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Synthesis of a full-length infectious cDNA clone of cucurbit aphid-borne yellows virus and its use in gene exchange experiments with structural proteins from other luteoviruses.

A full-length cDNA of cucurbit aphid-borne yellows virus (CABYV) has been constructed and expressed either as an in vitro transcript, under control of a bacteriophage T7 RNA polymerase promoter, or in vivo, under control of the cauliflower mosaic virus 35S promoter in an agroinfection vector. The biological activity of the cloned cDNA was demonstrated by the ability of its in vitro transcript to replicate in protoplasts and of the agroinfection vector to infect agroinoculated plants. Virus in the agroinfected plants cold be transmitted by the aphid vectors Myzus persicae and Aphis gossypii. The specificity of luteovirus RNA packaging was investigated by replacing (1) the CABYV coat protein gene (and the overlapping ORF5) by the corresponding region of potato leafroll luteovirus or (2) the CABYV readthrough domain by the readthrough domain of beet western yellows luteovirus. The resulting chimeric transcripts replicated in protoplasts and produced virions.

Animals↗

Light-dependent systemic infection of solanaceous species by cauliflower mosaic virus can Be conditioned by a viral gene encoding an aphid transmission factor.

Gene II of cauliflower mosaic virus (CaMV), which encodes an 18-kDa protein originally identified as an aphid transmission factor (ATF), influences host specificity in a light-dependent manner. A point mutation within the ATF gene that occurs in several CaMV strains was responsible for conditioning light-dependent systemic infections. A point mutant of CaMV strain W260 that carried the single mutation within the ATF gene was able to systemically infect Nicotiana bigelovii at low light intensity (100-180 micromol m-2 sec-1), but not at a higher light intensity level (350-450 micromol m-2 sec-1), while the wild-type W260 virus could systemically infect N. bigelovii under both light conditions. The same point mutation also affected the stability of the amorphous CaMV inclusions and previous studies have shown that it abolishes transmission of CaMV by aphids. The point mutation within the ATF gene that mediated light-dependent infections was complemented by transgenic N. bigelovii plants that express the CaMV gene VI product, a viral protein that has been identified as a translational transactivator. The complementation studies indicated that the ATF gene may influence systemic infections through an interaction with the CaMV gene VI product. The ATF gene of CaMV may contribute to viral infections by regulating expression of downstream genes or by influencing cell-to-cell or long distance movement within the plant.

Animals↗

The haplotype distribution of two genes of citrus tristeza virus is altered after host change or aphid transmission.

Genetic variability of citrus tristeza virus (CTV) was studied using the haplotypes detected by single-strand conformation polymorphism (SSCP) analysis of genes p18 and p20 in six virus populations of two origins. The Spanish group included a CTV isolate and subisolates obtained by graft-transmission to different host species. The other included two subisolates aphid-transmitted from a single Japanese isolate. The homozygosity observed for gene p20 was always significantly higher than that expected under neutral evolution, whereas only three populations showed high homozygosity for p18, suggesting stronger host constraints for p20 than for p18. Sequential transmissions of a Spanish isolate to new host species increased the difference between its population and that of the successive subisolates for gene p18, as estimated by the F statistic. Analysis of molecular variance indicated that variation between both groups of populations was not statistically significant, whereas variations between populations of the same group or within populations were significant for both genes studied. Our data indicate that selection affects the haplotype distribution and that adaptation to a new host can be as important or more as the geographical origin. Variation of the CTV populations after host change or aphid transmission may explain in part the wide biological variability observed among CTV isolates.

Analysis of Variance↗

Ultrastructure of the antennal sensilla of aphids. I. Coeloconic and placoid sensilla.

