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PubMed1994

Differentiation of aphid clones by arbitrarily primed polymerase chain reaction (AP-PCR) DNA fingerprinting.

Differentiation of aphid clones was attempted using AP-PCR which is a simple and rapid method to obtain DNA fingerprints of complex genomes. To establish optimal reaction conditions and examine reproducibility of the method, a laboratory-maintained clone of the pea aphid, Acyrthosiphon pisum, was used as test material. Under the reaction conditions employed, identical fingerprint patterns were obtained throughout a wide range of template DNA amount, from 5 to 800 ng, and irrespective of aphid instar. No changes in the patterns were seen throughout five parthenogenetic generations. When this method was applied to a wild population of the gall-forming aphid, Ceratovacuna nekoashi, five groups of insects originating from different galls formed on the same twig were successfully differentiated from one another by means of polymorphic fingerprint bands. In contrast, the fingerprints of the insects derived from the subgalls of the same gall were identical. These results indicated that in C. nekoashi: (i) members of a gall constitute a clonal population; (ii) a gall is founded by a single fundatrix; and (iii) intergall migration is absent or at least not frequent.

Animals↗
PubMed1977

Reproductive strategy of winged and wingless morphs of the aphids Sitobion avenae and Metopolophium dirhodum.

The reproduction of apterous and alate morphs of the aphids Sitobion avenae and Metopolophium dirhodum is compared on the basis of fecundity in 5- and 10-day periods of adult life. Apterae of both species are consistently more fecund than alatae of comparable weight, producing about three more nymphs on average in any 5-day period. The reproductive differences are related to the number and quality of embryos at eclosion and to ovulation rates, both of which in turn appear to be linked to wing-muscle maintenance. These relationships between weight, embryos and reproduction may be used to predict a newly moulted adult aphid's fecundity, a method which may facilitate the assessment of resistance to aphids in new cereal varieties, by obviating lengthy recording of reproduction. The strategies by which alatae of these and other aphid species minimize the difference between their fecundity and that of apterae are discussed.

Age Factors↗
PubMed1999

Plasmid-encoded anthranilate synthase (TrpEG) in Buchnera aphidicola from aphids of the family pemphigidae.

Buchnera aphidicola is an obligate intracellular symbiont of aphids. One of its proposed functions is the synthesis of essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. The genetic organization of the tryptophan pathway in Buchnera from proliferous aphids of the family Aphididae has previously been shown to reflect a capacity to overproduce this essential amino acid (C.-Y. Lai, L. Baumann, and P. Baumann, Proc. Natl. Acad. Sci. USA 91:3819-3823, 1994). This involved amplification of the genes for the first enzyme in the pathway, anthranilate synthase (TrpEG), on a low-copy-number plasmid. Here we report on the finding and molecular characterization of TrpEG-encoding plasmids in Buchnera from aphids of the distantly related family Pemphigidae. Buchnera from Tetraneura caerulescens contained a 3.0-kb plasmid (pBTc2) that carried a single copy of trpEG and resembled trpEG plasmids of Buchnera from the Aphididae. The second plasmid (pBPs2), isolated from Buchnera of Pemphigus spyrothecae, contained a different replicon. It consisted of a putative origin of replication containing iterons and an open reading frame, designated repAC, which showed a high similarity to the gene encoding the replication initiation protein RepA of the RepA/C replicon from the broad-host-range IncA/C group of plasmids. The plasmid population was heterogeneous with respect to the number of tandem repeats of a 1.8-kb unit carrying repAC1, trpG, and remnants of trpE. The two principal forms consisted of either five or six copies of this repeat and a single-copy region carrying repAC2, the putative origin of replication, and trpE. The unexpected finding of elements of the RepA/C replicon in previously characterized trpEG plasmids from Buchnera of the Aphididae suggests that a replacement of replicons has occurred during the evolution of these plasmids, which may point to a common ancestry for all Buchnera trpEG amplifications.

Amino Acid Sequence↗
PubMed2005

Horizontal transfer of bacterial symbionts: heritability and fitness effects in a novel aphid host.

