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PubMed2002

Acylated flavonol glycosides as probing stimulants of a bean aphid, Megoura crassicauda, from Vicia angustifolia.

A bean aphid, Megoura crassicauda, which feeds selectively on the plant genus Vicia (Fabaceae), was found to be stimulated to probe an extract solution of the host plant, narrowleaf vetch, Vicia angustifolia L., depositing characteristic stylet sheaths on a parafilm membrane. Two acylated flavonol glycosides were isolated as the specific probing stimulants from the extracts and characterized as quercetin 3-O-alpha-L-arabinopyranosyl-(1-->6)-[2"-O-(E)-p-coumaroyl]-beta-D-glucopyranoside and quercetin 3-O-alpha-L-arabinopyranosyl-(1-->6)-[2"-O-(E)-p-coumaroyl]-beta-D-galactopyranoside. A mixture of these compounds in the same equivalency strongly induced the probing response from M. crassicauda, suggesting their kairomonal roles during host recognition.

Acylation↗
PubMed2001

A novel usage of random primers for multiplex RT-PCR detection of virus and viroid in aphids, leaves, and tubers.

A multiplex reverse transcription polymerase chain reaction (m-RT-PCR) was developed for the simultaneous detection of five potato viruses and a viroid. The synthesis of cDNAs used for amplification was primed by hexanucleotides (random primers, RP). An RNA extraction procedure employing DNase I, is routinely used to isolate potato viruses and viroid (Potato virus S, PVS; Potato leafroll virus, PLRV; Potato virus X, PVX; Potato virus A and Y, PVA, PVY; and Potato spindle tuber viroid, PSTVd) from infected tissues. This extraction method produced deoxy-oligonucleotides, which in turn were used to prime the reverse transcription of RNA templates of all the viruses and the viroid. A time-course study from 15 s to 30 min showed optimal oligonucleotide generation by DNase I occurred at 10 min, an incubation time already incorporated in the extraction protocol. The presence of oligonucleotides capable of priming cDNA synthesis was also demonstrated in RNA preparations from aphids, leaves, and tubers. In order to duplicate the priming of templates by oligonucleotides, commercially available hexanucleotides were used as RP. When fragments were amplified from 5'- and 3'-ends of the random primed cDNA of PVY genome, bands of similar intensity were observed. In contrast, when two fragments (short and long) from the P1 gene region of the PVA genome were amplified, the yield of the short fragment was significantly higher in intensity than that of the long fragment in random primed cDNA. Irrespective of the origin of the primers (generated during extraction vs. commercially purchased), single or multiple viruses or the viroid were detected by amplification of random primed cDNAs present individually in the reaction or in a cDNA pool consisting of five viruses and the viroid. The cDNA produced by RP or virus specific primers (SP) was used to detect PLRV and PVY from infected tubers in a duplex reverse transcription polymerase chain reaction (d-RT-PCR). The RP cDNA gave increased detection. Comparison of RP primed cDNAs from dormant or sprouted tubers and leaves showed that for some cultivars, such as 'Shepody', leaves were more reliable for PVY and PLRV detection than the tubers, in both the d- and m-RT-PCR.

Animals↗
PubMed2003

Experimental layout, data analysis, and thresholds in ELISA testing of maize for aphid-borne viruses.

Several aspects of enzyme-linked immunosorbent assay (ELISA) procedures and data analysis have been examined in an attempt to find a rapid and reliable method for discriminating between 'positive' and 'negative' results when testing a large number of samples. A layout of ELISA plates was designed to reduce uncontrolled variation and to optimize the number of negative and positive controls. A transformation using the fourth root (A(1/4)) of the optical density readings corrected for the blank (A) stabilized the variance of most ELISA data examined. Transformed A values were used to calculate the true limits, at a set protection level, for false positive (C) and false negative (D). Methods are discussed to reduce the number of undifferentiated samples, i.e. the samples with response falling between C and D. The whole procedure was set up for use with an electronic spreadsheet. With the addition of few instructions of the type 'if em leader then em leader else' in the spreadsheet, the ELISA results were obtained in the simple trichotomous form 'negative/undefined/positive'. This allowed rapid analysis of more than 1100 maize samples testing for the presence of seven aphid-borne viruses-in fact almost 8000 ELISA samples.

