Thesis (MScAgric)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Abalone production is the largest financial contributor to aquaculture in South Africa and practically all of the abalone produced is exported to Asia. This means that the product must be globally competitive and many technologies have been applied to this cause. One that specifically shows great promise for bivalve mollusc production is triploidy; more precisely, sterility due to the induction of aneuploidy. Under normal maturation, energy is diverted from somatic growth through sexual maturation, therefore inhibiting or retarding gametogenesis through a process such as aneuploidy is expected to increase growth and decrease the time to marketing. Two studies preceding this one investigated the induction of triploidy through hydrostatic shock (De Beer, 2004) and the comparative growth rate of triploid genotypes from 8 to 24 months, prior to the onset of sexual maturation (Schoonbee, 2008). During this comparative growth stage, no convincing statistical evidence of faster growth or of seasonal environmental effects could be obtained. It was recommended that growth between triploid and diploid variants be compared during the age period when sexual maturity becomes a factor to determine whether triploidy in Haliotis midae is a useful biotechnological tool to improve biological productivity and global competiveness of the abalone industry. The growth measured as shell length and wet weight in the period from 29 to 62 months showed a statistically significant difference in mean weight and mean length with diploids showing a superior growth rate compared to their triploid siblings. This difference of 1.99 mm and 5.13 g was however not perceived as being commercially significant. Important production parameters including canning yield percentage and gonadosomatic index were also measured during this trial. For both these parameters, the triploid genotype showed statistically and commercially significant improvement of 10.68% increased canning yield and 28.42% reduction in gonadosomatic index when compared to their diploid counterparts. Triploid abalone was found to be not completely sterile; gametes and even mature gonads were observed in some instances. Even though complete sterility was not achieved there appeared to be a retarded gonadosomatic development in triploid variants. The delay in sexual maturation, together with the improvement in canning yield, may justify triploidy’s commercial application, despite its reduced growth rate. / AFRIKAANSE OPSOMMING: Perlemoen produksie lewer die grootste finansiële bydra tot akwakultuur in Suid Afrika en feitlik al die Perlemoen word uitgevoer na Asië. Dit beteken dat die produk moet kompeteer op die wêreld mark en verskeie tegnologieë word reeds aangewend vir die spesifieke doel. Een so tegnologie wat potensiaal toon ten opsigte van akwakultuur produksie is triploïedie; meer spesifiek, sterieliteit veroorsaak deur aneuploïedie induksie. Onder normale volwassewording, word energie weggeneem van somatiese groei wanneer geslagsrypheid intree en daarom kan groeitempo verhoog word deur gametogenese te inhibeer of te vertraag deur ‘n proses soos aneuploïedie en, word korter tydperk benodig om bemarkingsgrootte te bereik. Twee voorafgaande studies het gehandel oor die induksie van triploïedie deur hidrostatiese druk skok (De Beer, 2004) en die vergelykende groeitempo van triploïede genotipes vanaf ouderdom 8 tot 24 maande (Schoonbee, 2008) alvorens geslagsrypheid intree. Tydens hierdie vergelykende groeifase kon geen statisties betekenisvolle aanduidings van vinniger groei of seisoenale omgewingseffekte aangetoon word nie. Die studie handel vervolgens oor die uitbreiding van die vergelykende groeistudies tussen triploïede en diploïede genotipes tot ouderdom van 62 maande wat die intrede van geslagsrypheid insluit, ten einde te bepaal of die induksie van triploïedie in Haliotis midae voordele inhou ten opsigte van produksiedoeltreffendheid en mededingendheid op wêreldmarkte. Groei gemeet in terme van skulplengte en lewende massa oor die tydperk van 29 tot 62 maande het statisties betekenisvolle verskille getoon in gemiddelde massa en lengte van diploïede genotipes bo die van triploïede verwante individue. Die verskille van 1.99 mm en 5.13 g kan egter nie as kommersieel betekenisvol beskou word nie. Belangrike produksie eienskappe waaronder persentasie opbrengs van eindproduk en die gonadosomatiese indeks is ook bepaal. Vir beide die produksie eienskappe het die triploïede genotipe statisties sowel as kommersieel betekenisvolle verbetering van 10.68% getoon vir opbrengs en 28.42% verlaging in gonadosomatiese indeks in vergelyking met die diploïede genotipe. Triploïede genotipes was nie volledig steriel nie, gegewe die aanwesigheid van gonades en gamete in sommige individue. Selfs al is totale steriliteit nie bereik nie, het dit wel voorgekom asof daar vertraging in gonadosomatiese ontwikkeling plaasgevind het in triploïede genotipes. Die vertraging in geslagsrypheid tesame met die verhoogde persentasie opbrengs van die eindproduk hou voordele in bo die andersins effense stadiger groei van triploïede genotipes.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Haliotis midae (commonly referred to as perlemoen) is the only one of five endemic species in South Africa that is commercially valued both locally and internationally. Unfortunately, natural perlemoen populations have become a dwindling resource due to commercial exploitation, poaching and the influx of natural threats, such as the West Coast rock lobster, Jasus lalandii. To preserve the natural diversity and sustainability of natural populations as well as commercial stocks, genetic management and improvement of perlemoen is critical. Genetic management requires the utilisation of molecular markers, which aid in the construction of linkage maps and the identification of quantitative trait loci (QTL) associated with economically significant traits. This will allow improvement of commercial stock management in terms of broodstock selection as well as provide valuable insight into natural population dynamics. Single Nucleotide Polymorphisms (SNPs) were selected as the marker of choice due to their successful employment as molecular markers and their wide distribution and abundance within the genomes of various marine species. This study focuses on the characterisation of novel SNPs from transcript sequences generated by Next Generation Sequencing technology. Approximately 40% of the transcripts facilitated the isolation of 105 putative markers, indicating a SNP frequency of ~1% within the H. midae genome. A subset of 24 markers, in addition to 24 previously developed markers, was characterised using the Illumina GoldenGate genotyping assay with the VeraCode technology, a medium to high-throughput genotyping technology. This is the first reported medium- to highthroughput characterisation of SNPs in H. midae. The selected markers were used to determine the efficiency and overall success rate of the GoldenGate platform. Marker characterisation was completed in both natural and commercial populations to determine the utility of these markers for genetic diversity and population structure inference. An 85% genotyping success rate was achieved with the platform. Statistical analysis indicated that the markers developed in this study are suitable for applications including population genetic structure inference, genetic diversity estimation and possibly other downstream applications such as linkage mapping. These markers are considered to be invaluable for future work regarding the genetic management and conservation of H. midae. / AFRIKAANSE OPSOMMING: Haliotis midae (ook bekend as perlemoen) is die enigste van vyf inheemse spesies in Suid-Afrika wat noemenswaardige kommersiële waarde toon plaaslik sowel as internasionaal. Ongelukkig het kommersiële uitbuiting, wildstropery en natuurlike bedreiging (bv. die Weskus kreef Jasus lalandii), wilde perlemoen populasies noemenswaardig verminder. Dus, om natuurlike diversiteit en die voortbestaan van beide wilde en kommersiële populasies te beskerm, is genetiese bestuur en verbetering absoluut noodsaaklik. Genetiese bestuur vereis die gebruik van molekulêre merkers as ’n hulpmiddel in die opstellingvan koppelingskaarte, en die identifisering van die relevante kwantitatiewe eienskap loki (QTL) tipies geassosieer met ekonomies belangrike eienskappe. Die laasgenoemde beoog om kommersiële voorraad bestuur te verbeter, kragtens deur broeidier seleksie sowel as om insig te verskaf m.b.t. wilde bevolking dinamika. Enkel Nukleotied Polimorfismes (SNPs) is gekies as die toepaslike merker vanweë die omvattende toepaslikheid van hierdie merkers binne die genome van verskeie mariene spesies. Hierdie studie fokus op die karakterisering van nuwe SNPs vanuit transkript volgordes ontwikkel deur middel van Volgende Generasie Volgordebepaling (“Next Generation Sequencing”). ’n Beraamde 40% van transkripte het gelei tot die ontwikkeling van 105 potensiëlemerkers, aanduidend van ’n SNP frekwensie van ~1% binne die H. midae genoom. ’n Sub-versameling van 24 merkers, tesame met 24 bestaande merkers, is gekarakteriseer deur die Illumina GoldenGate genotiperings toets met die VeraCode tegnologie, ’n medium tot hoë deurvloei genotiperingstegnologie. Hierdie is die eerste berig van medium tot hoë deurvloei karakterisering van SNPs in H. midae. Die geselekteerde merkers is gebruik om die doeltreffendheid van die GoldenGate platform te bepaal. Merker karakterisering is uitgevoer in beide wilde en kommersiële bevolkings om die effektiewe bruikbaarheid van hierdie merkers m.b.t. genetiese diversiteit, en bevolking struktuur bepaling, te ondersoek. Die platform het ’n 85% genotiperingsukses syfer getoon. Statistiese analise dui daarop dat merkers ontwikkel tydens hierdie studie toepaslik is vir bevolking genetiese struktuur bepaling, genetiese diversiteitberaming en moontlik ook genetiese koppelingskartering. Hierdie merkers word bestempel as onmisbaar vir toekomstige navorsing in genetiese bestuur en bewaring van H. midae.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Haliotis midae, or more commonly known as Perlemoen, is an abalone species found along the coast of South Africa. It is the only cultured abalone species in South Africa and has a high demand abroad. Due to its popularity as a seafood delicacy, illegal harvesting has taken its toll on Perlemoen numbers. This increases the need for sustainable farming efforts and efficient implementation of law enforcement practices against poachers. Abalone farms make use of a limited number of broodstock for breeding, so it is necessary to ensure that genetic effects such as inbreeding and bottlenecks do not interfere with the viability of the offspring. Research that focuses on the genetics of Perlemoen will greatly aid the farms to continue sustainable production of this species as well as enhance their breeding efficiency. This study focuses on the construction of a linkage map for H. midae that will allow the future identification of markers associated with genes important to production, such as growth and disease resistance. Identification of these genes will allow breeders to select genetically superior abalone that will be used for breeding programmes in which the phenotype of the offspring will be enhanced. For the construction of a linkage map it is necessary to have enough informative markers for mapping. In this study, gene-linked microsatellite markers were developed by screening a contig assembly of H. midae’s transcriptome. Ninety-eight primer pairs could be developed from the contigs and 60 loci produced amplification products. Twenty-six microsatellites were found to be polymorphic (27% success rate). In addition to these markers, 239 previously developed microsatellites and 48 gene-linked SNPs were used to develop sex-average and sex-specific linkage maps in four full-sib families consisting of approximately 100 offspring each. Of these markers 99 were informative in family DS1 (31% success rate), 81 in family DS2 (26%), 77 in family DS5 (24%) and 71 in family DS6 (23%). These markers were used for linkage analysis (LOD>3). The average number of linkage groups for the sex-average maps ranged from 17-19. The average genome length for these maps ranged from 700cM to 1100cM with an average marker spacing of 8cM. The sex-specific maps’ linkage groups ranged from 13-17 with an average genome length of 600cM to 1500cM. The average marker spacing was approximately 16cM. The integrated map was constructed by merging the sex-average maps. This map contained 25 linkage groups with an average genome length calculation of 1700cM and an average marker spacing of 9.3cM. The linkage maps created in this study are the first to utilize SNPs in H. midae. Further incorporation of SNPs into linkage maps will enhance the density. The maps created in this study are of medium-density (65%) and provide a link to the development of high-density linkage maps to facilitate associations of phenotypic traits to certain markers, to so that QTL mapping can be performed. This information can be used for marker-assisted selection to produce genetically superior abalone. / AFRIKAANSE OPSOMMING: Haliotis midae, of meer algemeen bekend as Perlemoen, is 'n klipkous spesie wat langs die kus van Suid-Afrika voorkom. Dit is die enigste gekweekte klipkous spesie in Suid-Afrika en het 'n hoë aanvraag in die buiteland. As gevolg van sy gewildheid as 'n seekos lekkerny, het onwettige stropery sy tol geneem op Perlemoen getalle. Hierdie verhoog die behoefte vir volhoubare boerdery pogings en doeltreffende implementering van wetstoepassing teen stropers. Perlemoenplase maak gebruik van 'n beperkte aantal broeidiere vir teling, dus is dit nodig om te verseker dat genetiese effekte soos inteling en genetiese bottelnekke nie inmeng met die lewensvatbaarheid van die nageslag nie. Navorsing wat fokus op die genetika van Perlemoen sal grootliks die plase steun om die volhoubare produksie van hierdie spesie voort te sit, sowel as hul teling doeltreffendheid te verbeter. Hierdie studie fokus op die ontwikkeling van 'n genetiese koppelingskaart vir H. midae, wat die toekomstige identifisering van die merkers wat verband hou met die gene wat belangrik is vir die produksie, soos groei en weerstand teen siektes sal verbeter. Identifisering van hierdie gene sal toelaat dat telers genetiese voortreflike Perlemoen kan kies vir teelprogramme waartydens die fenotipe van die nageslag sal verbeter word. Vir die ontwikkeling van 'n genetiese koppelingskaart is dit nodig om genoeg informatiewe merkers vir die kartering te hê. In hierdie studie, is geen-gekoppelde mikrosatelliet-merkers ontwikkel deur ‘contig’ data van H. midae se transkriptoom te ondersoek. Agt en negentig inleier pare kon ontwikkel word uit die ‘contigs’ en 60 loki kon ‘n amplifiseringsproduk lewer. Ses-en-twintig mikrosatelliete was polimorfies (27% suksessyfer). Bykomend tot hierdie ontwikkelde merkers is 239 voorheen ontwikkelde mikrosatelliete en 48 geen-gekoppelde SNPs gebruik om geslagsgemiddelde en geslagspesifieke koppelingskaarte in vier volsib families, wat uit ongeveer 100 nageslag elk bestaan, te ontwikkel. Van hierdie merkers was 99 informatief in familie DS1 (31%), 81 in die familie DS2 (26%), 77 in die familie DS5 (24%) en 71 in die familie DS6 (23%). Hierdie merkers is gebruik vir 'n koppelingsanalise (LOD>3). Die gemiddelde aantal koppelingsgroepe vir die geslagsgemiddelde kaarte het gewissel van 17-19. Die gemiddelde genoom lengte vir hierdie kaarte het gewissel van 700cM tot 1100cM met 'n gemiddelde merker spasiëring van 8cm. Die koppelingsgroepe van die geslagspesifieke kaarte het gewissel van 13-17 met 'n gemiddelde genoom lengte van 600cM tot 1500cM. Die gemiddelde merker spasiëring was ongeveer 16cm. Die geïntegreerde kaart is saamgestel deur die samesmelting van die geslagsgemiddelde kaarte. Die kaart toon 25 koppelingsgroepe met 'n gemiddelde berekende genoom lengte van 1700cM en' n gemiddelde merker spasiëring van 9.3cM. Die genetiese koppelingskaarte wat in hierdie studie ontwikkel is, is die eerste om SNPs te gebruik in H. midae. Verdere insluiting van SNPs in koppelingskaarte sal die digtheid verhoog. Die kaarte wat in hierdie studie ontwikkel is, is van medium digtheid (65%) en bied 'n stap nader aan die ontwikkeling van hoë digtheid koppelingskaarte om fenotipiese eienskappe met sekere merkers te assosieer, vir kwantitatiewe kenmerk lokus kartering. Hierdie inligting kan gebruik word vir merker bemiddelde seleksie om geneties verbeterde Perlemoen te produseer.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The indigenous abalone Haliotis midae is one of the most remarkable and highly exploited species of marine molluscs in South Africa. It is the only species of southern African Haliotidae to be commercially reared and has been successfully cultured for almost two decades. Its short history of domestication along with market demands and the need to develop efficiency in the production process has resulted in an increased interest in the possible genetic improvement of this species. The unhurried growth rate associated with H. midae is a cause of particular concern to the industry, predominantly with regards to profitability and competitiveness in the market place. A modest amount of work has so far been directed at establishing a means of enhancement for selective breeding on the commercial level. Genetics plays a key role in the establishment of successful improvement programmes in various aquaculture species. The aim of this study was to develop species-specific microsatellite markers for the abalone and subsequently perform parentage assignment on farm produced animals entered into a growth performance recording scheme. Animals were obtained from the hatcheries of three commercial abalone farms situated in the Walker Bay region in the Western Cape. Microsatellites were isolated using the enrichment-based FIASCO method, and characterised into perfect, imperfect and compound repeats according to the structural nature of their repetitive units. From the partial gDNA libraries obtained and 365 screened colonies, a total of 54 loci were located. PCR primers were designed for 36 markers and the 15 primer pairs that displayed loci with the highest level of polymorphism were subsequently chosen for fluorescent labelling. The markers were tested on a subset of 32 wild H. midae individuals to determine their usefulness and efficiency in genotyping. Five markers, along with five others that were previously designed, were chosen for assigning parentage to the animals submitted to the performance recording scheme. Three thousand offspring from each of the three participating farms were equally divided and reared at five different locations. From each location 20 fast growing and 20 slow growing juveniles, as well as the broodstocks, were sampled and genotyped using the ten chosen microsatellite loci. Two farms had 60% of offspring unambiguously assigned to a single parental couple. Assignments showed patterns of dominant male and female brooders, but no trend in brooders specifically contributing to fast or slow growing offspring. Parentage assignment for the third farm was, however, unsuccessful due to lack of broodstock data. In future, screening of all available broodstock will ensure acquisition of relevant pedigree information. The results obtained in this study are an initial step in the development of a genetic improvement programme for commercial Haliotis midae. / AFRIKAANSE OPSOMMING: Die inheemse skulpvis Haliotis midae is een van die mees merkwaardige en hoogs oorbenutte mariene slakspesies in Suid-Afrika. Dit is die enigste suidelike Afrika Haliotidae spesie wat kommersieel benut word en dit word al meer as twee dekades suksesvol geteel. Die spesie se kort domestiseringsgeskiedenis, toenemende mark aanvraag en die behoefte om meer effektiewe produksie daar te stel, het gelei tot toenemende belangstelling in die moontlike genetiese verbetering van die spesie. Die stadige groeitempo geassosieer met H. midae is veral ‘n punt van kommer vir die industrie, veral in terme van winsgewendheid en kompetering in die markplek. Minimale werk is sover gedoen in die daarstelling van verbetering deur selektiewe teling op ‘n kommersiële skaal. Genetika speel ’n sleutelrol in die daarstelling van suksesvolle verbeteringsprogramme van verskeie akwakultuur spesies. Die doel van hierdie studie was om spesie-spesifieke mikrosatelliet merkers vir perlemoen te ontwikkel en vervolgens ouerskapsbepaling van kommersiële diere, wat deelneem aan ‘n groeiprestasie aantekenstelsel, uit te voer. Diere is voorsien deur die teelstasies van drie kommersiële perlemoenplase geleë in die Walker Bay omgewing in die Wes-Kaap. Mikrosatelliete is geïsoleer deur die verrykings-gebaseerde FIASCO metode, en gekarakteriseer as perfekte, onderbroke of saamgestelde herhalings gebaseer op die strukturele aard van die herhalings eenhede. Vanaf die gedeeltelik gDNA biblioteke wat bekom is en 365 gesifte kolonies, is ‘n totaal van 54 loki opgespoor. PKR inleiers is ontwerp vir 36 merkers en die 15 inleierpare, wat loki met die hoogste polimorfisme geamplifiseer het, is vervolgens geselekteer vir fluoreserende merking. Die merkers is getoets op ’n kleiner groep van 32 natuurlike H. midae individue om hulle bruikbaarheid en genotiperingseffektiwiteit te bepaal. Vyf merkers is saam met vyf reeds ontwikkelde merkers gekies vir ouerskapsbepaling van die diere in die prestasie aantekenstelsel. Drieduisend nageslag diere vanaf elkeen van die drie deelnemde plase is gelykop verdeel en grootgemaak op die vyf verskillende lokaliteite. ‘n Monster van 20 vinnig groeiende en 20 stadig groeiende jong perlemoen, sowel as broeidiere, is vanaf elke lokaliteit geneem en gegenotipeer deur middel van die 10 geselekteerde mikrosatelliet loki. Sestig persent van twee van die plase se nageslag is onteenseglik toegesê aan ‘n enkele ouerpaar. Ouerskapstoekenning het patrone van dominante vroulike en manlike broeidiere getoon, maar geen tendens in terme van bydrae tot vinnig en stadig groeiende nageslag kon gevind word nie. Ouerskapstoekenning vir die derde plaas was onsuksesvol as gevolg van ’n gebrek aan data vir die broeidiere. In die toekoms sal genotipering van alle beskikbare broeidiere die daarstelling van relevante stamboominligting verseker. Die resultate verkry in hierdie studie verteenwoordig ‘n eerste stap in die ontwikkeling van ’n genetiese verbeteringsprogram vir kommersiële Haliotis midae.

