Abalone Haliotis midae farming in South Africa is highly intensive, employing pump-ashore, flow-through systems. Despite the known sensitivity of abalone to water quality, there is only a rudimentary understanding of water quality dynamics on South African abalone farms and its effects on abalone production. Furthermore, the potential for reusing the relatively dilute abalone farm effluent to culture other animal species has not been investigated. This study investigated the dynamics of water quality and growth on a South African abalone farm and assessed the suitability of the effluent for the culture of silver kob Argyrosomus inodorus and bloodworm Arenicola loveni loveni. Monitoring of water quality and abalone growth in abalone tanks revealed that oxygen concentrations decreased, while H⁺ ion and free-ammonia (NH₃) concentration increased in a gradient between the inflow and outflow. Abalone growth was positively correlated with oxygen concentration and negatively correlated with free-ammonia and H⁺ ion concentration. The oxygen (O) concentration of the farm influent was dependent upon the influents’ temperature (T) and was described by the relationship O (mg L⁻¹) = 11.244 – 0.208T (r²=0.74). Linear regression analysis of data collected from abalone farm tanks revealed that the concentration of total ammonia at the outflow of abalone tanks (μg TAN L⁻¹) was dependant upon temperature (°C), flow-rate (L s⁻¹ kg⁻¹ H. midae), abalone size (g) and length of time since the tank was last cleaned (d) (n = 125, r² = 0.80). The production of total ammonia (μg TAN s⁻¹ kg⁻¹) was related to temperature, abalone size and days that the tanks remained un-cleaned (n = 125; r² = 0.81). A diurnal cycle of respiration was evident in abalone tanks with higher oxygen consumption and H+ ion production at night. The oxygen concentration of farm effluent was related to temperature, farm biomass and flow rate by means of a linear regression equation (n = 40; r² = 0.69). The results demonstrated the importance of optimising the flow-rate per unit of biomass for various temperatures and sizes of abalone. As abalone size and temperature cannot be controlled under farm conditions, the flow-rate per unit of biomass which the abalone culture system receives will determine the quality of the culture water. The specific growth rate (0.48 ± 0.01 % BW d⁻¹), mortality (1.8 ± 0.5 %), feed conversion ratio (3.0 ± 0.2) and protein efficiency ratio (1.0 ± 0.1) of silver kob kept in either abalone farm effluent or control seawater for 120 days did not differ significantly (t-test, P>0.05). A 90 day growth trial indicated that abalone farm effluent is a suitable culture medium for bloodworm. Bloodworm supplied with control seawater lost weight at 0.19 ± 0.04 % BW d-1, while those given abalone effluent grew at 0.39 ± 0.07 % BW d⁻¹. Mortality was 6 ± 3 % in effluent and 11 ± 8 % in seawater. The bloodworm were efficient at processing solid waste. Abalone farm effluent initially contained 7.7 ± 13 mg L⁻¹ more suspended solids than control seawater, which contained 3.5 ± 0.5 mg L⁻¹, but after passing through bloodworm systems the concentration in abalone effluent was reduced to only 1.4 ± 3.5 mg L⁻¹ above that in control seawater. Therefore, abalone farm effluent could be reused as a culture medium for both silver kob and bloodworm. Future work is needed to investigate aspects of the feasibility of such systems such as growth rates at different sizes and stocking densities.

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.

According to farm records, cultured Haliotis midae (50-70 g.abalone⁻¹) were growing 10% slower in winter when compared to summer. This reduction in growth rate also coincided with enlarged gonads. Initial trials showed that there were differences in mean monthly growth rates ranging from 1.97 – 5.14 g abalone⁻¹ month⁻¹, and gonad bulk index (GBI) also varied between months (GBI range: 26.88 ± 12.87 to 51.03 ± 34.47). The investment of energy into gonad tissue growth did not compromise whole body growth as the abalone continued to gain weight throughout the reproductive periods, probably due to gonadal growth. Growth of this size class of abalone was not influenced by water temperature or day length, suggesting favourable on-farm culture conditions (regression analyses, p > 0.05). There is no need to implement a seasonal dietary regime. Cultured H. midae were fed artificial diets with different protein sources, including only soya, only fishmeal, a combination of soya and fishmeal, and these were compared to kelp-fed abalone. Kelp-fed abalone grew slower than those fed artificial feeds (p>0.05). Gonad growth was the greatest when soya meal was included in the diet (average GBI: 74.91 ± 23.31), while the average gonad size of abalone fed the fishmealbased diet had gonads which were 38% smaller, and kelp-fed abalone had gonads which were 75% smaller than those of the abalone fed on diets containing soya meal. The increased gonad mass in abalone fed on diets including soya meal could be attributed to phytoestrogenic activity, as a result of the presence of isoflavones found in the soya plant; this remains to be tested. The use of soya in brood stock diet development is advised. The influence of dietary protein to energy ratio (1.41 – 2.46 g MJ⁻¹) on growth and gonad size was tested. Protein and energy levels within the ranges tested (22 and 33% protein; 13.5 and 15.6 MJ kg⁻¹) did not interact to influence growth rates of cultured H. midae. GBI increased from 50.67 ± 4.16 to 83.93 ± 9.35 units as a function of dietary protein to energy ratio (y = 42.02 x⁰·⁸¹; r² = 0.19; regression analysis: F₁¸₃₈ = 8.9; p = 0.005). In addition, protein level influenced gonad size, with gonad growth being greater in abalone fed the high protein diet (factorial ANOVA: F₁¸₃₂ = 7.1, p = 0.012). Canning yields were reduced by 7% when the protein content was increased, while increasing the quantity of dietary energy improved canning yields by ~ 6% (one-way ANOVA: F₁¸₂₈ = 14.4, p= 0.001). The present study provided evidence that although growth rates are varying seasonally, reproductive investment is not hindering weight gain. Gonad growth can be influenced if desired by farms, depending on the level of soya inclusion, as well as the protein to energy ratio in the diet. Monthly variation in growth and gonad size, as well as the influence of diet on gonad growth were highlighted, and the implications for farm application and further research were discussed.

Abalone, Haliotis midae, farmers in South Africa that feed formulated diets reported a periodic drop in abalone growth during periods of increased gonad development. A large drop in abalone biomass was noticed after presumed spawning events. This study was aimed to determine the effect of diet and sex-sorting on gonad development in abalone. Experiments were conducted on a commercial abalone farm from July 2012 to the end of June 2013. Isonitrogenous and isoenergetic diets were formulated with two protein sources. A fishmeal and soybean meal (S-diet) diet and a fishmeal only (F-diet) diet were fed to abalone (50 - 70 g abalone⁻¹) over 12 months. Weight and length gain, gonad bulk index (GBI), visceral index (%) and meat mass index (%) were determined monthly and seasonally. A histological study on the female gonads was conducted. This study also included an experiment to test the effect of sex-sorting (70 - 80 g abalone⁻¹) on growth and body composition with treatments including males (M), females (F) and equal numbers of males and females (MF). Weight gain and length gain were faster in S-diet-fed abalone (RM-ANOVA, F ₍₁, ₁₆₎ = 7.77, p = 0.01; F ₍₁, ₆₉₎ = 49.9, p < 0.001, respectively). Gonad development was significantly affected by the inclusion of soybean meal with S-diet-fed abalone showing higher GBI-values than F-diet-fed abalone (RM-ANOVA, F ₍₁, ₃₃)= 16.22, p = 0.0003). Male abalone had higher GBI-values than females (RM-ANOVA, F ₍₁, ₃₃₎ = 39.87, p < 0.0001). There was no significant difference in average feed conversion ratio (FCR) between diets over time (RM-ANOVA, F ₍₁, ₂₁₎ = 0.008, p = 0.97). However, average FCR-values were significantly highest between November 2012 and March 2013, the presumed spawning season. The visceral mass (gut and gonad) as a proportion of whole mass (visceral index, %) was significantly higher in abalone fed the S-diet (RM-ANOVA; F ₍₁, ₆₉₎ = 68.06, p < 0.0001). There was no difference in meat mass index (%) between diets for both male and female abalone (RM-ANOVA; F ₍₇, ₂₄₈₎ = 0.80, p = 0.60; F ₍₇, ₂₄₁₎ = 1.7, p = 0.11,respectively). Meat mass index significantly decreased from September 2012 to February 2013 coinciding with the period of high GBI-values. The distribution of oocyte maturity stages differed between diets. The majority of oocytes within S-diet-fed abalone were fully mature stage 8 oocytes compared to a majority of stage 7 oocytes in F-diet-fed abalone. Histology corroborated peaks in GBI-values for abalone fed both diets. There was no significant difference in growth, GBI, visceral index (%) and meat mass index (%) between abalone sorted into monosex and mixed-sex populations. Thus, the presence of the opposite sex did not have an effect on growth and gonad mass in H. midae. The phytoestrogens daidzin, glycitin, genistin, daidzein, glycitein and genistein were present in soybean meal and only traceable amounts were found in the F-diet. This study provided evidence that soybean meal present in formulated feed affected growth and gonad development in H.midae. The difference in the distribution of the maturity stages of oocytes was affected by diet. Sex-sorting abalone into monosex and mixed-sex populations had no influence on weight and length gain and gonad development.

Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The genetic status of H. midae broodstock in the South African aquaculture industry reflects that of random samples originating from undomesticated wild populations. The nature of growth in abalone is very slow, taking between three and four years to reach a marketable size of between 60 to 100 grams. It is therefore of paramount importance to improve this trait in order to ensure global competitiveness and economic viability within the industry. Improving this negative characteristic through conventional selection methods is a long-term venture and alternative means that would yield instantaneous results had to be considered. Crossbreeding was identified as an alternative, short-term strategy to improve growth rate. A crossbreeding experiment was performed between two populations of the abalone, Haliotis midae, from the East (E) and West (W) Coast of South Africa. This was done to investigate the occurrence of heterosis for growth among the crossbred genotypes (East x West, West x East). Fifteen males and females from both the East and the West Coast populations were mated in a complete dialelle crossbreeding experiment to produce four progeny groups (WW, EE, EW and WE). Progeny groups were evaluated for weight (bW) and length gain (bL) over a specific growth period of 9 months. The results provided no evidence of significant differences in weight (P = 0.085) or length gain (P = 0.244) between the four progeny groups, giving no indication of significant heterosis for weight and length gain among the crossbred progenies of these East and West Coast populations. It is recommended that further efforts to obtain improved growth rate in the abalone, Haliotis midae, through crossbreeding only be considered in light of clear evidence of substantial genetic differentiation between such populations. / AFRIKAANSE OPSOMMING: Die huidige status van perlemoen, soos dit voorkom in akwakultuur bedrywighede in Suid Afrika, weerspieël dié van ‘n ewekansige monster vanuit wilde, natuurlike populasies. Perlemoen is inherent ‘n stadig groeiende organisme wat tussen drie en vier jaar neem om tot ‘n bemarkbare grote van 60 tot 100 gram te groei. Dit is dus uiters noodsaaklik om hierdie eienskap te verbeter ten einde die bedryf ekonomies lewensvatbaar en mededingend op wêreld markte te maak. Konvensionele seleksie as ‘n metode om hierdie negatiewe eienskap te verbeter is ‘n langtermyn onderneming wat die identifisering van ‘n korttermyn metode, wat ondmiddellike resultate lewer, noodsaak. Kruisteelt is geïdentifiseer as geskikte korttermyn oplossing aangesien dit onmiddellike resultate lewer. ‘n Kruisteel eksperiment is uitgevoer tussen twee populasies van die perlemoen, Haliotis midae, van die Ooskus (E = East) en die Weskus (W = West) van Suid Afrika. Dit is gedoen om die omvang van heterose vir groeitempo in die gekruisde nageslag (East x West, West x East) te bepaal. Fyftien mannetjies en wyfies van beide die Oos- en Weskus populasies is met mekaar gepaar in ‘n volledige dialleel kruising om vier nageslag groepe (WW, EE, EW en WE) te vorm. Die nageslag is geëvalueer ten opsigte van massa (bW) en lengte (bL) toename oor ‘n spesifieke groei tydperk van 9 maande. Die eksperimentele resultate dui daarop dat die vier nageslag groepe nie betekenisvol van mekaar verskil het ten opsigte van massa (P = 0.085) en lengte (P = 0.244) toename nie en dat daar dus geen aanduiding van heterose vir massa en lengte toename in die nageslag van kruisings tussen die Ooskus en Weskus populasies bestaan nie. Daar word aanbeveel dat kruisteling as ‘n metode van genetiese verbetering van groeitempo in Haliotis midae slegs oorweeg word in die lig van nuwe molekulêre bewyse van genoegsame genetiese differensiasie tussen sulke populasies.

M.Sc. / Abalone are among the world’s leading shellfish consumed by human populations. Harvesting in California began in the late 1800s from intertidal zones and in the early 1900s wild abalone were collected by diving. Popular demand for abalone products in the Far East then led to extensive harvesting of wild abalone and a drastic decline in population numbers. This problem was overcome to a degree by the development of land-based abalone farms. At these farms it was possible to breed abalone on a large scale. Currently twelve abalone farms operate in South Africa and the estimated production for 2006 was 537 tons of meat, worth R 80 mil. Parasites and diseases pose threats to the production of abalone, especially under farmed conditions, and can cause considerable financial loss. Labyrinthuloides haliotidis, Haplosporidium nelsoni and Terebrasabelle heterouncinata are a few parasites that contribute to the above mentioned problems. Lately, a new protozoan parasite was discovered in the digestive glands of Haliotis midae farmed in the Western Cape Province, during routine health assessments. For the purposes of this dissertation it is designated an unidentified digestive gland parasite (UDP). The aims of this study are thus to undertake a comprehensive literature review of parasites infecting wild and farmed abalone, as well other shellfish species, describe and characterise the UDP infecting the digestive gland of Haliotis midae based on its structure and ultrastructure, evaluate the role of this parasite in disease by analysing data from histological studies, provide a preliminary indication of the life cycle of this parasite, attempt analysis of DNA from the UDP, and identify potential areas for further research into control of the parasite. A total of 180 abalone, (Haliotis midae) were collected from three abalone farms in the Western Cape during May 2005, October 2005, January 2006 and January 2007. To establish whether this parasite also occurs in wild abalone, a single sampling (six H. midae and 28 H. spadicea) took place during 2006 in Tsitsikamma National Park. Collected farmed and wild abalone were weighed and measured, removed from their shells and then killed according to accepted methods before their digestive glands were removed.

