Independent action and contrasting phenotypes of resistance genes against spotted alfalfa aphid and bluegreen aphid in Medicago truncatula

Host resistance to aphids is poorly understood. Medicago truncatula, a model legume and cultivated pasture species, was used to elucidate defense against two aphid species, Therioaphis trifolii f. maculata (spotted alfalfa aphid, SAA) and Acyrthosiphon kondoi (bluegreen aphid, BGA). Aphid performance and plant damage were compared between near-isogenic cultivars, Mogul and Borung, that differ in resistance to both aphids. Analyses of aphid resistance in Mogul x Borung F2 plants and their progeny revealed modes of action and chromosome locations of resistance genes. Separate genes were identified for SAA resistance (TTR) and BGA resistance (AKR); both mapped to chromosome 3 but were found to act independently to reduce survival and growth of their target aphid species. The TTR locus controls distinct, and contrasting, local and systemic plant responses between the near-isogenic cultivars. TTR-mediated plant responses imply interaction between a resistance factor(s) in vascular tissue and a bioactive component(s) of SAA saliva. Features of both resistance traits suggest homology to aphid resistance in other legumes; elucidation of their molecular mechanisms will likely apply to other aphid-plant interactions.     

Responses of a compatible lucerne variety to attack by spotted alfalfa aphid: Changes in the redox balance in affected tissues

The spotted alfalfa aphid,Therioaphis trifolii maculata (Buckton), caused local browning of cells surrounding feeding sites on lucerne plants (cv. Hunter River). Aqueous extracts of infested leaves underwent a marked browning process that did not occur in extracts of healthy leaves. The process was accelerated by addition of tyrosinase and peroxidase and reversed by reducing agents such as ascorbate and glutathione. In the presence of added reducing agents, the extracts produced brown precipitates, probably conjugates of phenolics with leaf proteins similar to those involved in the sealing of damaged tissuesin vivo. Partially autoxidised catechin (PAC) solutions showed an absorbance peak at 438 nm that was increased by polyphenol oxidase and decreased by ascorbate and glutathione. When extraction of tissues into PAC was used to assess redox activities, healthy tissues showed a rapid, short lived oxidising activity combined with a much more persistent reducing activity, whereas infested leaves had even greater oxidising activity and no detectable reducing activity. Soluble phenolics increased in infested leaves and stems. Total protein decreased, but the specific activity of peroxidase, catechol oxidase and superoxide dismutase relative to protein content increased. The ability of extracts to reduce cytochrome c increased, indicating an overall increase in superoxide radicals in attacked tissues. These results are consistent with a general disturbance of redox balance induced in tissues by aphid feeding, including accumulation of oxidases and phenolic substrates and loss of reducing activity and protein.     

Fitness effects of two facultative endosymbiotic bacteria on the pea aphid,Acyrthosiphon pisum,and the blue alfalfa aphid,A. kondoi

The effects of two bacterial endosymbionts, designated PASS and PAR, were evaluated on the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera:Aphididae), in which they occur facultatively, and on the blue alfalfa aphid, A. kondoi Shinji, in which these bacteria have not been found in natural populations. Subclones of pea aphids and blue alfalfa aphids, derived from parent aphid clones that did not contain PASS or PAR, were infected with one or both bacteria, generating PASS- and/or PAR-positive subclones with minimal genetic differences from the parent clones. Under laboratory conditions at 20 °C, PAR consistently reduced the fecundity (by between 19 and 60%) of subclones derived from three different parent pea aphid clones on bur clover, Medicago hispida Gaertn. PAR had intermediate effects on pea aphids reared on sweet pea, Lathyrus odoratus L., and had no significant effect on pea aphids on alfalfa, Medicago sativa L. The effect of PASS was either neutral or negative, depending on parent clone as well as host plant. Also at 20 °C, PASS reduced fecundity (70–77%) and longevity (40–48%), and increased the age of first reproduction (by up to 1.5 days) of blue alfalfa aphid reared on alfalfa and clover. PAR had a less dramatic effect (e.g., 30–39% reduction in fecundity) on these traits of blue alfalfa aphid. In contrast, PAR and PASS increased the fitness of pea aphid subclones of one parent clone reared for three generations at 25 °C on each of the three test plants. Without facultative bacteria, fecundity of the parent clone was reduced to a mean total of < 6 offspring per adult at this elevated temperature, but with PASS or PAR, mean total fecundity of its subclones was > 35. However, this ameliorative effect of facultative bacteria at 25 °C was not found for two other sets of parent clones and their derived subclones. Alate production in pea aphids was significantly increased in large populations of two PASS- and PAR-positive subclones relative to their parent clones. Attempts to transmit PASS or PAR horizontally, i.e., from aphid to aphid via feeding on host plants (bur clover), were unsuccessful.     