Magister Scientiae (Biodiversity and Conservation Biology) / Land-based abalone aquaculture in South Africa started in the early 1990s and is based on the local species Haliotis midae. This industry expanded with great success over the last decade. In 2006 abalone exhibiting typical clinical signs of tubercle mycosis was discovered for the first time in South African abalone culture facilities,posing a significant threat to the industry. Halioticida noduliformans, a fungus belonging to the Peronosporomycetes (formerly Oomycetes), has been identified as the causative agent of abalone tubercle mycosis (ATM). While diagnoses of this disease are currently done by gross observation and histopathology, these methods fail to be sensitive enough to identify the causative agent accurately and reliably.Molecular confirmation could provide for quicker more accurate diagnostic information. The aim of this study was to develop a DNA based molecular diagnostic test. Polymerase chain reaction (PCR) has been used to rapidly detect, characterise and identify a variety of organisms. Nucleotide sequences of the smalland large-subunit ribosomal ribonucleic acid (rRNA) and mitochondrial cytochrome oxidase subunit II (cox2) genes of H. noduliformans were compared with closely related Peronosporomycete gene sequences to identify potential PCR primer sites. H. noduliformans specific real-time quantitative PCR (Q-PCR) primer sets were designed and optimised for each of the selected genes. Results indicate that, although all tested primers sets could amplify fungal DNA, only the LSU and cox2 primer sets - v -demonstrated no cross-amplification with the closely related Peronosporomycete and non-fungal DNA tested in the present study. The H. noduliformans specific LSU primer set was chosen for further analysis and used for all subsequent real-time PCR assays. The lowest detection limit for the LSU primer set was evaluated by running Q-PCR on serial dilutions of known quantities of extracted H. noduliformans DNA.Serial dilutions were made in PCR grade water as well as in an abalone tissue matrix.The sensitivity of the Q-PCR reaction was determined to be 266 pg of H.noduliformans DNA per 25 μL reaction volume. However, inclusion of a nested PCR step, utilising universal fungal outer primers, followed by Q-PCR with the H.noduliformans LSU specific primers improved sensitivity to 0.266 pg of H.noduliformans DNA per 25 μL reaction volume. This equates to approximately 2.4spores per 25 μL reaction volume. DNA extraction protocols were optimised to ensure efficient and repeatable extraction of high quality fungal DNA from pure fungus and tissue samples spiked with known quantities of fungal DNA. PCR amplification efficiency and potential inhibition were examined for each extraction method. Results suggest that real-time PCR has great potential in monitoring and quantifying H. noduliformans on abalone culture facilities in South Africa.

The profitability of abalone farms is heavily influenced by their production per unit of grow-out space. With farms having physically expanded to the maximum, and with increasing production costs, one of the most realistic ways for farms to increase their production is through optimizing stocking densities. The effect of stocking density on Haliotis midae performance is undocumented and optimal stocking densities for this species have not been determined. Experiments were conducted under farm conditions to investigate the effects of four different stocking densities (16 %, 20 %, 22 % and 24 % of available surface area) on growth, production and health of three different size classes of abalone (15-35 g, 45-65 g, and 70-90 g start weight). Each treatment was replicated four times and trials ran over a period of eight months with measurements being made at four month intervals. Abalone behaviour was observed during the trials in the experimental tanks. Weight gain per abalone decreased with an increase in density for all tested size classes (5.04 ± 0.18 to 2.38 ± 0.17; 5.35 ± 0.21 to 4.62 ± 0.29; 7.97 ± 0.37 to 6.53 ± 0.28 g.abalone-1.month-1 for the 15-35, 45-65 and 70-90 g classes respectively, with an increased density of 16 to 24 %). Individual weight gain of 15-35 g abalone was similar at stocking densities of 16 % and 20 % while weight gain of 45-65 g and 70-90 g abalone decreased when density was increased above 16 %. Biomass gain (kg.basket-1.month-1) was not affected by stocking density in the 15-35 g and 45-65 g size classes (1.29 ± 0.02 and 0.97 ± 0.02 kg.basket-1.month-1 respectively). However, the biomass gained by baskets stocked with 70-90 g abalone increased with stocking density (1.08 ± 0.02 to 1.33 ± 0.02 kg.basket-1.month-1) with an increased density of 16 to 24 %) and did not appear to plateau within the tested density range (16 to 24 %). Food conversion ratio did not differ significantly between densities across all size classes. Stocking density did not have a significant effect on abalone condition factor or health indices. The proportion of abalone above the level of the feeder plate increased with density (7.26 ± 1.33 to 16.44 ± 1.33 with an increased density of 16 to 24 %). As a proportion of abalone situated in the area of the basket, the same proportions were situated on the walls above the feeder plate and on the feeder plate itself irrespective of stocking density (p > 0.05). Higher proportions of animals had restricted access to feed at higher stocking densities (p = 0.03). The amount of formulated feed available on the feeder plate did not differ between stocking densities throughout the night (p = 0.19). Individual abalone spent more time above the feeder plate at higher stocking densities (p < 0.05). The percentage of time above the feeder plate, spent on the walls of the basket and on the feeding surface was not significantly different at densities of 20 %, 22 % and 24 % (p > 0.05) but abalone stocked at 16 % spent a greater percentage of time above the feeder plate on the feeding surface (83.99 ± 6.26 %) than on the basket walls (16.01 ± 6.26 %). Stocking density did not affect the positioning of abalone within a basket during the day or at night. Different size H. midae are affected differently by increases in stocking density in terms of growth performance. Findings from this research may be implemented into farm management strategies to best suit production goals, whether in terms of biomass production or individual weight gain. The fundamental mechanisms resulting in reduced growth at higher densities are not well understood, however results from behaviour observations suggest that competition for preferred attachment space and feed availability are contributing to decreased growth rates. With knowledge of abalone behaviour at different densities, innovative tank designs may be established in order to counter the reduction in growth at higher densities.

