Marine forensics : a molecular tool for trade monitoring and compliance in southern African fisheries, with focus on commercially exploited elasmobranch species

Date
2019-12
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Marine forensic science can be described as protecting fisheries resources, marine mammals and endangered species based on enforcement of the nation's laws. Species identification becomes challenging when morphological features (such as fins, scales and heads) are removed, or if confiscated samples are already in a processed state. The harvesting of elasmobranchs (sharks, rays and skates) is driven by the international shark meat and shark fin trade. In recent decades, a combination of increasing demand and economic globalisation has created a global market for elasmobranch products, especially the highly prized shark fins for Asian markets. In this study, marine forensics was assessed as a tool for complementing traditional identification methods – through the development of a mini-barcoding assay as well as investigating High Resolution Melting (HRM) analysis – for species identification, with focus on elasmobranch species occurring in the southern African region. Firstly, this involved the testing and optimisation of the standard barcoding region of the cytochrome oxidase c subunit I (COI) gene, and then using traditional barcoding primers as well as nested polymerase chain reaction (PCR) primers in a multiplex assay. Preliminary results (only a 22 % species identification success rate) indicated the limitations of using only the traditional COI primers and warranted the inclusion of alternate COI gene fragments for species identification in future related forensic cases. A mini-barcoding multiplex assay, comprising three primer sets, was optimised and applied to a wide range of forensic case studies involving confiscated shark fins, possibly for illegal trade. A significant number of CITES-listed and endangered species were identified when confiscated specimens from various regions in southern Africa were tested. Secondly, PCR amplification of a 16S ribosomal RNA (16S rRNA) gene fragment was optimised based on six southern African houndshark species and seven other commercially exploited species, including: hammerhead sharks Sphyrna lewini and Sphyrna zygaena, copper shark Carcharhinus brachyurus, dusky shark Carcharhinus obscurus, bull shark Carcharhinus leucas, blacktip shark Carcharhinus limbatus and blue shark Prionace glauca. High Resolution Melting analysis using the 16S rRNA gene region was investigated as a species identification method for these species. The HRM assay was successfully applied for the identification of seven commercially exploited shark species, including some of the top commercially important sharks and one endemic houndshark Scylliogaleus quecketti. Although further optimisation is required, this relatively fast and cost-effective approach will be a valuable tool for the initial screening of detained shipments, for possible illicit trade. Accordingly, this research presents species identification assays suitable for various shark related forensic case studies, and in future could be applied to identify most, if not all, elasmobranch species involved in trade regionally.
AFRIKAANSE OPSOMMING: Mariene forensiese wetenskap kan beskryf word as die beskerming van visserye, mariene soogdiere en bedreigde spesies gebaseer op die handhawing van die land se wette. Spesies identifikasie word ‘n uitdaging wanneer morfologiese kenmerke (vinne, skubbe en koppe) verwyder word of in die geval waar gekonfiskeerde monsters reeds in 'n verwerkte toestand is. Die oes van spesies in sub-klas elasmobranchii (haaie en rogspesies) word deur die internasionale haaivleis- en haaivin-handelmark aangedryf. In onlangse dekades het ‘n kombinasie van toenemende vraag en ekonomise globalisering ‘n globale mark vir elasmotak-produkte geskep, veral die hooggewaardeerde haaivinne vir Asiatiese markte. In hierdie studie was mariene forensiese wetenskap beoordeel as ‘n instrument om tradisionele identifikasiemetodes te ondersteun – deur die ontwikkeling van ‘n mini-vingerafrdukmerker toets asook ‘n ondersoek van Hoë Resolusie-Smelting (HRS) analise – vir die identifisering van spesies, met klem op elasmobranchii wat in die suider-Afrika streek voorkom. Eerstens was die standaard strepieskode-streek van die sitokroom oksidase c subeenheid I (COI) geen geanaliseer en optimiseer, daarna is tradisionele vingerafdrukmerker inleiers sowel as geneste polimerase kettingsreaksie (PKR) inleiers in 'n veelvoudige metode getoets. Beperkings in die gebruik van slegs die tradisionele COI inleiers was beklemtoon deur voorlopige resultate (22 % spesies identifikasie sukses) wat die insluiting van alternatiewe COI geenfragmente vir die identifisering van spesies in toekomstige forensiese verwante gevalle geregverdig. ‘n Minivingerafdrukmerker veelvoudige toets, bestaande uit drie inleier-stelle, was geoptimiseer en op ‘n wye reeks forensiese gevallestudies toegepas, insluitend die betrokkenheid van gekonfiskeerde haaivinne – moontlik betrokke vir onwettige handel. ‘n Beduidende aantal CITES-gelysde en bedreigde spesies was geïdentifiseer toe monsters, gekonfiskeer van verskeie streke in suider-Afrika, getoets was. Tweedens, PKR amplifisering van 'n 16S ribosomale RNA (16S rRNA) geen fragment was geoptimiseer op grond van ses Suider-Afrikaanse sloothaaispesies en sewe ander spesies wat kommersieel ontgin word. Die sewe spesies van belang sluit in: hamerkophaaie Sphyrna lewini en Sphyrna zygaena, bronshaai Carcharhinus brachyurus, skemerhaai Carcharhinus obscurus, bulhaai Carcharhinus leucas, swarttiphaai Carcharhinus limbatus en blouhaai Prionace glauca. Hoë Resolusie-Smelting analise gekoppel met die 16S rRNA geenstreek was ondersoek as ʼn moontlike spesiesidentifiseringsmetode vir die spesies van belang. Die HRS analise was suksesvol toegepas op die identifisering van sewe haaispesies wat kommersieel ontgin is, met van die mees kommersiële belangrike haaie en een endemiese sloothaai Scylliogaleus quecketti ingesluit. Alhoewel verdere optimiseering benodig is, is hierdie relatiewe vinnige en koste-effektiewe benadering 'n waardevolle hulpmiddel vir die aanvanklike keuring van aangehoude skeepsvrag vir moontlike onwettige handel. Gevolglik bied diè navorsing spesiesidentifikasie analises aan wat geskik is vir verskeie haai-verwante forensiese gevallestudies, en kan in die toekoms toegepas word om die meeste, indien nie alle, soort elasmobranchii-spesies wat by streekhandel betrokke is te identifiseer.
Description
Thesis (MScAgric)--Stellenbosch University, 2019.
Keywords
Marine mammals -- South Africa, Fishes -- Molecular genetics, Polymerase chain reaction, High Resolution Melting -- Analysis, Fisheries -- South Africa, Fishes -- Speciation, UCTD
Citation