Animals↗
PubMed2003

Multiplex RT-PCR detection of four aphid-borne strawberry viruses in Fragaria spp. in combination with a plant mRNA specific internal control.

The principal aphid-borne viruses infecting Strawberry (Fragaria spp.) Strawberry crinkle virus (SCV), Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV) and Strawberry vein banding virus (SVBV) can cause serious crop losses. In this paper, a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) method is described for the simultaneous detection of all four viruses in combination with a plant mRNA specific internal control which can be used as an indicator of the effectiveness of the extraction and RT-PCR. In total, 18 strawberry isolates infected naturally were analysed by this method. Every combination of RNA virus was able to be detected and a full complement of all four viruses were found together in three isolates, all taken from wild strawberry (Fragaria chiloensis (L.) Duch.) in Chile. The upper detection limit for the four viruses was at an extract dilution of 1/200. The broad applicability of the RNA specific internal control primers-which produced a PCR fragment of the expected size in 25 of 27 plant species tested-combined with improvements, made in extraction methods described provides potentially a standard method for comparable RT-PCR analyses in a wide variety of plant species.

Animals↗
PubMed1998

Reverse-transcription polymerase chain reaction for the detection of viruses from plants and aphids.

A reverse transcription polymerase chain reaction (RT-PCR) protocol used for the detection of potato viruses in dormant tubers and leaves and in an aphid vector is described. Problems in plant sample preparation from different hosts, uneven distribution or low concentration of viruses and the presence of PCR inhibitors in plant extracts are discussed and various ways to eliminate their effect are described. Using Potyviridae viruses, it has been shown that RT-PCR in various modified forms can be used to differentiate viruses at the level of family, genus, species, strains and their subtypes or serotypes. The specificity of primer pairs and PCR modifications has been used to separate closely related potato viruses A and PVY strains (PVY(O), PVY(N) and PVY(NTN)) from a known mixture.

Animals↗
PubMed2000

Control of aphid-borne Lily symptomless virus and Lily mottle virus in Lilium in the Netherlands.

An overview is presented on the management of viruses of Lilium crops in the Netherlands since the 1960s. This mainly concerns Lily symptomless virus (LSV) and Lily mottle virus (LMoV). Various factors which affect the efficiency of control are considered. The variable symptoms of LMoV in the many vegetatively propagated cultivars grown (c. 340) pose problems in the efficient roguing of diseased plants of some cultivars. Additionally the acceptable incidence of viruses such as LSV which are generally symptomless in field-grown plants may cause problems under the unfavourable light and growing conditions of year-round cut-flower production in greenhouses. The reduction in bulb yield and quality caused by viruses of lilies necessitates a further decrease in the already low virus levels still tolerated in lily bulb stocks. The routine detection of LSV and LMoV by ELISA has been developed extensively over the years. The impact of testing bulbs ('bulb test') during storage has been important in achieving an overall decrease in virus incidence in many stocks of lily cultivars, e.g. in Asiatic hybrids. The 'leaf test' used to assess many other cultivars, e.g. oriental hybrids, in which the bulb test for LMoV is not applicable, will be developed so as to eliminate at an early stage severely infected stocks that were initially intended for further propagation. The spread of the aphid-borne LSV and LMoV generally occurs very rapidly. The low virus incidence in the initially virus-tested stocks obtained by tissue culture procedures is effective in decreasing the access of vectors to virus-infected lilies. The viruses spread mainly in June and July, considerably less in May and least in August/September. Consequently the routine spraying of mixtures of mineral oil and pyrethroid insecticide is generally done weekly in May, June and July and fortnightly in August and September. There are differences in efficacy of the different brands of mineral oil. The rapid propagation of lilies, as done routinely by the scaling of bulbs and by tissue culture procedures, enables the rapid bulking of virus-tested and other stocks of high quality which have the health status required. The impact of the different factors in the management and control of viruses in Lilium crops in the Netherlands is discussed.

Animals↗
PubMed2000

Mitigating epidemics caused by non-persistently transmitted aphid-borne viruses: the role of the pliant environment.