An electron microscopical study was made of the coeloconic and placoid sensilla on the antennae of the aphids Aphis pomi, Macrosiphum euphorbiae, Nasonovia ribis-nigri, and Pemphigus bursarius. Scanning electron microscopy revealed some variation in morphology which may be functionally important but is more likely to reflect the evolution of these species. The placoid sensilla were shown by tranwo or three neurons is surrounded by two ensheathing cells. The ciliary regions of the dendrites pass through a vacuole into a cavity between an outer and an inner cuticle where they may be connected to the dendritic branches although such connections were no seen. Small pores (8 nm diameter) partially penetrate the cuticle implying that these sensilla have an olfactory function. They are suggested to be important in host selection by alate aphids. The coeloconic sensilla are poreless pegs with nonsensory cuticular projections at their tips. The distal portions of their dendrites contain densely packed microtubules and the cellular arrangement of the sensila is similar to that of the placoid sensilla. It is suggest that they may function as thermoreceptors.

Animals↗

A comparative study on the cell-free translation of the genomic RNAs of two aphid picorna-like viruses.

The genomic RNAs of aphid lethal paralysis virus (ALPV) and Rhopalosiphum padi virus (RhPV)--two distinct picorna-like viruses found in aphids [Williamson et al. (1988) J Gen Virol 69: 787-795]--were both efficiently translated in rabbit reticulocyte lysates. ALPV RNA was translated into primary products with molecular weights ranging from 92 kDa to 170 kDa. These underwent time-dependent post-translational cleavage to produce smaller polypeptides including some with molecular weights comparable to those of the viral structural proteins. A 92 kDa polypeptide as well as smaller proteins were immunoprecipitated with capsid protein antisera, indicating the presence of at least one large capsid subunit protein precursor. RhPV RNA was translated into products of molecular weights ranging from 45 kDa to 175 kDa. There was no evidence for time-dependent post-translation cleavage of RhPV translation products. However, a 60 kDa polypeptide was precipitated with antiserum to RhPV virions, indicating that at least one capsid protein of RhPV is derived by proteolysis of a precursor protein, like those of ALPV and most other picornaviruses.

Animals↗

The intestine is a site of passage for potato leafroll virus from the gut lumen into the haemocoel in the aphid vector, Myzus persicae Sulz.

Four detection techniques, three of which gave reliable identification of the virus particles, were used to locate potato leafroll virus (PLRV) in the alimentary canal of its main aphid vector, Myzus persicae Sulz: immunofluorescence on cryostat sections, conventional transmission electron microscopy on ultrathin sections and immune electron microscopy with gold labeling, either prior to or after fixation-embedding. Each method clearly showed the presence of the virus in the intestine epithelium and its absence in cells of the other parts of the alimentary canal. Under the experimental conditions used, the intestinal cells seemed to be the pathway for PLRV transport from the gut lumen into the haemocoel. Electron microscopy examinations showed many virus particles close to the apical plasmalemma of the epithelial cells in the gut lumen of the intestine. Other particles were seen in shallow pit-like regions or surrounded by coated vesicles in the apical part of these cells. Thus the virus particles seemed to enter the epithelial cells of the intestine by a mechanism of endocytosis. In the cytoplasm of these cells, virions were also frequently observed in isolated--or more often aggregated--tubular vesicles. The latter could be involved in PLRV transport through the cell since they were observed fusing with different cell organelles. A few viral particles were also detected in lysosomes as well as in multivesicular bodies. Virus particles were observed between the plasmalemma and basal lamina of the intestine cells but not in the haemocoel, where probably they were quickly dispersed. Our results are discussed in relation to other reports which have shown hindgut and stomach as sites of passage from the gut lumen into the aphid's body cavity for PLRV and other circulative viruses.

Animals↗

A single amino acid substitution at N-terminal region of coat protein of turnip mosaic virus alters antigenicity and aphid transmissibility.

