Development of a molecular detection assay for accurate identification of five economically important tephritid species of commercial fruit in South Africa

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dc.contributor.advisorMaree, H. J.en_ZA
dc.contributor.advisorBester, Rachelleen_ZA
dc.contributor.authorAndrews, Kelsey Jayneen_ZA
dc.contributor.otherStellenbosch University. Faculty of AgriSciences. Dept. of Genetics.en_ZA
dc.date.accessioned2022-11-22T10:26:27Z
dc.date.accessioned2023-01-16T12:51:21Z
dc.date.available2022-11-22T10:26:27Z
dc.date.available2023-01-16T12:51:21Z
dc.date.issued2022-12
dc.descriptionThesis (MScAgric)--Stellenbosch University, 2022.en_ZA
dc.description.abstractENGLISH ABSTRACT: South African fresh fruit production and export are plagued by five major fruit fly pests (Diptera: Tephritidae). These fruit flies: Ceratitis capitata (Wiedemann) Mediterranean fly, Ceratitis cosyra (Walker) Marula fly, Ceratitis quilicii (de Meyer, Mwatawala & Virgilio) Cape fly, Ceratitis rosa (Karsch) Natal fly, and Bactrocera dorsalis (Hendel) the Oriental fly, are of quarantine significance in certain export markets. Apart from economic loss incurred due to limitations in export markets, these flies also cause physical damage to fresh fruit through oviposition. Accurate identification of these fruit flies can be tricky as this fruit fly family consists of multiple morphologically cryptic species and species complexes. Morphological identification of these fruit flies is unreliable when larvae or closely related species are intercepted. This has highlighted a need for accurate methods to distinguish between these species that do not rely on morphological traits. Therefore, this study aimed to develop a molecular identification assay that can differentiate between the five fruit flies of concern to South Africa accurately and timeously. To achieve this, two colony-reared insects from each species underwent DNA extraction and high throughput sequencing (HTS). HTS data were subjected to de novo assembly and used to construct ten complete mitochondrial genomes using a combination of de novo and reference-based assembly methods. From this, two identification assays were developed: a sequencing- based assay targeting a mitochondrial intergenic region and a multiplex PCR assay targeting the gene opsin Rh4. Regarding the sequencing-based assay, a single primer set was designed to amplify a mitochondrial region between tRNAⁱˡᵉ and tRNAᵐᵉᵗ. The intergenic region between tRNAⁱˡᵉ and tRNAᵍˡⁿ (designated intergenic region I) within the amplicon is species-specific in size and proposed as a potential tool for species differentiation of the five species of interest in this study. In the multiplex PCR assay, five sets of species- specific primers with varying sizes were designed and optimised for use in a multiplex format. The resulting species-specific amplicons can be separated using a 2% agarose-TAE gel, providing accurate species identification. Both assays were validated using larval stages and wild, trap-collected specimens. The assays developed in this study can be applied in pest surveillance and monitoring activities and during fruit inspection at packhouses and ports of entry (PoE).en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Suid-Afrikaanse varsvrugteproduksie en -uitvoer word geteister deur vyf groot vrugtevliegplae (Diptera: Tephritidae). Hierdie vrugtevlieë: Ceratitis capitata (Wiedemann) Mediterreense vlieg, Ceratitis cosyra (Walker) Marula vlieg, Ceratitis quilicii (de Meyer, Mwatawala & Virgilio) Kaapse vlieg, Ceratitis rosa (Karsch) Natalse vlieg, en Bactrocera dorsalis (Hendel) die Oosterse vlieg, is van kwarantynbelang in sekere uitvoermarkte. Afgesien van ekonomiese verliese wat gely word as gevolg van beperkings in uitvoermarkte, veroorsaak hierdie vlieë ook fisiese skade aan vars vrugte deur oviposisie. Akkurate identifikasie van hierdie vrugtevlieë is moeilik aangesien hierdie vrugtevliegfamilie uit veelvuldige morfologies kriptiese spesies en spesiekomplekse bestaan. Morfologiese identifikasie van hierdie vrugtevlieë is onbetroubaar wanneer larwes of naverwante spesies onderskep word. Dit het die behoefte aan akkurate metodes om te onderskei tussen hierdie spesies beklemtoon wat nie op morfologiese eienskappe staatmaak nie. Daarom het hierdie studie daarop gefokus om 'n molekulêre identifikasietoets te ontwikkel wat akkuraat en iiiining kan onderskei tussen die vyf vrugtevlieë wat vir Suid-Afrika kommerwekkend is. Om dit te bereik, het twee kolonie-insekte van elke spesie DNA-ekstraksie en hoë deurvloei-volgordebepaling ondergaan (HTS). HTS-data is aan de novo-samestelling onderwerp en gebruik om tien volledige mitochondriale genome te konstrueer deur 'n kombinasie van de novo- en verwysingsgebaseerde samestellingsmetodes te gebruik. Hieruit is twee identifikasietoetse ontwikkel: 'n volgordebepaling-gebaseerde toets wat 'n mitochondriale intergeniese gebied en 'n multipleks PCR-toets gerig op die geen opsin Rh4. Met betrekking tot die volgordebepaling- gebaseerde toets, is 'n enkele inleier-stel ontwerp om 'n mitochondriale gebied tussen tRNAⁱˡᵉ en tRNAᵐᵉᵗ te vermenigvuldig. Die intergeniese gebied tussen tRNAⁱˡᵉ en tRNAᵍˡⁿ (aangewese intergeniese streek I) binne die amplikon is spesie-spesifiek in grootte en kan as 'n potensiële hulpmiddel vir spesie-differensiasie van die vyf spesies van belang in hierdie studie. In die multipleks PCR-toets is vyf stelle spesiespesifieke inleiers met verskillende groottes ontwerp en geoptimiseer vir gebruik in 'n multipleks-formaat. Die gevolglike spesie- spesifieke amplikone kan geskei word met behulp van 'n 2% agarose-TAE-gel, wat akkurate spesie- identifikasie verskaf. Beide toetse is bekragtig deur gebruik te maak van larwes en wilde vlieë. Die toetse wat in hierdie studie ontwikkel is, kan toegepas word in plaagtoesig- en moniteringsaktiwiteite en tydens vruginspeksies by pakhuise en hawens of lughawens (PoE).af_ZA
dc.description.versionMastersen_ZA
dc.format.extentxi, 89 pages : illustrations (some color)en_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/126125
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subjectMolecular identification assayen_ZA
dc.subjectFruit-flies -- Economic aspectsen_ZA
dc.subjectMolecular detectionen_ZA
dc.subjectFruit-flies (Diptera: Tephritidae) -- Speciation -- Genetic aspectsen_ZA
dc.subjectFruit trade -- Economic aspects -- South Africaen_ZA
dc.subjectUCTDen_ZA
dc.titleDevelopment of a molecular detection assay for accurate identification of five economically important tephritid species of commercial fruit in South Africaen_ZA
dc.typeThesisen_ZA
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