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PubMed2004

Rack-1, GAPDH3, and actin: proteins of Myzus persicae potentially involved in the transcytosis of beet western yellows virus particles in the aphid.

Beet western yellows virus (BWYV) is a Polerovirus that relies on the aphid Myzus persicae for its transmission, in a persistent-circulative mode. To be transmitted, the virus must cross the midgut and the accessory salivary glands (ASG) epithelial barriers in a transcytosis mechanism where vector receptors interact with virions. In this paper, we report in vitro interaction experiments between BWYV and aphid components. Using the M. persicae clone from Colmar, we showed that a set of aphid polypeptides, separated by SDS-PAGE or 2D electrophoresis (2DE), can bind in vitro to purified wild type or mutant particles. Using subcellular fractionation, we showed that the 65-kDa polypeptide identified as symbionin is a soluble protein whereas the other polypeptides seem to be associated more or less strongly to the membrane. We hypothesize that three polypeptides, identified by mass spectrometry as Rack-1, GAPDH3, and actin, may be involved in the epithelial transcytosis of virus particles in the aphid vector.

Actins↗
PubMed2004

The evolution of host plant manipulation by insects: molecular and ecological evidence from gall-forming aphids on Pistacia.

One of the most striking characteristics of gall-forming insects is the variability in gall position, morphology, and complexity. Our knowledge of the driving forces behind the evolutionary divergence of gall types is limited. Natural enemies, competition, and behavioral constraints might be involved. We present a cladogram, based on sequences of COI and COII (1952bp), of mitochondrial DNA for the evolution of 14 species of gall-forming aphids (Fordinae). These insects induce five gall types with remarkable morphological variation on Pistacia spp. hosts. The parsimony cladogram divides the Fordinae into three lineages, Fordini and Baizongiini, and a third (new) sister group including the previously Fordini member, Smynthurodes betae (West). We then use ecological data to trace and explain the evolution of gall morphology. The aphids seem to have evolved gradually towards better ability to manipulate their host plant, induce stronger sinks, and gain higher reproductive success. We suggest that the ancestral gall type was a simple, open, "pea"-sized gall located on the leaflet midvein. Some Fordini and S. betae evolved a two-gall life cycle, inducing a new gall type on the leaflet margin. The Baizongiini improved the manipulation of their host by inducing larger galls near the midvein, with stronger sinks supporting thousands of aphids. Similar gall types are induced at similar sites on different Pistacia hosts suggesting control of the aphids on gall morphology and frequent host shifts. Thus, even extreme specialization (specific gall and host) is flexible.

Animals↗
PubMed2001

Purification and characterisation of a non-plant myrosinase from the cabbage aphid Brevicoryne brassicae (L.).

Plant myrosinases and glucosinolates constitute a defence system in cruciferous plants towards pests and diseases. We have purified for the first time a non-plant myrosinase from the cabbage aphid Brevicoryne brassicae (L.) to homogeneity. The protein was N-terminally blocked and protease (trypsin and lys c) degradation gave peptides of which five were sequenced. The protein is a dimer with subunits of mass 54 kDa+/-500 Da. Western blot analysis with an anti-aphid myrosinase antibody showed a strong cross reaction with a protein extract from the Brassica specialist, B. brassicae. The anti-aphid myrosinase antibody does not cross react with plant myrosinase neither does an anti-plant myrosinase antibody cross react with aphid myrosinase.

Animals↗
PubMed2003

Comparison of newly isolated cuticular protein genes from six aphid species.

This paper reports on the first aphids' cuticular proteins. One gene (Mpcp1) was obtained by screening a cDNA library of Myzus persicae with antibodies to a lepidopteran cuticle protein. MpCP1 presents a putative signal peptide, a central extended R&R domain, flanked by N- and C-terminal repeats of alanine, tyrosine and proline. The mRNA of Mpcp1 could be detected in a larval and in adult stages. Primers based on Mpcp1 allowed isolating and comparing cuticle protein genes from five aphid species, but not from whitefly or thrips. Comparison revealed a high degree of similarity. Data from this paper suggest that this cuticle protein family is typical and predominant to aphids. The conformation of these cuticle proteins and the significance on particular properties of aphid cuticle is discussed.

Amino Acid Sequence↗
PubMed1999

Amplified detection, using a monoclonal antibody, of an aphid-specific epitope exposed during digestion in the gut of a predator.

Monoclonal antibodies are invaluable tools for identifying and quantifying prey remains in the fore-guts of predators. However, they must be target-specific, detect an epitope that is well replicated within the prey (to enhance assay sensitivity) and, critically, recognise a site that can resist digestion. A monoclonal antibody is reported that proved to be aphid-specific and capable of detecting, and accurately identifying, as little as 16.5 ng of aphid protein within a heterologous mixture of invertebrate material. The antibody was selected by screening hybridoma lines for antibodies that bound with semi-digested aphid proteins. The antibody detected an epitope that was found, against expectation, to significantly increase in concentration with time (by approx. 50% over 6 h) in the gut of the carabid predator Pterostichus melanarius. The resultant extended antigen detection period and half-life, and the high specificity of this antibody, showed it to be an enhanced tool for studying interactions between aphids and their predators in the field. It was concluded that the antibody was initially generated to a surface epitope on a high molecular weight native protein (> 200 kD). This epitope, however, was then either replicated on internal sites progressively revealed by digestion, or new epitopes became available as the conformation of the protein changed during digestion.

Animals↗
PubMed2006

Effect of 6-methoxybenzoxazolin-2-one (MBOA) on the reproduction rate of the grain aphid (Sitobion avenae F.).

Partial host-plant resistance could make a substantial contribution to reducing the damage caused by economically important grain aphids and, therefore, to reduced insecticide use. Naturally occurring hydroxamic acids, in particular 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), have been shown to be involved in the resistance of cereals to insects. DIMBOA is unstable in aqueous solutions and has been reported to decompose to 6-methoxybenzoxazolin-2-one (MBOA). MBOA was tested on grain aphids living on artificial diet incorporated with MBOA. From 0 to 0.1 mM, the intrinsic rate of increase (rm) increased. From 0.1 to 0.3 it decreased by 73%. The rm was calculated to be 0 at 1.0 mM. Consequently, even low concentrations of MBOA may reduce the aphid multiplication to a level below the economic damage threshold. Therefore, it is possible to breed wheat varieties with a sufficiently high content of DIMBOA to decrease grain aphid populations.

Animals↗
PubMed1998

Orientation of the 'stretched' univalent X chromosome during the unequal first meiotic division in male aphids.

In situ hybridization was used to label the ends of the X chromosomes of two aphid species, Myzus persicae and Amphorophora tuberculata, in order to study the peculiar behaviour and orientation of the univalent X in aphid spermatogenesis. Anaphase I begins with the long axis of the X chromosome at right angles to the spindle and its two chromatids closely associated, but as the division proceeds the chromatids separate along most of their lengths, retaining only a midway connection as the X chromosome becomes stretched on the spindle. Both ends of one chromatid move towards one pole, while both ends of the other chromatid move towards the other pole. However, the midway connection is retained and the whole X chromosome eventually passes into one daughter cell. This form of X chromosome behaviour is common to all aphids and therefore presumably dates back to the Permian. It is independent of the type of meiosis, which in aphids can be 'normal' (reductional first division) or 'inverted' (reductional second division).

Animals↗
PubMed2003

Transformation of tobacco with genes encoding Helianthus tuberosus agglutinin (HTA) confers resistance to peach-potato aphid (Myzus persicae).