Members of several bacterial lineages are known only as symbionts of insects and move among hosts through maternal transmission. Such vertical transfer promotes strong fidelity within these associations, favoring the evolution of microbially mediated effects that improve host fitness. However, phylogenetic evidence indicates occasional horizontal transfer among different insect species, suggesting that some microbial symbionts retain a generalized ability to infect multiple hosts. Here we examine the abilities of three vertically transmitted bacteria from the Gammaproteobacteria to infect and spread within a novel host species, the pea aphid, Acyrthosiphon pisum. Using microinjection, we transferred symbionts from three species of natural aphid hosts into a common host background, comparing transmission efficiencies between novel symbionts and those naturally infecting A. pisum. We also examined the fitness effects of two novel symbionts to determine whether they should persist under natural selection acting at the host level. Our results reveal that these heritable bacteria vary in their capacities to utilize A. pisum as a host. One of three novel symbionts failed to undergo efficient maternal transmission in A. pisum, and one of the two efficiently transmitted bacteria depressed aphid growth rates. Although these findings reveal that negative fitness effects and low transmission efficiency can prevent the establishment of a new infection following horizontal transmission, they also indicate that some symbionts can overcome these obstacles, accounting for their widespread distributions across aphids and related insects.

Animals↗
PubMed2005

Aphid-symbiotic bacteria cultured in insect cell lines.

The cells and tissues of many aphids contain bacteria known as "secondary symbionts," which under specific environmental circumstances may be beneficial to the host insect. Such symbiotic bacteria are traditionally described as intractable to cultivation in vitro. Here we show that two types of aphid secondary symbionts, known informally as T type and U type, can be cultured and maintained in three insect cell lines. The identities of the cultured bacteria were confirmed by PCR with sequencing of 16S rRNA gene fragments and fluorescence in situ hybridization. In cell lines infected with bacteria derived from aphids harboring both T type and U type, the U type persisted, while the T type was lost. We suggest that the two bacteria persist in aphids because competition between them is limited by differences in tropism for insect tissues or cell types. The culture of these bacteria in insect cell lines provides a new and unique research opportunity, offering a source of unibacterial material for genomic studies and a model system to investigate the interactions between animal cells and bacteria. We propose the provisional taxon names "Candidatus Consessoris aphidicola" for T type and "Candidatus Adiaceo aphidicola" for U type.

Animals↗
PubMed1993

Molecular cloning and nucleotide sequence of a putative trpDC(F)BA operon in Buchnera aphidicola (endosymbiont of the aphid Schizaphis graminum).

A 8,392-nucleotide-long DNA fragment from Buchnera aphidicola (endosymbiont of the aphid Schizaphis graminum) contained five genes of the tryptophan biosynthetic pathway [trpDC(F)BA] which code for enzymes converting anthranilate to tryptophan. These genes are probably arranged as a single transcription unit. Downstream of the trp genes were ORF-V, ORF-VI, and P14, three open reading frames which in Escherichia coli are also found downstream of the trp operon. Upstream of the B. aphidicola trp genes were two unidentified open reading frames, one of which potentially codes for a membrane-spanning protein with a leader sequence. Evidence for the presence of trpB in the endosymbionts of eight additional species of aphids and two species of whiteflies was obtained. These results as well as those of A. E. Douglas and W. A. Prosser (J. Insect Physiol. 38:565-568, 1992) suggest that aphid endosymbionts are capable of synthesizing tryptophan, which is required by the aphid host.

Amino Acid Sequence↗
PubMed1997

Putative evolutionary origin of plasmids carrying the genes involved in leucine biosynthesis in Buchnera aphidicola (endosymbiont of aphids).

An 8.5-kb plasmid encoding genes (leuABCD) involved in leucine biosynthesis and a small plasmid of 1.74 kb of yet unknown function were found in the intracellular symbiont, Buchnera aphidicola, of two divergent aphid species, Thelaxes suberi and Tetraneura caerulescens, respectively. The leuABCD-carrying plasmid (pBTs1) was amplified from total aphid DNA by inverse long PCR, using outwardly oriented oligonucleotide primers specific to leuA. The resulting 8.2-kb PCR fragment as well as the 1.74-kb plasmid (pBTc1) were cloned and sequenced. pBTs1 differed from a previously described B. aphidicola plasmid (pRPE) of the aphid Rhopalosiphum padi by the presence of a small heat shock gene (ibp) and in the order of the leuABCD and repA genes. Comparison of both leucine plasmids to the small plasmid pBTc1 revealed extensive similarity with respect to putative replication functions as well as in the presence of a highly conserved open reading frame that was found to be homologous to Escherichia coli YqhA and Haemophilus influenzae HI0507 and which may encode an integral membrane protein. The three B. aphidicola plasmids most likely evolved from a common ancestral replicon, which in turn may be distantly related to IncFII plasmids. Phylogenetic affiliations of the B. aphidicola strains of the two aphid species were assessed by sequencing of their 16S rRNA genes. Evaluation of the distribution of the leuABCD-encoding plasmids within a phylogenetic framework suggests independent origins for pBTs1 and pRPE from an ancestral replicon resembling pBTc1. The implications for symbiotic essential amino acid biosynthesis and provisioning are discussed.

