Diuraphis noxia Kurdjumov (Hemiptera: Aphididae: Macrosiphini) is a major agricultural pest that causes extensive economic losses to the wheat and barley industries. Resistant cultivars were relatively successful in controlling this pest until the recent development of new D. noxia (Russian wheat aphid, RWA) biotypes. The aim was to investigate the role of the aphid endosymbiont, Buchnera aphidicola, in the RWA-host interaction. It was hypothesized that variations in the endosymbiont’s key essential amino acid biosynthetic pathway genes, their copy numbers, and/or expression levels, maybe a determining factor influence the RWA’s success in the aphid-host interaction. Aphid symbiont species content, key essential amino acid biosynthetic gene variation, plasmid copy numbers and expression levels of ten different RWA biotypes were determined, using DGGE, RT-PCR, RT-qPCR, 5’-RACE and sequencing. The RWA biotypes were shown to be monosymbiotic, with plasmid copy numbers varying between biotypes. Only a single CCC-insert in a non-coding region of the leucine plasmid differed between the biotypes. Similar variations were identified in the family Aphididae, suggesting a regulatory function for this region. The presence of this CCC-insert in a plasmid led to an increase in the leader sequence length of the leuA gene. The insert may also have a functional role through gene regulation, since it increased the expression levels of subsequent genes (leuA and leuB). An endosymbiont that upgrade the host’s diet with the required essential amino acids will be beneficial to RWAs when feeding on resistant wheat cultivars as it will enhance aphid fitness. This suggests selective pressure of resistant wheat cultivars on the aphid, i.e. the incapability to change resistant cultivar essential amino acid content, could select for individuals with beneficial endosymbionts. B. aphidicola could therefore play a role in the development of RWA biotypes. The influences that statistical normalization methods have on the final identification of differentially regulated Affymetrix probe sets in RWA-plant interactions were also investigated. The hypothesis was that a subset of the probe sets determined as differentially regulated would be consistent, regardless of the normalization and background method utilized, if all the other analyses are kept constant. This subset would be normalization-method-independent. The data of two Affymetrix RWA-plant interaction experiments were analyzed with five different normalization and background correcting methods and at three different confidence levels, with the results subjected to FDR and FWER correction algorithms. The results showed that on average a third of the regulated genes were only selected after normalization by a single method and that the total number of genes deemed regulated was highly normalization method dependent. Normalization-method-biases could also not be countered by increased confidence levels and these biases eventually determined the probe sets deemed differentially regulated, even after FDR and FWER corrections. Both these strategies actually increased normalization-method-biases and these could only be corrected by using multiple normalization methods to identify the normalization-method-biases-independent probe set subset. / Thesis (PhD)--University of Pretoria, 2011. / Genetics / unrestricted

This is a study of transcriptional gene regulation in wheat (Triticum aestivum, L.) in response to Russian wheat aphid (RWA) (Diuraphis noxia, Kurdjumov) infestation. The Russian wheat aphid feeds on the phloem sap in the leaves of wheat plants, and causes the leaves of susceptible wheat plants to curl. This forms a protective barrier for the RWA from insecticides and natural enemies. Chlorosis also results from the RWA feeding. In cases of high infestation, death of susceptible plants can also occur. Eleven wheat genes that confer resistance to the Russian wheat aphid have been identified, but their mechanism at molecular level is still not clearly understood. Wheat near-isogenic lines (NILs) were used in a genome-wide, transcriptome analysis using cDNA-AFLP technology. RWA-resistant cultivar ‘Tugela DN’ and RWA-susceptible cultivar ‘Tugela’ were infested with the RWA and leaves were collected from the infested plants at different (0-, 1-, 2-, 6-, 12-, 24-, 48- and 120-) hours post infestation. cDNA samples derived from these leaves was then analyzed by cDNA-AFLP which revealed 18 clusters of differential gene regulation between the two NILs. The results of this experiment show that differential regulation of transcripts occur even within the first hour of infestation. All types of regulation were observed within the clusters. Differentially expressed transcript derived fragments (TDFs) that were randomly isolated from PAGE gels and sequenced (41 TDFs) included sequences in the functional groups similar to those observed in the microarray analysis. The functional categories are cell structure and maintenance [protein synthesis (14%), chaperone (2%), protein degradation (2%), transcription factor (5%)]; photosynthesis [sugar metabolism (5%), carbohydrate metabolism (2%), energy related (7%)]; defenserelated [signaling (7%), defense-related (10%)] while the rest did not have any significant homology to any known or characterized proteins. Previous suppressive subtractive hybridization experiments identified transcripts that are differentially expressed in wheat in response to RWA feeding. More transcripts were identified by PCR from cDNA pools derived from RWA-infested plants as having conserved motifs common in pathenogenesis related proteins. The isolated transcripts were used to generate a defense response-biased microarray chip that was used to investigate the regulation of these transcripts during infestation of RWA resistant wheat plants (‘Tugela DN’) in a time trial. Dual hybridization of CyDye labeled probes derived from the induced ‘Tugela DN’ plants to the microarray chips revealed differential regulation of the immobilized transcripts in wheat, at different time points post infestation with the RWA. Statistical analysis of the CyDye intensities on the 380 spots mounted on the cDNA microarray slides showed 29 transcripts to be significantly regulated (P≤0.05) during the time of the experiment. These included ESTs that were grouped into four functional categories, namely cell structure and maintenance (9 ESTs); photosynthesis (8 ESTs); defense-related (4 ESTs) and those with no significant homology found or proteins with unknown function (8 ESTs). Patterns of regulation of these transcripts in all of the functional categories included all types of regulation e.g. mainly down-regulation, mainly up-regulation, and a combination of up-/up-/down-regulation in response to RWA feeding. In conclusion, data obtained utilizing cDNA microarray and cDNA-AFLP analyses in infested wheat suggest that the ability to maintain structures involved in photosynthesis by regulating the relevant transcripts through-out infestation is an important determinant in plant survival during RWA feeding. The timing of regulation is also important as some of the transcripts are also regulated in RWA susceptible ‘Tugela’ plants but not in a timely manner which leads to loss of energy and subsequent death of susceptible plants. / Dissertation (MSc)--University of Pretoria, 2011. / Genetics / unrestricted

