Characterization of the mitochondrial genomes of Diuraphis noxia biotypes

De Jager, Laura-Ellen (2014-12)

Thesis (MSc)--Stellenbosch University, 2014.

Thesis

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.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/96007
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