Amino Acid Sequence↗
PubMed2004

Coexistence of Wolbachia with Buchnera aphidicola and a secondary symbiont in the aphid Cinara cedri.

Intracellular symbiosis is very common in the insect world. For the aphid Cinara cedri, we have identified by electron microscopy three symbiotic bacteria that can be characterized by their different sizes, morphologies, and electrodensities. PCR amplification and sequencing of the 16S ribosomal DNA (rDNA) genes showed that, in addition to harboring Buchnera aphidicola, the primary endosymbiont of aphids, C. cedri harbors a secondary symbiont (S symbiont) that was previously found to be associated with aphids (PASS, or R type) and an alpha-proteobacterium that belongs to the Wolbachia genus. Using in situ hybridization with specific bacterial probes designed for symbiont 16S rDNA sequences, we have shown that Wolbachia was represented by only a few minute bacteria surrounding the S symbionts. Moreover, the observed B. aphidicola and the S symbionts had similar sizes and were housed in separate specific bacterial cells, the bacteriocytes. Interestingly, in contrast to the case for all aphids examined thus far, the S symbionts were shown to occupy a similarly sized or even larger bacteriocyte space than B. aphidicola. These findings, along with the facts that C. cedri harbors the B. aphidicola strain with the smallest bacterial genome and that the S symbionts infect all Cinara spp. analyzed so far, suggest the possibility of bacterial replacement in these species.

Animals↗
PubMed2000

Effects of point mutations in the readthrough domain of the beet western yellows virus minor capsid protein on virus accumulation in planta and on transmission by aphids.

Point mutations were introduced into or near five conserved sequence motifs of the readthrough domain of the beet western yellows virus minor capsid protein P74. The mutant virus was tested for its ability to accumulate efficiently in agroinfected plants and to be transmitted by its aphid vector, Myzus persicae. The stability of the mutants in the agroinfected and aphid-infected plants was followed by sequence analysis of the progeny virus. Only the mutation Y201D was found to strongly inhibit virus accumulation in planta following agroinfection, but high accumulation levels were restored by reversion or pseudoreversion at this site. Four of the five mutants were poorly aphid transmissible, but in three cases successful transmission was restored by pseudoreversion or second-site mutations. The same second-site mutations in the nonconserved motif PVT(32-34) were shown to compensate for two distinct primary mutations (R24A and E59A/D60A), one on each side of the PVT sequence. In the latter case, a second-site mutation in the PVT motif restored the ability of the virus to move from the hemocoel through the accessory salivary gland following microinjection of mutant virus into the aphid hemocoel but did not permit virus movement across the epithelium separating the intestine from the hemocoel. Successful movement of the mutant virus across both barriers was accompanied by conversion of A59 to E or T, indicating that distinct features of the readthrough domain in this region operate at different stages of the transmission process.

Amino Acid Sequence↗
PubMed2005

The polerovirus minor capsid protein determines vector specificity and intestinal tropism in the aphid.

Aphid transmission of poleroviruses is highly specific, but the viral determinants governing this specificity are unknown. We used a gene exchange strategy between two poleroviruses with different vectors, Beet western yellows virus (BWYV) and Cucurbit aphid-borne yellows virus (CABYV), to analyze the role of the major and minor capsid proteins in vector specificity. Virus recombinants obtained by exchanging the sequence of the readthrough domain (RTD) between the two viruses replicated in plant protoplasts and in whole plants. The hybrid readthrough protein of chimeric viruses was incorporated into virions. Aphid transmission experiments using infected plants or purified virions revealed that vector specificity is driven by the nature of the RTD. BWYV and CABYV have specific intestinal sites in the vectors for endocytosis: the midgut for BWYV and both midgut and hindgut for CABYV. Localization of hybrid virions in aphids by transmission electron microscopy revealed that gut tropism is also determined by the viral origin of the RTD.

Animals↗
PubMed2005

A surface loop of the potato leafroll virus coat protein is involved in virion assembly, systemic movement, and aphid transmission.

