Thesis (MScAgric)--Stellenbosch University / ENGLISH ABSTRACT: Triploidy is the genetic state of containing three sets of chromosomes per cell in stead of two as in diploid organisms. The South African abalone (Haliotis midae) is naturally a diploid organism that sexually matures between four to eight years of age. Early sexual maturity is a disadvantage in cultured abalone stock, as the process of gonad development and spawning is energy demanding, causing energy to be diverted away from somatic growth. This same problem has been extensively experienced in diploid bivalve molluscs, where triploidy has since been applied as a means to prevent sexual maturation from occurring, thereby speeding up the growth process and shortening the time to marketing. Because triploidy was effective in bivalves, it was thought that it could contribute to faster growth in abalone as well. A procedure for the induction of triploidy in the abalone, Haliotis midae, was developed by De Beer (2004) and yielded up to 100 percent triploidy in treated abalone larvae. The next step was to compare the growth of the diploids and triploids to establish whether there was indeed a growth advantage on the part of the triploids, in view of commercial application. By using the same techniques as described by De Beer (2004), three groups consisting of triploid and diploid siblings were produced and subscribed to a comparative growth trial. The groups were spawned in three different seasons. The main objective was to establish whether there was in fact a difference in growth between diploid and triploid siblings, and whether seasonal effects were associated with growth advantages for either triploids or diploids. The two growth parameters measured were shell length and body weight. Measurements commenced at eight months of age, when the abalone could be individually tagged and continued up to the age of 24 months. The over-all results provided no convincing evidence of statistically significant faster growth of triploid juveniles compared to that of diploids up to two years of age. Growth differences were detected between seasons, but could not confidently be ascribed to seasonal environmental effects. The regression of shell length to body weight was similar for diploids and triploids. / AFRIKAANSE OPSOMMING: Triploiede organismes bevat drie stelle chromosome per sel in plaas van twee soos dit normaalweg in diploiede diere voorkom. Die Suid Afrikaanse perlemoen (Haliotis midae) is van nature ‘n diploiede organisme wat tussen die ouderdom van vier tot agt jaar seksueel aktief word. Vroeë seksuele aktiwiteit is ongewens in kommersiële akwakultuur aangesien energie spandeer word aan gonade ontwikkeling in plaas van somatiese groei. Dieselfde probleem is vroeër in die oester bedryf ondervind waar dit deur middel van triploiede induksie aangespreek is. Triploiedie veroorsaak steriliteit en kan gebruik word as ’n metode om steriliteit op groot skaal te induseer. Steriliteit sou dan meebring dat meer energie beskikbaar is vir somatiese ontwikkeling, wat verhoogde groeitempo en n verkorte tyd tot bemarking beteken. Op soortgelyke wyse is dus gepostuleer dat triploiedie in perlemoen ook tot steriliteit kon lei. ‘n Triploiede induksie metode was ontwikkel deur Mathilde de Beer (2004) wat ‘n hoë persentasie triploidie in geinduseerde perlemoen opgelewer het. Die volgende logiese stap was om die groei van diploiede diere met die van triploiede diere te vergelyk om te bepaal of triploiedie wel ’n groei voordeel tot gevolg het met die oog op kommersiële toepassing. Deur van dieselfde tegnieke as De Beer (2004) gebruik te maak, is drie groepe, elk bestaande uit verwante diploiede en triploiede diere, geproduseer en ingeskryf aan n vergelykende groei proef. Die groepe was in drie verskillende seisoene geproduseer. Die hoof doelstelling van die proef was om groeitempo van diploiede en triploiede diere te vergelyk, asook om die invloed van seisoen op groei van diploide en triploide te bepaal. Twee groei eienskappe naamlik skulp lengte en liggaamsmassa is gemeet vanaf ‘n ouderdom van agt maande (wanneer die diere individueel gemerk kon word) tot ‘n ouderdom van 24 maande. Die algehele resultate het gedui op geen betekenisvolle verskil tussen die groei van triploiede en diploiede perlemoen tot op die ouderdom van twee jaar. Verskille het voorgekom in die groei tussen seisoene, maar daar kon nie bewys word dat die verskille die gevolg van seisoenale omgewingseffekte was nie. Diploiede en triploiede het dieselfde skulp lengte tot liggaamsmassa verhouding getoon tot op twee jaar ouderdom.

Thesis (MSc)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Haliotis midae, is the only commercially important species of the six abalone species found in South African coastal waters and has become a lucrative commercial commodity. Wild stocks of H. midae are, however, no longer commercially sustainable due to a combination of environmental factors and poaching. The solution to the crisis is artificial production systems in the form of abalone farms. An abalone enhancement programme was initiated in South Africa in 2006, funded by industry and government. This programme focuses on the elucidation of the abalone genome and genetic factors contributing to increased productivity, thereby aiding the commercial production of abalone. The aims of this study, the first of its kind concerning H. midae, were to develop AFLPbased markers (specifically fluorescent AFLP analysis); to monitor the segregation of these markers in a single full-sib family and to use the markers and additional microsatellite markers to generate the first preliminary linkage map for H. midae. Genomic DNA of sufficient quality and purity for fluorescent AFLP analysis was obtained from 3.5-month-old H. midae juveniles. Preliminary linkage maps were constructed using AFLP and microsatellite markers segregating in an F1 family following a pseudo-testcross mapping strategy. Twelve AFLP primer combinations, producing 573 segregating peaks, and 10 microsatellite markers were genotyped in the parents and 108 progeny of the mapping family. Of the 573 segregating AFLP peaks genotyped, 241 segregated in a 1:1 ratio and 332 in a 3:1 ratio. Of these AFLP markers, 90 segregated according to the expected 1:1 Mendelian ratio and 164 segregated according to the expected 3:1 Mendelian ratio at the P = 0.05 level and were used for linkage analysis. Of the 10 microsatellite markers genotyped, nine were informative for linkage mapping analysis. Preliminary male and female genetic linkage maps were developed using markers segregating in the female or male parent. A total of 12 and 10 linkage groups were detected for the female and male maps respectively. The female map covered 1473.5cM and consisted of 56 markers, and the male map covered 738.9cM consisting of 30 markers. Markers with segregation distortion were observed as previously reported in other abalone species and potential homology between one of the linkage groups of the male map and two of the linkage groups of the female map were identified using the 3:1 segregating AFLP markers. In conclusion, the genetic linkage map presented here, despite the fact that it has relatively low genome coverage and low marker