Host-dependent association and its implications on the relative abundance of blue alfalfa aphid and pea aphid (Homoptera: Aphididae) on alfalfa in Oklahoma

Field populations of blue alfalfa aphid and pea aphid on alfalfa were sampled during 1985 and 1986 to determine the association of co-occurrence, interspecific interactions and comparative temporal variations in the spatial dispersion patterns of these species in Oklahoma. Relative abundance of these species is discussed in the light of above analyses.Cole' s coefficient revealed a high degree of association between these species in terms of their occurrence on the same alfalfa stems in the field. Regression analyses indicated that the species populations tended to increase in concert on the same stems without evidence of competitive displacement. Spatial dispersion patterns of both species were highly aggregated at low population densities early in the season. Over time, both species tended to disperse and became less aggregated as numbers increased. It was concluded that magnitude of interspecific interactions between the blue alfalfa aphid and the pea aphid were not of a nature that they could be termed as competing species. On the contrary, a concept of an “ecospecies” is proposed for practical applications such as sampling plans and economic threshold determinations.     

Myzus persicae (green peach aphid) salivary components induce defence responses in Arabidopsis thaliana

Myzus persicae (green peach aphid) feeding on Arabidopsis thaliana induces a defence response, quantified as reduced aphid progeny production, in infested leaves but not in other parts of the plant. Similarly, infiltration of aphid saliva into Arabidopsis leaves causes only a local increase in aphid resistance. Further characterization of the defence-eliciting salivary components indicates that Arabidopsis recognizes a proteinaceous elicitor with a size between 3 and 10 kD. Genetic analysis using well-characterized Arabidopsis mutants shows that saliva-induced resistance against M. persicae is independent of the known defence signalling pathways involving salicylic acid, jasmonate and ethylene. Among 78 Arabidopsis genes that were induced by aphid saliva infiltration, 52 had been identified previously as aphid-induced, but few are responsive to the well-known plant defence signalling molecules salicylic acid and jasmonate. Quantitative PCR analyses confirm expression of saliva-induced genes. In particular, expression of a set of O -methyltransferases, which may be involved in the synthesis of aphid-repellent glucosinolates, was significantly up-regulated by both M. persicae feeding and treatment with aphid saliva. However, this did not correlate with increased production of 4-methoxyindol-3-ylmethylglucosinolate, suggesting that aphid salivary components trigger an Arabidopsis defence response that is independent of this aphid-deterrent glucosinolate.     

蚜虫唾液蛋白研究进展

蚜虫属于半翅目蚜科,多为重要的农业害虫,通过刺吸式口器吸食植物汁液,传播病毒,其爆发常常造成重大经济损失。在漫长的协同进化历程中,植物建立了高效的防御系统以应对蚜虫威胁。为了克服植物的防御反应,蚜虫也发展了相应的反制手段,其中蚜虫在取食过程中分泌的唾液蛋白能调控植物防御反应,降解植物次生物质,从而在蚜虫与植物互作中发挥着至关重要的作用。本文综述了蚜虫唾液蛋白的组分鉴定方法和相关蛋白的功能,并对唾液蛋白在蚜虫防治的应用和今后的研究方向进行了展望。常见的蚜虫唾液蛋白组分的鉴定和分析方法包括唾液蛋白的酶活性分析、唾液蛋白组学分析、唾液腺转录组学和蛋白组学分析等。但这些方法各有利弊,仅采取一种分析方法不能客观全面地反映蚜虫唾液蛋白分泌谱,多种技术手段联合分析方可提供更为逼真详实的信息。蚜虫唾液蛋白种类繁多,可分为解毒酶、保护酶、水解酶、结合功能蛋白以及分类未知的效应蛋白等。蚜虫唾液蛋白功能多样,能参与唾液鞘的形成,诱导植物防御反应,促进蚜虫取食,提高蚜虫繁殖力等。通过RNAi干扰唾液蛋白编码基因会显著改变蚜虫取食行为,并降低蚜虫存活率、产蚜量和适合度。因此,唾液蛋白是防控蚜虫的理想靶标。目前,采用寄主诱导的基因沉默(host-induced gene silencing, HIGS)技术已培育了数种靶向唾液蛋白基因的高效抗蚜作物品系,展示出了良好的应用前景。从目前研究来看,各种蚜虫唾液蛋白谱急需采用多组学手段联合分析的方法来进行完整解析。各种唾液蛋白的具体功能方面的研究还严重缺乏,需从蚜虫、植物、两者之间的互作等多维度探究唾液蛋白的作用及相关的分子机制,为发展基于蚜虫唾液蛋白调控的蚜虫防治新策略打下基础。     

Effect of annual and/or biennial burning of seed alfalfa stubble on populations of alfalfa weevil and pea aphid

The effects of five burning treatments of alfalfa (Medicago sativa L.) stubble combined with two insecticide treatments on populations of alfalfa weevil, Hypera postica (Gyllenhal), and the pea aphid, Acyrthosiphon pisum (Harris) were evaluated over an eight year period. The effects of the burn treatments were dependent on insect species, time of burn and year. Immature alfalfa weevil population levels were significantly reduced by the burn-every-autumn treatment in 3 of the 8 years, in 2 out of 8 years by the burn-e very-spring treatment, and by the alternate-year-burn treatment in 1 out of 4 years. Burning had little or no effect on pea aphid populations.     

Effect of alfalfa saponins and flavonoids on pea aphid

We studied the development and feeding behaviour of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae), on the Radius and Sapko alfalfa ( Medicago sativa L.) (Fabaceae) cultivars. Three saponins and flavones were identified in the alfalfa cultivars after thin layer chromatography separation. Cultivar Radius differed from Sapko in that it had a higher level of saponins, including zanhic acid tridesmoside and 3-GlcA,28-AraRhaXyl medicagenic acid glycoside. The flavones identified, including 7- O -β-D-glucuronopyranosyl-4'- O- [2'- O- E-feruloyl- O -β-D-glucuronopyranosyl(1→2)- O -β-D-glucuronopyranoside] apigenin, 7- O -{2- O- E-feruloyl-[β-D-glucuronopyranosyl(1→3)]- O -β-D-glucuronopyranosyl(1→2)- O -β-D-glucuronopyranoside} apigenin, and 4'- O- [2'- O -E-feruloyl- O -β-D-glucuronopyranosyl(1→2)- O -β-D-glucuronopyranoside] apigenin, occurred in tissues of both alfalfa cultivars. However, cv. Radius had very low mean flavonoid concentrations in comparison to cv. Sapko. Pea aphids that fed on cv. Radius plants showed a reduction in reproduction and survival. The aphid pre-reproductive period on cv. Radius was prolonged and the reproductive and post-reproductive periods on cv. Radius were reduced, compared to those on cv. Sapko. Cultivar Radius also negatively influenced aphid probing behaviour. This was especially the case during the initial period of the pathway phase. The results suggested that alfalfa cv. Radius, which had a higher level of saponins and a lower level of flavonoids, was less accepted by the pea aphid.     