Two acidic domains of the Potato leafroll virus (PLRV) coat protein, separated by 55 amino acids and predicted to be adjacent surface features on the virion, were the focus of a mutational analysis. Eleven site-directed mutants were generated from a cloned infectious cDNA of PLRV and delivered to plants by Agrobacterium-mediated mechanical inoculation. Alanine substitutions of any of the three amino acids of the sequence EWH (amino acids 170 to 172) or of D177 disrupted the ability of the coat protein to assemble stable particles and the ability of the viral RNA to move systemically in four host plant species. Alanine substitution of E109, D173, or E176 reduced the accumulation of virus in agrobacterium-infiltrated tissues, the efficiency of systemic infection, and the efficiency of aphid transmission relative to wild-type virus, but the mutations did not affect virion stability. A structural model of the PLRV capsid predicted that the amino acids critical for virion assembly were located within a depression at the center of a coat protein trimer. The other amino acids that affected plant infection and/or aphid transmission were predicted to be located around the perimeter of the depression. PLRV virions play key roles in phloem-limited virus movement in plant hosts as well as in transport and persistence in the aphid vectors. These results identified amino acid residues in a surface-oriented loop of the coat protein that are critical for virus assembly and stability, systemic infection of plants, and movement of virus through aphid vectors.

Animals↗
PubMed1994

Mitochondrial DNA diversity in the pea aphid Acyrthosiphon pisum.

The pea aphid Acyrthosiphon pisum has been established in North America for at least a century and occurs on a broad range of host plants. Despite its importance as a crop pest, there is little understanding of the genetic structure of its populations or the extent of genetic divergence associated with different host plant utilization. This study examined the extent of mitochondrial DNA (mtDNA) diversity among 35 clones of pea aphids from alfalfa. Polymorphisms were detected at only 2 of 126 restriction sites, suggesting the same impoverished level of genetic diversity found in prior allozyme studies. However, length variation was common and apparently arose as a result of tandem repeats in two regions of the mtDNA molecule. Region 1 occurred in proximity to the control region of the molecule, while region 2 was close to a cluster of tRNA genes flanking the NAD-dehydrogenase subunit 3 gene on the opposite side of the mtDNA molecule. Each of the aphid clones was homoplasmic for a single length variant in region 1, which varied with respect to the number of copies of a 120-bp repeat. By contrast, one-third of the clones were heteroplasmic at region 2, where they possessed a variable number of copies of a 210-bp repeat. Reanalysis of clones after 30 generations of parthenogenetic reproduction established the stability of length variants over this interval, suggesting their value in studies of the genetic structure of aphid populations.

Animals↗
PubMed1999

The role of rDNA genes in X chromosome association in the aphid Acyrthosiphon pisum.

Silver staining of mitotic metaphases of the aphid A. pisum reveals the presence of argentophilic bridges connecting the two X chromosomes. The presence of nucleolar material connecting sex chromosomes seems to be quite a common phenomenon in organisms belonging to very different phyla, and suggests a role of nucleolar proteins in chromosome association and disjunction. In somatic cells of A. pisum, bridges connecting X chromosomes are detectable not only after silver staining but also after CMA3 staining. This finding suggests that GC rich DNA is involved in this type of association. Molecular analysis of rDNA intergenic spacers shows several 247 bp repeats containing short sequences having a high level of homology with the chi sequence of Escherichia coli and with the consensus core region of human hypervariable minisatellites. Moreover, each 247 bp repeat presents a perfect copy of a promoter sequence for polymerase I. These aphid repeats show structural homologies with a 240 bp repeat, which is considered to be responsible for sex chromosome pairing in Drosophila, not only in view of their common presence within rDNA spacers but also for their length and structure. The presence of chi sequences in the IGS of A. pisum, by promoting unequal crossing-over between rDNA genes, could thus give rise to the nucleolar organizing region (NOR) heteromorphism described in different aphid species. Although X pairing at NORs is fundamental in aphid male determination, the presence of heteromorphism of rDNA genes does not inhibit male determination in the A. pisum clone utilized for our experiments.

Animals↗
PubMedDate not supplied

[Pathogenicity of hyphomycet fungi to aphids Aphis gossypii Glover and Myzus persicae (Sulzer) (Hemiptera: Aphididae)].