density, forms an ideal starting point for more detailed study of the H. midae genome and will provide a scaffold for basic and applied studies in abalone. A high-density linkage map of H. midae should in future be developed with additional co-dominant molecular markers, such as microsatellites, to improve the transferability of the linkage map between different laboratories and among populations. A high-density linkage map will facilitate the mapping of QTL of commercially important traits (i.e. growth) and future MAS breeding programmes. / AFRIKAANSE OPSOMMING: Perlemoenspesie, Haliotis midae, is die enigste spesie van kommersiële belang van die ses wat in die kuswater van Suid-Afrika aangetref word en het ‘n winsgewende handelskommoditeit in Suid-Afrika geword. Die ontginning van natuurlike H. midae populasies is egter, as gevolg van ‘n kombinasie van omgewingsfaktore en stropery nie meer kommersieel volhoubaar nie. Die perlemoenkrisis kan die hoof gebied word deur kunsmatige produksiesisteme op perlemoenplase tot stand te bring. ‘n Perlemoen verbeteringsprogram is in 2006 in Suid-Afrika geïnisieer en word deur die industrie en regering befonds. Die program focus op die ontrafeling van die perlemoen genoom en die genetiese faktore wat bydrae tot verhoogde produksie. Sodanige inligting kan gebruik word om kommersiële perlemoenproduksie te bevorder. Die doel van hierdie studie, die eerste met H. midae, is om AFLP-gebaseerde merkers (spesifiek fluoresserende AFLP analise) te ontwikkel; die segregasie van hierdie merkers te monitor in ‘n enkel volledige verwante familie en die merkers en addisionele mikrosatelliet merkers te gebruik om die eerste voorlopige koppelingskaart vir H. midae te genereer. Genomiese DNS van genoegsame kwaliteit en suiwerheid vir fluoresserende AFLP analise is ge-ekstraeer uit 3.5-maand-oue H. midae individue. Voorlopige koppelingskaart is gekonstrueer deur van segregerende AFLP en mikrosatelliet merkers in ‘n F1 familie gebruik te maak deur ‘n pseudo-kruistoets karteringstrategie te volg. Twaalf AFLP inleier kombinasies, wat 573 segregerende fragmente geproduseer het, en 10 mikrosatelliet merkers is gegenotipeer in die ouers en 108 individue van die nageslag van die karteringsfamilie. Van die 573 segregerende AFLP merkers wat gegenotipeer is, het 241 in ‘n 1:1 verhouding en 332 in ‘n 3:1 verhouding gesegregeer. Van hierdie AFLP merkers, het 90 volgens die verwagte 1:1 Mendeliese verhouding en 164 volgens die 3:1 Mendeliese verhouding by die P = 0.05 gesegregeer vlak en is vir die koppelingsanalise gebruik. Van die 10 mikrosatelliet merkers gegenotipeer, was 9 informatief vir koppeling karteringsanalise. Voorlopige manlike en vroulike genetiese koppelingskaarte is ontwikkel met gebruik te maak van merkers wat in die manlike of vroulike ouer segregeer het. ‘n Totaal van 12 en 10 koppelingsgroepe is onderskeidelik in die vroulike en manlike karate gegenereer. Die vroulike kaart dek 1473.5cM and bestaan uit 56 merkers, terwyl die manlike kaart 738.9cM beslaan het met 30 merkers. Merkers wat segregasie distorsie toon is waargeneem soos voorheen in ander perlemoenspesies gerapporteer. Potensiële ooreenstemming tussen een van die koppelingsgroepe van die manlike kaart en twee van die koppelingsgroepe van die vroulike kaart is aangetoon deur van die 3:1 segregerende AFLP merkers gebruik te maak. Die genetiese koppelingskaarte verskaf wel ‘n relatiewe lae genoomdekking en ‘n lae merkerdigtheid, maar is ‘n ideale vertrekpunt vir meer gedetailleerde studie van die H. midae genoom en dien as ‘n raamwerk vir toekomstige basiese en toegepaste studies in perlemoennavorsing. ‘n Hoëdigtheid koppelingskaart van H. midae moet in die toekoms ontwikkel word met gebruik van bykomstige ko-dominante molekulêre merkers, soos mikrosatelliete. Dit sal die oordraagbaarheid van die koppelingskaart tussen verskillende laboratoria asook tussen populasies verbeter. ‘n Hoëdigtheid koppelingskaart sal die kartering van kwantitatiewe kenmerk loki (KKL) vir kommersieel belangrike kenmerke (onder andere groeikrag) en toekomstige merker bemiddelde seleksie (MBS) teelprogramme moontlik maak.

Thesis (MSc)--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: The five leading abalone producers in South Africa have initiated a genetic enhancement program for Haliotis midae in a collaborative effort to improve economically valuable traits. Several independent objective-specific studies were initiated, including the establishment of a Performance Recording Scheme (PRS), utilised in this study, and necessary to monitor the ongoing performance of individuals as the move from mass-selection to marker assisted selection (MAS) is implemented. The primary objective of this study was parentage assignment of F1 offspring mass-selected for size at approximately one year and allocated to either a “faster” or a “slower” growth group. Nine microsatellite markers were used to genotype juveniles and potential parents, with assignment completed using CERVUS 2.0. Average growth results for Abagold and HIK were comparable for both growth groups. Slight environmental effects, although not statistically significant, were evident as growth advantages for juveniles within the faster growth group at two of the five locations and for juveniles within the slower growth group at one of the five rearing locations. Despite measures to standardise environmental influences, variables are difficult to control within the reality of a production environment; and potential genotype x environment interactions may require further investigation and factoring into future breeding programs. The additional costs associated with MAS often make the technology prohibitive to most aquaculture operations, despite the significant genetic gains to be realised from its implementation. Cost-optimising routine processes such as DNA extractions may be one approach to reduce these additional costs. Chelex®100 appears to be a suitable alternative to the CTAB method – being quick and cost-effective to perform. Applying this method in combination with the high throughput of a robotic platform warrants further evaluation. For the microsatellite development, 50% of positive recombinant clones contained inserts. Sequencing of these clones produced 16% perfect repeats and 47% imperfect repeats for which 52 primer sets were designed and tested. In total, 31 polymorphic microsatellite loci of different motifs and composition were developed. Sixty-one percent of sequenced clones were deemed redundant and pre-screening for both uniqueness and the presence of microsatellites would reduce unnecessary sequencing thus improving the efficiency of the FIASCO method and reducing costs. Nine loci were selected for parentage assignments. Null alleles were present for all the selected markers; however, frequencies were below the critical level of 5%. Parentage yielded 91% and 90% successful assignment for Abagold and HIK respectively; however, observations indicate that a measure of relatedness may exist between breeders. Recommendations with regards to future family breeding include, for both Abagold and HIK, retaining selected breeders based on their respective contributions to the F1 progeny while reassessing the potential of remaining breeding stock under more controlled breeding conditions. No obvious trends were observed for growth with most individuals producing both faster and slower growing offspring. Juveniles will be reassessed at two years to determine whether the size advantage or disadvantages were maintained and to ascertain whether growth advantages/disadvantages may be gender specific. / AFRIKAANSE OPSOMMING: Die vyf mees toonaangewende perlemoen produseerders in Suid Afrika het „n genetiese verbeteringsprogram vir Haliotis midae geinisieer in „n gesamentlike poging om ekonomiese belangrike eienskappe te verbeter. Verskeie onafhanklike fokus-spesifieke studies is geinisieer, insluitend die totstandkoming van „n groeiprestasie aantekenstelsel, soos gebruik in hierdie studie, en wat noodsaaklik is om die aaneenlopende prestasie van individue te moniteer soos daar beweeg word van massa seleksie tot merker bemiddelde seleksie. Die primêre fokus van hierdie studie was die ouerskapsbepaling van F1 nageslag wat massa geselekteer is op ouderdom 1 jaar vir grootte en as of “vinniger” of “stadiger” groeiers geklassifiseer is. Nege mikrosatelliet merkers is gebruik om jong perlemoen individue en moontlike ouers te genotipeer, met die ouerskapstoekenning bereken deur CERVUS 2.0. Groei resultate vir Abagold en HIK was vergelykbaar vir beide groei groepe op drie van die lokaliteite. Geringe omgewingseffekte, alhoewel nie statisties betekenisvol nie, was sigbaar as „n groei voordeel vir jong individue op twee van die vyf lokaliteite. Ongeag maatstawe om omgewingsinvloede te standardiseer, is varieerbares moeilik om te beheer in die produksie omgewing en genotipe x omgewings interaksies mag verdere navorsing vereis en behoort in ag geneem te word in toekomstige telingsprogramme. Die onkoste wat met merker bemiddelde seleksie geassosieer word, maak die tegniek soms onaantreklik vir die meeste akwakultuur operasies; nie teen staande die genetiese voordele wat die gebruik daarvan veroorsaak. Die koste-optimiseering van roetine prosesse, soos byvoorbeeld, DNA ekstraksies, is dalk een aanslag om die addisionele koste te verminder. Chelex®100 blyk „n geskikte alternatief tot die CTAB metode te wees – die tegniek is vinnig en koste-effektief om uit te voer. Die gebruik van hierdie metode in kombinasie met die hoë deurvloei van ‟n robotiese sisteem behoort verder ondersoek te word. Vir die mikrosatelliet ontwikkeling het slegs 50% van die positiewe rekombinante klone invoegings bevat. Nukleotiedvolgorde bepaling van hierdie klone het 16% perfekte herhalings en 47% onderbroke herhalings bevat waaruit 52 inleierstelle ontwikkel en getoets is. In totaal is 31 polimorfiese mikrosatelliet loki van verskillende motiewe en samestelling ontwikkel. Een-en-sestig persent van die volgorde bepaalde klone is oortollig geag en vooraf sifting vir beide uniekheid en die teenwoordigheid van mikrosatelliete sal onnodige volgorde bepaling verhoed, die effektiwiteit van die FIASCO tegniek verhoog sowel as addisionele koste verminder. Nege loki is geselekteer vir ouerskapsbepaling. Nul allele was teenwoordig vir al die geselekteerde merkers, maar die frekwensies was egter laer as die 5% kritieke waarde. Ouerskap is 91% en 90% suksesvol bepaal vir Abagold en HIK onderskeidelik. Waarnemings dui egter daarop dat daar verwantskappe mag wees tussen van die broeidiere. Voorstelle in terme van toekomstige familie teling sluit is, vir beide Abagold en HIK, om geselekteerde broei diere te behou gebaseer op hulle onderskeie bydraes tot die F1 nageslag asook die herevaluaring van die potensiaal van die oorblywende broei diere onder meer beheerde teling toestande. Geen voor-die-handliggende tendense is waargeneem vir groei nie met die meeste individue wat beide vinniger en stadiger groeiende nageslag geproduseer het. Jong individue moet geherevalueer word op tweejarige ouderdom om te bepaal of die groei voordeel of nadele behou is en om te bepaal om groei voordele/nadele geslagspesifiek is.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science. / ENGLISH ABSTRACT: Haliotis midae, or Perlemoen, is the only cultured species of abalone in South Africa and is under great international demand. This species is considered endangered, making sustainable farming practises and law enforcement against poaching essential for maintaining wild stocks. A limited amount of broodstock animals are provided to each farm from which thousands of offspring are grown and exported. The prevention of inbreeding and preservation of genetic diversity within farmed stocks is necessary for future sustainable farming and production of genetically stable offspring. Further research into the genetic dynamics of Perlemoen will provide the knowledge for advanced management programs for optimal farming practises and essentially sustainable production. This study focuses on genetic linkage map development with the intention of future identification of markers associated with genes of economic importance, such as growth rate. Identification of markers linked to genes responsible for such phenotypic traits will ultimately allow farming practises to select naturally genetically superior animals for breeding, thereby enhancing production. For the construction of a genetic linkage map of H. midae, microsatellite markers were developed using two strategies: FIASCO and screening of next generation sequence-bysynthesis contig data. The FIASCO-derived markers were characterised by genotype screening in 32 individuals from a full-sib family and analysed using Mendelian segregation expectations. The Illumina-derived markers were characterised by genotype screening in 32 individuals from wild populations and analysed against Hardy-Weinberg expectations. Forty four microsatellite-family combinations were obtained from FIASCO of which 28 provided informative genotype results (32% success). Twenty two markers were developed from sequence-by-synthesis screening. Fourteen provided reliable genotypes (37%) and six conformed to Hardy-Weinberg expectations. These markers were used, in addition to 156 previously developed markers, to develop sex-specific and sex-average linkage maps in two full-sib families consisting of approximately 100 offspring each. One hundred and six polymorphic loci were used for linkage analysis (LOD>3) in both families. The number of linkage groups obtained from sex-specific maps ranged from 13-16. The average genome length ranged from 500 cM to 800 cM with an average marker spacing of 10 cM. The sex-average linkage map provided 18 linkage groups with an average genome length calculation of 1800 cM and average marker spacing of approximately 13 cM. The linkage maps created in this study are preliminary but provide a stepping stone towards a high density map incorporating high throughput markers. This also provides a base for QTL mapping studies, in which phenotypic traits of interest can be identified and associated to specific locations in the H. midae genome for marker-assisted selection. / AFRIKAANSE OPSOMMING: Haliotis midae, ook bekend as Perlemoen, is in groot internasionale aanvraag en is ook die enigste klipkous spesie waarmee in Suid Afrika geboer word. Hierdie spesie word as bedreig beskou en daarom is volhoubare boerdery bedrywe en wetstoepassing teen stroping noodsaaklik om wilde populasies te beskerm. Elke perlemoenplaas word met ‘n beperkte aantal broeidiere verskaf, waarvan die nageslag dan gekweek en uitgevoer word. Voorkoming van inteling en handhawing van genetiese diversiteit binne gekweekte populasies is noodsaakllik vir toekomstige volhoubare kweking en produksie van ń geneties stabiele nageslag. Verdere ondersoeke na die genetiese dinamika van Perlemoen sal die nodige kennis verskaf om sodoende gevorderde bestuursprogramme te ontwikkel, wat tot optimale kweek praktyke en effektiewe volhoubare produksie sal lei. Hierdie studie fokus op die ontwikkeling van ‘n genetiese koppelingskaart met die voorneme om toekomstige merkers te identifiseer wat met gene van ekonomiese belang, soos byvoorbeeld groei tempo geassosieerd is. Identifisering van merkers wat vir sulke fenotipiese eienskappe verantwoordelik is sal sodoende toelaat dat boerdery praktyke kan selekteer vir diere vir verbeterde teling en produksie. Mikrosatelliet merkers is ontwikkel om die genetiese koppelingskaart saam te stel. Die volgende twee strategieë is benut: FIASCO en sifting van volgende generasie volgordebepaling-deur-sintese “contig” data. Die FIASCO-afgeleide merkers is gekarakteriseer deur genotipiese sifting in 32 individue van ‘n volsib familie en is deur Mendeliese segregasie verwagtinge ge-analiseer. Die Illumina-afgeleide merkers is gekarakteriseer deur genotipiese sifting in 32 individue van wilde populasies en is met Hardy-Weinberg ewewig ge-analiseer. Vier en veertig mikrosatelliet-familie kombinasies is deur FIASCO verky, waarvan 28 informatiewe genotipiese resultate gelewer het (32% sukses). Twee en twintig merkers is vanaf volgordebepaling-deur-sintese sifting ontwikkel. Veertien van hierdie merkers het betroubare genotipes (37%) verskaf en ses het aan Hardy-Weinberg verwagtinge voldoen. Hierbenewens is 156 voorheen ontwikkelde merkers gebruik om geslagspesifieke en geslagsgemiddelde koppelingskaarte in twee volsib families saam te stel. Hierdie volsib families het uit ń naslag van 100 elk bestaan. Een honderd en ses polimorfiese lokusse is vir koppelingsanalise gebruik, waar ‘n LOD waarde groter as drie statisties betekenisvol geag was. Die aantal koppelingsgroepe verkry van geslagspesifieke kaarte het tussen 13 en 16 gewissel. Die gemiddelde genoom lengte het van 500 cM tot 800 cM met ‘n gemiddelde merker spasiëring van 10 cM. Die geslagsgemiddelde koppelingskaart het 18 koppelingsgroepe gehad met ‘n gemiddelde genoom lengte berekening van 1800 cM en ‘n gemiddelde merker spasiëring van ongeveer 13 cM. Die koppelingskaarte wat in hierdie studie geskep is, is voorlopig en verskaf ‘n grondslag vir die ontwikkeling van ‘n hoër digtheidskaart, wat hoë deurset merkers inkorporeer. Dit verskaf ook ‘n basis vir kwantitatiewe kenmerk lokus karteringstudies. Hierdie karteringstudies kan fenotipiese eienskappe van belang identifiseer en assosieer met spesifieke posisies binne die H. midae genoom vir merker bemiddelde seleksie.

Thesis (MSc (Genetics))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: Haliotis midae is the most important aquaculture species in South Africa, with abalone farming contributing 80% of the Rand value of the aquaculture industry. Although genetic research has benefited the abalone industry, several issues still hinder increases in abalone production. Progress towards an increase in H. midae growth rate by utilizing conventional genetic studies and selective breeding has been relatively slow. Gene transfer has therefore become a plausible option to address this problem. Genes that code for certain desirable traits, such as increased growth rate, could be incorporated into the genome of commercial abalone. The current study undertook the optimization of a chemically-mediated gene transfer technique using Polyethylenimine (PEI) as transfection reagent and fluorescent proteins as reporter genes. Before gene transfer could be undertaken, several complementary studies also needed to be undertaken due to the novel nature of the study. The auto fluorescence of H. midae, the suitability of several H. midae tissues as targets for gene transfer and the cytotoxic effect of transfection reagents and selection antibiotics were assessed before gene transfer optimization could be attempted. Also, genes linked to an increase in growth rate were characterized for differential expression in different abalone age-groups to determine the suitability of these genes for incorporation into a homologous gene construct in future transfection studies. The auto fluorescence of ova, embryos and larvae were found to be comparable to that of the fluorescent reporter genes, EGFP and DsRed. A PCR-based transfection validation method was therefore employed to confirm the presence of internalized transgenes. It was established that sperm, ova, larvae and haemocyte cell culture were the most suitable target tissues for transfection. The transfection reagents, a 25kDa PEI and ExGen 500, were not cytotoxic to sperm, embryos and haemocyte cell cultures. The minimum lethal concentration of the selection antibiotics, neomycin and zeocin, was determined for larvae and haemocytes. After transfection treatment of sperm and fertilization of untreated ova, the presence of internalized transgenes could be verified for larvae. The presence of internalized transgenes could not be detected after transfection treatment of ova and larvae. Fluorescent flow cytometry and microscopy analysis of haemocytes could not detect the expression of the fluorescent reporter genes. Expression of two of the growth related genes was found to differ between age-groups. The perlustrin gene was upiv regulated in older animals, while the insulin related peptide receptor gene was down regulated in older animals. The third gene, a thrombospondin-1 precursor was stably expressed in all age-groups. This study represents the first report of transfection studies carried out on H. midae. Future studies will benefit from the groundwork established in H. midae transfection. / AFRIKAANSE OPSOMMING: Haliotis midae is die belangrikste akwakultuur spesie in Suid-Afrika met perlemoen boerdery wat 80% van die Rand waarde van die akwakultuur industrie bydrae. Alhoewel genetiese studies die perlemoen industrie ‘n hupstoot gegee het, is daar steeds sekere struikelblokke wat verdere toename in produksie verhoed. Vooruitgang ten opsigte van ‘n toename in H. midae se groei tempo deur gebruik te maak van konvensionele genetiese studies en selektiewe teling was tot dusver relatief stadig. Genetiese transformasie het daarom ‘n wesenlike alternatief geword wat moontlik hierdie probleem kan oplos. Gene wat kodeer vir sekere eienskappe, soos ‘n toename in groeitempo, kan in die genoom van kommersiële perlemoen inkorporeer word. Die huidige studie het onderneem om ‘n chemies-gemedieerde genetiese transfeksie tegniek te optimiseer en van Polyethylenimine (PEI) as transfeksie reagens en fluoresserende proteine as verklikkers gebruik te maak. As gevolg van die oorspronklikheid van die studie moes verskeie bykomende ondersoeke ook aangepak word voordat genetiese transfeksie uitgevoer kon word. Die outofluoressensie van H. midae, die geskiktheid van verskeie H. midae teiken weefsels en die sitotoksiese effek van die transfeksie reagense en seleksie antibiotika is ondersoek voordat transfeksie uitgevoer is. Gene gekoppel aan ‘n toename in groeitempo is ook gekarakteriseer vir verskille in uitdrukking in verskillende perlemoen ouderdoms-groepe om te bepaal of hierdie gene moontlik in ‘n homoloë geen konstruk ingesluit kan word vir toekomstige transfeksie studies. Dit is gevind dat die outofluoressensie van ova, embrios en larwes vergelykbaar is met die fluoressensie van die verklikker proteïene, EGFP en DsRed. ‘n PKR-baseerde metode om die internalisering van die transgeen te kontroleer is daarom gebruik. Dit is vasgestel dat sperm, ova, larwes en haemosiete die mees geskikte teiken vir transfeksie sou wees. Die transfeksie reagense, ‘n 25kDa PEI en Exgen 500, is nie sitotoksies vir sperm, embrios of haemosiete nie. Die minimum dodelike konsentrasie van die seleksie antibiotika, neomycin en zeocin, is bepaal. Na transfeksie behandeling van sperm en bevrugting van onbehandelde ova, kon die teenwoordigheid van internaliseerde transgene bevestig word vir larwes. Die teenwoordigheid van internaliseerde transgene kon nie bevestig word na transfeksie behandeling van ova en larwes nie. Fluoressente vloei sitometrie en mikroskopiese analise kon nie die uitdrukking van die fluoressente verklikker gene bevestig in haemosiete nie. Die uitdrukking van twee van die gene gekoppel aan groei het verskil tussen ouderdomsgroepe. Die perlustrin geen is meer uitgedruk in ouer diere terwyl die insulien geassosieerde peptied reseptor geen minder uitgedruk is in ouer diere. Die thrombospondin-1 voorloper geen is stabiel uitgedruk in al die ouderdomsgroepe. Hierdie studie verteenwoordig die eerste verslag van transfeksie studies uitgevoer op H. midae. Toekomstige studies sal baat vind by die grondslag wat deur hierdie projek gelê is.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Haliotis midae (locally also known as perlemoen) is the largest of five endemic species found along the coast of South Africa. It is the only species with commercial value contributing to the exploitation of these animals. Due to declines of natural stocks, farming practices were established during the early 1990s in order to supply the international demand. To facilitate efficient breeding methods and ensure the sustainability of these commercial populations, genetic management, which can be accomplished with the use of molecular markers such as single nucleotide polymorphisms (SNPs), is necessary. Single nucleotide polymorphisms have become the markers of choice in various applications in aquaculture genetics due to their abundance in genomes, reduction in developmental costs and increased throughput of genotyping assays. Identification of SNPs in non-model species such as H. midae can be achieved by in silico approaches. In silico methods are suitable for de novo SNP identification and are both cost- and time-efficient. It is based on the analysis of multiple alignments where mismatches may be reported as candidate SNPs. Various medium-throughput genotyping methods are available to confirm putative SNPs, but the ideal method depends on factors such as cost, accuracy and multiplexing capacity. Although SNP markers can have various applications within the aquaculture environment the focus for this current study was saturating the linkage map of H. midae with additional markers. This would assist in the identification of quantitative trait loci associated with economically important traits, which in turn could ultimately be employed for marker-assisted selection and improved molecular breeding programs. In order to identify in silico SNPs, sequenced transcriptome data from a previous study was used and subjected to a series of criteria: minor allele frequency 10%, minimum coverage 80, 60 bp flanking regions. Selected loci were genotyped using a 192-plex assay with the Illumina GoldenGate genotyping assay with the VeraCode technology on the BeadXpress platform, in individuals from six mapping families. A conversion rate of 69.35% and global success rate of 76.34% was achieved. Polymorphic loci were subjected to linkage analysis using JoinMap® v.4.1 to create sex-average and sex-specific maps and to saturate the current linkage map for H. midae. Along with previously developed markers, 54% of the newly developed SNPs could be successfully incorporated into the linkage map of H. midae. A total of 18 linkage groups were observed with an average marker spacing of 6.9 cM and genome coverage of 79.1%. Bioinformatic analyses and setting stringent criteria to identify SNPs from sequenced transcriptomic data proved to be an efficient way for SNP discovery in the current study. Genotyping of the identified loci with the GoldenGate genotyping assay demonstrated a high success rate; providing a genotyping assay adequate for species with little genomic information. The linkage map created in this study illustrated the utility of SNP markers in conjunction with microsatellite markers for linkage map construction and the adequate marker spacing obtained provides a step closer to quantitative trait loci mapping in this species. / AFRIKAANSE OPSOMMING: Haliotis midae (plaaslik ook bekend as perlemoen) is die grootste van vyf inheemse spesies wat langs die kus van Suid-Afrika aangetref word. Dit is die enigste spesie van kommersiële waarde wat bydraend is tot die uitbuiting van hierdie diere. As gevolg van die afname in hierdie natuurlike hulpbron het boerdery praktyke gedurende die vroeë 1990's ontstaan om in die internasionale aanvraag te voorsien. Ten einde doeltreffende teelmetodes te beoefen en die volhoubaarheid van hierdie kommersiële populasies te verseker is genetiese bestuur, wat bewerkstellig kan word deur die gebruik van molekulêre merkers soos enkel nukleotied polimorfismes (ENPs), baie belangrik. Enkel nukleotied polimorfismes is gewilde merkers in verskeie toepassings in akwakultuur genetika as gevolg van hul oorvloed in genome, verlaagde ontwikkelingskoste en verhoogde deurset van ENP-genotiperingstoetse. Identifisering van ENPs in nie-model spesies soos H. midae kan uitgevoer word deur in siliko benaderings te gebruik wat geskik is vir de novo ENP identifisering en ook tyd- en koste-effektief is. Dit word gebaseer op die analise van veelvuldige inlynstellings waar nukleotiedes wat nie ooreenstem nie as kandidaat ENPs gerapporteer kan word. Om kandidaat ENPs te bevestig, kan verskeie medium-deurset genotiperingsmetodes uitgevoer word, maar die ideale metode word bepaal deur faktore soos koste, akkuraatheid en multipleks kapasiteit. Alhoewel ENP merkers in verskeie toepassing binne die akwakultuur omgewing gebruik kan word was die fokus van die huidige studie om die koppelingskaart van H. midae te versadig. Dit sal bydrae tot die identifisering van kwantitatiewe eienskap lokusse wat gekoppel kan word aan ekonomies belangrike eienskappe wat dan op die beurt weer vir merkerbemiddelde seleksie gebruik kan word en uiteindelik ten opsigte van die verbetering van molekulêre teelprogramme aangewend kan word. Ten einde in siliko ENPs te identifiseer is transkriptoomdata van 'n vorige studie gebruik en onderwerp aan 'n reeks kriteria: geringste alleelfrekwensie 10%, minimum dekking 80, 60 bp gebiede weerskante van polimorfisme. Geïdentifiseerde lokus-genotipering is met behulp van 'n 192-pleks toets uitgevoer met die Illumina GoldenGate genotiperingstoets met die VeraCode tegnologie op die BeadXpress-platform, in individue afkomsitg vanaf ses karteringsfamilies. 'n Omskakelingskoers van 69.35% en 'n algehele sukseskoers van 76.34% is bereik. Polimorfiese lokusse is onderwerp aan koppelings-analise met behulp van JoinMap® v.4.1 om geslags-gemiddelde en geslags-spesifieke kaarte te skep asook om die kaart wat beskikbaar is vir H. midae te versadig. Saam met voorheen ontwikkelde merkers is 54% van die nuut ontwikkelde ENPs suksesvol opgeneem in die kaart van H. midae. 'n Totaal van 18 koppelingsgroepe is verkry met 'n gemiddelde merker-spasiëring van 6.9 cM en 'n genoomdekking van 79.1%. Die gebruik van bioinformatiese analises en streng kriteria om ENPs vanaf transkriptoomdata te identifiseer blyk doeltreffend te wees in hierdie studie. Genotipering van die geïdentifiseerde lokusse met die GoldenGate genotiperingstoets dui op 'n hoë suksessyfer en verskaf 'n voldoende genotiperingstoets aan spesies met min genomiese inligting. Die koppelingskaart in hierdie studie het geïllustreer dat die ENP merkers suksesvol saam met mikrosatelliet merkers gebruik kan word vir koppelingskaart konstruksie en dat die voldoende merker-spasiëring verkry 'n stap nader aan kwantitatiewe eienskap lokus kartering in hierdie spesie bied.

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The species Haliotis midae is of great commercial value to the South African abalone industry and is mainly exported to Asian markets, specifically China. Up to 50% is sold as canned products with H. midae registering an average canning yield of approximately 35%. The species is presently genetically undomesticated and breeding programmes are being introduced to improve a range of production traits of which growth and yield is of primary importance. The objective of the study was to determine genetic parameters such as heritability, genotypic and phenotypic correlations of yield-related traits to assess the potential genetic improvement through selective breeding. A series of yield-related parameters were identified that is of relevance to the standard abalone canning procedure. Low to moderate heritabilities where recorded for most traits, including pre-shuck/live weight (0.20 ± 0.06), post-shuck weight (0.15 ± 0.05), post-gut weight (0.15 ± 0.05), post-brine weight (0.19 ± 0.06), pre-canning weight (0.19 ± 0.06), post-canning weight (0.21 ± 0.06), shell weight (0.16 ± 0.05), canning yield percentage (0.08 ± 0.03) and shell weight to post-gut weight ratio (SW: PGW) (0.09 ± 0.04). Weight related parameters are phenotypically highly correlated (0.86 ≤ r ≤ 0.99) but show negative correlation with canning yield percentage (-0.38 ≤ r ≤ 0.04). The nett yield of abalone shows a relatively strong positive correlation with the live weight (r = 0.66). Shell length is highly heritable (h2 ≈ 0.48) and show a strong positive correlation with live weight (r = 0.94). Shell weight is also highly correlated with live weight (r = 0.80) and the SW: PGW ratio does not show a significant correlate with live weight (r = 0.03). Weight-related traits show heritability values ranging from 0.15 to 0.20 that could allow a positive genetic response. Shell length (as a linear growth parameter) shows a high heritability (h2 ≈ 0.48) and a strong positive correlation with live weight (r = 0.94) which also makes it suitable for use as a selection criterion in breeding programmes for improved growth rate. Direct selection for canning yield is compromised by the destructive nature of measurement and the low heritability (h2 < 0.10). The negative correlations between yield as a percentage and growth traits (-0.38 ≤ r ≤ 0.04) further complicate its use as a direct breeding objective. Although the canning yield as a percentage shows a decrease with an increase in live weight, the nett canning yield increases (r = 0.66) with the live weight. It is therefore recommended to use shell length as a criterion for selection for increased growth rate and nett yield, thereby optimising profitability. / AFRIKAANSE OPSOMMING: Die spesie Haliotis midae is van groot kommersiёle waarde tot die Suid-Afrikaanse perlemoenindustrie en word meestal uitgevoer na markte in Asiё, spesifiek China. Tot 50% van die perlemoen wat in Suid-Afrika geproduseer en uitgevoer word, word verblik en huidiglik is die verblikkingsopbrengspersentasie ongeveer 35%. Haliotis midae is tans geneties onderontwikkeld en die gebruik van teelprogramme word nou geimplementeer met die doel om 'n verskeidenheid eienskappe te verbeter, waarvan groei en opbrengs van primêre belang is. Die doelwit van die studie was om genetiese parameters soos oorerflikheid en ook die genotipiese en fenotipiese korrelasies van obrengsverwante eienskappe te bepaal om sodoende die potensiёle genetiese verbetering as gevolg van selektiewe teeling te assesseer. 'n Reeks obrengsverwante eienskappe is geidentifiseer wat relevant is binne bestaande en standaard kommersiёle perlemoenverblikkingsprotokolle. Lae tot matige oorerflikheidswaardes is waargeneem en sluit in lewende, of voor-ontskulpingsgewig (0.20 ± 0.06), na-ontskulpingsgewig (0.15 ± 0.05), na-oopvlekkingsgewig (0.15 ± 0.05), na-pekelgewig (0.19 ± 0.06), voor-verblikkingsgewig (0.19 ± 0.06), na-verblikkingsgewig (0.21 ± 0.06), skulpgewig (0.16 ± 0.05), verblikkingsopbrengspersentasie (0.08 ± 0.03) en 'n skulpgewig tot na-oopvlekkingsgewig verhouding (SW: PGW) (0.09 ± 0.04). Gewigsverwante parameters is fenotipies hoogs gekorreleerd met mekaar (0.86 ≤ r ≤ 0.99) maar toon 'n negatiewe korrelasie met die verblikkingsopbrengspersentasie (-0.38 ≤ r ≤ 0.04). Die netto opbrengs van perlemoen dui op 'n relatiewe sterk positiewe korrelasie met lewende gewig (r = 0.66). Skulplengte is hoogs oorerflik (h2 ≈ 0.48) en toon 'n sterk positiewe korrelasie met lewende gewig (r = 0.94). Skulpgewig is ook hoogs gekorreleerd met lewende gewig (r = 0.80) en die SW: PGW verhouding toon geen beduidende korrelasie met lewende gewig nie (r = 0.03). Gewigsverwante eienskappe toon oorerflikheidswaardes wat varieer tussen 0.15 en 0.20 en kan 'n moontlike genetiese respons lewer. Skulplengte (as 'n liniêre groeiparameter) toon 'n hoё oorerflikheid (h2 ≈ 0.48) en 'n sterk positiewe korrelasie met lewende gewig (r = 0.94) wat dit gepas maak vir gebruik as 'n seleksiekriterium in 'n teelprogram met verbeterde groeitempo as doel. Direkte seleksie in terme van verblikkingsopbrengs word ingeboet danksy die destruktiewe natuur van die metingsmetodiek asook 'n lae oorerflikheid (h2 < 0.10). Die negatiewe korrelasies tussen verblikkingsopbrengs (uitgedruk as 'n persentasie) en groeieienskappe (-0.38 ≤ r ≤ 0.04) dien as 'n verdere komplikasie in die gebruik van dié eienskap as direkte teeldoelwit. Alhoewel die verblikkingopbrengs 'n afname toon soos lewende gewig toeneem, is daar steeds 'n positiewe korrelasie tussen die netto verblikkingsopbrengs en die lewende gewig (r = 0.66). Dit word dus aangeraai om skulplengte as seleksiekriterium vir verbeterde groeitempo en netto opbrengs te gebruik om sodoende wins te maksimaliseer.