The antigenic activity of the N-terminal region of coat protein of turnip mosaic virus (TuMV) aphid transmissible strain 1 and non-transmissible strain 31 was examined by using a panel of monoclonal antibodies (MAbs) raised against the two virus strains as well as antisera raised against several synthetic peptides from the N-terminal region of the protein. The reactivity of these antibodies was tested in ELISA and in a biosensor system (BIAcore Pharmacia) using virus particles, dissociated coat protein and synthetic peptides as antigens. Substitution of a single amino acid at position 8 in the coat protein of TuMV strain 1 abolished any cross-reactivity between MAbs to strain 1 and the substituted peptide (strain 31) in ELISA although some cross-reactivity was apparent in BIAcore inhibition experiments. In reciprocal tests with MAbs to strain 31 no cross-reactivity with the heterologous peptide was detected in either type of assay. The amino acid residue present at position 8 appears to play a critical role in the binding capacity of MAbs specific for the N-terminal region of TuMV. Antiserum to a synthetic peptide corresponding to residues 1-14 of the protein of TuMV strain 1 was found to react strongly with dissociated coat protein and intact virus particles and was able to inhibit the aphid transmission of the virus. Antiserum to the corresponding peptide of strain 31 did not have this capacity.

Amino Acid Sequence↗

Improved diagnosis of cowpea aphid-borne mosaic virus in Africa: significance for cowpea seed-indexing, breeding programs and potyvirus taxonomy.

Large-scale surveys in Africa for blackeye cowpea mosaic (B1CMV) and cowpea aphid-borne mosaic (CABMV) showed that several CABMV isolates from Southern Africa were either not or poorly recognized by monoclonal antibodies prepared to isolates collected in West Africa. Selection of three new monoclonal antibodies prepared against the Maputo (Mozambique) isolate of CABMV, and their incorporation into a revised panel of monoclonal antibodies, resulted in the assignment of four of these new CABMV isolates to existing serotypes (II, IV, and V) and three others to a new serotype (VI). The South African isolate of passiflora mosaic virus was shown to be related to CABMV isolates in serotype IV. It is proposed that CABMV isolates be assembled into a distinct species in the legume-infecting, aphid-transmissible potyviruses.

Africa↗

Ultrastructural study of acquisition and retention of potato leafroll luteovirus in the alimentary canal of its aphid vector, Myzus persicae Sulz.

The chronology of PLRV acquisition and retention by Myzus persicae was investigated using electron microscopy. Examination demonstrated a rapid translocation of the virus through the intestine into the haemocoel. Indeed, viral particles could be observed in the intestinal epithelial cells, then in the haemocoel, 4 and 8 h, respectively, after their arrival in the lumen of the alimentary canal. However, the virus accumulated in the intestinal epithelial cells. In these cells, the first viral particles were seen enclosed in isometric or tubular isolated vesicles; a few hours later, they were present in tubular aggregated vesicles and also in lysosomes or multivesicular bodies. After a 40 h acquisition period, all studied intestinal epithelial cells exhibited high numbers of viral particles which were consistently distributed throughout these cell structures. When aphids were removed from viral source, viral particles were detected in intestinal lumen for a further three days and in intestinal epithelial cells for a total of eight days. Virus content in these cells began to decrease from the second day. Areas with tubular aggregated vesicles were maintained for seven days following aphid removal from viral source, but progressively became smaller and fewer. The accumulation and the persistence of PLRV in the intestine are discussed.

Animals↗

Attraction of two lacewing species to volatiles produced by host plants and aphid prey.

It is well documented that host-related odors enable many species of parasitoids and predatory insects to locate their prey and prey habitats. This study reports the first characterization of prey and prey host odor reception in two species of lacewings, Chrysoperla carnea (Say) and Chrysopa oculata L. 2-Phenylethanol, one of the volatiles emitted from their prey's host plants (alfalfa and corn) evoked a significant EAG response from antennae of C. carnea. Traps baited with this compound attracted high numbers of adult C. carnea, which were predominantly females. One of the sex pheromone components (1R,4aS,7S,7aR)-nepetalactol of an aphid species, Acyrthosiphon pisum (Harris) attracted only C. oculata adults. Single sensillum recordings showed that the olfactory neurons of C. carnea responded to both 2-phenylethanol and aphid sex pheromone components, but those of C. oculata only responded to the latter.