Acceptance of low-saponin lines of alfalfa with varied phenolic concentrations by pea aphid (Homoptera: Aphididae)

This research aims to examine the effect of phenolics on pea aphid (Acyrthosiphon pisum) (Homoptera: Aphididae) development and feeding behaviour, on leaves of selected low-saponin lines of Radius alfalfa (Medicago sativa). There was a slight, negative correlation (Spearman rank correlation r _s = −0.80) between concentrations of saponins and phenols. Lines with higher concentrations of saponins had less phenolics. Levels of phenolics in low-saponin lines of alfalfa cv. Radius were related to their acceptance by the pea aphid. Our data revealed an inverse relationship between level of phenolics and the aphid abundance and its biology on studied alfalfa lines. Larval development of the pea aphid was longer, reproduction period was shorter, and the fecundity was lower on low-saponin lines with higher level of phenolics. There were observed some tendencies in the pea aphid feeding behaviour on these lines: prolonging the probing of the peripheral tissues (epidermis and mesophyll) and shortening the period of phloem sap ingestion. The better hosts for the pea aphid were low-saponin lines with low levels of phenolic compounds.     

Effect of low and high-saponin lines of alfalfa on pea aphid

Pea aphid fed on a high-saponin line of alfalfa showed reduction of reproduction and survival, and disturbance in development of its population. This line negatively influenced aphid probing behaviour, particularly prolonging the non-probing period and probing of the peripheral tissues (epidermis and mesophyll) and shortening the period of phloem sap ingestion. The high-saponin line of alfalfa differed from the low-saponin one by the presence of zanhic acid tridesmoside and a higher level of 3-GlcA,28-AraRhaXyl medicagenic acid glycoside. The saponins incorporated into sucrose-agarose gels significantly reduced number of the aphid probes into the gels and extended their duration in comparison to the control gels (without tested compounds). Role of zanhic acid tridesmoside and 3-GlcA,28-AraRhaXyl medicagenic acid glycoside as potential factors for partial resistance of alfalfa towards the pea aphid is discussed.     

Insight into watery saliva proteomes of the grain aphid,Sitobion avenae

The grain aphid, Sitobion avenae, is an economically important cereal pest worldwide. Aphid saliva plays an essential role in the interaction between aphids and their host plants. However, limited information is available regarding the proteins found in the saliva of S. avenae. Here, the watery saliva proteins from S. avenae were collected in an artificial diet and identified using a liquid chromatography–mass spectrometry/mass spectrometry analysis. A total of 114 proteins were identified in S. avenae saliva, including several enzymes, binding proteins, and putative effectors, as well as other proteins with unknown functions. In comparison with salivary proteins from nine other aphid species, the most striking feature of the salivary protein from S. avenae was the different patterns of protein functions. Several orthologous proteins secreted by other aphid species such as glucose dehydrogenase, elongation factors, and effector C002 were also detected in S. avenae saliva and speculated to play a significant role in aphid–plant interactions. These results provide further insight into the molecular basis between aphids and cereal plant interactions.     

Comparisons of salivary proteins from five aphid (Hemiptera: Aphididae) species

Aphid (Hemiptera: Aphididae) saliva, when injected into host plants during feeding, causes physiological changes in hosts that facilitate aphid feeding and cause injury to plants. Comparing salivary constituents among aphid species could help identify which salivary products are universally important for general aphid feeding processes, which products are involved with specific host associations, or which products elicit visible injury to hosts. We compared the salivary proteins from five aphid species, namely, Diuraphis noxia (Kurdjumov), D. tritici (Gillette), D. mexicana (Baker), Schizaphis graminum (Rondani), and Acyrthosiphon pisum (Harris). A 132-kDa protein band was detected from the saliva of all five species using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Alkaline phosphatase activity was detected from the saliva of all five species and may have a universal role in the feeding process of aphids. The Diuraphis species cause similar visible injury to grass hosts, and nine electrophoretic bands were unique to the saliva of these three species. S. graminum shares mutual hosts with the Diuraphis species, but visible injury to hosts caused by S. graminum feeding differs from that of Diuraphis feeding. Only two mutual electrophoretic bands were visualized in the saliva of Diuraphis and S. graminum. Ten unique products were detected from the saliva of A. pisum, which feeds on dicotyledonous hosts. Our comparisons of aphid salivary proteins revealed similarities among species which cause similar injury on mutual hosts, fewer similarities among species that cause different injury on mutual hosts, and little similarity among species which feed on unrelated hosts.     