Marine ranching has been identified as an alternative to traditional aquacultural rearing and growing organisms for consumption. In the Eastern Cape, abalone ranching is a new and experimental industry. The aims of the research were to: first develop a GIS model to assist management in site selection for abalone seeding; and secondly to develop and standardize the sampling methodology in order to ground truth the sites, and assist in the monitoring and habitat identification of abalone. The GIS model developed in Chapter 3 was created using an unsupervised classification and fuzzy logic approach. Both vector and raster datasets were utilized to represent 7 different layers. Predominantly satellite imagery was used to classify the different substrate groups according to pixel colour signatures. The basic process was to apply a fuzzy rule set (membership) to rasters which gave an output raster (Fuzzification). The membership output rasters were overlaid which creates a single model output. It was found that model accuracy increased significantly as more layers were overlaid, due to the high variability within each of the individual layers. Model ground-truthing showed a strong and significant correlation (r2 = 0.91; p < 0.001) between the model outputs and actual site suitability based on in situ evaluation. Chapter 4 describes the investigation towards the optimal sampling methods for abalone ranching habitat assessments. Both destructive sampling methods and imagery methods were considered as methods of data collection. The study also evaluated whether quadrat and transects were going to be suitable methods to assess sites, and what size or length respectively they should be to collect the appropriate data. Transect length showed great variation according to the factor assessed. A transect of 15 metres was found to be optimal. Abalone counts showed no significant (p = 0.1) change in the Coefficent of Variance (CV) for transect lengths greater than 15m, and had a mean of 0.2 abalone per metre. Quadrat size showed a significant difference in functional group richness between quadrat sizes of 0.0625m2, and 0.25m2 but no difference between 0.25m2 and 1m2 quadrats for both scape and photographic quadrats. It was also found that between 5 and 10 replicates (p = 0.08) represents the functional groups appropriately using quadrats and that a 0.25m2 quadrat is most suitable for sampling. Chapter 5 describes the benthic community structure of Cape Recife shallow water reefs. Using the standardized methodology previously mentioned, 45 sites were assessed to identify the community structure. These sites were grouped into 5 different groups influenced by depth and substrate, as well as functional group composition according to a Wards classification. The community structure showed that depth and substrate play a significant role (p < 0.05) in the community type. There is also a significant relationship (p < 0.05) between complexity, rugosity, abalone presence and substrate. During this study the basic protocols for site selection and benthic community monitoring have been developed to support the abalone ranching initiative in the Cape Recife area. It has also provided a baseline of the benthic community in the ranching concession area which will be used as a benchmark for future monitoring efforts. The site selection, sampling, and monitoring methods developed during the course of this work have now been rolled out as Standard Operating Procedures for the ranching programme in this area.

Thesis (PhD) -- tellenbosch University, 2014. / ENGLISH ABSTRACT: This study is part of the continuing global research effort dedicated to finding alternative aquafeed ingredients, which not only replace fish meal and fish oil as sources of essential nutrients in aquafeeds, but also provide specialist functional properties when included in feeds. Due to constraints in supply of fish meal and fish oil originating from wild capture fisheries, the continually growing aquafeed industry requires new sources of raw materials for the production of high quality feeds. The aim of the study was to investigate the specialist functional properties of feed ingredients (with emphasis on immune-stimulation potential) derived from fish processing waste after enzymatic hydrolysis by endogenous proteolytic enzymes (autolysis). Further aims were to (i) quantify effects of these feed ingredients on production performance of two species relevant to the South African aquaculture industry, namely Mozambique tilapia Oreochromis mossambicus and South African abalone Haliotis midae, (ii) compare performance to commercially available enzymatically produced feed ingredients, and (iii) separate the functional effects of these fish processing waste derived feed ingredients from the acid used to preserve them against bacterial spoilage during the autolysis process. Oil recovered after autolysis of rainbow trout viscera proved to be an attractive feed ingredient due to favourable effects on the non-specific cellular immune function of both Mozambique tilapia and South African abalone. However, in South African abalone, increased immune function due to inclusion of fish oil was accompanied by a significant decrease in production performance. The inclusion of hydrolysed proteins, obtained by autolysis of fish waste, in aquaculture feeds also improved non-specific immunity and survival of Mozambique tilapia significantly – independently of the preserving acid – although the same was not observed for South African abalone. Production performance was dependent on dietary hydrolysed protein inclusion levels in both species; excessive inclusion resulted in decreased production performance. The performance of dietary hydrolysed protein from autolysis and those from commercial production processes were significantly different, possibly as a result of different raw material origins and production processes. It is