The effects of the hta gene encoding Helianthus tuberosus agglutinin (HTA) on an insect in the order Homoptera were investigated. Homologous cDNAs of hta-a, hta-b, hta-c and hta-d with CaMV35S as promoter were introduced into tobacco via Agrobacterium tumefaciens. Southern blot results showed that the exogenous hta gene was inserted into the genome of host plants, and northern blot analysis confirmed that hta was expressed in transgenic plants. A bioassay with peach-potato aphid (Myzus persicae) demonstrated that transgenic plants had deleterious effects on the insect. The average population of aphids fed on transgenic T0 plants during an 11-day assay decreased by 70%, compared controls. In transgenic plants of T1 generation, aphid fecundity inhibitions were 53.0% (hta-b) and 64.6% (hta-c), respectively. The development of aphids was notably retarded. We conclude that hta could be a novel and promising candidate for plant transgenic engineering against homopteran insect pests.

Agglutinins↗
PubMed2004

Identification of two sex pheromone components of the potato aphid, Macrosiphum euphorbiae (Thomas).

Females of the potato aphid Macrosiphum euphorbiae exhibit typical calling behavior, with virgin female oviparae raising their back legs off the substrate to release sex pheromone from glands on the tibia. Airborne collections from calling oviparae were analyzed by GC and GC-MS to determine if, like the majority of aphids examined to date, they produced (1R,4aS,7S,7aR)-nepetalactol (1) and (4aS,7S,7aR)-nepetalactone (2). Both components were present and produced in ratios that varied with age from 4:1 to 2:1. The relative stereochemical configurations of these components were determined by GC-coinjection of the aphid-derived sample with synthetic standards on both HP-1 and DB-Wax GC columns. The absolute stereochemical configuration of the nepetalactol (determined from approximately 15 microg of material in an air entrainment sample) was determined as (1R,4aS,7S,7aR)-1 by derivatization of the aphid sample with (S)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride (Mosher's acid chloride) to generate a diastereoisomer that was compared to synthetic samples by NMR spectroscopy and GC. In bioassays in the wind tunnel, M. euphorbiae males responded to potato plants with oviparae but not to unattacked plants or those infested with parthenogenetically reproducing apterae. In no-choice laboratory bioassays, the same level of male response was observed to virgins and to the 3:1-5:1 synthetic blends of nepetalactol (1):nepetalactone (2). However, the time taken to reach the source was significantly less to virgin females than to the synthetic pheromone blends. In all cases, males walked rather than flew to the source. Males showed lower responses to a 1:1 synthetic mixture and did not respond to either of the components when presented alone. Under field conditions, few M. euphorbiae males were captured in traps baited with different ratios of the synthetic pheromone. Possible reasons for the different responses under laboratory and field conditions are discussed.

Animals↗
PubMed1977

The properties of a carboxylesterase from the peach-potato aphid, Myzus persicae (Sulz.), and its role in conferring insecticide resistance.

Carboxylesterases from different strains of Myzus persicae were examined to try to understand their contribution to insecticide resistance. Preliminary evidence that they are involved comes from the good correlation between the degree of resistance and the carboxylesterase and paraoxon-degrading activity in aphid homogenates. Furthermore the carboxylesterase associated with resistance could not be separated from the insecticide-degrading enzyme by electrophoresis or ion-exchange chromatography. Homogenates of resistant aphids hydrolysed paraoxon 60 times faster than did those of susceptible aphids, yet the purified enzymes from both sources had identical catalytic-centre activities towards this substrate and also towards naphth-1-yl acetate, the latter being hydrolysed by both 2x10(6) times faster than paraoxon. These observations provide evidence that the enzyme from both sources is identical, and that one enzyme hydrolyses both substrates. This was confirmed by relating the rate of paraoxon hydrolysis to the rate at which paraoxon-inhibited carboxylesterase re-activated. Both had the same first-order rate constant (0.01min(-1)), showing clearly that the hydrolysis of both substrates is brought about by the same enzyme. Its K(m) for naphth-1-yl acetate was 0.131mm, and for paraoxon 75pm. The latter very small value could not be measured directly, but was calculated from substrate-competition studies coupled with measurements of re-activation of the diethyl phosphorylated enzyme. Since the purified enzymes from resistant and susceptible aphids had the same catalytic-centre activity, the 60-fold difference between strains must be caused by different amounts of the same enzyme resulting from mutations of the regulator gene(s) rather than of the structural gene.