Animals↗

Preliminary mapping of wheat (Triticum aestivum L.) tolerance genes to the English grain aphid (Sitobion avenae Fabricius) by genome-wide association study.

Six Sitobion avenae-tolerant wheat accessions, mapped 110 associated SNPs and six candidate genes were identified, providing valuable genetic resources for breeding wheat with tolerance to S. avenae. Wheat tolerance to the English grain aphid (Sitobion avenae) is rarely incorporated into integrated pest management strategies for wheat fields. The scarcity of tolerant accession and insufficient mapping of tolerance-related gene are key limiting factors. To address these gaps, 640 wheat accessions were evaluated for S. avenae tolerance, combined with genome-wide association study (GWAS) and qPCR validation. Six wheat accessions with stable tolerance were identified: Lerma Rojo 64, AC Vista, Hanxuan 10, Zimai, Ningnuomai 1, Louguding. A total of 110 single nucleotide polymorphism (SNP) loci associated with tolerance to S. avenae were mapped, and six candidate genes (TraesCS2D03G0041800, TraesCS2Dnew048215, TraesCS2D03G0046300, TraesCS6B03G0655800, TraesCS2Dnew048223, TraesCS2D03G0040800) were examined for transcriptional responses following aphid infestation via qRT-PCR. These genes are involved in cellular redox homeostasis, ADP-binding-mediated defense, and photosystem II (PSII) functionality. This study provides valuable genetic resources for breeding wheat with tolerance to S. avenae and lays a foundation for subsequent functional validation of these tolerance genes and its molecular mechanism exploration.

Animals↗

A new rickettsia from a herbivorous insect, the pea aphid Acyrthosiphon pisum (Harris).

An undescribed, maternally heritable, rod-shaped bacterium (or "tertiary symbiont") was detected by microscopy in hemolymph of about half (59/122) of pea aphid [Acyrthosiphon pisum (Harris)] clones collected from widely separated locations in California. On the basis of molecular phylogenetic analysis of 16S rDNA sequences, the bacterium was clearly placed among other Rickettsia in the alpha-subgroup of Proteobacteria, close to Rickettsia bellii-a rickettsia found in ticks. A PCR assay was developed to detect this bacterium in pea aphid clones with specific 16S rDNA PCR primers. Results of PCR-based assays completely correlated with detection by microscopy. This is the first confirmed detection of a Rickettsia in a herbivorous insect.

Animals↗

Endosymbionts (Buchnera) from the aphids Schizaphis graminum and Diuraphis noxia have different copy numbers of the plasmid containing the leucine biosynthetic genes.

The prokaryotic endosymbiont (Buchnera) of the aphid Schizaphis graminum contains 24 copies of a plasmid that has genes encoding enzymes of the leucine biosynthetic pathway while the endosymbiont of the related aphid Diuraphis noxia has only one copy of this plasmid. These results, in conjunction with similar results for the trpEG-containing plasmids, suggest that D. noxia has a reduced demand for endosymbiont-derived essential amino acids.

Animals↗

Buchnera aphidicola (Aphid endosymbiont) contains genes encoding enzymes of histidine biosynthesis.

Buchnera aphidicola is an endosymbiont of aphids. One of its functions appears to be the synthesis of essential amino acids for the aphid host. A 12.8-kilobase B. aphidicola DNA fragment has been cloned and sequenced. It contains genes encoding all of the enzymes required for the biosynthesis of the essential amino acid histidine. The order of the genes, hisGDCBHAFI, is the same as that found in Escherichia coli and is consistent with their constituting a single transcription unit. The DNA fragment also contained genes involved in aromatic amino acid biosynthesis (aroC), the oxidative pentose pathway (gnd), and 2'-deoxyribonucleotide metabolism (dcd), as well as a tRNA synthase (metG).

