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Abalone Haliotis midae are distributed from the cold, hypercapnic waters of the dynamic Benguela Current Large Marine Ecosystem to the relatively warm, normocapnic waters of the Agulhas Current. The species supports an important fishery as well as a thriving aquaculture industry. Due to the relatively low capacity to regulate their acid–base balance and their need to calcify shell and radula, abalone are especially vulnerable to increasing ocean acidification. Exposure to acidified seawater, i.e., hypercapnia, also occurs during the farming operation and can originate from (a) changes in influent seawater, (b) pH decrease by accumulation of waste products, and (c) intentional hypercapnia for anaesthesia using CO2-saturated seawater for size grading. Currently, these are acute exposures to hypercapnia, but increasing ocean acidification can cause chronic exposure, if not mitigated. Wild South African abalone are already exposed to periodic hypercapnia during ocean upwelling events and will be more so in the future due to progressive ocean acidification. This study investigated the acute pH effects in isolation as an initial step in studying the acute physiological response of H. midae to provide a mechanistic basis for the design of complex multifactorial studies, imitating more closely what occurs on farms and in the natural habitat. The major findings relevant to the above conditions are as follows: 1. Acute exposure to hypercapnia induces a reversible, unbuffered respiratory acidosis. 2. The impact of acute hypercapnia is size-dependent and potentially fatal. 3. Exposure to extreme, short hypercapnia during anaesthesia causes a rapid imbalance in the acid–base state but a rapid subsequent recovery. LC50 for small, medium and large abalone range from pH 6.27 to 6.03, respectively, and sub-lethal levels from pH 6.8 to 6.2. These results can be used by abalone aquaculture farms to mitigate/avoid the impact of acute (and chronic) hypercapnia but also to standardise their anaesthesia method. They are also a proxy to estimate the effects on wild populations. [ABSTRACT FROM AUTHOR]

Using stable isotope analysis, we assessed the relative contributions of the main protein contributors fishmeal, soya and sunflower meal to mature abalone muscle and gonad tissue over 1 year. Prerequisites for the use of stable isotope mixing models were established in this species. The δ13C and δ15N discrimination factors and turnover rates were influenced by diet composition, and there were tissue‐specific and sex‐specific differences. Abalone were fed the two combination diets (fishmeal–soya and fishmeal–sunflower meal) grew at similar rates. However, feed was most efficiently utilised in abalone fed a combination of fishmeal and soya. The relative contribution of fishmeal, soya and sunflower meal to female and male abalone tissue growth fluctuated throughout the 1‐year study. Overall, fishmeal contributed over 50% of the gonad (54%–82%) and muscle (54%–84%) tissue growth in females. Similarly, fishmeal contributed over 50% of gonad tissue growth of male abalone, ranging from 59% to 87%, while the contribution to meat tissue growth in males ranged from 44% to 94%. These findings support the importance of laboratory studies when using stable isotope analysis and mixing models for nutrient allocation in aquaculture. [ABSTRACT FROM AUTHOR]

Abstract Using stable isotope analysis, we assessed the relative contributions of the main protein contributors fishmeal, soya and sunflower meal to mature abalone muscle and gonad tissue over 1 year. Prerequisites for the use of stable isotope mixing models were established in this species. The δ13C and δ15N discrimination factors and turnover rates were influenced by diet composition, and there were tissue‐specific and sex‐specific differences. Abalone were fed the two combination diets (fishmeal–soya and fishmeal–sunflower meal) grew at similar rates. However, feed was most efficiently utilised in abalone fed a combination of fishmeal and soya. The relative contribution of fishmeal, soya and sunflower meal to female and male abalone tissue growth fluctuated throughout the 1‐year study. Overall, fishmeal contributed over 50% of the gonad (54%–82%) and muscle (54%–84%) tissue growth in females. Similarly, fishmeal contributed over 50% of gonad tissue growth of male abalone, ranging from 59% to 87%, while the contribution to meat tissue growth in males ranged from 44% to 94%. These findings support the importance of laboratory studies when using stable isotope analysis and mixing models for nutrient allocation in aquaculture.

Abalone Haliotis midae are distributed from the cold, hypercapnic waters of the dynamic Benguela Current Large Marine Ecosystem to the relatively warm, normocapnic waters of the Agulhas Current. The species supports an important fishery as well as a thriving aquaculture industry. Due to the relatively low capacity to regulate their acid–base balance and their need to calcify shell and radula, abalone are especially vulnerable to increasing ocean acidification. Exposure to acidified seawater, i.e., hypercapnia, also occurs during the farming operation and can originate from (a) changes in influent seawater, (b) pH decrease by accumulation of waste products, and (c) intentional hypercapnia for anaesthesia using CO2-saturated seawater for size grading. Currently, these are acute exposures to hypercapnia, but increasing ocean acidification can cause chronic exposure, if not mitigated. Wild South African abalone are already exposed to periodic hypercapnia during ocean upwelling events and will be more so in the future due to progressive ocean acidification. This study investigated the acute pH effects in isolation as an initial step in studying the acute physiological response of H. midae to provide a mechanistic basis for the design of complex multifactorial studies, imitating more closely what occurs on farms and in the natural habitat. The major findings relevant to the above conditions are as follows: 1. Acute exposure to hypercapnia induces a reversible, unbuffered respiratory acidosis. 2. The impact of acute hypercapnia is size-dependent and potentially fatal. 3. Exposure to extreme, short hypercapnia during anaesthesia causes a rapid imbalance in the acid–base state but a rapid subsequent recovery. LC50 for small, medium and large abalone range from pH 6.27 to 6.03, respectively, and sub-lethal levels from pH 6.8 to 6.2. These results can be used by abalone aquaculture farms to mitigate/avoid the impact of acute (and chronic) hypercapnia but also to standardise their anaesthesia method. They are also a proxy to estimate the effects on wild populations.