Inadequate information of the nutritive physiology and the dietary requirements of abalone are the principle factors that currently limit the development of a formulated feed for the commercial culture of Haliotis midae. The need to develop a method to determine apparent digestibility co-efficient's for abalone in order to facilitate further applied nutritional research was identified. Animals between 50 and 80 mm were collected from natural stocks along the east Cape coast of South Africa at Port Alfred and Great Fish point, and acclimated to laboratory conditions. Initial trials demonstrated that H. midae accepted and preferred a semi-purified diet to the seaweed Plocamium corallorhiza, one of the main components of it's natural diet. A technique of determining apparent digestibility co-efficient's (ADC) using the indirect method with chromic oxide as an inert marker was developed. Digestibility trials yielded higher dry matter (DMADC) and crude protein apparent digestibility co-efficient's (CPADC) for the semi-purified diet than for two species of algae, Gelidium amanzii and P. corallorhiza (83.7% and 95.6%, 70.7% and 80.0%, and 29.9% and 57.3% respectively). The ability of the animals to utilize terrestrial animal and plant ingredients efficiently makes it feasible to use conventional feed ingredients in formulated feeds for H. midae. Trials to determine the effect of different temperatures (15°C, 18°C and 22°C) on DMADC and CPADC of the semipurified diet showed that peak digestibility occurred at 18°C. There was also a positive relationship between temperature and consumption rate. Although no enzyme studies with H. midae have been conducted, the peak ADC's at 18°C is attributed to an increase in enzyme activity at this temperature. Transit time, an inverse function of temperature and consumption, is considered to be responsible for the decrease in the ADC' s at 22°C in conjunction with a possible decrease in enzyme activity at this temperature. A photoperiod trial to investigate the effect of darkness on DMADC and CPADC of the semi-purified diet revealed that digestive efficiency decreased with increasing hours of darkness. There was also a positive relationship between duration of darkness and the rate of consumption. The decrease in ADC's is attributed to decreased transit times as the duration of darkness increased . The contribution of this project to the understanding of abalone nutrition, the development of a formulated abalone feed and systems design for abalone farms is discussed.

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.

ENGLISH ABSTRACT: The first successful captive spawning of the South African abalone Haliotis midae occurred in the 1980’s and subsequently the commercial abalone industry in South Africa has developed, with an estimated investment of US$ 12 million and annual output of 500 to 800 tons by 2001, making South Africa the biggest abalone producer outside of Asia. Natural kelp is currently the major feed and the development of a suitable substitute, and improved disease management in abalone culture are seen as the primary factors limiting expansion of the industry in South Africa. Further, abalone growth rates are very slow and improvements in growth rate will lead to shortened production times with benefits to producers. Diseases in aquaculture have traditionally been combated using antibiotics as treatment (therapeutic usage) and preventative measure (prophylactic usage). In terrestrial livestock management, antibiotics are also used as growth promoters. The use of antibiotics in aquaculture has recently sparked concerns about the development of antibiotic resistance in pathogens of humans and aquaculture organisms, and alternative strategies to using antibiotics mainly focus on manipulating the microbial composition in the host organism, in order to establish a beneficial microbial population to prevent disease. The role that organic acids and their salts can play as growth promoters in the South African abalone Haliotis midae, and as manipulators of the gut microflora of this species of abalone was investigated and compared to the effects of antibiotics. Three different treatments were tested against a negative control and a positive control containing 30ppm avilamycin, a commercial antibiotic growth promoter (AGP) used in the pig and poultry industry. The 3 treatments consisted of 1% acetic and 1% formic acid (treatment AF), 1% sodium benzoate and 1% potassium sorbate (treatment SBPS), and 1% benzoic and 1% sorbic acid (treatment BS). Three different experiments were conducted to test the effects of the different acids and salts. The first experiment was under controlled optimum water temperature conditions (16.5ºC), another at elevated water temperature (20.5ºC) in order to test response during temperature stress conditions, and the final trial was conducted under uncontrolled practical production conditions. In an attempt to establish the mechanism by which the treatments have their effects (if any), the composition of the gut microflora of the abalone was monitored. It was found that the organic acids and salts investigated can enhance the growth rate of Haliotis midae in the size class 23 mm to 33 mm mean length significantly when compared to both control treatments. It was further found that the tested AGP had no effect on growth rate. None of the treatments had a significant effect on feed conversion ratio (FCR), Incidence cost (IC) or feed intake. It could also not be shown that the treatments affected the intestinal microflora of the abalone, although this might be due to inadequate microbiological methods. The mechanism by which the acids and salts have their effects could not be established. It was found that the animals in the controlled system underwent an initial adaptation period, which led to improvement in specific growth rate (SGR), FCR and IC as the experiment progressed during the controlled optimal conditions experiment. Large differences in FCR and IC was seen for controlled optimal conditions and production conditions which means that there is still a large scope for developing methods to improve practical on-farm feed utilisation by abalone. SGR, FCR and IC were negatively influenced by raising water temperature from 16.5ºC to 20.5ºC. The composition of the gut microflora of the abalone also changed significantly after the water temperature was raised. It appears that animal weight gain and shell growth respond differently to changing water temperatures, which is reflected in a change in Fulton condition factor. A relationship between the length and weight of abalone between 15 mm and 47 mm was established and it was found that Haliotis midae does not follow an isometric growth relationship. This relation can be used as a tool to improve farm management and therefore also profitability. Various micro-organisms were isolated from Haliotis midae during the trial, but their relationship and interaction with abalone is not clear. Clear dominance by specific species of bacteria was observed during certain periods. The current research has clearly showed the potential of organic acids and their salts to act as growth promoters in the South African abalone Haliotis midae, with application in both the local aquaculture and feed manufacturing industries. The possibility further exists that some aspects of the current research can be adapted to be applicable in other abalone species and even in other aquaculture species. / AFRIKAANSE OPSOMMING: Die eerste suksesvolle aanteel van die Suid-Afrikaanse perlemoen Haliotis midae in gevangeskap is in die 1980’s gerapporteer, waarna ‘n suksesvolle akwakultuur industrie ontwikkel het met ‘n geskatte produksievermoë van 500 tot 800 ton en kapitaalbelegging van US$ 12 miljoen in 2001. Suid-Afrika is tans die grootste perlemoen-produserende land wat buite Asië geleë is. Die ontwikkeling van ‘n geskikte alternatiewe voedselbron vir natuurlike kelp (tans die algemeenste voedselbron wat gebruik word in die kweek van perlemoen), sowel as verbeterde siektebestryding word tans gesien as die hooffaktore wat verdere uitbreiding in die Suid-Afrikaanse industrie beperk. Perlemoen het verder baie stadige groeitempo’s en enige verbetering in hierdie verband sal produksietye verkort en dus produsente bevoordeel. Siektes in akwakultuur word tradisioneel bestry deur gebruik te maak van antibiotiese behandeling (terapeutiese bestryding) of van voorkomende behandeling (profilaktiese bestryding). In gewone diereproduksie-sisteme (bv. varke en hoenders) word antibiotika ook gebruik as groeistimulante. Die gebruik van antibiotika in akwakultuur het onlangs die bekommernis laat ontstaan dat sekere menslike en dierepatogene weerstand kan ontwikkel teen sommige middels, wat die behoefte laat ontstaan het om siektebestryding sonder die gebruik van antibiotika te ontwikkel. Alternatiewe strategieë fokus grootliks daarop om die samestelling van die mikrobiese bevolking van die gasheer te manipuleer en sodoende ‘n voordelige bevolking in die gasheer te vestig, wat dan siektes voorkom. Daar is ondersoek ingestel na die rol van organiese sure en hul soute as groeistimulante en manipuleerders van die mikrobiese bevolking in die Suid-Afrikaanse perlemoen Haliotis midae. Drie verskillende behandelings is getoets en vergelyk met beide ‘n negatiewe- en positiewe kontrole (wat 30 dele per miljoen van ‘n kommersiële antibiotiese groeistimulant bevat het). Die drie formulasies het onderskeidelik bestaan uit ‘n mengsel van 1% etanoëen 1% metanoësuur (behandeling AF), 1% bensoë- en 1% sorbiensuur (behandeling BS) en 1% natriumbensoaat en 1% kaliumsorbaat (behandeling SBPS). Om die effekte van hierdie formulasies te toets, is daar 3 proewe gedoen. Een proef is gedoen onder temperatuurbeheerde toestande teen ‘n optimum watertemperatuur van 16.5ºC terwyl ‘n ander gedoen is onder onbeheerde, praktiese produksie-omstandighede. ‘n Verdere beheerde proef is gedoen teen ‘n watertemperatuur van 20.5ºC om die effek van die verskillende formulasies te toets wanneer die diere aan temperatuur-spanning blootgestel word. Die samestelling van die mikrobiese bevolking in die dunderm van die perlemoen is deurentyd gemonitor in ‘n poging om die meganisme vas te stel waarvolgens die sure en soute hul effek het, indien daar enige effek waargeneem word. Daar is gevind dat die onderskeie sure en suursoute die groeitempo van Haliotis midae met ‘n gemiddelde lengte van 23 mm tot 33 mm beduidend kan verhoog in vergelyking met die groeitempo’s van beide kontroles. Daar is gevind dat die antibiotiese groeistimulant geen effek het op die groei van die diere nie en dat geen behandelings ‘n beduidende effek op voeromsetting, voerkoste of voerinname gehad het nie. Daar kon nie bewys word dat enige van die formulasies of die antibiotika ‘n effek gehad het op die mikrobes in die spysverteringskanaal van die perlemoene in die sisteem nie, alhoewel die gebrek aan ‘n effek moontlik toegeskryf kan word aan die onakkurate en onvoldoende mikrobiologiese metodes wat gebruik is tydens die studie. Die meganisme waarvolgens die sure werk kon nie vasgestel word nie. Daar is verder gevind dat die diere in die temperatuur-beheerde eksperiment aanvaklik deur ‘n aanpassingsperiode gegaan het, wat tot gevolg gehad het dat die spesifieke groeitempo, voeromsetting en voerkoste verbeter het met die verloop van die eksperiment. Daar is groot verskille gevind in die voeromsetting van beheerde optimale toestande en onbeheerde produksietoestande, wat impliseer dat daar nog baie ruimte en geleenthede is om metodes te ontwikkel wat beter voeromsetting bewerkstellig tydens perlemoenproduksie. Spesifieke groeitempo, voeromsetting en voerkoste is nadelig beïnvloed toe die watertemperatuur verhoog is vanaf 16.5ºC na 20.5ºC. Die samestelling van die mikrobiese bevolking in die spysverteringskanaal van die perlemoen het ook beduidende veranderinge ondergaan tydens hierdie temperatuur verhoging. Dit wil voorkom asof die lengtegroei van die dop en die toename in massa verskillend reageer op ‘n verandering in watertemperatuur en hierdie effek word weerspieël in die verandering in Fulton-kondisiefaktor. ‘n Verwantskap tussen totale doplengte en totale gewig van Haliotis midae kon vasgestel word vir diere tussen 15 mm en 47 mm en daar is gewys dat H. midae nie ‘n isometriese groeipatroon volg nie. Hierdie verwantskap kan aangewend word tydens produksiebestuur om produksie te verbeter en daardeur ook winsgewendheid te verhoog. Verskeie mikrobes is tydens die verloop van die proef geïsoleer, maar die rol van en interaksie tussen hierdie mikrobes en die Suid-Afrikaanse perlemoen is nie duidelik nie. Sekere bakterieë het die mikrobiese bevolking in die spysverteringskanaal van die perlemoen in hierdie proef oorheers tydens sekere groeiperiodes. Die huidige navorsing het duidelik aangetoon dat organiese sure en hul soute as groeistimulante kan optree in die Suid-Afrikaanse perlemoen Haliotis midae, met toepassings in die plaaslike akwakultuur- en voervervaardigins-industrieë. Dit beskik verder oor die potensiaal om aangepas te word sodat dit toepaslik is in ander perlemoenspesies en selfs ander akwakultuur organismes.

Chan, Wing Hin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 126-143). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Table of Content --- p.viii / List of Figures --- p.xiv / List of Tables --- p.xvi / Abbreviations --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Food and herb authentication --- p.1 / Chapter 1.1.1 --- Background and definition --- p.1 / Chapter 1.1.2 --- Importance of species identification in food and herb authentication --- p.2 / Chapter 1.1.2.1 --- Primary health care --- p.2 / Chapter 1.1.2.2 --- Food and herb safety --- p.3 / Chapter 1.1.2.3 --- Conservation --- p.4 / Chapter 1.1.3 --- Methods for species identification in food and herb authentication --- p.4 / Chapter 1.1.3.1 --- Morphological identification --- p.5 / Chapter 1.1.3.2 --- Chemical analysis --- p.6 / Chapter 1.1.3.3 --- Molecular analysis --- p.9 / Chapter 1.1.4 --- Legislation --- p.11 / Chapter 1.1.4.1 --- Labeling ´ب --- p.11 / Chapter 1.1.4.2 --- Chinese medicine : --- p.12 / Chapter 1.1.4.3 --- Conservation --- p.12 / Chapter 1.2 --- Dongchongxiacao --- p.13 / Chapter 1.2.1 --- Background information of Dongchongxiacao --- p.13 / Chapter 1.2.2 --- Classification of fungal part of Dongchongxiacao --- p.14 / Chapter 1.2.3 --- Dongchongxiacao as a Traditional Chinese Medicine. --- p.15 / Chapter 1.2.4 --- The Dongchongxiacao market --- p.16 / Chapter 1.2.5 --- Adulteration and contamination of Dongchongxiacao --- p.18 / Chapter 1.2.6 --- Authentication of Dongchongxiacao --- p.19 / Chapter 1.2.6.1 --- Morphological identification --- p.19 / Chapter 1.2.6.2 --- Chemical analysis --- p.20 / Chapter 1.2.6.3 --- Molecular analysis --- p.22 / Chapter 1.2.6.3.1 --- "FINS analysis with genomic ITS, nrLSU, EF-lα and rpbl regions for fungal analyses" --- p.22 / Chapter 1.2.6.3.2 --- FINS analysis with mitochondrial CytB and COI regions for caterpillar analyses --- p.24 / Chapter 1.3 --- Abalone --- p.26 / Chapter 1.3.1 --- Background information of abalone --- p.26 / Chapter 1.3.2 --- Abalone as food --- p.27 / Chapter 1.3.3 --- The abalone market --- p.28 / Chapter 1.3.4 --- Adulteration of abalone --- p.31 / Chapter 1.3.5 --- Authentication of abalone --- p.32 / Chapter 1.3.5.1 --- Morphological identification --- p.32 / Chapter 1.3.5.2 --- Chemical analysis --- p.32 / Chapter 1.3.5.3 --- Molecular analysis --- p.33 / Chapter 1.3.5.3.1 --- FINS analysis with mitochondrial COI and 16S rDNA --- p.33 / Chapter 1.3.5.3.2 --- Haliotis-specific detection --- p.34 / Chapter 1.4 --- Aim and Objectives --- p.35 / Chapter Chapter 2 --- Materials and Methods --- p.36 / Chapter 2.1 --- Materials used in this sutdy --- p.36 / Chapter 2.1.1 --- Dongchongxiacao and Cordyceps samples --- p.36 / Chapter 2.1.2 --- Downloaded sequences from NCBI database included in Dongchongxiacao study. --- p.45 / Chapter 2.1.3 --- Abalone and gastropod samples --- p.48 / Chapter 2.1.4 --- Downloaded sequences from NCBI database included in abalone study --- p.54 / Chapter 2.2 --- Reagents and equipments : --- p.56 / Chapter 2.2.1 --- Chemical test on the presence of potassium alum in Dongchongxiacao --- p.56 / Chapter 2.2.2 --- Sample preparation and DNA extraction --- p.57 / Chapter 2.2.3 --- Polymerase Chain Reaction --- p.57 / Chapter 2.2.4 --- Agarose gel electrophoresis and Gene Clean --- p.57 / Chapter 2.2.5 --- Cloning --- p.58 / Chapter 2.2.6 --- Cycle sequencing --- p.58 / Chapter 2.3 --- Experimental procedures --- p.58 / Chapter 2.3.1 --- Morphological observation of Dongchongxiacao and abalone --- p.59 / Chapter 2.3.2 --- Chemical test of potassium in Dongchongxiacao --- p.59 / Chapter 2.3.3 --- Sample preparation and DNA extraction --- p.60 / Chapter 2.3.4 --- Polymerase Chain Reaction --- p.61 / Chapter 2.3.5 --- Agarose gel electrophoresis and Gene Clean --- p.64 / Chapter 2.3.6 --- Cloning --- p.65 / Chapter 2.3.7 --- Cycle sequencing --- p.67 / Chapter 2.3.8 --- Sequence analyses --- p.67 / Chapter 2.3.9 --- Haliotis-specific primer design and PCR test --- p.68 / Chapter Chapter 3 --- Results --- p.71 / Chapter 3.1 --- Dongchongxiacao --- p.71 / Chapter 3.1.1 --- Morphological observations --- p.71 / Chapter 3.1.2 --- Chemical test of potassium alum --- p.77 / Chapter 3.1.3 --- Sequence analyses --- p.79 / Chapter 3.1.4 --- The dendrograms --- p.81 / Chapter 3.2 --- Abalone --- p.91 / Chapter 3.2.1 --- Morphological observations --- p.91 / Chapter 3.2.2 --- Sequence analyses --- p.92 / Chapter 3.2.3 --- The dendrograms --- p.94 / Chapter 3.2.4 --- Haliotis-specific PCR --- p.96 / Chapter Chapter 4 --- Discussion --- p.98 / Chapter 4.1 --- Dongchongxiacao --- p.98 / Chapter 4.1.1 --- Species identification of Dongchongxiacao and related Cordyceps species --- p.98 / Chapter 4.1.1.1 --- Ophiocordyceps sinensis --- p.98 / Chapter 4.1.1.2 --- Cordyceps gunnii --- p.100 / Chapter 4.1.1.3 --- Metacordyceps taii --- p.102 / Chapter 4.1.1.4 --- Cordyceps militaris --- p.103 / Chapter 4.1.2 --- Adulteration of Dongchongxiacao and labeling --- p.104 / Chapter 4.1.3 --- Hosts of Dongchongxiacao fungi and relationship between them --- p.107 / Chapter 4.2 --- Abalone --- p.109 / Chapter 4.2.1 --- Species identification of abalones and other gastropod species by FINS analysis --- p.109 / Chapter 4.2.1.1 --- Haliotis species --- p.109 / Chapter 4.2.1.1.1 --- Haliotis diversicolor --- p.110 / Chapter 4.2.1.1.2 --- Haliotis discus --- p.110 / Chapter 4.2.1.1.3 --- Haliotis asinina --- p.111 / Chapter 4.2.1.1.4 --- Haliotis rufescens --- p.111 / Chapter 4.2.1.1.5 --- Haliotis midae --- p.111 / Chapter 4.2.1.1.6 --- Haliotis madaka --- p.112 / Chapter 4.2.1.1.7 --- Haliotis rubra --- p.113 / Chapter 4.2.1.1.8 --- Haliotis iris --- p.113 / Chapter 4.2.1.1.9 --- Haliotis corrugata --- p.114 / Chapter 4.2.1.2 --- Concholepas concholepas --- p.114 / Chapter 4.2.1.3 --- Hemifusus species --- p.115 / Chapter 4.2.1.4 --- """Dried abalone slice"" samples (D1 to D3) and canned top-shell (E5)" --- p.115 / Chapter 4.2.2 --- Haliotis-speciflc PCR --- p.115 / Chapter 4.2.3 --- Adulteration of abalone and labeling --- p.116 / Chapter 4.3 --- Significance and limitation of molecular approaches in authentication of food and herbs --- p.117 / Chapter 4.3.1 --- FINS analysis --- p.117 / Chapter 4.3.1.1 --- High interspecific variability but low intraspecific variations --- p.118 / Chapter 4.3.1.2 --- Amplification with universal primers --- p.118 / Chapter 4.3.1.3 --- Insufficient DNA sequence available in database --- p.119 / Chapter 4.3.1.4 --- Contamination by foreign DNA and amplification of undesirable DNA in sample mixture --- p.120 / Chapter 4.3.1.5 --- Amplification of degraded DNA --- p.121 / Chapter 4.3.1.6 --- Suggested regions for authentication of Dongchongxiacao and abalone based on FINS analysis results --- p.121 / Chapter 4.3.2 --- PCR with specific primers for targeted amplicons --- p.122 / Chapter 4.3.3 --- Other limitations of molecular approaches in authentication of food and herbs --- p.123 / Chapter 4.4 --- Further investigation --- p.124 / Chapter 4.5 --- Conclusion --- p.124 / References : --- p.126 / Chapter Appendix 1 --- Sequence alignment of 16S rDNA gene sequences of abalone for Haliotis-specific primer design --- p.144 / Chapter Appendix 2 --- Accession numbers of sequences of Dongchongxiacao and Cordyceps samples in this study --- p.149 / Chapter Appendix 3 --- Search results of CytB sequences of caterpillar host of Cordyceps samples based on BLAST search results from GenBank --- p.150 / Chapter Appendix 4 --- Search results of COI sequences of caterpillar host of Cordyceps samples based on BLAST search results from GenBank --- p.151 / Chapter Appendix 5 --- Search results of COI sequences of caterpillar host of Cordyceps samples based on BLAST search results from GenBank --- p.152 / Chapter Appendix 6 --- Sequence alignment of ITS sequences of Cordyceps samples and related sequences --- p.153 / Chapter Appendix 7 --- Sequence alignment of nrLSU sequences of Cordyceps samples and related sequences --- p.161 / Chapter Appendix 8 --- Sequence alignment of EF-lα sequences of Cordyceps samples and related sequences --- p.168 / Chapter Appendix 9 --- Sequence alignment of rpbl sequences of Cordyceps samples and related sequences --- p.173 / Chapter Appendix 10 --- "Sequence alignment of combined dataset of three regions (nrLSU, EF-lα and rpbl) of Cordyceps samples and related sequences" --- p.179 / Chapter Appendix 11 --- Sequences alignment of CytB sequences of caterpillar host of Cordyceps samples and related sequences --- p.188 / Chapter Appendix 12 --- Sequence alignment of COI sequences of caterpillar host of Cordyceps samples and related sequences --- p.191 / Chapter Appendix 13 --- Sequence alignment of COI sequences of Cordyceps samples D12-2 and D14 and related sequences --- p.195 / Chapter Appendix 14 --- Sequence distance matrix of ITS sequences of Cordyceps samples and related samples based on K2P algorithm --- p.196 / Chapter Appendix 15 --- Sequence distance matrix of nrLSU sequences of Cordyceps samples and related samples based on K2P algorithm --- p.203 / Chapter Appendix 16 --- Sequence distance matrix of EF-lα sequences of Cordyceps samples and related samples based on K2P algorithm --- p.208 / Chapter Appendix 17 --- Sequence distance matrix of rpbl sequences of Cordyceps samples and related samples based on K2P algorithm --- p.213 / Chapter Appendix 18 --- "Sequence distance matrix of combined dataset of three regions (nrLSU, EF-lα and rpbl) sequences of Cordyceps samples and related samples based on K2P algorithm" --- p.217 / Chapter Appendix 19 --- Sequence distance matrix of CytB sequences of caterpillar host of Cordyceps samples and related samples based on K2P algorithm --- p.219 / Chapter Appendix 20 --- Sequence distance matrix of COI sequences of caterpillar host of Cordyceps samples and related samples based on K2P algorithm --- p.223 / Chapter Appendix 21 --- Sequence alignment of chloroplast trnH-psbA sequences of Cordyceps sample D12-2 and related sequences --- p.226 / Chapter Appendix 22 --- Accession numbers of sequences of abalone and gastropod samples in this study --- p.227 / Chapter Appendix 23 --- Search results of 16S rDNA sequences of the abalone and gastropod samples based on BLAST search results from GenBank --- p.228 / Chapter Appendix 24 --- Search results of COI sequences of the abalone and gastropod samples based on BLAST search results from GenBank --- p.229 / Chapter Appendix 25 --- Search results of COI sequences of the abalone and gastropod samples based on BOLD-IDS --- p.230 / Chapter Appendix 26 --- Sequence alignment of 16S sequences of abalone samples and related sequences --- p.231 / Chapter Appendix 27 --- Sequence alignment of COI sequences of abalone samples and related sequences --- p.234 / Chapter Appendix 28 --- Sequence alignment of COI sequences of abalone product sample D2 and related sequences --- p.238 / Chapter Appendix 29 --- Sequence distance matrix of 16S sequences of abalone samples and related samples based on K2P algorithm --- p.239 / Chapter Appendix 30 --- Sequence distance matrix of COI sequences of abalone samples and related samples based on K2P algorithm --- p.243