Recent Technological Innovations in Aquaculture

Rohana P. Subasinghe1, David Curry2, Sharon E. McGladdery3 and Devin Bartley4

1
Senior Fishery Resources Officer (Aquaculture)
FAO Fisheries Department
Rome, Italy

2
Old Farmhouse, Carnbo
Kinross, KY13 0NX
United Kingdom

3
Aquatic Animal Health, Oceans and Aquaculture Science
Department of Fisheries and Oceans Canada
200 Kent Street (8N180)
Ottawa, Ontario K1A 0E6
Canada

4
Senior Fishery Resources Officer (Inland Fisheries)
FAO Fisheries Department
Rome, Italy

Introduction
Aquaculture faces many challenges over the next decade, notably, combating diseases and
epizootics, broodstock improvement and domestication, development of appropriate feeds and
feeding mechanisms, hatchery and grow-out technology, as well as water-quality management.
These all present considerable scope for biotechnological and other technology interventions.
Aquaculture biotechnology can be described as the scientific application of biological concepts
that enhance the productivity and economic viability of its various industrial sectors (Liao and
Chao, 1997). The Convention on Biologicial Diversity defines Biotechnology as, “any technological
application that uses biological systems, living organism, or derivatives thereof, to make or modiy
products or processes for specific use”. Biotechnology encompasses a wide range of approaches
that can improve subsistence and commercial aquaculture production and management.
Although some biotechnologies are modern and novel, others have a long history of application,
e.g. fermentation and fertilization of ponds to increase feed availability. Many modern
biotechnologies are based on rapidly evolving knowledge of molecular biology and genetics.
The major biotechnology sectors involved in aquaculture are similar to those for agricultural
sectors. Development of the knowledge required to optimise safe biotechnological innovation in
aquaculture is of particular significance, and presents a unique set of challenges, due mainly to
the diversity of species cultured and production systems used. A key consideration behind all
technology transfer to the aquaculture sector is that it should be used with due consideration to
the protection of wild aquatic diversity and potential impacts on the autonomy and economy of
rural and subsistence populations. The emphasis on biotechnology and its contribution to food
security, poverty alleviation, and income generation is increasing and we need to be prepared
to address the challenges this will bring, and develop these technologies in a responsible manner.

59
Reproduction Innovations
The application of genetic principles to increase production from aquatic animals currently lags
far behind that of the plant and livestock sectors. Only a small percentage of farmed aquatic
species have been subject to genetic improvement programmes (Gjedrem 1997); however,
biotechnology and genetics have great potential to increase production and enhance ecological
sustainability. Biotechnology can be applied to enhance reproduction and early development
success of cultured organisms, as well as expand periods of gamete and fry availability. Genetics
also have the potential to satisfy new markets for farmed products, e.g., for specific market
tastes or aesthetics. Likewise, biotechnology may provide avenues for improving the reproductive
success and survival of endangered species, thereby helping to identify and conserve aquatic
biodiversity. Transgenic technologies can enhance growth rates and market size, feed conversion
ratios, resistance to disease, sterility issues and tolerance of extreme environmental conditions.
In the shrimp aquaculture sector, transgenic shrimp have been reported (Mialhe et al., 1995),
but there has been no successful development to date for commercial culture (Bachère et al.,
1997; Benzie, 1998). However, the use of transgenic organisms in aquaculture (as in other sectors)
is controversial and issues of consumer education and acceptance must be addressed.

Carp and tilapia culture in Asia is benefiting from genetics research in a number of areas,
including genetic sequencing and the development of specific genetic markers. Markers are
short unique pieces of genetic code that can help locate genes that are important for growth, sex
determination factors or disease susceptibility (Kocher et al., 1998). Such techniques have already
resulted in genetic improvements in some fish being cultured. The traditional technique used
for many generations by farmers throughout Asia has been selecting fish by desirable phenotypic
traits for breeding, on an ad hoc basis. This has led, in many cases, to in-breeding and suppression
of optimum production performance (Chen Defu and Shui Maoxing, 1995). Improving genetic
understanding across millions of small-scale farms in the Asia region is a difficult challenge,
especially since traditional approaches have focussed on improvement of core stocks that can
then be distributed to farmers.

The GIFT (Genetic Improvement of Farmed Tilapia) project in Asia is an example of a programme
aimed at examining the genetics of an important farmed fish species. The GIFT project has been
working with Nile tilapia hybrids and strains in culture around the region, with a view to
development of pure-bred lines and the distribution of strains of improved performance to
farmers. The programme is a collaborative effort between ICLARM (International Centre for
Living Aquatic Resources Management) headquartered in Malaysia and research institutions
in Malaysia, Philippines, UK and USA. The programme has not yet reached the fully commercial
phase and the ‘improved’ tilapia in most of the participating countries are still under evaluation
by fisheries scientists. The program has, however, shown considerable potential for improving
farm production (http://www.iclarm.org/resprg_1f.htm. Similar breeding programmes for
commercially important carps could bring comparable benefits; indeed, because carp fry
production is typically more centralised than is the case for tilapia, the spread of improved
stocks could occur more readily.

For many species of farmed freshwater fish, there are differences in growth rate between the
sexes. Consequently, the development of techniques to produce monosex populations continues
to be important. Historically, farmers have mainly depended on the use of hormones to trigger
sex-reversal, or on the use of particular hybrid crosses that give skewed sex distributions in their
offspring, in order to produce fish of all one sex, e.g., in tilapias. However, these techniques both
have drawbacks. The use of hormones in food animals is increasingly being questioned by
consumers and hybrid crosses that give skewed sex distributions may not be the best hybrids for
farm productivity. Alternative methods for producing monosex populations include cloning by
nuclear transplantation and gynogenesis. Cloning has been possible for carp for more than
thirty years (Zhu et al., 1985) and can form a useful basis for producing all female fry. In several

60
commercially important carp species, females grow faster than males for the first years of life,
so farmers prefer all-female populations. All-female offspring can be produced from certain
carp species, such as the silver crucian carp (Carassius auratus gibelio), which can reproduce
gynogenetically (monosex female reproduction). Artificially induced gynogenesis has also been
used successfully for many years in China to produce pure lines of common carp, silver carp
and ornamental colour carp (Cyprinus carpio) (Jian-Fang Gui and Qi-Ya Zhang, 2000).

In the case of tilapia, males are preferred for culture as they grow faster than females. Recently
all male populations of tilapia fry have been produced through use of YY chromosome male
fish, sometimes termed ‘supermales’. These are the offspring of a normal male, bred with a
female produced by hormonal sex reversal of genetic male. A quarter of the offspring from such
a mating typically have a YY configuration of their sex chromosomes, instead of the normal XY.
When a YY male is crossed with a normal XX female it produces a high percentage of XY (male)
offspring (Figure 1).

Figure 1. Production of all male offspring via YY males.

Sex differentiation does not depend

entirely on the XY/XX chromosomes, so
a small percentage (normally less than
5%) of the offspring are female, but this
technique allows breeders the freedom
to work with the best culture species
and to avoid the use of hormones in the
production of food fish (Mair et al.,
1999). This technology is now well
developed for tilapia and research is
ongoing for a number of other fish
species. Since consumer resistance to
‘hormone’ treated fish is unlikely to
disappear, technologies such as that of
the ‘supermale’ will become increasingly
important, especially for fish produced
for export markets. The production of
‘superfemales’ by a complimentary
technique to that used for ‘supermales’
may also be feasible.

In some farmed fish species, early

maturation and breeding before
attaining market size is a significant
constraint on production. Energy is used
for egg production at the expense of
growth and, in some cases, such as with
tilapia, ponds can become filled with
undersized fish. This is a notable
problem in Africa with the Nile tilapia.
In such cases, stocking with sterile fry
would be useful. Techniques used to
achieve this include the production of

61
fish with extra sets of chromosomes, i.e., polyploid (triploid and tetraploid) fish (Thorgaard,
1986), or shock treatment using temperature or pressure during early embryo development to
cause retention of multiple sets of chromosomes in each cell and, in most cases, sterility. Artificial
triploids and tetraploids have been induced in many farmed fish species, including crucian
carp (Carassius auratus), bighead carp (Aristichthys nobilis), silver carp (Hypophthalmichthys
molitrix) and common carp (Cyprinus carpio) (Jian-Fang Gui & Qi-Ya Zhang, 2000).

Snapper (mainly Lutjanus spp.) culture has been limited by the availability of supplies from wild
fisheries. Researchers in the southern USA, however, have made considerable recent advances
in hatchery production of one species, the mutton snapper, L. analis, and are proceeding to
ongrowing trials (Benetti et al., 2001). At the University of Miami egg production by this species
has been achieved for the first time using environmental manipulation, rather than hormone
injection. It is hoped that this technique will allow year-round egg production. A similar advance
has been reported by the Hawaii-based Oceanic Institute with the red snapper L. campechanus
(Oceanic Institute News, 2000).