Using Soybean mosaic virus as a model system, the rate, magnitude and timing of epidemics caused by non-persistently transmitted, aphid-borne viruses are examined under various field conditions. Emphasis is placed on the behavioural responses of vectors to environmental cues, although all three biotic components (host plant, vector and virus) are considered. Both single and double manipulations of the cropping system environment are explored using a computer model developed earlier by Ruesink and Irwin (Plant Virus Epidemics: Monitoring, Modelling and Predicting Outbreaks.

Animals↗
PubMed2000

Cloning, heterologous expression and co-assembly of Mpbeta1, a nicotinic acetylcholine receptor subunit from the aphid Myzus persicae.

Nicotinic acetylcholine receptors (nAChRs) play a major role in excitatory synaptic transmission in insects and are also the target site for chloronicotinyl insecticides such as imidacloprid. Here we report the cloning and characterization of a novel nAChR beta subunit, Mpbeta1, from the aphid Myzus persicae, an economically important pest species. Sequence analysis has identified an open reading frame of 509 amino acids with features typical of nAChR subunits. The Mpbeta1 gene is expressed as a single major transcript of 4.6 kb, considerably larger than the predicted length of the Mpbeta1 open reading frame (1527 bp). By heterologous expression in Drosophila S2 cells, the Mpbeta1 subunit has been shown to co-assemble with the previously cloned nAChR subunits Mpalpha1 and Mpalpha2. In contrast, no co-assembly of Mpbeta1 could be detected with either Mpalpha3 or Mpalpha4. With the aim of gaining a clearer insight into the influence of subunit composition upon assembly, the ability of M. persicae nAChR subunits to co-assemble with vertebrate nAChR subunits has also been examined.

Animals↗
PubMed2003

New use of broomcorn millets for production of granular cultures of aphid-pathogenic fungus Pandora neoaphidis for high sporulation potential and infectivity to Myzus persicae.

Glutinous broomcorn millets from the crop Panicum miliaceum were first used as substrate to produce granular cultures of Pandora neoaphidis, an obligate fungal pathogen specific to aphids. Carrying a water content of 36.5% after being steamed in a regular autoclaving procedure, millet grains of each 15 g (dry weight) in a 100-ml flask were mixed with 3 ml modified Sabouraud dextrose broth containing half a mashed colony of P. neoaphidis grown on egg yolk milk agar and then incubated at 20 degrees C and a light/dark cycle of 12 h/12 h for 21 days. Based on individually monitoring conidial production potential of 20 millet grains sampled from an arbitrarily taken flask at 3-day intervals, the millet cultures incubated for 6-15 days were capable of producing 16.8-23.4 x 10(4) conidia per millet grain with conidial ejection lasting for up to 6 days. The cultured millet grains individually produced significantly more conidia than apterous adults of Myzus persicae killed by P. neoaphidis (8.4 x 10(4) conidia per cadaver) and sporulated twice longer. The modeling of time-dose-mortality data from bioassays on M. persicae apterae exposed to conidial showers from the cultured millet grains and the mycelial mats produced in liquid culture resulted in similar estimates of LC(50) (millets: 21.4, 7.3, and 4.9 conidia mm(-2) on days 5-7 after exposure; mycelial mats: 22.1, 10.6, and 7.7 conidia mm(-2)) although the LT(50) estimated at a given conidial concentration was slightly smaller for the millet cultures than for the mycelial mats. This indicates that the millet grains cultured with P. neoaphidis produced conidia as infective as or slightly more infective to M. persicae than those from the mycelial mats. Based on the sporulation potential, infectivity, and ease and cost of the millet cultures, the method developed in this study highly improved in vitro cultures of P. neoaphidis and may adapt to culturing other entomophthoralean fungi for microbial control of insect pests.

Animals↗
PubMed2000

Genomic copy number of intracellular bacterial symbionts of aphids varies in response to developmental stage and morph of their host.

Buchnera, endosymbiotic bacteria of aphids possess many genomic copies per cell. In this study, we estimated genomic copy number per Buchnera cell from host insects at various developmental stages and of two different morphs, apterae and alatae, by fluorimetry and real-time quantitative PCR. The results indicated that the genomic copy number of Buchnera increased during postembryonic development of insects to adulthood, and that it decreased during the host's ageing. In Buchnera from alatae, the genomic copy number per cell was about twice as many as in those from apterae. DAPI-staining showed that the distribution of the genomic DNA in the Buchnera cells from old insects tended to aggregate, suggesting that intracellular structure of the genomic DNA of Buchnera varies in response to the physiological conditions of their host.

Animals↗
PubMedDate not supplied

Characterization and feeding deterrent effects on the aphid, Schizaphis graminum, of some derivatives of the sweet compounds, stevioside and rebaudioside A.

The intensely sweet compounds, stevioside [1] and rebaudioside A [2], and 15 of their derivatives, were tested for feeding deterrent activity against the aphid, Schizaphis graminum. Included with these compounds was steviol [4], the aglycone of stevioside and rebaudioside A that was one of the most active compounds in this investigation. Loss of feeding deterrent activity of steviol was observed on acetylation or glycosylation of the C-13 tertiary hydroxy group or on methylation of the C-19 carboxylic acid substituent. In contrast, the antifeedant activity of steviol was not greatly affected by modification of either the C-16 exomethylene group or the C/D-ring junction stereochemistry. 13C-nmr data have been obtained for 12 of the test compounds investigated.

Animals↗
PubMed1998

A 169-base pair tandem repeat DNA marker for subtelomeric heterochromatin and chromosomal rearrangements in aphids of the Myzus persicae group.

Numerous copies of a 169-base pair DNA sequence (Myzus persicae group repeat; MpR) occur at subtelomeric locations on all chromosomes of three members of the Myzus persicae species group (Myzus persicae, M. antirrhinii, M. certus). MpR occurs in large tandem arrays at both ends of all autosomes of the standard 2n = 12 karyotype, and near one end of the X chromosome (the end opposite to the nucleolar organizer) and is estimated to make up about 5% of the genome (a total of about 200000 copies). Locations of MpR were compared in various karyotypes to determine the likely nature of the rearrangements (fusions, dissociations, translocations) that are found in this species group which, like other Hemiptera, has holocentric chromosomes that are devoid of morphological markers. Aphid clones heterozygous for autosome dissociations do not have any detectable MpR at 'new' chromosome ends, indicating that this sequence is not involved in 'capping' of chromosomes. However, a clone with a de novo autosome fusion had an interstitial block of MpR marking the point of fusion, and clones heterozygous for an autosomal 1,3 translocation had MpR from autosome 1 translocated to a new site on autosome 3. The isolation from M. antirrhinii of the telomeric repeat TTAGG, which is found in several insect groups, is also reported.

Animals↗
PubMed1998

Silver staining as a new banding technique to identify aphid chromosomes.

The karyotype of the aphid Aphis sambuci has been studied by means of C-banding, fluorochrome staining and AgNO3 staining. Heterochromatic areas are confined exclusively to the X chromosomes. Interestingly, silver staining produces a clear-cut longitudinal differentiation of all chromosomes, which allows reliable identification of homologues.

Animals↗
PubMed2001

Effect of Fusarium, Paecilomyces and Trichoderma formulations against aphid Schizaphis graminum.

Fungal strains belonging to the genera Fusarium, Paecilomyces and Trichoderma were tested in vitro in order to study their effects against Schizaphis graminum, one of the major pests of cereal crops around the world. Biological assays were performed using a solid formulation that was obtained from fungal cultures grown on rice and then finely ground (<0.2 mm). The occurrence of toxic secondary metabolites (fumonisin B1 and beauvericin) produced by these fungi was also investigated. In each experiment, three groups of aphids: 15-hour old larvae, 5-day old nymphs with wing buds and wingless morphs were treated with a suspension of a fungal formulation. Some strains belonging to the genera Fusarium and Trichoderma significantly controlled the specimens of the three groups of S. graminum. The F proliferatum strain ITEM 1407, producing a high level of fumonisin B1 in the culture (1250 microg/g), and F larvarum strain ITEM 2139 had high insecticidal activity (>60%) within 10 minutes after application. As F. larvarum ITEM 2139 did not produce metabolites toxic to mammals, it might be a good candidate as a biocontrol agent of S. graminum in the field.

Animals↗
PubMed2002

Subcellular location of the helper component-proteinase of Cowpea aphid-borne mosaic virus.

The helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic virus (CABMV) was expressed in Escherichia coli and used to obtain HC-Pro antiserum that was used as an analytical tool for HC-Pro studies. The antiserum was used in immunofluorescence assays to study the subcellular location of HC-Pro expressed with other viral proteins in cowpea protoplasts in a natural CABMV infection, or in protoplasts transfected with a transient expression construct expressing HC-Pro separately from other viral proteins under the control of the 35S promoter. In both cases the protein showed a diffuse cytoplasmic location. Similar localisation patterns were shown in live protoplasts when the transient expression system was used to express HC-Pro as a fusion with the green fluorescent protein as a reporter. In an alternative expression system, the HC-Pro coding region was subcloned in-frame between the movement protein and large coat protein genes of RNA2 of Cowpea mosaic virus (CPMV). Upon transfection of protoplasts with this construct, HC-Pro was expressed as part of the RNA2 encoded polyprotein from which it was fully processed. In this case, the protein localised in broad cytoplasmic patches reminiscent of the typical CPMV induced cytopathic structures in which CPMV replication occurs, suggesting an interaction of HC-Pro with CPMV proteins or host factors in these structures. Finally, recombinant CPMV expressing HC-Pro showed a strongly enhanced virulence on cowpea and Nicotiana benthamiana consistent with the role of HC-Pro as a pathogenicity determinant, a phenomenon now known to be linked to its role as a suppressor of host defense responses based on post-transcriptional gene silencing.

Animals↗
PubMed2003

Karyotype variation in the South American aphid genus Neuquenaphis (Hemiptera, Aphididae, Neuquenaphidinae).

The endemic South American aphid genus Neuquenaphis (Hemiptera, Aphididae, Neuquenaphidinae) forms an important component of the phytophagous insect fauna associated with southern beeches, Nothofagus (Nothofagaceae), but has not previously been studied cytologically. As part of ongoing studies of the taxonomy, evolution and host relationships of this genus, the karyotypes of 12 species are described and illustrated. Species are mostly distinguishable by differences in number and/or relative lengths of chromosomes, with 2n (female) numbers ranging from 6 to 16. The taxonomic and evolutionary significance of the karyotype variation in this group are discussed.

Animals↗
PubMed2000

Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS.

Almost all aphid species (Homoptera, Insecta) have 60-80 huge cells called bacteriocytes, within which are round-shaped bacteria that are designated Buchnera. These bacteria are maternally transmitted to eggs and embryos through host generations, and the mutualism between the host and the bacteria is so obligate that neither can reproduce independently. Buchnera is a close relative of Escherichia coli, but it contains more than 100 genomic copies per cell, and its genome size is only a seventh of that of E. coli. Here we report the complete genome sequence of Buchnera sp. strain APS, which is composed of one 640,681-base-pair chromosome and two small plasmids. There are genes for the biosyntheses of amino acids essential for the hosts in the genome, but those for non-essential amino acids are missing, indicating complementarity and syntrophy between the host and the symbiont. In addition, Buchnera lacks genes for the biosynthesis of cell-surface components, including lipopolysaccharides and phospholipids, regulator genes and genes involved in defence of the cell. These results indicate that Buchnera is completely symbiotic and viable only in its limited niche, the bacteriocyte.

Aerobiosis↗
PubMed2001

Genetic linkage of ecological specialization and reproductive isolation in pea aphids.

The evolution of ecological specialization generates biological diversity and may lead to speciation. Genetic architecture can either speed or retard this process. If resource use and mate choice have a common genetic basis through pleiotropy or close linkage, the resulting genetic correlations can promote the joint evolution of specialization and reproductive isolation, facilitating speciation. Here we present a model of the role of genetic correlations in specialization and speciation, and test it by analysing the genetic architecture of key traits in two highly specialized host races of the pea aphid (Acyrthosiphon pisum pisum; Hemiptera : Aphididae). We found several complexes of pleiotropic or closely linked quantitative trait loci (QTL) that affect key traits in ways that would promote speciation: QTL with antagonistic effects on performance on the two hosts are linked to QTL that produce asortative mating (through habitat choice). This type of genetic architecture may be common in taxa that have speciated under divergent natural selection.

Adaptation, Biological↗