The Russian wheat aphid (Diuraphis noxia, RWA), is a serious pest in most wheat producing countries around the world. Infestation of wheat fields by this pest has a severe economic impact, as a result of heavy losses in crop yield. Because of the importance of wheat as a food source and its ever growing supply demand, the study of wheat-Russian wheat aphid interactions on the molecular level are integral to the development of management strategies. This is highlighted by the fact that new RWA biotypes that overcome resistance in a number of wheat varieties, continually emerge. Therefore, this study aims to contribute to this endeavour, by elucidating the molecular mechanisms by which the RWA resistance gene Dn7 confers resistance to three different RWA biotypes (one from SA, and two from the USA). Firstly, suppression subtractive hybridization (SSH) was applied in order to isolate transcripts differentially expressed in the RWA resistant wheat line, 94M370, carrying the Dn7 gene. There are two main advantages to this technique. One is that the relative representation of rare transcripts is increased in the subsequent cDNA population, and it is these low abundance transcripts that are arguably the ones of particular interest. Secondly, this method allows for the isolation of unknown transcripts, without the need for existing sequence information. Experiments with this method however, failed, leading to an investigation as to probable causes. The various steps involved in the SSH procedure were individually assessed in an attempt to identify and correct the problem. Various adjustments were made to PCR procedures, template enzyme digestions and ligation reactions, without success. After creating a basic cDNA-AFLP fingerprint from the existing cDNA template, in order to confirm that the template is not responsible for experimental difficulties – it was decided to apply a different strategy in order to meet research objectives. Consequently, the study on Dn7 mediated defence responses was continued with cDNA-AFLP. In addition to studying the response by Dn7 to South African biotype RWA infestation, its responses to infestation by two United States RWA biotypes was also explored. This allowed us to gain a greater comprehension of the methods by which Dn7 activates defences against different aphid eliciting agents. Findings suggest that this gene activates responses that are unique to each of the different aphid interactions. Although the interactions between Dn7 and the two US biotypes were very similar, this can possibly be explained by the fact that the differences between these two biotypes on molecular level are minuscule. Dn7 responds to the South African biotype of the RWA in a completely different manner, as judged by the very dissimilar expression patterns obtained during cDNA-AFLP analysis. Reasons for this phenomenon could include molecular differences between the South African and US RWA biotypes, differences in response generating elicitor molecules (which has indeed been shown to be the case between South African and US aphid biotypes), or a combination of both. The sequencing of fragments displaying differential expression patterns during cDNA-AFLP fingerprinting, provides us with additional information as to the exact mechanisms potentially involved. As expected, various compounds related to plant defence were identified, such as a number of Leucine rich repeat (LRR) domain containing proteins, genes related to cell signalling and genes involved in protein processing (proteases, peptidases). Finally, these results are consistent with theories that Dn7 may recognise and interact with its distinct aphid elicitors either directly, by the presence of multiple bindings sites on the same protein, or indirectly. In that case, in accordance with the guard hypothesis, Dn7 may simply monitor interactions between aphid elicitors and other recognition factors- after which a response cascade is activated. Useful potential research would focus on Dn7 itself, including mapping, isolation as well as structural and functional characterization. / Dissertation (MSc)--University of Pretoria, 2007. / Genetics / unrestricted