further shown that formic acid can contribute to improved water stability in abalone feeds, a novel mode of action not previously described. The study concludes that the simple autolysis process for processing of fish waste can provide aquafeed ingredients with immune stimulatory potential, which can contribute to improved production performance in the Mozambique tilapia and the South African abalone. The result can contribute to improved sustainability of the aquafeed industry, through substitution of fish meal and fish oil derived from capture fisheries with processed fish waste components. / AFRIKAANSE OPSOMMING: Hierdie studie vorm deel van die voortdurende en wêreldwye soeke na nuwe akwakultuur voerbestanddele wat nie net vismeel en –olie kan vervang as bron van noodsaaklike voedingstowwe nie, maar wat ook gespesialiseerde funksionele eienskappe openbaar wanneer dit in akwavoere ingesluit word. As gevolg van beperkings in die voorsiening van vismeel en –olie afkomstig vanaf wilde visserye, word die voortdurend groeiende akwavoer bedryf genoodsaak om nuwe grondstowwe te benut vir die vervaardiging van hoë kwaliteit voere. Die doelwit van die studie was om moontlike gespesialiseerde funksionele eienskappe (met spesifieke klem op potensiële immuun-stimulasie) van voerbestanddele te ondersoek wat herwin is vanaf reënboogforel proseseringsafval, na ensiematiese hidrolise d.m.v. endogene ensieme teenwoordig in die afval (outolise). Verdere doelwitte was om (i) effekte op produksie prestasie van hierdie bestanddele te kwantifiseer in twee akwakultuur spesies relevant tot die Suid- Afrikaanse bedryf (naamlik die Mosambiekse kurper Oreochromis mossambicus en die Suid- Afrikaanse perlemoen Haliotis midae), (ii) om produksie prestasie te vergelyk met kommersieel beskikbare voerbestanddele voorberei d.m.v. ensiemtegnologie, en (iii) om die moontlike effekte van die voerbestanddele te skei van die van die sure gebruik om die bestanddele te preserveer tydens die outolitiese proses. Daar is bevind dat olie herwin na outolise van reënboogforel ingewande ‘n goeie voerbestanddeel is wat gunstige effekte op die nie-spesifieke, sellulêre immuniteit van beide Mosambiekse kurper en Suid-Afrikaanse perlemoen gehad het. In die Suid-Afrikaanse perlemoen het verbeterde immuunfunksie gepaard gegaan met ‘n verswakking in produksie prestasie. Die gehidroliseerde proteïen komponent van ge-outoliseerde prosesseringsafval het ook beduidende verbetering in nie spesifieke immuniteit en oorlewing van Mosambiekse kurper tot gevolg gehad - onafhanklik van die preserverende suur - maar dieselfde is nie in die Suid-Afrikaanse perlemoen waargeneem nie. Produksie prestasie was afhanklik van die insluitingsvlakke van gehidroliseerde proteïen in beide spesies en dit is bevind dat oormatige insluiting produksie nadelig beïnvloed. Prestasie van proteïen afkomstig van outolise en die van ‘n kommersiële produksieproses het beduidend verskil, moontlik as gevolg van verskillende grondstowwe en prosesseringstegnieke. Daar is verder vir die eerste keer getoon dat mieresuur ‘n beduidende verbetering in waterstabiliteit in sekere perlemoenvoere teweeg kan bring. Die studie kom tot die slotsom dat die eenvoudige outolise proses funksionele akwavoerbestanddele kan produseer wat kan bydra tot ‘n verbetering in produksie prestasie in die Mosambiekse kurper en Suid-Afrikaanse perlemoen. Die resultaat kan bydra tot verbeterde volhoubaarheid van die akwavoer bedryf, deur vismeel en –olie afkomstig van wilde visserye, te vervang.

A gradient of sea temperatures is created along the South African coastline by the confluence of the cold Benguela Current on the West coast with the warm Agulhas Current on the East coast. This temperature gradient allows for an assortment of species to occupy the variety of microenvironments occurring in this area. Amongst these species is commercially important South African abalone, Haliotis midae, which although being capable of existing across this wide range of temperatures grows larger on the cooler West coast. Abalone reared on the warmer East coast however, experience greater mortalities especially during the more thermally variable summer months. The aim of the study was thus to assess the zone of tolerance for H. midae by exposing abalone to fluctuating temperatures in an attempt to model environmental temperature instability, a scenario which may likely be worsened by global climate change. Animals from the West and East coasts were exposed to two thermal treatments of fluctuating temperatures with the first group being kept at 16°C±2 and the second group kept at 16°C±4. The control group was maintained at a constant 16°C indicating that the mean temperature experienced by all three groups was 16°C. Oxygen consumption, nitrogen excretion and O:N ratio were assessed at the organismal level to give an indication of metabolic rate, amount of protein excreted and type of metabolic substrate utilized respectively. At the biochemical level, D-lactate accumulation was quantified to indicate whether metabolism was proceeding aerobically or anaerobically. Heat shock protein 70 (Hsp70) expression and degree of carbonylation were analyzed at the proteomic level with Hsp70 also being assessed at the transcriptomic level. All biological responses were measured at days 1, 3, 7 and 14 of the two week exposure. Oxygen consumption rates were significantly elevated on day 14 when comparing treatment group animals to control group animals of the same biogeographic region. P < 0.05 for both treatment groups from the West coast, while P < 0.001 for the East coast treatment groups. The ammonia excretion rates of the West coast animals were significantly lower than those of the controls at day 14 with P < 0.001 for both treatment groups, while ammonia excretion rates were elevated in East coast animals at day 14, although not significantly. Trends similar to those seen for ammonia excretion