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Stanford L. Loeb Anne Spacie. Margaret S. Reproductive Biotechnology in Finfish Aquaculture. However, F1 Nile tilapia transgenic for a construct consisting of a sockeye salmon metallothionein promoter spliced to a sockeye salmon growth hormone gene exhibited no growth enhancement Rahman et al. An all-fish GH construct has been successfully introduced into the common carp, resulting in increased growth rate and more efficient feed conversion as compared to farmed fish controls. Middle-scale trials of these all-fish GH-transgenic common carp have shown high potential for successful commercial application in aquaculture Wu et al.
Transgenic lines of silver sea bream, an economically important cultivated species in Asia, were developed using a construct containing rainbow trout Oncorhynchus mykiss GH complementary DNA cDNA with a common carp promoter Lu et al.
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Insertion of a GH transgene in coho salmon results in accelerated growth, and increased feeding and metabolic rates. Whether other physiological systems within the fish are adjusted to this accelerated growth has not been well explored. Leggatt and co-workers examined the effects of a GH transgene and feeding level on the antioxidant glutathione and its associated enzymes in various tissues of coho salmon. When transgenic and control salmon were fed to satiation, transgenic fish had increased tissue glutathione, increased hepatic glutathione reductase activity, decreased hepatic activity of the glutathione synthesis enzyme g-glutamylcysteine synthetase, and increased intestinal activity of the glutathione catabolic enzyme g-glutamyltranspeptidase.
However, these differences were mostly abolished by ration restriction and fasting, indicating that upregulation of the glutathione antioxidant system was due to accelerated growth, and not to intrinsic effects of the transgene. Transgenic Fish: Issues and Applications 15 Transgenic tilapia containing the GH gene driven by the human cytomegalovirus CMV was compared to nontransgenic siblings on a number of metabolic and physiological parameters Martinez et al. The results showed several significant differences, with transgenic tilapia consuming 3. In addition, growth efficiency, average protein synthesis, anabolic stimulation, and synthesis retention were higher for the transgenic tilapia.
A further investigation into the use of CMV as a GH gene promoter showed no difference in the muscle composition of transgenic fish as compared to non-transgenic siblings Krasnov et al. However, the transgenic fish did show some metabolic features that are often observed in farmed salmonids, such as an increased metabolic rate and faster utilization of dietary lipids, particularly in the case of triglycerides.
In transgenic studies involving the Indian major carp L. Insertion of other GH constructs into tilapia has also yielded positive results, but not as dramatic as those with the salmon GH constructs. Two possible explanations for the difference in results are the type of construct and the type of tilapia studied was different. When introduced into coho salmon, cutthroat trout, Oncorhynchus clarki, rainbow trout, and Chinook salmon, GH gene constructs using either an ocean pout antifreeze promoter driving a Chinook salmon GH cDNA, or a sockeye salmon metallothionein promoter driving the full-length sockeye GH1 gene elevated circulating GH levels by as much as 40 folds Devlin, , resulting in up to 5- to fold increase in weight after one year of growth Devlin et al.
The largest of these P1 transgenics were mated and produced offspring with extraordinary growth. As was seen with three transgenic common carp and channel catfish, the effect of GH gene insertion was variable among families, and multiple insertion sites and multiple copies of the gene were observed. Results with the Atlantic salmon are not quite as impressive as with coho salmon. Transgenic Atlantic salmon also ingested more, exhibiting 2. The results of a recent study into the genetic expression and interactions of GH, the insulin-like growth factor I IGF-I , and their receptors indicate involvement of the hormones in the areas of vertebral growth and bone density Wargelius et al.
Even though the GH excess on fish metabolism is poorly known, several species have been genetically engineered for this hormone in order to improve growth for aquaculture. In some GH- transgenic fish growth has been dramatically increased, while in others high levels of transgene expression have shown inhibition of the growth response. Figueiredo et al. The results obtained here demonstrated that homozygous fish did not grow as expected and have a lower condition factor, which implies a catabolic state.
Varying results among species and families might be related to different gene constructs, coding regions, chromosome positions and copy numbers. Magnification effects can explain some of the growth differences between transgenic and control salmon, however, specific growth rates of the transgenic coho were approximately 2. GH levels were increased dramatically However, additional data on transgenic rainbow trout Devlin et al. When OnMTGH1 was transferred to another wild rainbow trout strain, F77, growth was enhanced 7-fold which was almost 4-fold greater growth than that observed in a non-transgenic domestic rainbow trout.
In this case, the wild transgenic is actually superior to the domestic selected strain indicating that genetic engineering can have a greater, rather than Transgenic Fish: Issues and Applications 17 equivalent effect to the domestication and selection. When F77 was crossbred with a domestic strain, growth of the crossbreed was intermediate to the parent strains, a typical result Dunham and Devlin, The combined effects of transgenesis and crossbreeding had a much greater growth enhancement than crossbreeding or transgenesis alone.
A recent study investigated the effect of supplementing feed with transgenic Synechocystis sp. PCC containing the Paralichthys olivaceus growth hormone GH gene on growth, feed intake and feed efficiency ratio, muscle composition, haematology and histology of turbot Scophthalmus maximus L. PCC was Haematological parameters, including red blood cell, white blood cell, haemoglobin, and serum biochemical indices, such as enzyme activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, concentrations of total protein, glucose, blood urea nitrogen, creatinine, triglyceride and cholesterol and ion levels of K, Na, Cl, P were not influenced by supplementing the transgenic Synechocystis sp.
Furthermore, no histopathological alterations were induced by transgenic alga treatment in the stomach, intestine, liver, spleen and kidney of the experimental fish Liu et al. Hallerman et al. Elevation of GH increases rates of protein synthesis and lipid mobilization, affecting not only growth, but also feed conversion efficiency, metabolic rate, body composition, head and body morphometrics, osmoregulation, and age at maturity, with other modifications particular to species and transgenic lines.
Because of heightened feeding motivation, transgenic fishes often prove more active, aggressive, and willing to risk exposure to predation. Swimming ability of some transgenic lines is reduced. While limited in scope, studies suggest that fish of some transgenic lines show lessened reproductive behaviour. Welfare issues posed by morphological, physiological, and behavioural alternation in GH-transgenic fishes have not been well characterized. The use of weaker promoters in expression vectors and by selection of transgenic lines with physiologically appropriate levels of GH expression may reduce welfare issues that do arise.
Also, markers of welfare for GH-transgenic fish under production Cold and Freeze Tolerance Most efforts in transgenic fish have been devoted to growth enhancement, although there are reports of improvement in cold resistance Fletcher and Davies, ; Shears et al. Early research also involved the transfer of the antifreeze protein gene of the winter flounder Fletcher et al.
The primary purpose of this research was to produce salmon that could be farmed under arctic conditions, but expression levels obtained have been inadequate for increasing cold tolerance of salmon. However, preliminary results with goldfish show some promise for increasing survival within the normal cold temperature range. Some Arctic fish species are known to resist freezing by producing an antifreeze protein which prevents ice crystal formation in the blood, thus lowering the freezing point of the blood plasma Davies et al.
This phenomenon was eventually attributed to a set of peptides and glycopeptides termed AFPs and antifreeze glycoproteins AFGPs , respectively. These proteins are synthesized primarily in the liver and secreted into the blood and extracellular space, where they bind and modify the structure of microice crystals, thereby inhibiting ice crystal growth and lowering the freezing point of body fluids Davies and Hew, ; Raymond, Fletcher et al. However, the level of AFP was about one one- hundredth of that in winter flounder. Thus, for the production of salmon with low-temperature tolerance, improvement on the gene construct to give higher efficiency of expression would be required Sin, Also, the number of copies and type of AFP genes varies with the fish species: for example, winter flounder Pleuronectes americanus have 30 to 40 copies of type I; sea raven Hemitripterus americanus have 12 to 15 copies of type II; and Newfoundland ocean pout have copies of type III Hew et al.
Transgenic Fish: Issues and Applications 19 transferred and expressed in goldfish Wang et al. The gene was microinjected into the goldfish oocytes and was inherited through two generations. The transgenic goldfish showed significantly higher cold tolerance as compared to controls, suggesting possible use of the transgene for promoting cold resistance in fish.
AFP transgenic technology could be highly beneficial to the aquaculture industry in countries with freezing and sub-zero coastline conditions. Therefore, research is currently under way to develop strains of Atlantic salmon that could be cultivated over a wider geographic range. This could be accomplished by i introduction of a set of AFP transgenes that allow the fish to survive lower water temperatures, or ii introduction of GH transgenes to produce a rapidly growing strain that does not require over- wintering Hew et al.
Although AFP transgenes have been successfully introduced into, expressed in, and inherited through germlines of Atlantic salmon, the cold-tolerant transgenic salmon do not produce AFP in sufficient quantities to achieve freeze resistance Hew and Fletcher ; Fletcher et al. Hew et al. The genomic clone 2A-7 coding for a major liver-type antifreeze protein gene wflAFP-6 was integrated into the salmon genome. From a transgenic founder, an F3 generation was produced. In this study, southern blot analysis showed that only one copy of the antifreeze protein transgene was integrated into a unique site in F3 transgenic fish.
The integration site was cloned and characterized. Northern analysis indicated that the antifreeze protein mRNA was only expressed in the liver and showed seasonal variation. All of the F3 offspring contained similar levels of the antifreeze protein precursor in the sera and the sera of these offspring showed a characteristic hexagonal ice crystal pattern indicating the presence of antifreeze activity. Expression of proAFP was liver-specific and showed seasonal variations not identical but very similar to those in winter flounder.
Although in Atlantic salmon the proAFP precursor may not be processed into mature protein due to the An increase in the copy number of the transgene or the use of constructs with other AFPs that have a higher antifreeze activity might be helpful for successful enhancement of freeze-resistance in farm fish. The intracellular, skin-type family of AFPs that are present in the external tissues of cold-water marine fish and that were recognized as the first line of defence against freezing is currently under consideration for gene transfer Low et al. Transgenic Sterilization Commercial production of transgenic fish will depend on the risk to wild aquatic species.
Fish are typically raised in the sea in netted pens, and escapes are quite common. If the escaped fish breed with their wild counterparts the consequences of spreading the modified genes into environment are unpredictable. For safety, genetically modified fish for human consumption should be made sterile. An alternative method could involve induced sterility in transgenic lines by blocking of the gonadotropin releasing hormone GnRH with antisense RNA. The idea came from experiments with hypogonadal mice Mason et al.
Although use of transgenic fish in aquaculture has the potential to increase food availability and reduce production costs, there is much concern over the possibility of escapement of transgenic fish and contamination of wild populations through interbreeding. Therefore, it is of great interest to develop techniques for preventing introduction of transgenes into wild stocks. Two concepts currently being researched include induction of sterility and gonad-specific transgene excision Hu et al.
Recently, sterility was reported using a transgenic method to inhibit expression of the gene coding for the gonadotropin-releasing hormone GnRH , an important component in gonad development and reproductive function Hu et al. In a series of pilot studies, reversible fertility was achieved in common carp by inserting a gene coding for an antisense RNA sequence that inhibits expression of the In fish, as in other vertebrate, GnRH is thought to play an important role both in sexual maturation and in reproductive behaviour.
They are present in the brain and the gonads and are separately regulated in both tissues von Schalburg et al. Generating transgenic fish with desirable traits e.
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These obstacles may be overcome by producing transgenic fish that are sterile, possibly by inhibiting hormones related to reproduction. In vertebrates, synthesis and release of gonadotropin GtH and other reproductive hormones is mediated by GnRH. Transcripts of both genes were detected in ovary and testis in mature and regressed, but not in juvenile carp. Furthermore, some antisense transgenic fish had no gonadal development and were completely sterile Hu et al. Carp beta actin-tilapia salmon type GnRH antisense construct was injected into Nile tilapia.
Transgenic females were crossed with wild type males. A reduction in fertility of about half that of non-transgenic control females was observed. Fertility was much more greatly reduced in transgenic males crossed to control females. Limited data on transgenic females crossed with transgenic males indicated near zero fertility.
Tilapia beta actin-tilapia seabream GnRH antisense construct was injected into Nile tilapia, but no reduction in fertility of heterozygous transgenic males and females was observed Dunham, Limited data on transgenic females crossed with transgenic males indicated no reduction in fertility. Reciprocal crosses between seabream However, although the transgene was integrated into the genome, transmitted through the germline and the antisense-sGnRH was expressed specifically in the brain of transgenic offspring, the presence of the antisense-sGnRH did not result in sterility of transgenic fish.
Preliminary data indicated that spermiation of transgenic males was only obtained after prolonged treatment with salmon pituitary extract, whereas control males spermiated naturally. Data is still needed for the females. Preliminary results show promise for this approach Thresher et al. To achieve gonad-specific transgene excision, two types of transgenic zebrafish were created Hu et al. One line of zebrafish contained the gene coding for Cre recombinase driven by the T7 promoter along with the desired transgene in this case fluorescent protein flanked by 2 loxP sites. When expressed, the Cre recombinase excises the transgene in between the 2 loxP sites.
The other line of zebrafish contained the T7 RNA polymerase driven by a protamine promoter specifically expressed in the gonad. When these 2 lines of zebrafish are crossed, the offspring specifically express the T7 RNA polymerase in the gonads, resulting in the expression of the Cre recombinase and excision of the foreign gene.
This concept would allow for the creation of transgenic individuals lacking the ability to pass on a foreign gene. Modification of Metabolism A distinct approach, currently not successful, has been to use gene transfer for the modification of metabolic pathways to permit better utilization of food Krasnov et al.
Secondly, an attempt to enhance the utilization of glucose by generation of trout transgenic for human glucose transporter hGLUT1 or rat hexokinase rHKII was undertaken. Glucose transporter and hexokinase have a rate-limiting role in glucose transport and phosphorylation, respectively, while GLO is an enzyme that catalyzes the terminal reaction of the L-AAB pathway. In fish the activities The Cytomegavirus CMV or the sockeye salmon metallothionein promoters were used for the targeted modification of these metabolic pathways Zbikowska, However, in the transgenic trout the GLO protein was not detectable and the interpretation of glucose utilization results was complicated by mosaicism.
Further development of these biotechnology applications will strongly depend on the better understanding of metabolic pathways in fish and their regulation on the molecular level. The omegapolyunsaturated fatty acids n-3 PUFAs eicosapentaenoic acid EPA and docosahexaenoic acid DHA are known to provide a number of benefits to human health, including promotion of visual and neurodevelopment, alleviation of diseases such as arthritis and hypertension, and reduction of cardiovascular problems Bao et al. Fish obtain high levels of these fatty acids through the aquatic food chain and are thus a major dietary source of EPA and DHA for humans.
Recently, a gene construct containing the ndesaturase-like gene from the masu salmon Oncorhynchus masuo linked to a b-actin gene promoter from the Japanese medaka was microinjected into the 1-cell embryos of zebrafish Alimuddin et al. The expression of the ndesaturase-like gene in the transgenic zebrafish resulted in a 1. Despite these changes in fatty acid composition in the transgenic fish, total lipid content remained constant.
This technology has potential in the aquaculture industry as a means to increase levels of n-3 fatty acids in farmed fish, thereby providing consumers with a healthier product. It could also reduce the reliance of the aquaculture industry on ocean-caught feed sources and allow for widespread use of cheaper, plant-based diets rich in ALA. Although these transgenic plants produce variable and limited levels of n-3 PUFAs, they do show potential for use in aquaculture feed. The use of terrestrial plant based diets in aquaculture has a number of advantages over more traditional marine-derived diets Naylor et al.
Plant products such as soybean meal and vegetable oils can supply high levels of protein and energy at a lower cost than marine products Also, some argue that use of plants helps to conserve marine ecosystems by reducing the need for the wild-caught small pelagic fish often used to produce fish feed. However, since plant- based diets differ in composition from the traditional marine diets, it is important to ensure that farmed organisms are able to maintain appropriate levels of nutrients and other beneficial ingredients.
In another effort to facilitate the use of plant-based diets in aquaculture, the gene coding for the enzyme phytase was recently expressed in the Japanese medaka Hostetler et al. Phytase breaks down phytate, the major form of phosphorus in plants. Attempts to add the phytase enzyme to the feed for reduction of phytate waste have shown limited success due to degradation of the enzyme at the high temperatures required for feed processing. Hostetler et al. The transgenic fish were compared to their nontransgenic siblings on three different diets all rich in phytate.
The benefits of polyploidy include better food convertibility, higher output with regard to production systems. In fish and shellfish, gametes can produce triploid, viable offspring with suitable handling Blanc et al. Triploid fish and shellfish are viable and tend to be sterile due to a lack of gonadal development.
This sterility allows for reproductive energy to be diverted toward somatic growth, resulting in higher growth rates for some triploid individuals. Although triploidy has been highly effective for enhancing growth in shellfish, results thus far in fish have shown variable, conflicting results, with reports of triploid fish growing slower, at the same rate, or faster than diploids Dunham, Polyploidy has been studied in China in close to 30 shellfish species, including Pacific oysters, Chinese scallop Chlamys farreri , pearl oyster, and abalone Haliotis discus hannai Beaumont and Fairbrother, The benefits of triploids vary with species, from larger adductor muscles in scallops to increased survival in the Chinese pearl oyster Pinctada martensi Allen, In aquaculture, research has been focused mainly in the development of techniques for producing triploid populations Exadactylos and Arvanitoyannis, Use of triploids in aquaculture can be advantageous when reproductive efforts negatively affect growth, survival, or product quality.
Transgenic Fish: Issues and Applications 25 flesh palatability is reduced as gonad replaces stored glycogen Shatkin et al. Processes that are used commonly can induce sterility in the next generation. Polyploidy in fish is because of individuals with more than two chromosome sequences Leggatt and Iwama, Besides advantages such as increased growth rates, use of sterile triploids in aquaculture can help protect the genetic diversity of native populations and prevent establishment of populations of escaped organisms.
This could be particularly relevant in addressing concerns over use of transgenic organisms in the aquaculture industry and their possible escapement and subsequent mating with wild broodstocks. To prevent unauthorized breeding of farmed shrimp, research into shrimp polyploidy is currently under way, with successful production of all- female, sterile triploids with comparable growth to diploids Sellars et al. Although most triploids are sterile, they still exhibit sexual behaviour, participating in mating rituals and thereby disrupting natural spawning processes within populations Dunham, A potential alternative to the frequent problematic and expensive production of triploids by mechanical induction is the crossing of tetraploids with diploids.
Tetraploids are induced in a similar way as triploids, but during a more advanced stage of embryonic development. Appropriate risk analyses must be carried out in order to evaluate possible detrimental effects on both the environment and human health Kapuscinski, ; Rasmussen and Morrissey, Production of transgenic animals has raised concern regarding their potential ecological impact should they escape or be released to the natural environment.
This concern has arisen mainly from research on laboratory-reared animals and theoretical modelling exercises. In this study, biocontained naturalized stream environments and conventional hatchery environments were used to show that differences in phenotype between transgenic and wild genotypes depend on rearing conditions Genetically wild and growth hormone transgenic coho salmon Oncorhynchus kisutch were reared from the fry stage under either standard hatchery conditions or under naturalized stream conditions. When reared under standard hatchery conditions,the transgenic fish grew almost three times longer than wild conspecifics and had under simulated natural conditions strongerpredation effects on prey than wild genotypes even after compensation for size differences.
A major cause of concern regarding aquaculture is the escapement of farmed organisms into the wild and subsequent interaction with native populations, possibly leading to significant alterations in the properties of the natural ecosystem Ramirez and Morrissey, The escapees can be particularly harmful to wild populations, especially when farming occurs in the native habitat of the escaped fish, when there is a proportionally high number of farmed fish compared to the wild stock, or when the wild population is exposed to pathogens occurring in farmed fish Naylor et al. When transgenic fish breed with wild populations, the resulting fish may acquire transgenes that could alter natural behaviour in areas such as reproduction, anti-predator response, and feeding Galli, Fish containing artificial genes such as those coding for AFPs, increased growth, or increased disease resistance have the potential to outcompete native populations.
Studies involving comparisons of GH-transgenic and non-transgenic coho salmon have shown contradictory results, with reports of GH-transgenic salmon showing increased competitive feeding abilities Devlin et al. Besides the possibility of increased survivability, disease resistant transgenic fish also have the potential of carrying certain bacteria, parasites, or viruses that may be harmful to natural populations.
Although advocates of transgenesis argue that genetically modified organisms GMOs are not too different from species that have been genetically altered by breeding techniques, the general population and many environmental groups remain wary of the concept of artificial gene insertion FAO, Application of transgenic fish in environmental toxicology remains at an early stage. However, while theoretically feasible, the idea of using fish as canaries for detection of contaminants in water sounds intriguing.
Progress in this field has been reviewed by Carvan et al. Transgenic Fish: Issues and Applications 27 Several transgenic lines of zebrafish carrying reporter genes driven by pollutant-inducible DNA response elements were tailored to be utilized as aquatic biomonitors for detection of hazardous substances in water Carvan et al. Briefly, the fish is placed in water containing the environmental pollutant to be tested. Following uptake, distribution, and accumulation of the pollutant in fish tissues, then integrated with the genome response elements that respond to the selected substance are capable of activating a reporter gene.
The reporter gene activity that is proportional to the concentration of the chemical can be easily assayed in the intact live zebrafish. Transgenic fish have been also developed to perform mutation assays to assess potential DNA damage after exposure to chemicals in aquatic environments Amanuma et al.
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On account of the ever growing demand of live, dried and canned abalone, already high prices of this seafood delicacy continue to escalate. Abalone family Haliotidae belongs to a class of marine vetigastropod molluscs, which are distributed along rocky shores and reefs of coastal temperate and tropical waters [ 11 , 12 ]. The abalone family consists of about 56 species all belonging to the genus Haliotis [ 13 , 14 ]. Live abalone achieves higher revenues however, it does deem problematic in terms of transportation and related logistics [ 15 ].
Due to the demand of this prestigious seafood, supply of abalone is under severe pressure; and has led to the increase in the occurrence of abalone farming facilities around the world. Globally farmed abalone species and their location adapted from [ 16 , 17 ]. Cultivation of abalone is widespread in many countries, including USA, Mexico, South Africa, Australia, New Zealand, Japan, Taiwan, China, Ireland, Chile and Iceland [ 17 - 20 ] China is the largest producer in the world with over farms and a total production of approximately metric tonnes [ 9 ].
China, Taiwan and South Africa are considered as the key production powerhouses in the abalone industry Table 2. China is the highest contributor of live product annually, and is still the major market for abalone produced world-wide [ 8 ]. This occurrence is closely related to the economic growth and the increase in personal wealth exhibited by the Chinese population as well as the growth of the Chinese middle class population [ 4 , 15 ]. It has been reported that the total abalone produce reaching markets through harvesting, illegal poaching and natural supply, does not meet demand for this seafood delicacy [ 9 , 18 ].
Global production of abalone indicating world leaders in abalone production, quantity of produce legally harvested and sold, and quantity of product illegally harvested; data [ 20 ]. The South African abalone industry continues to establish itself as a premium brand in Asia, and is a good example of mariculture in a developing country. Abalone farming in SA is a relatively new but dynamic industry and has demonstrated a high production capacity [ 15 ].
One of the main challenges faced by the SA industry is the loss in revenue experienced due to poaching. Reports suggest that approximately tonnes are lost to the economy [ 4 ]. As a result, an economic environment whereby abalone aquaculture has become increasingly attractive as a financial investment has been established [ 17 ]. Abalone rearing facilities employs an intensive system in which abalone is reared at high densities in shore-based aquaculture systems [ 21 ]. The abalone H. Even under farmed conditions, abalone growth is slow and often varies with size and age [ 23 ].
Mariculture of abalone is thus important to ensure market supply and it is for these reasons that alternate approaches involved in the promotion of abalone growth and an increased immunity to disease of farmed abalone are required. Land based aquaculture of abalone has increased over the last decade in South Africa Figure 1 , and commercially produced abalone has almost completely replaced the wild harvested product [ 4 ].
In the output of all facets of abalone harvest totalled The former status of abalone aquaculture in South Africa is outlined in Table 3. These farms produced tonnes of abalone, and created direct employment to about people. There was an increase in skilled individuals of approximately 7. Due to the high demand for this seafood delicacy, a gross turnover of approximately R million per annum was achieved [ 26 ]. The industry has demonstrated continued growth. It was estimated that by the production of abalone would amount tons with a value of R million, making abalone mariculture the leading subsector contributor in the aquaculture industry [ 4 ].
This growth has had a direct impact on the socio-economic growth of the country, whereby more than people with necessary skills are currently employed in the industry. Global aquaculture initiatives have shown that the success of the technology is largely dependent on government sectors for support to enable the creation of a robust and sustainable industry [ 15 ].
The mariculture of abalone and on-going growth of this industry is extremely important, as it addresses a number of challenges faced by the South African nation, which are also common to many developing countries. This practice will contribute to a number of strategic imperatives including economic and enterprise development, job creation, food security as well as the adoption of sustainable mariculture practices [ 15 ]. The status of abalone aquaculture and total investment in the South African abalone industry between and [ 26 ].
Many aquaculture farmers, including those in the abalone mariculture sub-sector are faced with a myriad of challenges [ 28 ]. The challenges are further exacerbated as abalone mariculture activities become more intensified to optimise efficiencies in land usage and productivity. Adversities faced include slow growth rate of abalone, the outbreak of diseases, waste accumulation and deterioration of environmental conditions within the culture system [ 29 , 30 ].
Disease occurrence is usually associated with primary invasion by pathogenic strains as well as mechanical injury coupled to stressful environmental conditions such as physiochemical changes and poor water quality [ 31 ]. These factors, in an interactive way, challenge the health and immune response of the abalone and can lead to poor growth, ill health and increased mortality.
This predicament has become one of the main barriers towards the successful development in the aquaculture industry, given that it limits the production of aquaculture products in terms of quality, quantity, and regularity [ 23 ]. Disease control is an inherent part of any animal production system, however, in the aquatic environment, the intimate relationship between bacteria and their host, and the use of open production systems adds to this challenge [ 5 ]. Unpredictable mass mortalities still occur in the early life stages as a result of the proliferation of pathogens and opportunistic microorganisms, which are responsible for major economic losses [ 1 ].
Abalone like other aquatic species is susceptible to common marine pathogenic organisms such as Vibrio parahaemolyticus, Vibrio anguillarum and Vibrio carchariae , as well as prokaryotes and viruses [ 23 , 32 , 33 ]. When pathogenic bacteria or viruses are detected, farmers usually apply antimicrobial compounds to the feed and the rearing water [ 34 ]. Broad-spectrum anti-microbials have been extensively used as a means of disease control on many aquaculture facilities and unfortunately remains the method of choice for many farmers [ 23 ].
Some farmers also use antibiotics as prophylactics in large quantities, even when pathogens are not evident. This ill-advised practice has led to an increase in Vibrios , and other opportunistic pathogens, which possess multiple antibiotic resistance and as a result leads to the emergence of more virulent pathogens [ 28 , 35 ]. Plasmid-carrying resistance determinants have been transferred in-vitro from aquatic pathogens to human pathogens, such as from V.
Furthermore, the presence of antibiotic residues in the tissues of animals, an imbalance of microorganisms in the gastrointestinal tract of aquatic species and the release of antibiotics into natural waters, and thereby poses further challenges. Consequently, the indiscriminate use of antibiotics confers a negative effect on the health of aquatic host species, the environment and consumers of food products [ 37 ]. Due to these concerns, more stringent regulation of antibiotic use in aquaculture has been imposed by the European Union [ 38 ].