Growth rate is one of the most important factors in the farming of Haliotis midae, and somatic growth rates decline after abalone reach sexual maturity. Artificial diets are suspected to accelerate maturation, in particular when soya meal is used as a protein source, because of this plant's high concentration of phytoestrogens. The effect of diet on the gonad development of 27-month-old farmed Haliotis midae, raised at two stocking densities, was analysed. We fed two artificial diets and a natural diet, kelp, to cultured abalone. The first artificial diet had fishmeal as its main source of protein, while in the other, Abfeed® S34, 32% of the fish meal was replaced with soya meal. For each gonad sample, the development phase was determined based on both histological criteria and the gonad bulk index (GBIn). The hypothesised link between dietary protein source and gonad development could not be established by either morphological criteria or GBIn. Similarly, stocking density did not have a significant effect on GBIn-values. GBIn values were significantly correlated to the results from the histological analyses in males and females. The results from this study suggest that artificial diets including those using soya meal are a valid alternative in the feeding of abalone. The research provides a database for future research on factors influencing gonad development in this species. [ABSTRACT FROM AUTHOR]

As a result of increasing CO 2 emissions and the prevalence of global climate change, ocean acidification (OA) is becoming more pervasive, affecting many trophic levels, particularly those that rely on succinctly balanced ocean chemistry. This ultimately threatens community structures, as well as the future sustainability of the fishing/aquaculture industry. Understanding the molecular stress response of key organisms will aid in predicting their future survivability under changing environmental conditions. This study sought to elucidate the molecular stress response of the South African abalone, Haliotis midae , an understudied organism with high economic value, utilising a high throughput iTRAQ-based proteomics methodology. Adult abalone were exposed to control (pH 7.9) and experimental (pH 7.5) conditions for 12, 72 and 168 h, following which protein was isolated from sampled haemocytes and subsequently processed. iTRAQ-labelled peptides were analysed using mass spectrometry, while an array of bioinformatics tools was utilised for analysing the proteomic data. COG analysis identified "Cytoskeleton", "Translation, ribosomal structure and biogenesis", "Post-translational modification, protein turnover, chaperones", and "Intracellular trafficking, secretion and vesicular transport" to be the most enriched functional classes, while statistical analysis identified a total of 33 up-regulated and 23 down-regulated effectors of OA stress in abalone. Several of the up-regulated proteins that were identified function in central metabolism (ENO1, PGK, DUOX1, GPD2), the stress/immune response (CAMKI, HSPA5/GRP78, MAPKI), and cytoskeleton, protein sorting and signal transduction (IQGAP1, MYO9B, TLN1, RDX, TCP-1/CCT, SNX6, CHMP1a, VPS13a). Protein-protein interactions were predicted using STRING DB, Cytoscape and Ingenuity Pathway Analysis, providing a model of the effects of OA on the H. midae haemocyte proteome. The data indicated that H. midae underwent a metabolic shift under OA conditions to utilize more energy-efficient mechanisms of ATP generation, while attempts at restoring haemocyte stabilisation and homeostasis were reflected by up-regulation of oxidative stress and cytoskeletal proteins. Our results support other molluscan studies that report a complex array of overlapping functions of both the stress and immune response systems. This interplay of the mounted stress and immune response is maintained and observed through the up-regulation of proteins involved in protein synthesis and turnover, as well as intracellular signalling and transport. The data presented in this study highlight the value of employing sensitive and robust -omics technologies for assessing the effects of changing environmental conditions on marine organisms. • 227 proteins were differentially expressed in response to ocean acidification conditions. • Under OA conditions, H. midae underwent a metabolic shift to generate ATP via energy-efficient mechanisms. • Haemocyte stabilisation and homeostasis in response to OA was reflected by up-regulation of oxidative stress and cytoskeletal proteins. • An interplay between the stress and immune response was observed through up-regulation of proteins involved in protein synthesis and turnover, as well as intracellular signalling and transport. [ABSTRACT FROM AUTHOR]

Macrocystis pyrifera (Linnaeus) C. Agardh, known in some regions as 'giant kelp', occurs along temperate coastal rocky reefs throughout the southern hemisphere, as well as along the north-east Pacific coastline. In Chile, it serves as a major source of abalone feed within the abalone industry, with considerable literature on cultivation of the species. Here we attempt to cultivate South African M. pyrifera through vegetative propagation and investigate its use as a feed for the abalone, Haliotis midae. Kelp holdfasts were attached to a rope-raft system in Saldanha Bay, South Africa, and monitored over 3 months. In addition, a 6-month H. midae feed inclusion trial was carried out, which compared the use of M. pyrifera with the predominant commercial kelp feed species, Ecklonia maxima. Young M. pyrifera grew well on the culture system, with mean upright length nearly doubling over the culture period (55.6–102.2 cm) and displayed mean upright relative growth rates between 0.99 and 1.22% day-1. Additionally, holdfasts developed new uprights and holdfasts increased in size, growing around culture lines. Simulated partial harvesting of samples stopped upright elongation, but samples survived, whereas complete removal of uprights caused mortality. Haliotis midae fed with M. pyrifera + formulated feed (SAF 3000®) displayed comparable growth rates to the E. maxima + SAF 3000® diet. No significant differences in mean specific growth rate (M. pyrifera 0.19, E. maxima 0.18% body weight day-1), daily increment in shell length (M. pyrifera 31.19, E. maxima 24.64 μm day-1), condition factor (M. pyrifera 1.37, E. maxima 1.40 g mm-1) and mortality rate were identified between the two diets. Additionally, H. midae consumed significantly less M. pyrifera than E. maxima, suggesting potential differences in feed conversion ratio/kelp nutritional composition among the kelp species. These findings provide baseline information for the potential cultivation and use of M. pyrifera as a new source of fresh seaweed feed in the South African abalone industry. [ABSTRACT FROM AUTHOR]

The South African abalone Haliotis midae is a commercially important species farmed at high densities in land-based aquaculture systems. Disease outbreaks have had a severe financial impact on the abalone industry yet the molecular mechanisms underlying the immune response of H. midae remain obscure. In this study, a comparative shotgun proteomics approach using iTRAQ coupled with LC-MS/MS was employed to investigate H. midae proteome changes in response to Vibrio anguillarum challenge. A total of 118 non-redundant, unique haemocyte proteins were identified and quantified, with 16 proteins significantly regulated. Hierarchical clustering and pathway analysis uncovered a coordinated response dominated by calcium and cAMP signalling via activation of MAPK cascades. Early up-regulated biological processes involve phagocytosis, nitric oxide production and ATP-synthesis, whilst down-regulated responses were predominantly involved in the regulation of apoptosis. The late up-regulated response involved protein kinase activity and detoxification processes. Expression of selected proteins was validated by Western blot. A putative allograft inflammatory factor-1 protein was further selected to establish its functional molecular role in haemocytes. Confocal imaging revealed that allograft inflammatory factor-1 regulates phagocytosis via a functional interaction with filamentous actin. This is the first time a high-throughput proteomics approach has been used to investigate the immune response of H. midae. • Comparative shotgun proteomics was used to investigate H. midae immune changes. • 118 unique haemocyte proteins were identified by iTRAQ and LC-MS/MS. • Early upregulation involved phagocytosis, endocytosis and ATP-synthesis. • The late immune response involved detoxification processes. • Allograft inflammatory factor-1 regulates phagocytosis and interacts with F-actin. [ABSTRACT FROM AUTHOR]

Abalone is currently considered South Africa's most successfully produced aquaculture export product, with a 76% share of the total value generated by the aquaculture sector. A major risk factor for this sector is slow growth rates experienced during farming. Abalone feeds are often supplemented with amino acids in an attempt to enhance abalone growth. This is a first investigation of the effect of added proline to standard abalone feed, on the metabolite profile of slow‐growing abalone. A targeted liquid chromatography tandem mass spectrometry metabolomics research approach was followed to recognise the metabolic response of abalone showing slower growth performance. The addition of proline to the standard abalone diet was found to serve as a substrate for amino acid catabolism in slower growing abalone, by means of proline breakdown to assist with energy production via the tricarboxylic acid cycle. Other amino acids and urea cycle intermediates, that is, arginine, asparagine, ornithine and creatine further support energy production via the action of protein catabolism in slow‐growing abalone. Additionally, the importance of understanding how abalone respond metabolically to modified feed highlights the use of metabolomics to answer abalone aquaculture farming questions. [ABSTRACT FROM AUTHOR]

Soya in formulated diets for abalone, Haliotis midae, can improve somatic growth. However, it may also influence gonad development, thereby limiting the potential benefit of including soya in feed. This is the first study to determine both the effect of dietary soya level on growth and reproduction of abalone and the role of crystalline isoflavones (ISO) at concentrations occurring in soya diets. Abalone (40-50 g individual−1) were fed seven isonitrogenous and isoenergetic diets. Three diets contained 5, 10 or 19% soya, while an additional three diets included graded levels of ISO. A fishmeal (FM)-only diet was included. The inclusion of ISO did not influence gonad development or abalone growth while soya inclusion level had an effect on both reproductive development and abalone growth. After 180 days, male abalone fed FM with soya inclusion had the highest whole body mass (69.0 ± 2.48 g abalone−1), meat mass index (60.5 ± 1.88%), visceral mass index (13.0 ± 0.27%) and gonad bulk index (42.7 ± 9.82 g abalone−1). Weight loss was observed in all treatments probably due to spawning events. The frequency distribution of the predominant oocyte stage, stage 7 was influenced by soya concentration. The increase in the frequency of stage 7 oocytes in abalone fed FM with soya did not reduce the number of oocytes (45.0 ± 3.01 oocytes mm−2), while the number of oocytes (o) in abalone fed the FM-only diets decreased with increasing abundance of stage 7 oocytes, possibly due to an increase in oocyte size. [ABSTRACT FROM AUTHOR]

The South African abalone farming industry is entirely based on Haliotis midae , which has been commercially cultured with great success the last 20 years. Even though abalone are cultivated under identical farming conditions, originating from homogenous genetic stocks, variation is experienced in individual abalone growth rates, justifying further research into the mechanisms related to these growth differences. Insights into the biochemical processes of abalone would help to identify the various metabolic factors related to varied abalone growth rates. Metabolomics, aims to investigate the metabolism holistically, and is considered a powerful tool for better elucidation of observed phenotypical changes. A metabolomics approach including the use of untargeted gas chromatography-time of flight spectrometry, semi-targeted liquid chromatography-quadrupole time of flight mass spectrometry and targeted liquid chromatography-tandem mass spectrometry were used to indicate and better describe the metabolic variation associated with slow and fast growing abalone. The results obtained by metabolomics analysis of H. midae adductor muscle samples showed that faster growing individuals utilise energy pathways and reserves (via elevated insulin production) in such a way that they promote protein synthesis. In contrast the metabolic profile of slow growing individuals supports protein catabolism, where energy allocation for breakdown of metabolic products has priority over mechanisms utilised for abalone growth. Overall these results sets the stage for future work highlighting metabolic pathways which should be investigated in a qualitative manner ensuring reference values which can furthermore be monitored to assist with abalone growth predictions. [ABSTRACT FROM AUTHOR]

The study aimed to investigate the effects of formic acid inclusion in abalone diets, which contained ingredients produced from fish processing by-products, and to quantify effects on production performance. Four diets (a commercial reference and iso-nutrient diets containing fish silage oil, unprocessed rainbow trout viscera or shrimp protein hydrolysate) were evaluated both with and without addition of 2 g kg−1 dietary formic acid, thus yielding a total of eight treatments, in feeding trials with cultured South African abalone Haliotis midae. Results indicated that inclusion of unprocessed rainbow trout viscera without formic acid decreased production performance of experimental animals, as evidenced by significantly decreased final animal weight and daily weight gain, and significantly increased feed conversion ratio compared to the iso-nutrient control. The decreased production performance was attributed to decreased feed water stability of the diet incorporating untreated trout viscera. Dietary formic acid supplementation partially mitigated the negative impacts of viscera inclusion on production performance of the experimental animals, as seen in significantly higher final animal weight and daily weight gain, and lower feed conversion ratio when compared to non-formic acid-supplemented, viscera-containing diet. However, no beneficial effects on production performance or water stability were found upon formic acid inclusion in diets not containing trout viscera. In conclusion, dietary formic acid can partially mitigate negative impacts on production performance when unprocessed trout viscera are included in formulated abalone diets by improving feed water stability, but the exact mechanism by which this is achieved was not established. [ABSTRACT FROM AUTHOR]

The effect of kelpEcklonia maximainclusion in formulated feeds on abalone growth and gut bacterial communities has not been previously investigated in South Africa. An eight-month on-farm growth trial was conducted with sub-adultHaliotis midae(∼43 mm shell length) fed graded levels of kelp in formulated feeds. Kelp inclusion (0.44–3.54% of pellet dry mass) promoted faster growth (65.7–74.5% total mass gain), with better feed and protein conversions (apparent feed conversion ratio [FCR] 1.4–1.8, apparent protein efficiency ratio [PER] 2.3–2.7), as compared with the non-supplemented feed (52.3% total mass gain, FCR 2.1, PER 1.9;p< 0.001). Abalone-gut bacterial DNA was sequenced using 16S rRNA gene pyrosequencing and the sequences were clustered into operational taxonomic units (OTUs) at a 97% similarity level. A supplementary 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) analysis was employed. The dominant OTUs differed in terms of their relative abundances, with an autochthonousMycoplasmastrain being significantly more abundant (p= 0.03) in the gut of abalone fed a kelp-supplemented feed. The DGGE band patterns displayed higher within-group variability for abalone fed the control diet, suggesting that dietary kelp inclusion promotes gut-bacteria homeostasis. This may contribute to better feed utilisation and growth in abalone fed kelp-supplemented feeds. [ABSTRACT FROM AUTHOR]

Previous studies have found that macroalgal inclusion in formulated diets for cultured abalone improves feed utilisation, and it was suggested that seaweed-associated bacteria supply enzymes that aid in the animal's digestion. In the present study, we hypothesised that kelp supplementation in formulated feed affects the profile of digestive enzymes in farmedHaliotis midae. Commercially reared sub-adult abalone fed a kelp-supplemented (Ecklonia maxima; 0.88% dry weight) or kelp-free control feed were collected for analysis of the amylase, alginate lyase, laminarinase, fucoidanase and protease activities in the gut. Levels of polysaccharidase and protease activity did not significantly differ between the diet treatments. However, enzyme-activity levels were more variable in abalone fed the kelp-free diet as compared with those fed the kelp-supplemented diet (coefficients of variation: 73%, 48.3–60.2% and 31.9% [control diet] versus 42.7%, 13.6–33.8% and 14.6% [KS diet] for amylase, macroalgal polysaccharidases and acid protease activity, respectively). We suggest that the presence of dietary kelp modulates the abalone's gut bacteria and their nutrient exchange. Proteomic identification of proteins in abalone gut sections showed that exogenous enzymes associated with the gut microbiome aid in bacterial utilisation of digested molecules, whereas abalone endogenous enzymes degrade the macronutrients in formulated feeds. [ABSTRACT FROM PUBLISHER]

The abalone, Haliotis midae, is an important aquaculture species in South Africa and the largest generator of revenue for the mariculture sector. Despite domestication of this species still being in the initial stages, significant differentiation has been observed between wild and cultured populations. The genetic consequences of founder effects have been well documented in many aquaculture species; however, the effects of selection remain under-investigated. Previous studies in this species identified several loci thought to be under divergent selection between wild and cultured populations. The aim of this study was therefore to investigate the influence of artificial selection on genetic variation by determining whether these candidate loci are associated with larger size (primary production trait) in a commercial F population. Thirteen microsatellite markers, putatively identified as being under directional selection, were chosen for association analysis. Various statistical tests were used to detect significant genotype-phenotype associations within a family-bias corrected population cohort and two family cohorts. Two loci demonstrated significant evidence for association with size, with both loci possessing alleles that correlated significantly with either increased or decreased size. As size is currently the only trait actively selected for in terms of production, the current results suggest that natural selection for adaptation to the novel aquaculture environment is the predominant selective force shaping genetic variation during the initial stages of domestication in abalone. Furthermore, whilst it is currently unclear as to whether these loci represent causative variants for size traits, they may be useful in future molecular-assisted breeding programmes for H. midae. [ABSTRACT FROM AUTHOR]

The South African abalone, Haliotis midae L., is an important mariculture species. In the natural environment, abalone move and migrate to new sites to escape from disturbances. This is the first study that tested the effect of disturbances on movement and feeding activity of farmed abalone for 6 days post-disturbance. Disturbances were (a) turning over the feeder plate, (b) moving and shaking the feeder plate and (c) briefly lifting the basket containing the abalone out of the water. Abalone in the control treatment were not subjected to disturbance. Activity levels of abalone were elevated in the first 1-2 nights post-disturbance with 2-3 times more abalone appearing on the walls of the baskets and larger numbers on the feeder plate than in the control treatment. Feed uptake was not influenced by treatment. Thus, even minor disturbances resulted in increased activity. As it takes at least 1 year to conduct growth experiments in this slow-growing mollusc, the results provide a motivation for studies of growth and metabolic rate in this species under a range of husbandry conditions, such as regular handling, sorting and moving of abalone. [ABSTRACT FROM AUTHOR]

The South African Abalone Haliotis midae is a vital aquaculture species, accounting for the highest percentage of total aquaculture production revenue. Improving the growth rate and thus, reducing operational costs with associated benefits in production efficiency and profitability is a priority of producers. The industry has been reluctant to implement anything but the most rudimentary breeding practices, due to unreliable tagging methods and the cost of using DNA markers. As such, the current study aimed to estimate genetic parameters for growth traits in the absence of individual tagging or DNA markers using the ‘internal reference’ method. The requirements of the internal reference method are evaluated and the merits and demerits considered. Heritability estimates were low to moderate in magnitude for wet weight (0.06–0.21) and shell length (0.11–0.16). It is concluded that substantial genetic gains can be achieved through selection and further work into developing strategies employing individual identification and DNA markers are warranted. Statement of relevance We adapted an existing statistical control method in an innovative way to estimate genetic parameters for growth traits in abalone, without individual tagging or DNA markers. This method facilitates the transition from simple family selection programs to combined selection programs by providing the necessary inputs for bio-economic modelling to inform the design of more advanced breeding programs. [ABSTRACT FROM AUTHOR]

The South African abalone ( Haliotis midae) is a gastropod mollusc of economic importance. In recent years natural populations have come under considerable pressure due to overharvesting and ecological shifts. The spatial genetic structure of H. midae has been determined; however there has not been a temporal assessment of abalone population dynamics around the South African coast. Using a population genomics approach this study aimed to assess fluctuations in genetic diversity among wild and cultured South African abalone populations through time and space. Various estimates of genetic diversity and population differentiation were calculated using EST-derived SNP markers. All populations had comparable levels of genetic diversity and the long-term effective population size appears to be sufficiently large for the wild populations, despite evidence of recent bottlenecks. Population differentiation was for the most part geographically correlated, with spatial genetic structure maintained across temporal samples. Significant genetic differentiation was, however, detected among temporal samples taken from the same locality. There was evidence for comparatively small short-term effective population sizes that could explain large changes in allele frequencies due to stochastic effects. Temporal heterogeneity could also be explained by changes in selection pressures over time. H. midae populations could, therefore, be more dynamic than previously estimated and this could have implications for effective conservation and fisheries management. [ABSTRACT FROM AUTHOR]

Next generation sequencing platforms have recently been used to rapidly characterize transcriptome sequences from a number of non-model organisms. The present study compares two of the most frequently used platforms, the Roche 454-pyrosequencing and the Illumina sequencing-by-synthesis (SBS), on the same RNA sample obtained from an intertidal gastropod mollusc species, Haliotis midae. All the sequencing reads were deposited in the Short Read Archive (SRA) database are retrievable under the accession number [SRR071314 (Illumina Genome Analyzer II)] and [SRR1737738, SRR1737737, SRR1737735, SRR1737734 (454 GS FLX)] in the SRA database of NCBI. Three transcriptomes, composed of either pure 454 or Illumina reads or a mixture of read types (Hybrid), were assembled using CLC Genomics Workbench software. Illumina assemblies performed the best de novo transcriptome characterization in terms of contig length, whereas the 454 assemblies tended to improve the complete assembly of gene transcripts. Both the Hybrid and Illumina assemblies produced longer contigs covering more of the transcriptome than 454 assemblies. However, the addition of 454 significantly increased the number of genes annotated.

The abalone, Haliotis midae (commonly referred to as ' perlemoen'), is an important aquaculture species in South Africa, accounting for the highest percentage of total aquaculture production revenue. The industry largely makes use of wild, undomesticated broodstock and thus a consortium of top producers was formed to develop a genetic improvement program to increase efficiency and sustainability of the industry. As such, the current study aimed to evaluate growth trait performance (wet weight and shell length) of four hatchery-derived cohorts of South African abalone, across four differing production environments. No significant cohort-by-location effects were found for weight and length traits; however, differences were detected between respective cohorts and locations. It is concluded that a single synthetic population comprised of all hatchery cohorts under a single breeding goal is suitable for production at all test locations. Furthermore, advances in artificial reproductive technologies, genetic markers, and individual identification hold promise for genetic improvement of the species. [ABSTRACT FROM AUTHOR]

The sensitivity of abaloneHaliotis midaelarvae to low levels of toxicants was used to conduct a laboratoryscale experiment to determine the possibility of using chemical containment to prevent the escape of larvae from land-based culture systems, thereby reducing the environmental impact of farming operations. The cryoprotective agent, dimethyl sulphoxide or DMSO (Me2SO), was used as a toxicant and embryos were exposed to increasing concentrations (0–25.60 mol l–1) of DMSO for a period of 24 hours. Hatch-out rates of the control group (0.0% DMSO) did not differ from those obtained at 0.2% (2.56 mol l–1) and 0.4% (5.12 mol l–1) DMSO. An exposure level at and above 0.6% DMSO resulted in a significant decrease in the number of normal larvae. At an exposure concentration of 0.6% (7.68 mol l–1) DMSO, 65% (SE 3.0) of the fertilised eggs hatched, compared to only 10% (SE 2.0) at a concentration of 1.0% (12.80 mol l–1) DMSO. This study indicates the potential for use of DMSO as a form of chemical containment of abalone larvae, even at very low doses. It presents South African abalone farms with a chemical containment method that could potentially be used to prevent the unintentional escape ofH. midaelarvae from land-based operations. [ABSTRACT FROM AUTHOR]

A Rickettsiales-like prokaryote was observed in digestive gland epithelium of farmed South African abalone Haliotis midae sampled for routine surveillance. Histological examination showed basophilic inclusions typical of rickettsial infections in shellfish (Mouton, 2000). This study aimed to identify the Rickettsiales-like prokaryote (RLP) colonising H. midae . Total genomic DNA was extracted from infected digestive gland sampled from cultured H. midae . PCR performed with “ Candidatus Xenohaliotis californiensis ” specific primers RA 5-1 and RA 3-6 did not yield positive results, indicating that the H. midae RLP is not the withering syndrome agent. The 16S rRNA gene sequence of the intracellular bacterium was determined following nested PCR using universal eubacterial primers fD1/Rp2 and the broad spectrum primers EHR16SD/EHR16SR which are specific for members of the Anaplasmataceae family within the Rickettsiales order. Phylogenetic and evolutionary distance analysis showed that the H. midae RLP is a unique member of the α-proteobacteria. The identity and location of the histologically observed intracellular bacterium was confirmed by in situ hybridization performed on sequential sections of infected H. midae digestive gland using a combination of three digoxigenin-labelled probes specific to various regions of the 16S rRNA gene of the RLP. Statement of relevance The manuscript shows that farmed abalone, showing no disease symptoms, can remain healthy despite the presence of intracellular bacteria colonising their digestive glands. The study shows that Rickettsiales-like prokaryotes detected in farmed abalone are not necessarily “ Candidatus Xenohaliotis californiensis”, the causative agent of withering syndrome. [ABSTRACT FROM AUTHOR]

South Africa has a rapidly developing abalone aquaculture industry based largely on the cultivation of Haliotis midae . The prophylactic use of probiotic microorganisms has been shown to enhance the health, growth and nutritional status of a variety of cultured aquaculture species. Furthermore, research has shown that H. midae fed a high protein artificial diet supplemented with the proteolytic marine bacterium Vibrio midae SY9 have enhanced digestive protease activity levels, protein digestion and absorption, and growth. The predominant V. midae SY9 extracellular alkaline protease, VmproA, has been previously shown to be present in the crop/stomach and intestinal regions of H. midae fed a diet supplemented with V. midae SY9. Therefore, VmproA may be responsible for the probiotic properties of the bacterium in farmed abalone. In this study, the role of VmproA on abalone growth was investigated using the mutant strain V. midae SY9Mut2, which is unable to produce VmproA. Over the course of a 180 day laboratory-based growth trial the growth rate of small (28 mm) abalone improved by 6.5 and 14.5% following dietary supplementation with V. midae SY9 and V. midae SY9Mut2, respectively, in comparison to abalone fed a basal diet. In situ protease activity within the H. midae digestive tract was significantly increased in animals fed the V. midae SY9 supplemented diet compared to abalone fed either the basal feed or the V. midae SY9Mut2 supplemented diet. Thus, VmproA does not appear to be the factor responsible for increased growth of probiotic-supplemented abalone. Statement of relevance The manuscript addresses the mechanism of how probiotics function in a farmed abalone species. This is important since probiotic supplementation of feed is proving successful in increasing the growth of these molluscs, and yet very little is known regarding the underlying relationship between the host animal and the probiont. [ABSTRACT FROM AUTHOR]

This report describes the use of pyrosequencing technologies to generate the first comparative analysis of de novo assembled transcriptome data from cultured and wild specimens of the South African abalone. The transcriptome data and database described here provide a significant genomic resource for abalone research. The data set annotated 11,240 genes, which matched genes with known functions in other species. A large number of transmembrane protein domains (4087) that may indicate a high portion of undiscovered gene receptors were identified. Further, we detected an interesting set of transcription factors (516) that are valuable candidates for participating in regulatory events in developmental (such as cell proliferation and differentiation) and reproductive processes. [ABSTRACT FROM AUTHOR]

Although there has been an increase in our understanding of the shell-boring polydorids that infest abaloneHaliotis midaein South Africa, abalone from a limited number of farms, and wild populations from east of Cape Agulhas only, have been examined. To gain further knowledge and a more complete understanding of the local distribution of polydorids, we examined up to 30 abalone from each of 14 farms in the Northern, Western and Eastern Cape provinces, and five wild sites in the Western Cape, west of Cape Agulhas. Farm and wild communities were significantly different from each other (ANOSIM,r= 0.632,p< 0.002) and Bray–Curtis cluster analysis showed that most farms clustered separately from wild sites.Boccardia proboscideawas present on all but one of the infested farms, whereas some were also infested byDipolydora capensis. By contrast,D. capensiswas present at all wild sites sampled, whereasB. proboscideawas absent from these sites.Polydora hoplura, a common shell-boring pest, was present at only two farms and two wild sites. There appeared to be some exchange of polydorids among farms and between farms and wild abalone. The farm on the East Coast did not cluster with any other sites, indicating a unique composition of polydorids on that coast. No new, potentially problematic, species was encountered, although four species showed an increase in their known distribution. [ABSTRACT FROM AUTHOR]

Abstract: This study was conducted to evaluate the effects of rainbow trout silage oil on the immune function, growth performance and foot muscle fatty acid profile and proximate composition of South African abalone Haliotis midae. Rainbow trout silage oil was prepared from farmed rainbow trout processing waste, included at 25gkg−1 in a formulated diet and compared to a control diet in a 153day growth trial. It was found that rainbow trout silage oil addition significantly improved abalone cellular immune function by increasing hemocyte phagocytic activity from 48.8% in the control diet to 65.2% in the silage oil diet. However, production performance was negatively impacted: specific growth rate (SGR) and final animal weight were decreased 17% and 5.6% respectively, and feed conversion ratio (FCR) increased by 25.1%. Foot muscle fatty acid profile and proximate composition were unaffected. It is concluded that dietary fish silage oil inclusion can improve cellular immune function in H. midae, but that the optimal inclusion level should be determined in order to negate the negative effects on production efficiency. [Copyright &y& Elsevier]