High variation in host adaptation among clones of the pea aphid,Acyrthosiphon pisum on peas,Pisum sativum

The performance of one clone of the pea aphid,Acyrthosiphon pisum (Harris), was assessed on 37 different cultivars and species ofPisum L. In addition, random samples of 36 pea aphid clones collected on alfalfa and clover were tested on a selection of fivePisum sativum L. cultivars. Aphid performance was evaluated in terms of the mean relative growth rate (MRGR) during the first five days of life or other life history variables. The MRGR of the first-mentioned pea aphid clone differed little between cultivars. No significant differences in MRGR were found between wild and cultivatedPisum species or between modern and oldP. sativum cultivars. There was considerable variation in host adaptation among the 36 pea aphid clones within each sampled field. The pea aphid clones showed no consistent pattern in performance on four of the five pea cultivars i.e. there was a significant pea aphid genotype —pea genotype interaction. On one of the cultivars all clones performed well. Pea aphid clones collected from red clover generally performed relatively poorly on pea cultivars, in contrast to the pea aphid clones collected on alfalfa. There was no difference in performance between the two pea aphid colour forms tested. Possible reasons for the high variation and the observed adaptation patterns are discussed. The fact that all clones were collected in two adjacent fields indicates thatA. pisum shows high local intraspecific variability in terms of host adaptation.     

Alfalfa cyclitols in the honeydew of an aphid

The free cyclitols pinitol, ononitol and myo-inositol occur in the honeydew (excreta) of pea aphids (Acyrthosiphon pisum) which feed on pea aphid-susceptible alfalfa (Medicago sativa cv Caliverde). These cyclitols also occur in the leaves and stems of alfalfa. Aphids were incapable of de novo synthesis of these cyclitols. Honeydew production by the pea aphid results from ingesting phloem-sap, so the occurrence of cyclitols in honeydew results from their translocation in the phloem. The relatively high content of myo-inositol in honeydew indicates that it is selectively translocated. The most abundant alfalfa cyclitol, pinitol, had no effect on aphid feeding behavior at concentrations up to 1% (w/v; artificial diet).     

Clones of pea aphid, Acyrthosiphon pisum (Hemiptera: aphididae) distinguished using genetic markers, differ in their damaging effect on a resistant alfalfa cultivar

CUF 101, a resistant cultivar of alfalfa, was exposed to 15 clones of Acyrthosiphon pisum Harris collected from alfalfa fields in three regions of France (east, south, central west) to determine whether the level of resistance varied across the different clones. The survival of alfalfa seedlings infested at the cotyledon stage was assessed using a standardized method. Although no difference in seedling mortality was detected between clones grouped by region, there was a significant variation among the 15 pea aphid clones. In particular, two clones of southern origin were more aggressive. In addition, the different pea aphid clones were characterized using allozyme and RAPD-PCR markers. Among the 15 clones, seven allozyme genotypes (plus one when adding colour polymorphism) and 12 RAPD-PCR genotypes were distinguished. The two southern clones differing by their aggressiveness on the resistant alfalfa belonged to the same allozyme and RAPD genotype which was distinct from the other pea aphid clones. Our results reinforce the need to take into account aphid genetic diversity in breeding programmes for resistance in cultivated plants.     

