Establishment of a genetic database and molecular methods for the identification of fish species available on the South African market

Cawthorn, Donna-Maree (2011-12)

Thesis (PhD (Food Sc)--Stellenbosch University, 2011.

Thesis

ENGLISH ABSTRACT: Consumers have the right to accurate information on the fish products they purchase to enable them to make educated seafood selections that will not endanger their own wellbeing or the wellbeing of the environment. Unfortunately, marine resource scarcity, financial incentives and inadequate or poorly enforced regulations have all promoted the mislabelling of fish species on global markets, the results of which may hold economic, conservation and health consequences. The primary aims of this study were to determine the most commonly available fish species on the South African market, to establish and compare DNA-based methods for the unambiguous identification of these species and to utilise the most applicable methods to evaluate the extent of mislabelling on the local fisheries market. The results from surveys of n = 215 restaurants and n = 200 retail outlets in four South African provinces (Western Cape, Kwa-Zulu Natal, Eastern Cape and Gauteng) indicated that 34 and 70 nominal fish types were available in restaurants and retail outlets, respectively, the most common of which were kingklip, salmon and hake. Over 30% of the fish species being sold were of conservation concern, while several outlets marketed specially-protected, illegal-to-sell species in South Africa. Fish purveyors were poorly equipped to provide information on the identity, origin, production method (farmed/wild) and sustainability of the fish they were selling and the labelling of many packaged fish products was in contravention with South African regulations. Data were published for the first time comparing the efficiency of five methods (urea-SDS-proteinase K, phenol-chloroform, salt extraction, SureFood PREP kit and Wizard Genomic DNA Purification kit) for the extraction of DNA from the muscle tissue of fish species available in South Africa. The SureFood kit was identified as the most suitable method for DNA extraction from fish muscle, extracting significantly (P < 0.05) higher DNA yields than all other methods evaluated and being simple and safe to use. A comprehensive reference library of genetic information was compiled for the first time that contains sufficient DNA sequence data from different mitochondrial DNA loci (16S ribosomal RNA (rRNA), 12S rRNA and cytochrome c oxidase I (COI) genes, as well as the control region) to allow the explicit identification of 53 fish species in South Africa. Although 16S and 12S rRNA gene sequencing allowed the identification of most fish to the genus level, the discrimination of closely-related, congeneric species was problematic when based on these gene regions. Conversely, the vast majority (98%) of fish examined could be readily differentiated by their COI sequences, with only members of the genus Thunnus requiring supplementary control region sequencing for species confirmation. Lastly, sequencing of the COI region was used to show that 9% of fish samples collected from local seafood wholesalers and 31% of samples from retail outlets were mislabelled. This study has established that fish mislabelling is a reality on the South African market and that DNA-based methods should be applied by both industry and regulatory bodies to deter illegal activities and to promote transparency on the domestic fisheries market.

AFRIKAANSE OPSOMMING: Verbruikers het die reg tot akkurate informasie rakende die visprodukte wat hulle aankoop. Hierdie inligting sal hulle bemagtig om ingeligte seekos keuses te maak wat voordelig sal wees vir beide die verbruiker se eie, sowel as die omgewing, se voortbestaan. Ongelukkig het 'n gebrek aan seelewebronne, geldelike aansporings en onvanpaste of swak geïmplimenteerde regulasies gelei tot die verkeerde etikettering van visspesies op die wêreldmarkte. Dit mag ekonomiese-, bewarings- en gesondheidsgevolge inhou. Die primêre doelwitte van hierdie studie was om te bepaal watter visspesies die algemeenste beskikbaar is in die Suid-Afrikaanse mark, om DNS-gebaseerde metodes vir die duidelike identifisering van hierdie spesies te vind en te vergelyk, en om die mees gepaste metodes te gebruik om die omvang van verkeerde etikettering in die plaaslike vismarkte te evalueer. Die resultate van opnames van n = 215 restaurante en n = 200 winkels in vier Suid-Afrikaanse provinsies (Wes-Kaap, Kwa-Zulu Natal, Oos-Kaap en Gauteng) het gewys dat 34 en 70 nominale visspesies in onderskeidelik restaurante en kleinhandelaars beskikbaar was. Koningklip, salm en stokvis was die mees algemene spesies. Meer as 30% van die visspesies wat te koop was is van bewaringsbelang, terwyl verskeie winkels spesiaal-beskermde, onwettig-om-te-verkoop spesies in Suid-Afrika bemark het. Visverkopers was swak bemagtig om informasie oor die identiteit, oorsprong, produksiemetode (teel/wild) en volhoubaarheid van die vis wat hulle verkoop het te kon gee. Verder was die etikettering van baie verpakte visprodukte in stryd met Suid-Afrikaanse regulasies. Vir die eerste keer is data gepubliseer wat vyf metodes (ureum-SDS-proteïenase K, fenolchloroform, sout-ekstraksie, SureFood PREP stel en Wizard Genomic DNS suiwering stel) vergelyk in hul doeltreffendheid om DNS vanuit die spierweefsel van visspesies wat in Suid-Afrika beskikbaar is te ekstraheer. Die SureFood stel is as die mees geskikte metode vir DNS ekstraksie vanuit visweefsel geïdentifiseer aangesien die DNS opbrengs betekenisvol (P < 0.05) hoër was met hierdie metode, en dit ook 'n eenvoudige en veilige metode is om te gebruik. 'n Omvattende verwysingsbiblioteek van genetiese informasie wat voldoende DNS volgordebepalingsdata van verskillende mitokondriale DNS lokusse (16S ribosomale RNS (rRNS), 12S rRNS en sitochroom c oksidase I (COI) gene, sowel as die kontrolegebiede) bevat, is vir die eerste keer opgestel om die besliste identifisering van 53 visspesies in Suid-Afrika toe te laat. Alhoewel 16S en 12S rRNS geenvolgordebepaling die identifisering van meeste visse op genusvlak toegelaat het, was die diskriminasie van naby-verwante, gelyksoorting spesies problematies wanneer hierdie geengebiede gebruik is. Die oorgrote meerderheid (98%) vis wat ondersoek is geredelik onderskei op grond van hul COI volgordebepalings, met slegs lede van die genus Thunnus wat addisionele kontrolegebied volgordebepaling vir spesies bevestiging vereis het. Laastens, is volgordebepaling van die COI-gebied gebruik om te wys dat 9% van die vismonsters van plaaslike seekosgroothandelaars en 31% van die monsters van kleinhandelaars verkeerd geëtiketteer is. Hierdie studie het bevestig dat die verkeerde etikettering van vis in Suid-Afrika 'n realiteit is, en dat DNS-gebaseerde metodes gebruik moet word deur die industrie sowel as die regulerende liggame om onwettige aktiwiteite teen te werk en om deursigtigheid in plaaslike vismarkte te bevorder.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/18045
This item appears in the following collections: