Browsing by Author "Macey, Brett M"

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    Open Access
    Characterization of Ulva (Ulvaceae, Chlorophyta) species cultured in commercial abalone farms in South Africa, and comparison with closely related wild species, using morpho-anatomical and molecular methods
    (2021) Bachoo, Teejaswani; Bolton, John J; Macey, Brett M; Reddy, Maggie M
    Seaweeds are among the five marine sub-sectors of species cultured in South Africa, with Ulva species cultured at a commercial scale. In South Africa, the annual production of Ulva is approximately 2000 tonnes (wet weight), with the majority of Ulva being grown in landbased paddle raceway systems receiving abalone effluent water. Cultured Ulva is mainly used as abalone feed and for bioremediation of farm effluent water. It is not sold but rather is used either as fresh feed or dried and incorporated into formulated feeds. The main commercial abalone farms growing Ulva in paddle raceway systems in South Africa are Irvin & Johnson (I&J) Cape Abalone, Abagold, Buffeljags Abalone and Diamond Coast Aquaculture in the Western Cape province, and Wild Coast Abalone in the Eastern Cape province. The main aim of this study is to precisely identify the cultured Ulva species as their identity is not clearly understood. This will provide information on the genetic diversity in the cultured material and could enable farmers to select for a species/strain that has the desired traits such as high nutritional value, rapid growth rate, resistance to diseases, and the ability to grow vegetatively, amongst others, so that the best feed is given to abalone. Next, the cultured Ulva species will be compared with closely related seashore Ulva species and with Ulva specimens from the main farming area in Hermanus to see if they are genetically similar. Ulva specimens from these farms, nearby seashores, including the Hermanus abalone farming complex in the New Harbour were identified using morpho-anatomical and molecular methods. The molecular markers employed in this study were the plastid large subunit of Ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL), Internal Transcribed Spacer of nuclear ribosomal DNA (ITS nrDNA) and the elongation factor tufA. The 12 cultured Ulva specimens belonged in the U. lacinulata clade with weak support value of 0.57 for PP in the rbcL tree, high support value of 0.86 for PP in the ITS tree and high support value of 92% and 0.92 for BP and PP, respectively, in the tufA tree. The seashore U. capensis and farmed Ulva specimens belonged in the same large U. lacinulata clade in the rbcL tree. However, the U. capensis samples and the locally cultivated Ulva samples belonged in separate sister clades with a support value of 70% and 0.75 for BP and PP, respectively, in the ITS phylogenetic tree, and 97% and 1 for BP and PP, respectively, in the tufA phylogenetic tree. Therefore, the identity of the cultivated Ulva samples is U. lacinulata and the clade containing the U. capensis samples has now been labelled as U. uncialis as it is an older available name than U. capensis. Furthermore, the foliose U. lacinulata was also found growing attached near the inlets of the Hermanus abalone farming complex in New Harbour. There was no genetic variation within the farmed Ulva samples as they were collapsed as a single haplotype by the three molecular markers. The genetic distance between the U. uncialis and farmed U. lacinulata samples were 0.16%, 0.76% and 0.92% for the markers, rbcL, ITS and tufA, respectively. Even though the low sequence divergence between the farmed U. lacinulata and U. uncialis specimens fits within the range of variability, these two clades are separate species that are closely related. Incongruences between the molecular and morpho-anatomical identification methods were observed, as the morpho-anatomical identification method identified 9 of the 12 farmed Ulva specimens as U. lactuca and the remaining as U. rigida sensu Stegenga et al. (1997). Fewer Ulva species were resolved morphologically because of the overlap in morphological description within U. lactuca sensu Stegenga et al. (1997) and U. rigida sensu Stegenga et al. (1997). Additionally, three new records of Ulva species (U. ohnoi, U. australis and U. stenophylloides) for South African seashore specimens were molecularly identified in this study, and foliose U. compressa was recorded for the first time in the region. In this study, the molecular marker tufA, was the best marker to delimit species, as its internal clades were better supported compared to the other two markers and it was able to better separate the farmed U. lacinulata samples and the seashore U. uncialis samples into two different clades.
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    Open Access
    Chemosensory properties of different Ulva extracts and their effects on Tripneustes gratilla
    (2014) Du Plessis, Darren Scott; Cyrus, Mark Digby; Bolton, John J; Macey, Brett M
    Identifying the chemical compounds that attract urchins to seaweeds will increase sea urchin aquaculture efficiency as these compounds can be utilized in the production of artificial feeds enhancing their attractiveness and palatability. This study investigated the chemosensory preferences of the tropical sea urchin Tripneustes gratilla for four crude extracts of Ulva as well as Fresh Ulva and an artificial feed. The four crude extracts of Ulva (Methanolic, Ethanolic, Chloroform and Chloroform-Methanol extracts) were tested by pair-wise chemosensory trials in a Y shaped maze. Urchins were significantly deterred by both the Chloroform and Chloroform- Methanol extracts as well as the solvent controls and artificial feed, however T. gratilla was significantly attracted to both the Methanolic and Ethanolic extracts. These two extracts were tested individually against Ulva and the urchins could not distinguish between Ulva and the extracts. When these two extracts were tested against one another it was found that the urchins were significantly attracted to the Methanolic extract. The Methanolic extract was then added to the artificial feed and significantly improved the feeds attractability. This indicates that these extracts, or the compounds which are responsible for this effect, can be incorporated into artificial feeds thereby making them more attractive to the sea urchin. The percentage yield of Ulva that each solvent extracted was different, although only the Ethanolic extract was found to be significantly different from the Chloroform and Chloroform-Methanol extracts. Future studies are needed to examine the chemical composition of these specific chemosensory compounds. By incorporating these attractive extracts into artificial feeds, South Africa can potentially improve the cultivation success of T. gratilla therefore expanding the economy of the local aquaculture industry through the export of this highly valued seafood product.
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    Determining larval settlement, post-settlement and weaning substrates and regimes for the sea urchin Tripneustes gratilla in intensive aquaculture
    (2022) Bennett, Michael Andrew; Cyrus, Mark D; Bolton, John; Coyne, Vernon E; Macey, Brett M
    Sea urchins gonads are a highly valued and priced seafood. Demand is stable and unlikely to decline in the future. Successful echinoculture is limited by the ability to produce large enough quantities of viable larvae and/or juveniles economically and efficiently due to a bottleneck in production during the settlement and post-settlement phases. In this study, larval settlement, post-settlement growth, and weaning regimes were investigated in the collector urchin, Tripneustes gratilla. Two cohorts of urchins were spawned for two separate growth trials. Growth trials assessed settlement, post-settlement growth, and the timing of weaning onto macroalgae (Ulva lacinulata). Experimental substrates tested include: Ulvella lens, fresh Ulva, dried Ulva and alginate, dried Ulva and agar, Nitzschia sp. (undescribed diatom), dried Isochrysis galbana and alginate, probiotic Vibrio midae SY9 and alginate, V. midae SY9 and Ulva extract F9 and alginate, an ethanol-alcohol and alginate control, and a null-alginate control (replicates=4, n=35 individuals). The highest average settlement success was achieved on fresh Ulva (67.14% ± 8.45) followed by Ulvella lens (55.71% ± 12.26) and then Nitzschia sp. (40.71% ± 5.88). These treatments were significantly different from all the other treatments (p< 0.05) but not from each other (p< 0.05). U. lens facilitated the greatest significant change in test diameter in T. gratilla post-settlement (difference of 3013µm over 4 weeks) and maintained high survival over this time (61.43% ± 10.47). Weaning was successful at 4 weeks post-settlement but was accompanied by a lag-phase in observable growth that was not observed when urchins were subjected to delayed weaning (three weeks later). Survival of urchins with delayed weaning was significantly greater than that of juveniles subjected to early weaning (p< 0.05): 100.00% compared to 92.50%; and achieving a significantly greater size: ̴̴ 1.5 mm difference in test diameter over 6 weeks. Results suggest that U. lens can induce settlement while maintaining high survival. When inducing settlement using U. lens, fresh Ulva should be placed in the same tank to facilitate increased settlement, U. lens facilitating post-settlement growth thereafter. The timing of weaning is important in facilitating optimal growth.
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    Open Access
    Enrichment of rotifers (Branchionus plicatilis), Artemia nauplii and artificial dry feed with live yeast (Debaryomyces hansenii) for the growth of cultured dusky kob (Argyrosomus japonicus) larvae
    (2013) Rousseau, Meghan; Bolton, John J; Macey, Brett M
    Aquaculture has expanded dramatically over the last fifty years and now contributes almost half of the global total fish production. Since fish are a very important source of protein for humans, aquaculture is an important food source and economic practice. It is therefore important to minimise and manage any factors that may negatively impact on aquaculture operations. One of the major problems in marine fish aquaculture is the high rate of larval mortality in captivity. One way of combatting this problem is by improving overall health of larvae. Probiotics, especially yeasts, can be used as immune boosters, disease control agents and a source of additional nutrients to help minimise this problem. These probiotics can be effectively administered to larvae using live feeds, such as rotifers and Artemia, and formulated feeds. The dusky kob, Argryosomus japonicus, is emerging as a commercially viable marine aquaculture species in South Africa. However, commercial production of this species is being hindered by parasitic disease. Therefore, this study tested the efficacy of (1) incorporating live culturable Debaryomyces hansenii (yeast) cells into rotifers (Branchionus plicatilis), Artemia nauplii and a formulated feed (Nutroscience Pre-starter Crumble), and (2) investigated the effects of the probiotic supplemented feeds on growth and survival of dusky kob larvae. We demonstrated that live culturable yeast cells, incubated at a period of 0.5 hours, delivered significantly more yeast cells per rotifer than the longer incubation periods (p<0.001). For Artemia, the incubation period of 0.5 hours delivered more yeast cells per Artemia, but this incubation period was not significantly better than the longer incubation periods (p=0.204). The starting yeast concentration of 1 x 1012 yeast cells.ml-1 resulted in significantly more yeast cells per rotifer (p=0.042) and Artemia (p<0.001) than the lower starting yeast concentrations. The proliferation of Debaryomyces hansenii on the formulated (dry) feed is significantly higher at a storage temperature of 4 °C than it is at a storage temperature of 20 °C (p<0.001). This could however, be causing a deterioration in the stability of the feed. The number of yeast cells per g of feed significantly decreases after 14 days, indicating a possible loss of culturability and stability of the yeast in the feed, regardless of the storage temperature (p<0.001 for 4 °C; p<0.05 for 20 °C). Drying the feed after coating it in yeast also causes a significant reduction in the number of yeast cells per g of feed (p<0.001). With more replication and more cautionary experimental design, this experiment could be repeated in the future. Unfortunately, the mortality rate of the larvae was so high that the results obtained for the larval growth trials could not be statistically analysed, even though rotifer and Artemia (live feed) incubations were performed.
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    Molecular characterization of Flavobacterium spp. isolated from Rainbow trout (Oncorhynchus mykiss) farmed in Southern Africa and development of a PCR-based tool for differentiating between isolates
    (2021) Komane, Godfrey M; Coyne, Vernon E; Macey, Brett M; Flemming, Leornard
    Bacterial fish diseases caused by yellow-pigmented, filamentous bacteria of the genus Flavobacterium are among those that lead to significant losses in the international aquaculture industry. An increasing number of Flavobacterium spp. have been isolated in association with diseased fish within the aquaculture industry. In salmonids, well known flavobacterial diseases include bacterial cold-water disease (F. psychrophilum), rainbow trout fry syndrome (F. psychrophilum), bacterial gill disease (F. branchiophilum, F. aquatile), and columnaris disease (F. columnare). Conventional diagnosis of Flavobacterium spp. is based on physico-chemical tests, but these tests are time-consuming and labor-intensive, and they are unable to distinguish between closely related species due to morphological similarities. Furthermore, little information exists on the diversity of fish associated Flavobacterium spp. from southern Africa. Recently, numerous, yellow-pigmented, filamentous bacteria were isolated from diseased rainbow trout farmed in South Africa and neighboring Lesotho. The aim of the present study was to elucidate the genotypic and phylogenetic diversity of the Flavobacterium spp. associated with these fish and to develop a new molecular system for rapid and accurate identification and differentiation of the isolates. In order to do this, the genotypic and phylogenetic diversity of ninety bacterial isolates, mostly yellow-pigmented, obtained from the gills, skin ulcers, liver, and kidneys of diseased Oncorhynchus mykiss was assessed following PCR and sequencing of the 16S rRNA gene. A BLAST search of the GenBank database revealed that 47 of the 16S rRNA gene sequences showed high similarity to several Flavobacterium spp.; 6 showed high similarity to Chryseobacterium spp., and 19 non-Flavobacterium isolates were identified, which included, amongst others, Hafnia spp. and Aeromonas spp. Fifteen isolates were excluded from further analysis due to poor DNA sequence data, whilst four isolates showed high similarity to uncultured bacteria and they were also excluded from further analysis. Isolate OM-46 was excluded from the phylogenetic analysis of Flavobacterium spp. since only the forward 16S rRNA sequence was available. Phylogenetic analysis based on the maximum likelihood method confirmed the allocation of 46 isolates as Flavobacterium spp., and 6 isolates as Chryseobacterium spp. Differential identification of the Flavobacterium spp. was achieved following PCR amplification of a hypervariable region of the 16S rRNA gene followed by high-resolution melt (HRM) analysis. Nine Flavobacterium isolates, presumed to be different species based on the phylogenetic analysis were identified and successfully differentiated using high resolution melt analysis. The present study has identified new Flavobacterium spp. (OM-13, OM-39, OM-51, OM-82, and OM84) from rainbow trout farmed in southern Africa and developed a tool that may be useful for the management of flavobacterial diseases worldwide.
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    Open Access
    Probiotic effect of Vibrio midae SY9, Cryptococcus sp. SS1 and Debaryomyces hansenii AY1 on the growth and disease resistance of farmed Haliotis midae
    (2005) Macey, Brett M; Coyne, Vernon
    Although the South African abalone, Haliotis midae, has been commercially harvested since 1949, successful cultivation of this species only began in the 1980s. Since then, the abalone mariculture industry has expanded dramatically and currently produces between 500 and 800 tons of abalone per year with a net farm gate value of approximately R125 million. However, disease has had a severe impact on the international aquaculture industry and is anticipated to become an increasingly important factor, together with the slow growth rate of H. midae, that will negatively impact on the further development and success of the local abalone mariculture industry. Thus, the future of H. midae mariculture in South Africa depends in part on the development of methods to enhance the growth rate and disease resistance of farmed H. midae. Erasmus et al. (1997) showed that abalone enteric bacteria enhanced digestive efficiency by secreting polysaccharolytic enzymes and it was suggested from these results that these bacterial enzymes could affect the growth rate of abalone. Furthermore, an overwhelming body of evidence has shown that probiotic microorganisms can significantly improve the growth rate and disease resistance of aquacultured animals. The aim of this study was to isolate enteric microorganisms from H. midae that are capable of hydrolyzing the various protein and starch substrates included in formulated abalone feeds. Upon identification, the selected microbes would be tested for their ability to colonize the digestive tract, improve digestion, growth and immunity of farmed H. midae.
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    The use of ULVA as a feed supplement in the development of an artificial diet and feeding regimes to produce export quality roe from the sea urchin Tripneustes gratilla (Linnaeus)
    (2013) Cyrus, Mark Digby; Bolton, John J; Macey, Brett M
    Tripneustes gratilla is a fast growing, shallow water echinoid, which occurs across the Indo-Pacific, with its south-western limit in warm, temperate waters of South Africa. The success of T. gratilla cultivation depends, in part, on the development of a high quality, costeffective, gonad-conditioning diet that can produce large, marketable quality gonads. The aim of this research was to determine whether Ulva supplementation would improve palatability, consumption and digestibility of an artificial feed administered to T. gratilla and optimise gonad production and quality. At an inclusion level of 20% (20U), Ulva was shown to significantly (p < 0.05) improve the attractiveness and palatability of a formulated feed, compared to a nutritionally equivalent feed that had not been supplemented with dried Ulva (0U). Food consumption rates (FCR) and apparent digestibility coefficients (ADC %) for protein and energy, using insoluble ash as an indigestible marker, were measured for all experimental diets. FCR was significantly higher (p < 0.05) for urchins fed artificial feeds supplemented with Ulva, when compared to urchins fed non-supplemented feeds, suggesting that the inclusion of Ulva into the artificial diets acts as a feeding stimulant.Increased palatability and consumption subsequently led to significantly increased protein retention in urchins fed the 20U diet. The addition of Ulva to artificial feeds also significantly improved gonad colouration duringgonad enhancement trials. Gonad lightness (L*), redness (a*) and yellowness (b*) were quantified using a hand-held reflected-light, fibre-optic spectrophotometer and showed that, at an inclusion level of 20%, Ulva produced gonads that did not differ in colouration from those fed a natural diet of fresh Ulva (FU), and significantly improved gonad colour, particularly gonad yellowness (b*), compared to the same artificial diet without Ulva supplementation. This response was likely related to Ulva's high β-carotene content. The Ulva content of artificial feed significantly improved gonad colouration, while the formulated portion of the feed produced significantly larger gonads