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Diuraphis noxia (Kurdjumov, Hemiptera, Aphididae) commonly known as the Russian wheat aphid (RWA), is a small phloem-feeding pest of wheat (Triticum aestivum L). Virulent D. noxia biotypes that are able to feed on previously resistant wheat cultivars continue to develop and therefor the identification of factors contributing to virulence is vital. Since energy metabolism plays a key role in the survival of organisms, genes and processes involved in the production and regulation of energy may be key contributors to virulence: such as mitochondria and the NAD+/NADH that reflects the health and metabolic activities of a cell. The involvement of carotenoids in the generation of energy through a photosynthesis-like process may be an important factor, as well as its contribution to aphid immunity through mediation of oxidative stress. The complete mitochondrial genome of global Diuraphis noxia populations was characterised using Next Generation sequencing, and was found to be 15 721bp in size and consisting of 38 genes typically found within most insects. Single nucleotide polymorphism (SNP) analyses of the genomes of nine populations revealed 125 SNPs in the protein coding genes with the majority of the SNPs occurring in the ND genes, and the least in the ND4L gene. Low SNP variant frequency was found for the atp6 and atp8 genes, which differed from other reports in the Hemiptera. Variable ND5 expression levels were observed among the biotypes, although no correlation was apparent between ND5 expression and the virulence associated with each biotype. Whereas atp6 transcription was higher in the highly virulent biotype (SAM) under normal and stressful conditions in comparison to the least virulent biotype (SA1). A significantly higher NAD+/NADH ratio was also observed for the SAM biotype under stressful conditions in comparison to the lesser virulent biotypes. UPLC-MS analysis did not reveal any lycopene or β-carotene due to low compound concentrations in the extracted samples but various hydrophobic compounds were present in different concentrations among the biotypes. The carotene desaturase expression profile revealed that SA1 had the lowest relative expression of the gene involved in carotenoid products, while SAM had the highest, under normal and stressful conditions. The results indicate that sequence conservation in mitochondrial genes are associated with key energy processes to maintain a state of homeostasis under variable conditions and that the generation of energy is a contributing factor to the virulence development of D. noxia. The results also show that carotenoids may possibly contribute to fitness of D. noxia through reactive oxygen species scavenging or the production of additional energy, but further investigation is needed for confirmation. / AFRIKAANSE OPSOMMING: Diuraphis noxia (Kurdjumov, Hemiptera, Aphididae) algemeen bekend as die Russiese koringluis (RWA), is ‘n klein floëem-voedende pes van koring (Triticum aestivum L). Virulente D. noxia biotipes wat instaat is om op voorheen bestande koring kultivars te voed gaan ontwikkel voortdurend, en daarom is die identifisering van faktore wat kan bydrae tot virulensie so belangrik. Omdat energie-metabolisme ‘n sleutelrol in die oorlewing van organismes speel, kan gene en prosesse wat by die produksie en regulering van energie betrokke is belangrike bydraers tot virulensie lewer: soos onder andere mitokondria en die NAD+/NADH-verhouding wat die gesondheid en metaboliese aktiwiteit van ‘n sel reflekteer. Die betrokkenheid van karotenoïede in die produksie van energie deur 'n fotosintese-verwante proses kan 'n belangrike faktor bydraend tot luis fiksheid wees, asook die bydra daarvan tot plantluis-immuniteit deur bemiddeling van oksidatiewe stres. Die volledige mitochondriale genoom van globale Diuraphis noxia populasies is met behulp van volgende generasie DNA volgordebepaling gekarrakteriseer, en daar is bevind dat dit 15 721 bp in grootte is en uit 38 gene bestaan wat tipies binne insekte voorkom. Enkelnukleotied- polimorfisme (SNP) ontleding van die genome van nege populasies het onthul dat daar 125 SNPs in die proteïen-koderende gene voorkom, met die meerderheid van die SNPs in die ND-gene, en die minste in die ND4L-geen. Lae SNP-frekwensies is gevind vir die atp6- en atp8- gene, wat verskil van verslae oor ander Hemiptera. Veranderlike ND5-uitdrukkingsvlakke onder die biotipes is waargeneem, alhoewel geen korrelasie duidelik was tussen ND5-uitdrukking en die virulensie geassosieer met elke biotipe nie. Die transkripsie van atp6 was hoër in die hoogs virulente biotipe (SAM) onder normale en stresvolle toestande in vergelyking met die minste virulente biotipe (SA1). ‘n Aansienlike hoër NAD+/NADH-verhouding is ook waargeneem vir die SAM-biotipe onder spanningsvolle omstandighede in vergelyking met die minder virulente biotipes. UPLC-MS-analise het geen likopeen of β-karoteen geïdentifiseer nie as gevolg van lae verbinding konsentrasies in die onttrekte monsters, maar verskeie hidrofobiese verbindings was in verskillende konsentrasies tussen die biotipes teenwoordig. Die karoteen desaturase-uitdrukkingsprofiel het aangetoon dat SA1 die laagste relatiewe uitdrukking van gene betrokke by karotenoïed produksie het, terwyl SAM die hoogste relatiewe uitdrukking onder normale en spanningsvolle omstandighede het. Die resultate van die studie dui daarop dat die volgorde bewaring in mitochondriale gene verband hou met die sleutel energie prosesse om 'n toestand van homeostase onder wisselende omstandighede te handhaaf en dat die produksie van energie 'n bydraende faktor tot die ontwikkeling van virulensie in D. noxia is. Die resultate toon ook aan dat karotenoïede moontlik kan bydra tot fiksheid van D. noxia deur reaktiewe suurstofspesies te aas of deur die produksie van addisionele energie, maar verdere ondersoeke word benodig ter bevestiging.