Animals↗
PubMed2001

Secondary predation: quantification of food chain errors in an aphid-spider-carabid system using monoclonal antibodies.

"Secondary predation" occurs when one predator feeds on a second predator, which has in turn eaten a target prey. Detection of prey remains within predators using monoclonal antibodies cannot distinguish between primary and secondary predation, potentially leading to quantitative and qualitative food chain errors. We report the first fully replicated experiments to measure secondary predation effects, using an aphid-spider-carabid system. Aphids, Sitobion avenae, were fed to spiders, Lepthyphantes tenuis, which were allowed to digest their prey for a range of time intervals. The spiders were then fed to carabids, Poecilus (=Pterostichus) cupreus, which were again allowed to digest their prey for set periods. The anti-aphid monoclonal antibody used to identify S. avenae remains in P. cupreus was one that detected an epitope that increased in availability over the first few hours of digestion, amplifying the signal, extending detection periods and thus increasing the chances of detecting secondary predation. Despite this, and the fact that spiders are known to digest their prey more slowly than many other predators, detection of secondary predation was only possible if the carabids were killed immediately after consuming at least two spiders which were, in turn, eaten immediately after consuming aphids. As this scenario is unlikely to occur frequently in the field it was concluded that secondary predation is unlikely to be a serious source of error during field studies.

Animals↗
PubMed1997

Genetic structure of an aphid studied using microsatellites: cyclic parthenogenesis, differentiated lineages and host specialization.

In a previous study, samples of the grain aphid Sitobion avenae (F.) were collected from wheat and adjacent cocksfoot hosts in a population thought to be primarily parthenogenetic, and DNA from individual aphids was analysed with a multilocus technique. Here we have applied single-locus microsatellites and a mitochondrial DNA marker to a subset of the same DNA extracts, and have made several additional inferences about important genetic and population processes in S. avenae. Microsatellite analysis indicated very high levels of genic and genotypic variation. S. avenae fell into three genotypic groups inferred to be almost noninterbreeding, while analysis of linkage and Hardy-Weinberg equilibria suggested high levels of sexual recombination within each genotypic group. Host specialization was evident: one lineage was found only on wheat, and one (bearing many alleles inferred to be introgressed from the blackberry-grass aphid S. fragariae (Walker)) was found only on cocksfoot. The third group of interrelated genotypes was found commonly on both hosts. Although most genotypes were found only once, some were much more numerous in the sample than expected from the frequency of the alleles they contained. This, and rapid temporal changes in genotypic composition of samples, indicates strong selective differences between genotypes and lineages. In the major genotypic group, the commonest genotypes were significantly more homozygous than were rare ones: thus these data may help to explain the frequent observation of homozygous excess in aphid allozymes. The genotype group showing S. avenae-like as well as S. fragariae-like alleles also carried S. fragariae-like mitochondrial DNA in at least 25/31 cases, indicating gender-asymmetrical hybridization.

Animals↗
PubMed1999

Reproductive mode and population genetic structure of the cereal aphid Sitobion avenae studied using phenotypic and microsatellite markers.