Abstract: Dietary hydrolysed proteins have been shown to stimulate the non-specific immunity of various finfish species, whilst the potential for immune stimulation of these feed ingredients in abalone has not been investigated. The immune-stimulating potential of two hydrolysed protein sources (self-prepared fish silage and a commercial fish protein hydrolysate) at two dietary inclusion levels in abalone diets was therefore measured in the South African abalone Haliotis midae, using animals of initial mean weight of 123g–128g. Diets containing the high inclusion levels were also fed in two feeding regimes: continuous feeding, or phase feeding, where the hydrolysed protein diets were alternated monthly with the control diet. In low inclusion diets, hydrolysate inclusion contributed 6g·kg−1 protein in the final diet, whilst in high inclusion diets this was increased to 18g·kg−1. It was found that the low inclusion level of the commercial hydrolysate significantly increased the cellular immunity through increasing the phagocytic activity of haemocytes by 18% compared to the control diet, whilst none of the other diets showed any significant differences compared to the control. Both inclusion levels of the commercial hydrolysed protein significantly improved daily weight increase of animals. Phase feeding had no positive impacts on immunity or production performance. The inclusion of both types of hydrolysed protein sources significantly decreased the water stability of feeds compared to the control, except at the lowest inclusion level of the commercial hydrolysate. It is concluded that the use of dietary hydrolysed proteins can lead to improved cellular immunity and growth in abalone, however it is important to determine appropriate inclusion levels to prevent negative impacts on feed water stability and production performance. [Copyright &y& Elsevier]

The South African abalone Haliotis midae is a commercially valuable species, but its numbers are seriously depleted due to illegal fishing. Overfishing not only affects the targeted species, but also potentially influences the integrity and functioning of the ecosystem through associated changes in community composition. We assessed the herbivorous effects of H. midae on the benthos through a 6-month field experiment involving exclusion/inclusion plots. This showed that benthic community composition remained largely unchanged whether H. midae was present or absent and specifically revealed no evidence that H. midae diminishes algal biomass, probably because in the region where we worked, it feeds mainly by trapping drift kelp rather than grazing on attached algae. The absence of responses to the removal of abalone was additionally evident in comparison with indices of abundance and diversity, functional groups and individual species. In the absence of abalone, there were subtle increases in the green alga Codium stephensiae and the crustose brown alga Zeacarpa leiomorpha and decreases in the four species of foliose macroalgae, but none of these effects were significant. The presence of H. midae thus had no negative grazing effects on any elements of the epilithic community composition. It does, however, have other influences, including occupation of space and provision of habitat in the form of its shell, which supports a community significantly different from the surrounding rocks. [ABSTRACT FROM AUTHOR]

The farming of abalone, Haliotis midae L., can be intensified in serial-pass systems, but water re-use increases the concentration of NH3 (free ammonia nitrogen, FAN) and reduces water pH. Changing the percentage dietary protein from 33% to 26% reduced the concentration of FAN (F42, 252 = 2.79; P < 0.0001) in a serial-pass system and did not reduce weight gain (F1, 12 = 1.09; P = 0.31) or length gain (F1, 12 = 1.08; P = 0.31). Low water pH was the most important variable to contribute to a reduction in abalone growth (weight gain: F1, 19 = 64.5; P < 0.0001; r2 = 0.76; length gain: F1, 19 = 41.9; P < 0.0001; r2 = 0.67). In addition, supplemental oxygen (103% saturation) improved length gain (t = 3.45, P = 0.026) in abalone exposed to an average FAN concentration of 2.43 ± 1.1 μg L−1) and an average pH value of 7.6 ± 0.13, relative to a treatment with no oxygen supplementation. Thus, in an abalone serial-use raceway with three passes, FAN was not the first growth-limiting variable. It is suggested that future studies should examine the major causes of reduced water pH in serial-use systems and their effect on the growth and health of H. midae. [ABSTRACT FROM AUTHOR]

Probiotics have been widely reported to increase the growth rate of commercially important fish and shellfish by enhancing the digestion of ingested feed through the production of extracellular enzymes such as proteases and alginases. In order to investigate this further, the objective of this study was to localise the bacterial probiont Vibrio midae SY9 and one of the extracellular proteases it produces in the digestive tract of the South African abalone Haliotis midae. This was accomplished by inserting a promotorless gfp gene into the chromosome of the bacterium which was incorporated in an artificial, fishmeal-based abalone feed. In situ histological comparison of abalone fed either a basal diet or the basal diet supplemented with V. midae SY9::Tn10.52 using a cocktail of DNA probes to the gfp gene localised the probiont to the crop/stomach and intestinal regions of the H. midae digestive tract. Generally, the ingested probiotic bacterium occurred in association with feed and particulate matter within the crop/stomach and intestinal regions, as well as adhered to the wall of the crop/stomach. Histological immunohistochemical examination using polyclonal anti-VmproA antibodies localised an extracellular protease produced by V. midae SY9 to the H. midae crop/stomach and intestine where it appeared to be associated with feed and/or other particulate matter in the abalone gut. Thus the data suggests that V. midae SY9 colonises and/or adheres to the mucous lining of the abalone gut. Furthermore, the close association observed between the bacterium, its extracellular protease and ingested feed particles supports the theory that V. midae SY9 elevates in situ digestive enzyme levels and thus enhances feed digestion in farmed abalone. [ABSTRACT FROM AUTHOR]

Probiotics have been widely reported to increase the growth rate of commercially important fish and shellfish by enhancing the digestion of ingested feed through the production of extracellular enzymes such as proteases and alginases. In order to investigate this further, the objective of this study was to localise the bacterial probiont Vibrio midae SY9 and one of the extracellular proteases it produces in the digestive tract of the South African abalone Haliotis midae. This was accomplished by inserting a promotorless gfp gene into the chromosome of the bacterium which was incorporated in an artificial, fishmeal-based abalone feed. In situ histological comparison of abalone fed either a basal diet or the basal diet supplemented with V. midae SY9::Tn10.52 using a cocktail of DNA probes to the gfp gene localised the probiont to the crop/stomach and intestinal regions of the H. midae digestive tract. Generally, the ingested probiotic bacterium occurred in association with feed and particulate matter within the crop/stomach and intestinal regions, as well as adhered to the wall of the crop/stomach. Histological immunohistochemical examination using polyclonal anti-VmproA antibodies localised an extracellular protease produced by V. midae SY9 to the H. midae crop/stomach and intestine where it appeared to be associated with feed and/or other particulate matter in the abalone gut. Thus the data suggests that V. midae SY9 colonises and/or adheres to the mucous lining of the abalone gut. Furthermore, the close association observed between the bacterium, its extracellular protease and ingested feed particles supports the theory that V. midae SY9 elevates in situ digestive enzyme levels and thus enhances feed digestion in farmed abalone. [ABSTRACT FROM AUTHOR]