Molecular techniques also show significant promise for aquaculture application, in that they
help provide more accurate information on the genetic diversity of natural stocks and allow
genetic tagging of animals in breeding programmes (Subasinghe et al., 2000). Effective breeding
programmes need to identify and track the pedigrees of individual organisms. Physical tagging
of early life-history stages of many aquatic species is difficult, thus, non-invasive, genetic markers
using microsatellite DNA, and AFLP’s (amplified fragment length polymorphisms) have been
developed to track pedigrees and provide linkage maps to identify quantitative trait loci (QTL’s
– genes coding for characters that have production value, such as growht rates, disease resistance
or cold tolerance) (Garcia et al., 1996, Benzie, 1998, Moore et al., 1999; Agresti et al., 2000).

Increased attention is being directed towards domestication of shrimp species. In order to

minimise environmental impacts and optimise use of genetic diversity, shrimp culture must,
however, break its current dependence on wild post-larvae for stocking (Wang 1998). Wild
larvae may currently be more economical and perform better than some hatchery produced
post-larvae, but there is a constant (and inevitable) risk of introducing pathogens into the culture
environment. Furthermore, there is a significant by-catch of other aquatic organisms. Recent
improvements in husbandry, larval rearing, and larval nutrition, as well as genetic improvement
of farmed shrimp, all have the potential to significantly reduce dependence on wild-caught
postlarvae in the future. For example, considerable success has already been achieved with
shrimp species, such as Penaeus vannamei, in development of specific pathogen free (SPF)
broodstock and some of these broodstock are now becoming commercially available. Similar
work is being undertaken for domestication of giant tiger shrimp (P. monodon) but only
preliminary progress has been made to date.

Endocrine regulation of reproduction has been effectively applied across a broad range of cultured
fish species, however, advancements have been slow with shrimp and molluscs. Recent research
has shown that there is potential for chemical treatment of shrimp gonad inhibiting
neurohormone (GIH) that could promote reproduction without the negative side effects of eye
stalk ablation (Keeley 1991; Wang et al., 2000). Research on shrimp GIH isolation is still ongoing
but elucidation of the structure and function of shrimp GIH, using peptide biotechnology-based
approaches, shows promise for countering the reproductive inhibitory effects of GIH. Further
research in this area is needed, and collaboration between researchers, shrimp culturists, and
resource providers from different regions could expedite its achievement.

62
Disease Management
Production of specific pathogen free (SPF) and specific pathogen resistant (SPR) stocks are two
complementary objectives being developed through shrimp broodstock management
programmes. The specific pathogens for these programs are those listed as ‘notifiable’ by the
OIE, representing direct trade concerns, as well as, significant threats to optimal production
(OIE, 2000, 2001). SPF shrimp are produced by selecting animals shown to be free of specific
pathogens, using them as broodstock, and raising their offspring under strictly controlled sanitary
conditions. SPF shrimp are of value for trade to countries or areas that are free of the specific
disease agent, or for restocking ponds following a disease outbreak and disinfection. By contrast,
SPR shrimp are developed through selective breeding of individuals that have survived
challenges/infections by specific pathogens. These, therefore have great potential to enhance
production in waters endemic for the specific diseases, but are inappropriate for use in non-
endemic waters, as they may carry sub-clinical infections of the pathogen in question. These
specific pathogen approaches are now being applied to shrimp stocking in countries like the
USA, Venezuela and French Polynesia using shrimp species such as P. vannamei and P. stylorostris
(Bedier, 1998). Both approaches produce ‘high health’ (HH), however, many SPF stocks perform
poorly when challenged by other pathogens , since their production under sterile conditions
impedes development of acquired resistance to common, but normally less significant, pathogens
(Browdy, 1998). If the immune or physiological traits of SPR strains are heritable, this has the
potential to confer significant performance improvement at the farm level. Taking this technology
beyond specific pathogens, there is exciting potential for this approach to be adapted to sselection
of lines with high non-specific immunity or high tolerance of physiological stresses that facilitate
opportunistic infections or other pathology (Bedier, 1998). Considering the major contribution
of P. monodon to the global shrimp production and the economic losses encountered due to both
facultative and opportunistic disease outbreaks; it is appropriate and timely to concentrate further
research to develop specific and non-specific resistant broodstock – especially for P. monodon.

Infectious disease is currently the single most devastating problem in shrimp culture and presents
ongoing threats to other aquaculture sectors. In addition, there is increasing concern over the
consequences of newly emerging diseases in aquaculture. Conventional methods of controlling
such diseases, such as chemotherapeutants, are ineffective for many new pathogens (notably
viruses), thus, molecular techniques are receiving increasing attention for pathogen screening
and identification. In addition, these techniques are providing significant insights into
pathogenesis (disease development), showing strong potential for disease control and prevention
programs, as well as for treatments of diseases (e.g., DNA vaccines). The increased sensitivity
and specificity conferred by nucelic acid (DNA or RNA) based probes has provided significant
inroads for early detection of diseases and identification of sub-clinical carriers of infections.
This has had a direct effect on enhancing preventative management and control of disease in
cultured species. Concomitant with this has been a decrease in the need for reactive treatments
using traditional methodologies such as antibiotics, or culling and disinfection. This has been
particularly successful for shrimp broodstock selection and breaking the infection cycle
perpetuated for years by accidental broodstock transmission of viral pathogens to developing
offspring.

In shrimp aquaculture, commercially available molecular probes have been developed for
IHHNV and type-A baculovirus (Durand et al., 1996), whereas commercial probes for other
viral pathogens, such as white spot, SEMBV, MBV, TSV, HPV, YHV are still under development.
As noted above, nucleic acid probes are extremely sensitive and can detect microbial infections
before they progress to produce clinical signs. In addition, such probes can be designed to be
highly specific, thereby allowing more accurate identification of pathogens than was possible
with many non-molecular techniques (Walker and Subasinghe, 1999). This clearly helps with
differentiation between significant and closely related infectious agents, which in turn helps
focus disease management intervention and reduce control costs. Increased efficiency in detecting

63
early stages of pathogen development also reduces reliance on prophylactic and active use of
antibiotics to control disease under culture conditions.

In vitro tissue culture for detection and isolation of pathogenic viruses and intracellular bacteria
are currently available for many fish species (FAO and NACA, 2001; OIE, 2000; Groff and La
Patra, 2000; Chi et al., 1999), although these still require specialised maintenance and quality
assurance to ensure optimum application to fish health needs (Lorenzen et al., 1999; Ariel and
Olesen, 2001). No self-replicating cell-lines currently exist for aquatic invertebrates. Considerable
research has gone into the development and maintenance of crustacean cell cultures but success
has, so far, been marginal (Shimizu et al., 2001; Wang et al., 2000; Walton and Smith, 1999;
Ghosh et al., 1995; Toullec, 1995). Many researchers have managed to develop primary cell
cultures, but most have failed to subculture or maintain them (Le Groumellec et al., 1995). The
situation is similar for molluscan cell-lines (Buchanan et al., 1999, 2001; Cheng et al., 2001;
LaPeyre and Li, 2000). Further research efforts to develop and maintain crustacean and
molluscan cell cultures is required, in order to provide equitable culture options for study of
intracellular infectious agents to those possible for many finfish pathogens.

Transboundary movements of aquatic animals have in some cases lead to the spread of aquatic
animal diseases. Reliable and sensitive diagnostic techniques and standards are required to
ensure such movements of live aquatic animals does not also include the dispersion of their
pathogens. Once DNA probes are field validated and refined for non-specialist use, these will
be particularly valuable tools for this purpose (FAO 2000). For example, once appropriate DNA
probes are developed for specific shrimp pathogens, live and processed shrimp could be certified
to be free of specific pathogens, thus promoting confidence in the shrimp culture industry, and
facilitating access to wider international markets.

Besides screening for pathogens, biotechnological methods can be used to ascertaining other
health parameters, including haematocrits, leucocrits, blood cell differentials, neutrophil oxidative
radical production, myeloperoxidase activity and phagocytic functions. Such techniques can be
applied to quantitative protein, immunoglobulin, lysozyme, cortisol and ceruloplasmin analysis
from plasma samples. Methods such as agglutination tests to assay antibody after immunisation
can now be supplemented with immunoassays, such as fluorescent antibody test (FAT) and
enzyme-linked immunosorbent assay (ELISA) (e.g., Bachère et al., 1995; Noel et al., 1996; Austin
1998; Mishra, 1998; Crawford et al., 1999; Romalde, 1999; Pernas et al., 2000; Meloni and
Scapigliati, 2000; Munoz et al., 2000; Nadala and Loh, 2000: Shelby et al., 2001). Also leucocyte
samples from fish blood or haematopoietic organs can be assayed by haemolytic plaque assay
or an enzyme labelled tag (ELISPOT) to determine levels of antibody (plaque-forming cells).
ELISPOT can be used to accurately quantify numbers of immunoglobulin or non-specific
antibody-secreting cells (Anderson, 1995) and are used in immunodiagnosis.