rates were exhibited by O:N ratios. West coast animals showed lower than control O:N ratios at day 14 (P < 0.01 for both treatment groups) while East coast animals displayed higher than control values (P < 0.05 only for the 16°C±2 group) at day 14. D-lactate, having been detected only for the West coast animals, showed no significant differences but large degrees of variation were noted on days 1 and 7. Carbonylation was evident for animals from both biogeographic regions with baseline carbonyl accumulation for East coast animals being greater (non-significantly) than that of the West coast animals. The hsp70 gene expression remained low for both biogeographic groups with West coast animals appearing to show slight elevations in expression at days 1 and 7, days which also displayed high degrees of variability. The West coast animals appeared to be better suited to coping with the thermal fluctuations, as they not only transiently reduced oxygen consumption rate to reduce ROS production, but also utilized the assistance of the D-lactate pathway possibly to maintain metabolism, both of which were not observed in the East coast animals. Although West coast abalone seemed to have slightly elevated hsp70 expression (suggestive of a repair response) when compared to their East counterparts, both groups of abalone were shown to have incurred notable amounts of protein damage (i.e. carbonylation). This suggests impairments in both protective and repair responses for animals from both biogeographic regions. The lack or attenuation of physiological responses noted in East coast abalone may be due to limitations in thermal adaptation but subsequent studies are required to confirm this notion. The information obtained from this study may assist in providing an insight into the mechanisms responsible for thermal limitation in H. midae and how this species is likely to respond to future periods of thermal instability which may be worsened by global climate change. An understanding of the processes leading up to limitations may potentially assist the abalone aquaculture industry in altering culturing practices early on to support optimal performance in abalone. / M.Sc. University of KwaZulu-Natal, Durban 2014.

An understanding of species specific water quality requirements is essential for efficient production of aquaculture products, an aspect not well documented for the land-based culture of the South African abalone, Haliotis midae. In order for the industry to remain competitive in international markets, efficient use of water supplies and the development of water reuse technology is needed. This study assessed the changes in water quality between tanks in a tiered serial-use raceway in relation to accumulated biomass and water flow and estimated the flow index (FI) (L h⁻¹ kg⁻¹) at which growth becomes significantly affected. The effect of dietary protein level, supplementation of pure oxygen and addition of sodium hydroxide (NaOH⁻) on water quality and fundamental production parameters in the serial-use raceways was also assessed. The serial-use raceways were used as a tool to create a range of water quality conditions at which the growth, feed conversion ratio (FCR) and condition factor (CF) of "cocktail" size (60 – 70 mm) H. midae could be monitored. The metabolic activity of the abalone resulted in a deterioration in water quality between tanks in series. pH (r² = 0.99; p < 0.001) and dissolved oxygen concentration (r² = 0.99; p < 0.001) were positively correlated with flow index (pH = 7.38 FI°·°² ; dissolved oxygen = 6.92 FI°·°⁴), while free ammonia nitrogen (FAN) (r² = 0.99, p < 0.001) and nitrite (NO²⁻ - N) (r² = 0.93, p < 0.001) were negatively correlated with flow index (FAN = 8.02 FI⁻°·⁷¹). Nitrite concentrations increased over time indicating colonisation of Nitrosomonas bacteria on the basket surfaces. A flow index of 7.2 – 9.0 L h⁻¹ kg⁻¹ was estimated as the minimum to avoid significant reductions in weight and shell length gain and increases in FCR values. Total ammonia nitrogen (TAN) and FAN concentrations were significantly correlated to dietary protein (P) (t = 6.63, p < 0.0001 and t = 6.41, p < 0.0001, respectively) and flow index (t = 5.42, p < 0.0001 and t = 3.9, p < 0.0002, respectively) and could be estimated using the models TAN = 9.73 P – 110.3 log (FI), and FAN = 0.132 P – 1.10 log (FI). Mean FAN concentrations were 67 and 41 % lower in tanks fed a diet containing 22 and 26 % protein respectively, when compared to tanks fed a 33 % protein diet. Supplementation with pure oxygen (103 ± 8 % saturation) improved shell length gain (t = 3.45, p = 0.026) in abalone exposed to high FAN (2.43 ± 1.1 μg L⁻¹) and low pH (7.6 ± 0.13), relative to a treatment with no oxygen supplementation (92 ± 6 % saturation). Addition of a sodium hydroxide solution resulted in elevated mean pH in treatment raceways when compared to control raceways. The increased pH resulted in significantly higher weight gain (g abalone⁻¹) (F₁·₁₂ = 4.51; p = 0.055) and shell length gain (mm abalone⁻¹) (F₁·₁₂ = 4.56; p = 0.054) at an α-error level of < 5.5 %. In two trials, weight gain and shell length gain were significantly correlated to pH (p < 0.001), and multiple regression of pH, dissolved oxygen and FAN consistently revealed pH to be the best predictor of growth. It is therefore suggested that decreasing pH is the first limiting water quality variable for abalone in serial-use raceways. As a decrease in water pH is linked to respiration by the abalone and subsequent increase in dissolved carbon dioxide (CO₂) concentration, future studies should examine the effects of CO₂ on H. midae metabolic rate, calcification rate and health. The results of this study will contribute toward our understanding of the specific water quality requirements for H. midae in commercial aquaculture systems, and influence the design and management procedures for abalone water reuse systems.