In their natural distribution range and in on land-based mariculture facilities, Haliotis midae may be exposed to a relatively wide range of temperatures and oxygen levels, that can induce differential degrees of stress with concomitant metabolic responses. In the current study juvenile H. midae were exposed for one month to two constant temperatures (14°C and 19°C), with three oxygen levels (82%, 98% and 126% oxygen saturation) at each temperature. One additional treatment was included at 19°C, 126% oxygen, where feed was supplemented with l-proline. The results presented here suggest that long-term 19°C exposure is more stressful to H. midae juveniles than a 14°C exposure of the same duration, since exposure to higher temperature generally resulted in higher activities of the antioxidant enzymes measured but lower total antioxidant capacities. Haemolymph glucose levels responded to oxygen treatment at the 14°C exposure, but the same response was not initiated in the 19°C treatment, possibly because the animals exposed to the 19°C treatment relied on proteins to a larger extent. Increased oxygen levels at the 19°C treatment did not cause more DNA damage or protein carbonylation compared to lower oxygen levels. Supplementing feed with l-proline at the high temperature/high oxygen treatment caused a decrease in haemolymph haemocyanin (which may exhibit catalase function) content and a slight decrease in some of the antioxidant enzyme activities, while maintaining the same total antioxidant capacity compared with the same treatment where feed was not supplemented. The overall DNA integrity profile also changed with proline-supplementation, as did the induction level of hsp70. When projecting tissue growth over a 12month period under these conditions, we estimate that H. midae juveniles in this study may exhibit a significant increase in mass when their feed was supplemented with l-proline, indicating that l-proline supplementation provides some benefit to the individual. It might therefore warrant conducting long-term growth trials on land-based mariculture facilities to further investigate its potential benefit to the mariculture industry. [ABSTRACT FROM AUTHOR]

Illegal fishing activities are reported to be on the increase in South Africa, including in its marine protected areas (MPAs). Research is presented on the nature and the scale of illegal fishing in Table Mountain National Park (TMNP) by analysing the numbers of abaloneHaliotis midaeand West Coast rock lobsterJasus lalandiiconfiscated from fishers operating in the park's marine protected area between 2000 and 2009. Data were collected from offence logbooks maintained by South African National Parks rangers and managers, the South African Police Services, and interviews with alleged or self-confessed illegal fishers. The research findings indicate that the annual numbers of illegally fished abalone and rock lobsters have increased significantly over time. Spatial analysis suggests that confiscations of abalone occur predominantly on the east coast of the park, whereas higher confiscations of illegally fished rock lobsters occur on the west coast. It is clear from this research that new and more efficient approaches will need to be designed and implemented to minimise illegal fishing in the TMNP MPA. Context-specific conservation targets that acknowledge and integrate social as well as developmental needs are required, and may be essential for limiting biodiversity loss in the longer term, which will ultimately ensure the success of fisheries management and conservation in TMNP. [ABSTRACT FROM AUTHOR]

When ectotherms are exposed to high temperatures, cellular oxygen demand increases. When oxygen demand exceeds oxygen availability, animals may experience functional hypoxia regardless of environmental oxygen levels. In the current study, we aimed to assess if oxidative or thermal restrictions exist when winter-acclimated Haliotis midae juveniles are re-acclimated to temperatures within their thermal tolerance range, but with one temperature outside the average daily temperatures they would experience in winter. At each acclimation temperature (14, 16 and 19°C), we also manipulated dissolved oxygen levels to obtain oxygen saturation levels of 82%, 98% and 126%. Our results indicate growth restrictions and hypometabolism at all oxygen treatments at 19°C. Efficiency of mitochondrial respiration, as indicated by an increase in cytochrome c oxidase subunits 1 and 2 (cox1 and cox2) transcript level, was improved at 19°C, 98% oxygen. A change in substrate utilisation, from a glycogen/lipid-driven metabolism to almost exclusively protein-driven metabolism provided the fuel for more effective cellular respiration. A further decrease in metabolic rate at 19°C, 126% oxygen, this time without an increase in cox1 and cox2 transcripts, may indicate some improvement in gas exchange, but without improvement in growth. We propose that 19°C is nearing a long-term limiting temperature for winter-acclimated SA abalone, and that animals survive this condition by re-allocating available energy to survival mechanisms instead of growth. This species, already threatened by over-harvesting, may be further threatened by increases in surface seawater temperatures. [ABSTRACT FROM AUTHOR]

Haliotis midae is one of the most valuable commercial abalone species in the world, but is highly vulnerable, due to exploitation, habitat destruction and predation. In order to preserve wild and cultured stocks, genetic management and improvement of the species has become crucial. Fundamental to this is the availability and employment of molecular markers, such as microsatellites and single nucleotide (SNPs). Transcriptome sequences generated through sequencing-by-synthesis technology were utilized for the in vitro and in silico identification of 505 putative SNPs from a total of 316 selected contigs. A subset of 234 SNPs were further validated and characterized in wild and cultured abalone using two Illumina GoldenGate genotyping assays. Combined with VeraCode technology, this genotyping platform yielded a 65%-69% conversion rate (percentage polymorphic markers) with a global genotyping success rate of 76%-85% and provided a viable means for validating SNP markers in a non-model species. The utility of 31 of the validated SNPs in population structure analysis was confirmed, while a large number of SNPs (174) were shown to be informative and are, thus, good candidates for linkage map construction. The non-synonymous SNPs (50) located in coding regions of genes that showed similarities with known proteins will also be useful for genetic applications, such as the marker-assisted selection of genes of relevance to abalone aquaculture. [ABSTRACT FROM AUTHOR]