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The aim of this study was to critically assess the implementation of the internal reference method within the most recent 173 full-sibling growth trial of the Innovation Fund Abalone Breeding Project. The trial was conducted over two locations for a period of five years, with minimal replication for the majority of test families and a single full-sibling family was entered into each experimental unit (basket) as an internal reference group. The primary focus was firstly, to validate the performance of the internal reference group as a control for comparisons and correction of environmental variation in test family performances. Secondly, to identify areas of weakness and either make recommendations to remedy areas of weakness or justify devoting resources to alternative methods of reducing extraneous environmental variance with limitations on replication. The efficiency and statistical power associated with utilising internal reference information as a covariate and for manual correction respectively were examined for the 6 full-sibling test families that were replicated. This study reports on the evaluation of factors which are potential sources of bias in the internal reference method, the first of which, tag loss, was found to be significant after 6- 12 months. However, it was not found to bias internal reference group performances as the factors which contribute to tag loss were found to act randomly. Variability in size ratio of internal reference to test family at co-stocking proved a significant source of bias, as reference groups smaller than their test family counterparts had reduced performances. Testing for genotype by environment interactions was precluded due to the inherent lack of replication and the subsequent confounding of genotype effects with inter-rearing structure effects at one of the locations. However, significant differences were detected for both traits of interest of the internal reference group over the two locations. Significant antagonistic interactions were detected and identified as a source of bias for average daily weight gain of replicate test families. The evaluation of average daily length gain for the efficiency of adjustment when the internal reference is a covariate and the change in statistical power when the internal reference is used for a manual correction, yielded conflicting results. The latter shows a decrease in statistical power and the former shows an increase in efficiency, both resulting in poor goodness of fit in the respective models. There was however evidence that when no antagonistic interactions occurred “between replicate variance” decreased and therefore the internal reference method has statistical merit provided all critical success factors are satisfied. Recommendations were made for future implementation of the internal reference method to facilitate adequate statistical testing for sources of bias and the prevention thereof. Additionally, an alternative method which may have merit in decreasing environmental variance and the need for replication, is discussed. / AFRIKAANSE OPSOMMING: Die doel van die studie was om die gebruik van ʼn interne verwysingsgroep te ontleed, soos toegepas tydens die evaluering van 173 volsib families as deel van die Innovasiefonds Perlemoen Teelprogram. Die evaluering is gedoen op twee lokaliteite oor 'n tydperk van vyf jaar, met minimale replikasie van die toets families en die gebruik van ‘n enkele volsib familie as 'n interne verwysingsgroep in elke eksperimentele eenheid (mandjie). Die primêre fokus was eerstens om die gebruik van die interne verwysingsgroep vir die korreksie van omgewingsvariasie in die toets familie optredes te evalueer. Tweedens, om spesifieke gebreke te identifiseer ten opsigte van die gebruik van die interne verwysingsgroep en aanbevelings maak dit reg te stel en om die meriete van alternatiewe metodes te oorweeg. Die doeltreffendheid en statistiese onderskeidingsvermoë van die gebruik van interne verwysingsgroep as 'n kovariaat is ondersoek met betrekking tot die 6 volsib groepe wat oor voldoende replikasies beskik het. Die studie doen voorts verslag oor die evaluering van potensiële oorsake van sydigheid ten opsigte van die gebruik van die interne verwysingsgroep, insluitend die beduidende verlies van identifikasie vanaf 6 tot 12 maande. Geen aanduiding van sydigheid is egter gevind en die aanleidende oorsake van verlies van identifikasie blyk van ʼn ewekansige aard te wees. Verskille in die grootte tussen die interne verwysingsgroep en toets-families met aanvang van evaluering blyk 'n belangrike bron van sydigheid te wees, waar die kleiner groepering aan verminderde prestasie gekoppel word. Bepaling van genotipe-omgewing-interaksies kon nie uitgevoer word nie as gevolg van die inherente gebrek van replisering oor lokaliteite. Beduidende verskille is egter waargeneem tussen interne verwysingsgroepe oor die twee lokasies ten opsigte van die beide groei eienskappe. Beduidende antagonistiese interaksies is waargeneem en geïdentifiseer as 'n bron van sydigheid ten opsigte van die gemiddelde daaglikse gewigstoename van replikaat toetsfamilies. Die evaluering van gemiddelde daaglikse lengtetoename met die interne verwysingsgroep as is 'n kovariaat en die verandering in statistiese ontledingsvermoë tydens die gebruik van die interne verwysingsgroep het teenstrydige resultate opgelewer. Laasgenoemde toon 'n afname in statistiese ontledingsvermoë en die eersgenoemde toon 'n toename in doeltreffendheid, met beide swak passing op die onderskeie modelle. In die afwesigheid van antagonistiese interaksies tussen replikasies het variansie afgeneem en beskik die interne verwysingsgroep oor die nodige statistiese meriete indien daar aan al die kritiese vereistes voldoen word. Aanbevelings is gemaak ten opsigte van die toekomstige implementering van die interne verwysingsmetode met verwysing na voldoende statistiese toetsing vir bronne van sydigheid en die voorkoming daarvan. 'n Verdere metode wat oor die nodige meriete beskik om die omgewingsvariasie en die noodsaaklikheid vir replikasie te verminder, word bespreek.