The aphids Aphis gossypii and Myzus persicae are cosmopolitan, poliphagous and damage cultivated plants. The effects of the entomopathogenic fungi Beauveria bassiana (isolate IBCB 66), Metarhizium anisopliae (isolate IBCB 121), Paecilomyces fumosoroseus (isolate IBCB 141) and Lecanicillium (= Verticillium) lecanii (isolate JAB 02) on third instar nymphs of A. gossypii and M. persicae were evaluated in the laboratory at 25 degrees C, 70 +/- 10% RH and 12h photophase. The aphids were transferred to petri dishes with a foliar disk (cotton or pepper) with a layer of 1 cm tick of agar-water. The fungi were applied in a suspension containing 1.0 x 106 to 1.0 x 108 conidia/ml. In the control treatment 1 ml of sterilized water was added to the foliar disks. The mortality of aphids was evaluated daily. B. bassiana and M. anisopliae caused 100% mortality at the seventh day after inoculation, for both species. L. lecanii was the fungus that provided mortality later in the aphids and M. persicae was more susceptible to both fungi than A. gossypii.

Animals↗
PubMed2002

Projection pursuit nonparametric regression applied to field counts of the aphid Macrosiphum euphorbiae (Homoptera: Aphididae) on tomato crops in greenhouses.

The number of plants that sampling requires in aphid population studies often exceeds one hundred. Thus, only quick and nondestructive methods can be used to sample this pest in a reasonable time interval. We propose a visual method for estimating the density of the aphid Macrosiphum euphorbiae (Thomas) on tomato plants reared in greenhouses. After approximately 1min of visual observation plants can be assigned to abundance classes, the boundaries of which are roughly the powers of square root 10. Precise counts were collected simultaneously on sets of reference plants from the same greenhouse. Projection pursuit nonparametric regression was then used to provide unbiased estimates of aphid densities from the abundance classes and several easily gathered explanatory variables. The robustness of the method was evaluated by testing the models on the complementary data sets from plants in which the aphid densities were precisely counted. In both single and twin-row cultural conditions, for the reference and complementary data sets, the order of magnitude of the error was less than one class rank per plant. The investigation time was reduced by approximately 10-fold compared with the exact counting method. This easy-to-teach field method could be useful in large-scale population surveys and for optimizing integrated pest management strategies.

Animals↗
PubMed2000

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.

Agriculture↗
PubMed2001

[Cloning of AHA gene from Amaranthus hypochondriacus and it's aphid inhibitory effect in transgenic tabacco plants].

Using total DNA isolated from Amaranthus hypochondriacus as template, Amaranthus hypochondriacus agglutinin AHA gene was amplified by PCR and cloned. Sequence analysis results showed that this gene is consisted of 2453 base pairs including one 1538 bp intron and two exons of 212 bp and 703 bp respectively. After inverse PCR amplification, coding region of AHA gene was obtained. AHA gene with it's intron (AHAg) and withou intron (AHAc) were inserted downstream of 35S promotor in the binary vector pBin438 resulting in the construction of two plant expression vectors pBAHAg and pBAHAc repectively. Leave explants of Nicotinana tabacum var. SR1 were transformed with A. tumefaciens LBA4404 harbouring the above expression vectors. Results from PCR and Southern blot analysis showed that AHA genes were inserted into the genome of transformed tobacco plants. Immunodot blot analysis indicated that AHA was expressed in transgenic plants. The results from insect bioassay with peach aphid (Mizus persicae) showed that the transgenic plants of pBAHAg and pBAHAc were aphid resistant, evidenced by a 57%-48% reduction in insect population density, some plants were more than 85%. The aphid resistance of transgenic plants transformed with AHAg gene as judged by aphid inhibition rate was higher than that of plants transormed with AHAc gene indicating that the intron in AHAg may be favorable for expression of AHA in transgenic plants.

Animals↗
PubMed2002

Aphids (Hemiptera: Aphidoidea) of ornamental plants from São Carlos, São Paulo state, Brazil.

A total of 25 aphid species were collected from 49 ornamental plant species in São Carlos-São Paulo, Brazil; 12 aphids were monophagous, four oligophagous and nine polyphagous. A total of 58 aphid-plant associations are recorded, 43 unknown from Brazil. Eucarazzia elegans (Ferrari, 1872) (Aphididae: Aphidinae: Macrosiphini) is recorded for the first time from Brazil, and Nectandra megapotamica Spreng. (Lauraceae) is recorded for the first time as host plant for Lizerius tuberculatus (E.E. Blanchard, 1939) (Drepanosiphidae: Drepanosiphinae: Lizeriini). We also describe the injuries caused by aphids to the ornamental plants.

Animals↗