Animals↗

Detection of messenger RNA transcribed from genes encoding enzymes of amino acid biosynthesis in Buchnera aphidicola (endosymbiont of aphids).

The aphid Schizaphis graminum is dependent on Buchnera aphidicola, a prokaryotic endosymbiont. One of the functions of the endosymbiont is the synthesis of essential amino acids for the aphid host. Previously we have found that B. aphidicola has many of the genes that encode enzymes of amino acid biosynthesis. Using reverse transcriptase and the polymerase chain reaction, we have detected messenger RNA corresponding to genes involved in the synthesis of tryptophan, isoleucine, valine, leucine, and histidine.

Amino Acids, Essential↗

Inheritance and meiotic behaviour of a de novo chromosome fusion in the aphid Myzus persicae (Sulzer).

A de novo tandem fusion between autosomes 2 and 3 (A2+3), arising in the course of laboratory crosses of sexual morphs of two clones of the aphid Myzus persicae, was stable through more than 180 generations of parthenogenetic (clonal) reproduction. Studies of its inheritance through the sexual phase, and segregation from an amplified esterase marker gene, showed that crossing over occurred during oogenesis, but not in spermatogenesis, confirming previous cytological observations. Only a small number of progeny resulted from attempts at selfing fusion heterozygotes, and none of these was homozygous for the fusion. A2+3 paired in parallel alignment with the separate A2 and A3 to form a trivalent at prophase I of spermatogenesis. Fusion heterozygotes had a segregation problem at anaphase I of meiosis, A2+3 forming a chromatin bridge between the daughter spermatocytes in about 42% of dividing cells, which could be attributed to alternate orientation in the trivalent (A2 and A3 paired with opposite sides of A2+3) in the preceding metaphase I. Males heterozygous for an A2 dissociation were also studied and found to have much less of a segregation problem, despite showing similar orientation patterns at metaphase I. Possible reasons for this difference and the significance of the findings in relation to karyotype evolution in aphids are discussed.

Animals↗

Two-dimensional electrophoresis of proteins discriminates aphid clones of Sitobion avenae differing in BYDV-PAV transmission.

Aset of 39 F1 Sitobion avenae clones was obtained by selfing a poorly efficient BYDV-PAV vector clone. These clones were genetically typed by 11 microsatellite loci, and tested for BYDV-PAV4 transmission to barley. The 39 clones displayed a continuum in transmission percentages, from 0% to 88% with a significant clone effect. From this set, two highly efficient (HEV) and two poorly efficient (PEV) vectoring clones were more precisely characterized for transmission of two other PAV isolates. The molecular bases of the lower transmissibility of BYDV-PAV4 by PEV clones and of the aphid vectoring properties were investigated respectively by comparing the sequences corresponding to structural proteins (CP and RTD) of BYDV, and by using proteomic analysis of aphids in two dimensional electrophoresis (2-DE) with immobilized pH gradients (IPG) after an improved protein extraction. Four residues specific to BYDV-PAV4 located in the CP sequence (A(24) and L(130)) or in the RTD region (M(334) and S(456)) could be responsible for the lower transmissibility of this isolate by PEV clones. Among a total of 2150 well-resoluted spots scored on S. avenae proteinic pattern, only twelve proteins were qualitatively or quantitatively different between clones. Four out of them discriminated HEV and PEV groups.

Animals↗

Verticillium lecanii spore formulation using UV protectant and wetting agent and the biocontrol of cotton aphids.

Verticillium lecanii spores (10(8 )spores ml(-1)) suspended in 1% (w/v) montmorillonite SCPX-1374 and 1% (w/v) of the wetting agent, EM-APW#2, which is a polyoxyethylene, had approx. 80% survival after exposure to UV-C for 30 min and about 93% after exposure to UV-B for 6 h. In greenhouse testing, cotton aphid densities increased 14-fold over their initial density in 15 d without spore application. However, initial cotton aphid densities were decreased by 60% of the initial level when plants were treated with the spore formulation.

Animals↗