As French populations of the aphid Sitobion avenae exhibit a range of reproductive modes, this species provides a good opportunity for studying the evolution of breeding system variation. The present analysis combined ecological and genetic investigations into the spatial distribution of variation in reproductive mode. Reproductive mode was characterized in 277 lineages of S. avenae from France, and these aphids were scored for five microsatellite loci. The analyses revealed strong geographical partitioning of breeding systems, with obligate asexuals mostly restricted to the south of France, while lineages producing sexual forms were more common in the north. Contrary to what might be anticipated for organisms with frequent parthenogenesis, there was substantial genic and genotypic diversity, even in the obligately asexual lineages. More than 120 different genotypes were detected among the 277 aphid lineages, with an average of 5.9 alleles per locus (range four to 16) and heterozygosity of 56.7%. As with previous studies of allozyme variation in aphids, most loci showed heterozygote deficits, and disequilibrium was common among allelic variants at different loci, even after removal of replicate copies of genotypes that might have been derived through clonal reproduction. Our results suggest that selection is important in structuring reproductive systems and genetic variation in French S. avenae. Canonical correspondence analysis was employed to examine the associations between genotypic and phenotypic variables, enabling the identification of alleles correlated with life-history traits.

Animals↗
PubMed1999

Molecular markers linked to breeding system differences in segregating and natural populations of the cereal aphid Rhopalosiphum padi L.

The aphid Rhopalosiphum padi shows coexistence of sexual and asexual populations, providing an opportunity to study the evolution of breeding system variation in the context of theories on the origin and maintenance of sex. However, assessments of the distribution of sexual and asexual lineages of this aphid are complicated by the difficulties in rapidly characterizing their breeding system. To facilitate this task and to gain insight into the genetic relatedness between sexual and asexual genotypes, molecular markers linked to breeding system differences were recently developed. In this study, we have successfully converted a random amplified polymorphic DNA (RAPD) marker associated with life-cycle variation in R. padi into a codominant sequences-characterized amplified region (SCAR). Segregating and natural populations of known breeding systems were examined to evaluate the life cycle-SCAR marker association. Complete linkage was found in segregating populations while the association averaged 94% in field populations. Detailed analysis of allelic distribution and sequence divergence of the SCAR locus among sexual and asexual populations provides further evidence for a unique and apparently ancient loss of sexuality in this aphid. It also suggests that occasional gene flow occurs between populations differing in their breeding system, mediated by males produced by 'asexual' lineages. This system provides the possibility for the recurrent emergence of new asexual lineages, ensuring the longer persistence of asexuality, and would have important implications for the assessment of costs and benefits of sex in aphids.

Animals↗
PubMed2002

Genetic architecture of sexual and asexual populations of the aphid Rhopalosiphum padi based on allozyme and microsatellite markers.

Cyclical parthenogens, including aphids, are attractive models for comparing the genetic outcomes of sexual and asexual reproduction, which determine their respective evolutionary advantages. In this study, we examined how reproductive mode shapes genetic structure of sexual (cyclically parthenogenetic) and asexual (obligately parthenogenetic) populations of the aphid Rhopalosiphum padi by comparing microsatellite and allozyme data sets. Allozymes showed little polymorphism, confirming earlier studies with these markers. In contrast, microsatellite loci were highly polymorphic and showed patterns very discordant from allozyme loci. In particular, microsatellites revealed strong heterozygote excess in asexual populations, whereas allozymes showed heterozygote deficits. Various hypotheses are explored that could account for the conflicting results of these two types of genetic markers. A strong differentiation between reproductive modes was found with both types of markers. Microsatellites indicated that sexual populations have high allelic polymorphism and heterozygote deficits (possibly because of population subdivision, inbreeding or selection). Little geographical differentiation was found among sexual populations confirming the large dispersal ability of this aphid. In contrast, asexual populations showed less allelic polymorphism but high heterozygosity at most loci. Two alternative hypotheses are proposed to explain this heterozygosity excess: allele sequence divergence during long-term asexuality or hybrid origin of asexual lineages. Clonal diversity of asexual lineages of R. padi was substantial suggesting that they could have frozen genetic diversity from the pool of sexual lineages. Several widespread asexual genotypes were found to persist through time, as already seen in other aphid species, a feature seemingly consistent with the general-purpose genotype hypothesis.