The detection of salivary enzymes of phytophagous Hemiptera: a compilation of methods

The role of Hemipteran saliva and salivary enzymes is central to an understanding of the etiology of damage that these insects cause to plants. The dilute nature of the salivary secretions and the minute quantities in which they are often obtainable have made analysis and detection of salivary components very difficult. Such investigations in this laboratory have led us to formalise the techniques involved and we believe that the compilation of these methods presented herein may be useful to other research workers in this area. Methods are described for acid and alkaline phosphatase, esterase, /S-glucosidase, carbohydrases, invertase, amylase, proteinase, pectinase, cellulase, catalase, peroxidase, catechol oxidase, superoxide dismutase and ascorbic oxidase.     

豌豆蚜唾液蛋白家族的克隆及在不同发育阶段的表达

豌豆蚜Acyrthosiphon pisum是一种重要的刺吸式害虫, 其分泌的唾液对取食寄主植物和传播植物病毒有重要的作用。为了探讨蚜虫唾液蛋白的功能, 本研究克隆了在豌豆蚜唾液腺高表达的一个未知功能的蛋白家族, 该家族包括13个基因, 编码14种蛋白, 其中4个基因在唾液腺高表达。这个家族是蚜虫特有的蛋白家族, 富含半胱氨酸, 有14个半胱氨酸高度保守, 其中6个半胱氨酸形成3个保守的CXXC结构域。通过与基因组比对, 发现这个家族的基因没有内含子, 分布在基因组的9个scaffold上。用半定量逆转录PCR检测了每个成员在豌豆蚜不同发育阶段的表达, 结果显示这个家族没有发育阶段特异性。推测这个家族的表达可能具有组织特异性, 有氧化还原酶或DNA甲基化酶的功能。     

Ecological specialization correlates with genotypic differentiation in sympatric host-populations of the pea aphid

The pea aphid, Acyrthosiphon pisum, encompasses distinct host races specialized on various Fabaceae species, but the extent of genetic divergence associated with ecological specialization varies greatly depending on plant and geographic origins of aphid populations. Here, we studied the genetic structure of French sympatric pea aphid populations collected on perennial (pea and faba bean) and annual (alfalfa and red clover) hosts using 14 microsatellite loci. Classical and Bayesian population genetics analyses consistently identified genetic clusters mostly related to plant origin: the pea/faba bean cluster was highly divergent from the red clover and the alfalfa ones, indicating they represent different stages along the continuum of genetic differentiation. Some genotypes were assigned to a cluster differing from the one expected from their plant origin while others exhibited intermediate genetic characteristics. These results suggest incomplete barriers to gene flow. However, this limited gene flow seems insufficient to prevent ecological specialization and genetic differentiation in sympatry.     

The secreted salivary proteome of the pea aphid Acyrthosiphon pisum characterised by mass spectrometry

Nine proteins secreted in the saliva of the pea aphid Acyrthosiphon pisum were identified by a proteomics approach using GE‐LC‐MS/MS and LC‐MS/MS, with reference to EST and genomic sequence data for A. pisum. Four proteins were identified by their sequences: a homolog of angiotensin‐converting enzyme (an M2 metalloprotease), an M1 zinc‐dependant metalloprotease, a glucose‐methanol‐choline (GMC)‐oxidoreductase and a homolog to regucalcin (also known as senescence marker protein 30). The other five proteins are not homologous to any previously described sequence and included an abundant salivary protein (represented by ACYPI009881), with a predicted length of 1161 amino acids and high serine, tyrosine and cysteine content. A. pisum feeds on plant phloem sap and the metalloproteases and regucalcin (a putative calcium‐binding protein) are predicted determinants of sustained feeding, by inactivation of plant protein defences and inhibition of calcium‐mediated occlusion of phloem sieve elements, respectively. The amino acid composition of ACYPI009881 suggests a role in the aphid salivary sheath that protects the aphid mouthparts from plant defences, and the oxidoreductase may promote gelling of the sheath protein or mediate oxidative detoxification of plant allelochemicals. Further salivary proteins are expected to be identified as more sensitive MS technologies are developed.