Since the application of antibiotics is problematic, a strong demand for alternative methods of disease control is required in abalone mariculture. Abalones are generally regarded as opportunistic herbivores that readily accept a wide range of diets. In natural ecosystems, abalone feed primarily on seaweed or kelp. This food contains a high degree of alginolytic material that is not readily digestible; as a result, enteric microflora is relied upon to effectively digest this material.
If the host intestinal flora lacks the ability to produce beneficial enzymes, a very slow digestion process would result, and consequently hinder the growth of the abalone itself. The proper nutrition and resultant growth of cultured abalone are critical factors that require insight in order to successfully culture this mollusc. Appropriate mechanisms for feeding of abalone are therefore very important and it has been shown that different diets results in different growth rates [ 39 ]. Growth rates, especially at the early life stages of abalone are affected considerably by the diet and the ability of the individuals to utilize available food with a high resultant feed conversion ratio [ 40 ].
An optimum formulated diet should enable more efficient digestion consequently resulting in higher feed conversion ratios, and ultimately boost the growth of the abalone, but the reality is that diets are based on raw material availability and minimum cost formulation models. This presents a challenge in digestibility, feed conversion efficiency, animal health and waste generation into the culture environment. The development of artificial feeds and specialized feeding regimes to improve the growth of abalone has assisted in developing this practice into a more cost-effective and manageable industry [ 21 ].
Incorrectly formulated diets, may also lead to the accumulation of waste in the culture system which could cause the deterioration of water quality in the culture environment. The propensity of algal blooms and the proliferation of disease-causing parasites and pathogens increases in the event of waste accumulation due to poor husbandry and poor feed digestibility. The abalone itself then becomes highly susceptible to disease due to these negative conditions in the mariculture water and succumbs to such challenging conditions.
Additionally, the digestive systems of these aquatic hosts are in constant contact with the rearing water, making the host more prone to infection. In conventional mariculture operations, due to the high stocking densities, the generation of elevated stressful conditions in the culture environment is a frequent occurrence [ 41 ]. During the sorting process, abalones are presented with further stresses due to excessive handling and may sustain mechanical damage.
Both disease and the deterioration of the environmental conditions are the most significant contributors to mass mortalities in mariculture operations [ 42 ]. Most operations employ land-based cultivation systems and use pump ashore technology which is energy intensive and costly [ 15 ]. The dilution of culture water, to reduce waste concentrations, by increasing flow rates is therefore not a feasible option.
Regulatory authorities are also becoming more stringent on the poor quality of farm effluent that is returned to the sea, as a result, preservation of the surrounding environment also becomes a serious challenge to abalone farmers. Bearing in mind that these factors are interactive and ultimately; either as singular occurrences or in combination, may result in decreased production and potential negative impact on the entire aquatic system. Improving digestion, reducing the concentration of waste and disease causing agents in the surrounding water and a heightened immune response are logical mitigation considerations to address the challenges of abalone mariculture.
However, classical interventions are costly and mass mortalities continue to occur, resulting in severe setbacks on both economic and social fronts.
Aquaculture Microbiology and Biotechnology, Volume Two
In more serious instances, some farms have had no other option but to cease operations. The abalone mariculture industry is therefore in dire need of suitable interventions that can address these challenges in an affordable and sustainable manner. During the past two decades, the use of biological agents, particularly in feed and as water additives, as an alternative to the use of antibiotics and chemicals has shown to be promising in aquaculture, particularly in fish and shellfish larviculture [ 43 ]. Biological agents are emerging as a significant microbial supplements in the field of prophylaxis [ 36 ].
Many studies to date have revealed the potential of these beneficial organisms to combat disease in an aquaculture environment [ 5 , 45 - 51 ]. In aquatic ecosystems there is an intimate relationship between microorganisms and other biota in the environment [ 47 ]. Apart from the aquatic animal being surrounded by water, there is also a constant flow of water through the digestive tract of the aquatic animal.
This consequently affects the synergistic balance of indigenous microflora associated with the cultured animal. The classical definition of a probiotic being that of microbes added to food, has become modified with respect to aquaculture, whereby a biological agent is used as a wider term and is defined as "a live microbial adjunct which has a beneficial effect on the host by modifying the host-association or ambient microbial community, by ensuring improved use of the feed or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient environment" [ 47 ].
Some studies have shown that as a result of intensification of aquaculture farms a negative impact has been conferred on the composition of the different protective microbial flora interacting with the host [ 5 ]. This occurrence results in an increase in susceptibility of the host to diseases. It has become evident that augmentation of aquaculture systems with biological agents can lead to growth of beneficial bacteria thus improving overall health of the culture system and the host [ 5 ]. The use of biological agents in disease control and improvement of aquaculture is important as demand for environmentally friendly aquaculture practices is on the rise.
