response to secondary compounds generally dominates that to amino acids. Some scenarios of plant-range determination by secondary plant compounds and amino acids, suggested by results, are discussed.

Decision making by generalist and specialist aphids with the same genotype.

One idea to explain the high incidence of specialisation in phytophagous insects is that their nervous systems are simple and unable to efficiently recognise multiple host plants. Here this 'neural limitations' hypothesis is tested using a generalist (the summer winged virginopara) and specialist (the autumn gynopara) morph of a single clone of the black bean aphid, Aphis fabae, that are almost identical morphologically and have the same genotype. Electrical monitoring of stylet behaviour and non-invasive behavioural observations are used to compare initial plant recognition, first reproduction, 'first registered phloem contact' and 'phloem acceptance' of the generalist and specialist on their specific host plants (generalist--herbaceous hosts, Beta vulgaris, Papaver dubium, Rumex obtusifolius, Vicia faba; specialist--woody host, Euonymus europaeus) and on the same host (E. europaeus). Additional analyses of behaviour over the first few minutes of plant contact are carried out for three mutual non-host plants: Euonymus alatus, Prunus padus and Brassica pekinensis. Few examples are found where the host-selection behaviour of the specialist on its host plant is more efficient than the generalist across its hosts and there is little difference in the behaviour of the different forms on the same host. Indeed there are several instances where the generalist is behaviourally more efficient than the specialist. These include absolute time to first reproduction by the generalist on various herbaceous hosts and the same host, E. europaeus, absolute time to first registered phloem contact by the generalist on P. dubium, and incidence of phloem acceptance by the generalist on several of its herbaceous hosts. Rejection of non-hosts is carried out more efficiently by the specialist gynopara on P. padus and B. pekinensis but not on E. alatus. For generalist and specialist morphs of the same A. fabae genotype, therefore, broad host range does not appear to be associated with a reduced ability to make plant-use decisions.