Alleles↗
PubMed2003

Low and homogeneous copy number of plasmid-borne symbiont genes affecting host nutrition in Buchnera aphidicola of the aphid Uroleucon ambrosiae.

The bacterial endosymbiont of aphids, Buchnera aphidicola, often provides amino acids to its hosts. Plasmid amplification of leucine (leuABCD) and tryptophan (trpEG) biosynthesis genes may be a mechanism by which some Buchnera over-produce these nutrients. We used quantitative polymerase chain reaction to assess the leuABCD/trpEG copy variability within Uroleucon ambrosiae, an aphid with a wide diet breadth and range. Both leuABCD and trpEG abundances are: (i) similar for aphids across 15 populations, and (ii) low compared to Buchnera from other aphid species (particularly trpEG). Consequently, the plasmid location of trpEG combined with Buchnera's chromosomal polyploidy may functionally limit, rather than increase, tryptophan production within Uroleucon ambrosiae.

Animals↗
PubMed2003

Selection by phage display of a variant mustard trypsin inhibitor toxic against aphids.

The mustard trypsin inhibitor, MTI-2, is a potent inhibitor of trypsin with no activity towards chymotrypsin. MTI-2 is toxic for lepidopteran insects, but has low activity against aphids. In an attempt to improve the activity of the inhibitor towards aphids, a library of inhibitor variants was constructed and cloned into the pRlac3 phagemid vector. The library of 9.3 x 107 independent colonies was created by randomisation of a stretch of five consecutive codons in the reactive site. Repeated selection rounds against bovine trypsin and chymotrypsin allowed the identification of novel, MTI-2 derived, antitrypsin and antichymotrypsin inhibitors. Chy8, the selected variant with highest affinity for bovine chymotrypsin (Ki = 32 nm versus >1000 nm for the wild-type) represents the strongest known recombinant chymotrypsin inhibitor of the MTI-2 family. It is highly toxic to nymphs of the aphid Acyrthosiphon pisum, and moderately toxic to nymphs of Aphis gossypii and Myzus persicae. The LC50 of 73 microg ml-1 towards A. pisum is the lowest value known among chymotrypsin inhibitors. The aphicidal activity of Chy8 was improved eightfold compared to the wild-type inhibitor. This demonstrates, for the first time, that bovine chymotrypsin provides a useful template to select engineered proteins highly toxic against these aphids. The selected gene will allow the development of transgenic crops that are protected against sucking insect pests.

Amino Acid Sequence↗
PubMed2004

Venomous protease of aphid soldier for colony defense.

In social aphids, morphological, behavioral, and physiological differences between soldiers and normal insects are attributed to differences in gene expression between them, because they are clonal offspring parthenogenetically produced by the same mothers. By using cDNA subtraction, we identified a soldier-specific cysteine protease of the family cathepsin B in a social aphid, Tuberaphis styraci, with a second-instar soldier caste. The cathepsin B gene was specifically expressed in soldiers and first-instar nymphs destined to be soldiers. The cathepsin B protein was preferentially produced in soldiers and showed a protease activity typical of cathepsin B. The cathepsin B mRNA and protein were localized in the midgut of soldiers. For colony defense, soldiers attack enemies with their stylet, which causes paralysis and death of the victims. Notably, after soldiers attacked moth larvae, the cathepsin B protein was detected from the paralyzed larvae. Injection of purified recombinant cathepsin B protein certainly killed the recipient moth larvae. From these results, we concluded that the cathepsin B protein is a major component of the aphid venom produced by soldiers of T. styraci. Soldier-specific expression of the cathepsin B gene was found in other social aphids of the genus Tuberaphis. The soldier-specific cathepsin B gene showed an accelerated molecular evolution probably caused by the action of positive selection, which had been also known from venomous proteins of other animals.

Animals↗