than those produced using a natural diet of fresh Ulva. The 20% Ulva inclusion diet, in particular, increased the gonad somatic index (GSI) by 205%, in just nine weeks (7.6 to 23.3%), compared to a 57% increase in the control group (fresh Ulva). Full life-cycle growth trials were also conducted using two of theformulated feeds (20U & 0U), as well as fresh Ulva, to establish appropriate feeding regimes thatcould produce a harvestable product in the shortest time. During grow out, juvenile somatic growth needs to be maximised until an individual reaches marketable size and sexual maturity, which is followed by gonad enhancement, through nutritive cell development,aimed to maximise gonad yield and quality before harvest. The effects of 5 different feeding regimes on somatic and gonadal growth of juvenile T. gratilla were investigated, over a 32 week period. The feeding regimes used were: fresh Ulva (FU) only; fresh Ulva for 20 weeks and the 20U diet for 12 weeks (FU-20U); fresh Ulva – 0% Ulva (FU-0U); 20% Ulva – fresh Ulva (20U-FU) and 0% Ulva – fresh Ulva (0U-FU). Somatic growth was largely effected by the presence of Ulva within a diet. Similar growth, in diameter, of juvenile urchins was achieved using either an artificial diet containing Ulva (20U) or fresh Ulva (FU), during the somatic growth phase.An artificial diet with the same nutritional properties but without Ulva supplementation (0U) produced urchins that were significantly (p > 0.05) smaller. Gonad production in the somatic growth phaseof the trial was higher for urchins fed with artificial diets (0U & 20U), but the reduced size of urchins in the 0U diets significantly reduced gonad mass. After the diets were changed from artificial feeds (0U & 20U) to fresh Ulva (FU) and vice versa (week 20), both gonad size and colour were affected, with artificial diets promoting gonad growth, while FU improved gonad colour. At the end of the study, all feeding regimes produced similar amounts of gonad, except for those individuals that were fed the 0U feed during the somatic growth phase (which were significantly (p > 0.05) smaller). Gonad colourationof all treatments at the end of the study was of marketable quality. From these results it is recommendedthat FU be fed inthe somatic growth phase, while the 20U artificial diet should be used to increase gonad size, and optimise gonad colour, in the gonad enhancement phase. Through the use of stable isotope mass spectrometry and IsoSource, a mixing and mass balance model, the relative contribution and importance of specific feed ingredientsto gonad production was determined. Ulva was shown to be an important isotopic source for gonad production, accounting for an average of 33% of the isotopic signal across all Ulvacontaining, diets at the end of the trial. The final section of work focused on efforts to manipulate the gametogenic cycle of T. gratilla, by altering daylength, to attempt to reduce the production of large amounts of gametes, which would decrease gonad value. Histology indicated that urchins exposed to a Short day (8:16 h) were significantly more advanced reproductively, with the majority of urchins in a mature or spent state compared to urchins exposed to a Long day (16:8 h), which were mostly premature. Nutritive phagocyte (NP) density within the gonads supported the findings from histology, as gonads from urchins exposed to a short day had significantly less NP's (21.58 ± 4.35%), compared to the Long day treatment (65.26 ± 3.09%). The results from this study suggest that urchins of this species exposed to Long days progress through gametogenesis more slowly than those exposed to Short days, which, appeared to mature more rapidly. These findings could allow for the production of a high quality product for longer periods of the year, without the onset of gonad maturation. The research in this thesis clearly shows that the use of the macroalga Ulva as a feed, or feed additive, to artificially formulated, high protein feeds can have a number of significant benefits in echinoculture. The use of the artificial feeds and feeding regimes developed in this work, along with the ability to manipulate daylenth to suspend gametogenisis, could greatly facilitate the success of the newly developing T. gratilla industry, both in South Africa and worldwide.