Biological agents that may be applied in aquaculture comprise of isolates belonging to a wide range of yeast, bacteria and even phytoplankton species [ 52 ]. In abalone aquaculture, potential probionts listed to date include, Vibrio spp. Biological agents have been found to confer beneficial effects on the host by various modes of action. These may occur as a singular or combined effect, and thus far the following have been reported; 1 the production of antimicrobial products; 2 competitive exclusion; 3 colonisation of the gut and improving microbial balance; 4 enhancement of the host immune response; 5 detoxification of harmful compounds; 6 improved growth rate of the host; 7 antiviral effects, 8 provision of nutrients and enzymatic functions; and 9 improved water quality.
Further reports by [ 24 ] stated that the addition of probiotics to the diet of farmed abalone, could possibly lead to a boost in abalone growth by a number of potential strategies. Some of which include 1 increasing the nutrients accessible to the abalone for absorption in the gut, 2 increasing the pool of secreted digestive enzymes in the gut of abalone, and 3 use of bacterial supplements as an additional nutrient source. The production of antibiotics, bacteriocins, enzymes, hydrogen peroxide, siderophores and the altering of the pH levels due to the generation of organic acids are all traits displayed by biological agents [ 47 , 59 - 61 ].
In addition, these biological agents compete with pathogens based on intrinsic growth rate and spacial attachment. Microbial colonisation is characterised by the attachment of the biological agent to the mucosal surface and epithelial cells of the host. This prevents the proliferation of opportunistic pathogens thereby preventing infection [ 62 ]. It is common knowledge that for a pathogen to be active and replicate in a host system, it requires attachment to these surfaces [ 62 ]. When probiotics are administered over a long period, they successfully colonize the gastrointestinal tract, even after cessation of feed supplemented with probiotics.
This occurs since the multiplication rate of these probiotics is higher than the rate at which they are removed, thus a build-up in the intestinal mucosa of the host is observed [ 62 ]. Host nutrition is improved, as the applied probionts secrete high levels of hydrolytic enzymes such as amylases, proteases and lipases; as well as the provision of growth factors such as fatty acids, amino acids and vitamins [ 63 , 64 ]. Some isolates also have the ability to break down potentially indigestible components of the feed thus reducing toxicity and improving feed conversion efficiency [ 23 , 63 ].
Abalones are in most instances, fed a diet consisting mainly of kelp, which is a complex macroalgal polysaccharide deficient in many essential nutrients [ 64 ]. It is therefore imperative that enteric bacteria in the abalone gut are present in sufficient amounts which will adequately facilitate digestion by supplying highly effective polysaccharolytic enzymes [ 23 ].
Many bacteria displaying these properties have been found to exist throughout the digestive tract of H.
Some findings indicated that enteric bacteria isolated from the gastrointestinal tract of abalone were capable of degrading agar, carrageenan, laminarin, and alginate. Related studies have indicated that Debaryomyces hansenii HF1; isolated from larvae of European bass Dicentrarchus labrax demonstrated high levels of amylase and trypsin; which aided in the digestion of feed [ 65 ]. CUMA and Vibrio sp. FUMA showed significant increases in growth of abalone over a day period [ 58 ]. An average monthly improvement in growth of 9. Probiotic organisms persisted in the gut up to a concentration of 10 6 CFU.
Authors, [ 40 ] and [ 66 ] also reported that when probiotics were applied to a host, a higher growth rate was observed, as isolated gut bacteria produced enzymes that were able to aid in digestion thus improving the health of abalone. An inaugural application of probiotics in abalone aquaculture was demonstrated by [ 23 ]. They reported that microbes isolated from the gastrointestinal tract of H. From their study it was discovered that D. Further research demonstrated that these probionts were able to breakdown complex proteins and starches, hence making the subsequent assimilation by abalone easier.
They later formulated a mixture of probiotics using two yeasts and one bacterial strain SS1, AY1 and SY9 respectively for abalone. In addition, increases in intestinal proteolytic and amylolytic activity were observed, in probiotic fed abalone when compared to abalone fed the standard feed devoid of probiotics [ 30 ]. Lactic acid bacteria LAB from different sources and evaluated potential probiotic effects in abalones in-vitro , Lactobacillus sp. Furthermore these organisms were able to colonize the gut of Haliotis gigantea thus enhancing the production of volatile short chain fatty acids VSCFA such as acetic acid.
They later showed that by supplementing commercially available abalone feed with a potential probiotic organism, Pediococcus sp. Ab 1, a change in host intestinal flora was observed.