Haliotis midae is the cornerstone of the South African abalone fishery. For more than a decade, the wild abalone stock of South Africa has suffered decline due to over-exploitation and illegal activities such as poaching. Prior to 1970, no regulations were in place concerning the annual landings. As a result the fishery was exploited as if it were an infinite resource. It is this initial uncontrolled harvesting (regardless of age) and poaching that has driven the abalone resource decline. Due to the slow growth rate exhibited by abalone as a species, natural replenishment of wild stock following exploitation and poaching was far below the rate of exploitation of this resource. Studies on the growth of abalone have mainly been conducted under laboratory conditions. The purpose of this study was to measure the growth of abalone, fed different diets, under commercial culture conditions. Three food types were used namely; commercial pellets, seaweed (Ulva spp.) and dried kelp bars (Ecklonia maxima). Four diets were obtained from the three food types namely; combination of commercial pellets and seaweed (Diet A), commercial pellets only (Diet B), seaweed only (Diet C) and dried kelp bars only (Diet D). The food types used in this study represent both artificial (Commercial pellets) and natural feeds (seaweed and kelp) used in commercial abalone culture. The growth of two cohorts (40-50 mm and 50-60 mm) was followed over a 426 day period, with data for the first 183 days being used for statistical analysis to determine performance of a given diet. The best growth rates were found in abalone fed Diet A (40-50 mm: 2.64 mm.month-1; 50-60 mm 2.78: mm.month-1) and B (40-50 mm: 2.20 mm.month-1; 50-60 mm: 2.35: mm.month-1). These (Diets A and B) gave higher growth rates when compared to Diets C and D (natural diets), whose growth rates ranged between 0.50 mm.month-1 and 1.71 mm.month-1 for both cohorts. Also observed in this study was that, the mixture of formulated diet and seaweed gave better growth than formulated diet given exclusively.

The abalone (Haliotis midae) culture industry in South Africa is becoming increasingly dependent on the use of formulated feeds, due to limitations in the supply of kelp. The bulk of the feeds that are currently available were developed based on the requirements of juvenile abalone cultured within the optimal temperature range for growth (18 - 20 °C). However, most abalone farming facilities are land-based pump ashore operations and are thus mostly exposed to temperatures outside of this range. In addition, these feeds have been found to be unsuitable for abalone cultured at elevated water temperatures (> 20°C). The aim of the study was to develop size and temperature specific diets for H midoe through optimisation of dietary protein, energy and lipid levels. Abalone were cultured under farm-like conditions in three partially recirculating temperature controlled systems at either 18, 22 or 24°C and fed formulated diets containing graded levels of protein (18,22 and 26 %) and energy (11.6, 13.5 and 16.2 MJ.kg·I ). Abalone were stocked into baskets at 5 % of available of surface area (n=36) and each diet (n=9) was fed to four baskets of abalone at each of the three temperature regimes for ten weeks. Abalone growth was temperature dependent, with growth declining from 4.33 g.month-I for abalone cultured at 18°C to 0.77 g.month-I at 24°C. Dietary protein could be reduced from 26 to 18 % provided dietary energy levels were maintained at 13.5 MJ.kg- l • A dietary energy level of 11.6 MJ.kg-1 was insufficient to meet the energetic requirements of H midae regardless of the protein content of the diet. The effects of water temperature and body size on the protein requirements of H midae were investigated by culturing abalone at temperatures within the optimal range for abalone farming (i.e. 14, 16 and 18°C). Three size classes of abalone (15, 50 and 80 mm) were fed formulated feed containing graded levels of dietary protein (20, 26, 32, 38 and 44 %) under controlled laboratory conditions for 12 weeks, and, in a separate experiment, under commercial farm conditions for 24 weeks. It was not possible to convincingly define the optimal protein levels for abalone of different sizes in this experiment because growth rates fell below average commercial growth rates obtained on farms. Growth was temperature dependent in the laboratory trial, with the rate of weight gain of the 15 mm (ANOV A: p=0.002) and 50 mm abalone (ANOV A: p=0.02) increasing significantly with an increase in temperature from 14 to 18°C. In the farm trial, dietary protein content did not affect the growth rate of the 10-15 or 80 mm abalone (ANOVA: p>0.05), however, the 50 mm abalone displayed significantly higher weight gain on the 32 % (4.72±0.20 g.month-I ) and 38 % (5.01±0.34 g.month-I ) protein diets compared to those fed the 20 % protein diet (3.75±0.13 g.month-I ) (ANOVA: p=O.OI). Although definition of optimal dietary protein levels were not possible, the effects of dietary protein content and water temperature on the growth of H midae were independent signifying that the protein requirements of abalone are temperature independent. In addition, there was no evidence to indicate that abalone of the different sizes tested here had different dietary protein requirements. The size specific dietary lipid and protein requirements of H midae were investigated by feeding two size classes of abalone (30 and 60 mm initial shell length) diets containing graded levels of dietary lipid (4, 7, 10, 13 and 16 %) and protein (34 - 39 %) for 12 weeks. The 30 and 60 mm abalone were stocked at 7 (n=200) and 9 % (n=36) of the available basket surface area respectively and each diet was fed to four baskets of abalone of each size class. The protein requirements of H. midae are influenced by the amount of available dietary energy and thus it is possible that the ability of abalone to utilise lipids as a source of energy differs in the presence of varying levels of dietary protein. High levels of dietary lipid negatively affected the growth, condition factor and soft tissue glycogen content of both size classes of abalone. This negative effect was greater in the 30 mm size class compared to the 60 mm abalone. The corresponding increase in feed consumption and feed conversion ratio in response to increasing levels of dietary protein also provides evidence that abalone are unable to utilise dietary lipids as an energy source and high levels of dietary lipid probably inhibit the uptake of carbohydrates and protein. High dietary lipid levels did however appear to promote gonad maturation. It was possible to reduce dietary protein from 34 to 20 % without negatively affecting growth through the maintenance of dietary energy levels and thus it is recommended that future experiments on the energy content of formulated feeds should focus on the improved use of carbohydrates. Reductions in the protein portion of formulated feeds for H. midae are possible provided the diet contains sufficient levels of energy supplied from carbohydrates. As the ability of abalone to utilise dietary lipid is limited, lipids are unlikely to play a significant role as an energy source in abalone feeds. Further investigations should focus on the utilisation of various carbohydrate sources in abalone feeds.