Genetic analysis of 433 samples of serious crop pest aphid Diuraphis noxia was conducted with the use of 8 microsatellites loci. Statistical analysis revealed sexual reproduction of D. noxia in temperate regions. The linkage disequilibrium was detected because of the excess of heterozygotes. These results support the theory of RNDr. Starý about the invasion of D. noxia to American continent via states of North Africa, Spain and France.

The complete sequence of mitochondrial DNA from Diuraphis noxia was obtained and characterized. The mitogenome contains a standard set of 13 protein-coding genes, 19 tRNA genes, 2 ribosomal RNA genes. A+T-rich and ?repets? regions in the same order as those of the other analyzed aphids. Comparison to mtDNAs from other Sternorrhyncha species obtained from GenBank revealed possible markers for studies on population differentiation. Phylogenetic analysis using parsimony and maximum likelihood confirmed the classification of Diuraphis noxia into the Aphididae.

Master of Science / Department of Entomology / C. Michael Smith / The Russian wheat aphid Diuraphis noxia (Kurdjumov) is a serious pest of world cereal grain crops, primarily barley and wheat. A phenotypic characteristic of D. noxia feeding, leaf rolling, creates a leaf pseudo gall which protects aphids, making it difficult to treat infested plants with insecticides or biological control agents. Therefore, the use of D. noxia-resistant crops is a desirable aphid management tactic. Because of the development of virulent D. noxia biotypes, the identification of new sources of barley and wheat resistance is necessary. Virus-induced gene silencing (VIGS) utilizes the plant defense system to silence viruses in inoculated plants. The accumulation of virus RNA in plants triggers the defense system to silence sequences homologous to the introduced virus and sequences of interest from a plant are inserted into the virus and silenced along with the virus. The VIGS method was tested to determine the ability of barley stripe mosaic virus (BSMV) to serve as a VIGS vector in wheat plants containing the Dnx gene for resistance to D. noxia. Dnx leaves with silenced BSMV virus yielded D. noxia populations that were significantly no different from populations produced on healthy Dnx leaves. Thus, BSMV silencing does not interfere with Dnx resistance. Several different methods were examined to determine how best to confine aphids to the silenced leaf, and a modified plastic straw cage was chosen as the optimum cage type. Microarray and gene expression data were analyzed to select two NBS-LRR type disease resistance protein genes - TaAffx.104814.1.S1_at and TaAffx.28897.1.S1 - (NBS-LRR1 and NBSLRR2), in order to assess their role in Dnx resistance. NBS-LRR1 and NBSLRR2 were silenced by inoculating leaves of Dnx plants with barley stripe mosaic virus (BSMV) containing sequences of each gene. Controls included Dnx and Dn0 plants inoculated with BSMV and non-BSMV inoculated plants. Aphids were allowed to feed on control and treatment plants to assess aphid population and mean weight of aphids surviving at the end of the experiment. There were no differences among treatments based on aphid population, but there were significant differences the mean weights of aphids reared on several different treatments.

The aim of this work was a research of the genetic variability of natural populations of Russian wheat aphid Diuraphis noxia (Aphididae) by means of microsatellite markers and markers for EPIC-PCR. First goal was to introduce the methods and optimise them for Diuraphis noxia. In the follow-up pilot study, specimens from 47 lines representing 12 populations from all over the world were analysed. Having used microsatellite markers, I proved expected variability among individual populations and within them. The highest genetic variability was detected between Chile and Algeria using markers for cytochrome C in EPIC-PCR. These findings can be used for further studies of the genetic variability of the aphid Diuraphis noxia.