One of the most urgent needs for aquaculture health management is establishment of standards
for quantitative assessment of health status in the broad range of species under culture. Progress
in this regard is being made for certain finfish, however, knowledge of shrimp and molluscan
health (and stress) are still relatively undeveloped. The above mentioned techniques could be
used to develop simple and rapid predictive health tests for use under field conditions by field
technicians, veterinarians and the farmers themselves. Bearing in mind the extensive literature
applied to physiological indices of aquatic animals (especially molluscs) as indicators of
environmental quality (Handy and Depledge, 1999), such tests could provide an invaluable
early warning of stress under hatchery production conditions, where disease losses are usually
acute and catastrophic (e.g., Weirich and Reigh, 2001).

Harnessing the host’s specific and non-specific defence mechanisms in an effort to control aquatic
animal diseases has considerable potential for reducing the impact and losses from diseases.
Immunostimulants and non-specific immune-enhancers are being incorporated into diets to
boost protection. Such methods, however, are still very limited, especially for shrimp, however,

64
the large number of commercial immunostimulants available on the market clearly reflects the
interest in this area as an alternative method to enhance survival from disease challenges. To
date, however, results from biological trials of these commercial products have been highly
variable, thus, further research is required to determine the precise mechanisms of their action
and assess their cost-benefit value (Flegel, 1996; Subasinghe et al., 1998).

Probiotics are generally administered as live microbial feed supplements which affect the host
animal by improving the intestinal microbial balance to optimise the presence of non-toxic species.
A stable gut microflora helps the host resist pathogenic invasions, particularly via the gastro-
intestinal tract. Antibiotics reduce specific or broad-spectrum gut microflora and probiotics
may have post-antibiotic treatment potential for restoring the microbial balance. Probiotics are
widely used in animal husbandry but their use in aquaculture is still relatively new. However,
there are increasing reports of potential probiotics for shrimp aquaculture, which has been
plagued by opportunistic bacteria, such as the luminescent Vibrio harveyi, and, in some cases,
probiotics have been reported to significantly reduce antibiotic use in shrimp hatcheries.
Suppression of proliferation of certain pathogenic bacteria (e.g., Vibrio spp.) in shrimp hatcheries
has been achieved by introducing (inoculating) non-pathogenic strains or species of bacteria,
that compete for microbial metabolite resources. This procedure shows promise to be effective
and economical, however, further refinement of administration and concentration loads required
for effective pathogen suppression is required. Effective and economically viable probiotics also
require greater research into optimal strains of probiotic micro-organisms and stringent evaluation
under field conditions of their economic feasibility.

Beside producing aquatic organisms for food, aquaculture has other important purposes which
help human well-being. Aquatic organisms are often adapted to extreme environments, and
can, therefore, provide unique models for research on biological and physiological processes.
Furthermore, studies of the developmental, cellular, and molecular aspects of aquatic organisms
could provide insights into the basis of disease mechanisms and pathogenesis in humans (Wright
et al., 2000).

Feed Technologies
Currently, one of the most heated debates concerning aquaculture development is the use of
fishmeal and other animal proteins in aquafeeds (Naylor et al., 2000; Forster and Hardy, 2001).
Although fishmeal is used for its high quality protein content, it has several disadvantages,
including high cost and instability of supply. Wild fish catches are on the decline and there are
increasing environmental concerns (eutrophication, pollution associated with excess nutrient
waste), ethical concerns over feeding fish to non-piscivorous fish, and social concerns over using
aquatic protein to feed fish that could be used for human nutrition (especially in nutritionally-
deficient areas of the world) . Although, the major users of fishmeal is terrestrial agriculture,
and the salmon, bream, bass and shrimp farming sectors are using species that would not
normally be used for human consumption, the concerns of consumers provide a strong impetus
to find ways to replace fish meal with vegetable protein from more sustainable sources.
Biotechnology offers opportunities for development of alternatives to fishmeal, especially plant-
based protein sources, by enhancing production and processing techniques. Other technologies
also offer potential for enhancing the efficacy of feed delivery.

Plant protein has significant potential for addressing the problem of phosphorus pollution, since
plants do not contain the high levels of phosphorus found in animal protein. The use of plant
protein in aquafeeds also helps reduce pressure on wild fish stocks. Research in this area is
focusing on the investigation of various plant species and plant-animal protein mixes, as new
sources for protein for aquafeeds for shrimp (Mendoza et al., 2001); molluscs (Shipton and
Britz, 2000) and finfish (Ogunji anf Wirth, 2001). In addition, brewers yeast is another protein
source under investigation for finfish (Oliva-Teles and Goncalves, 2001), along with plant lipid

65
substitutes for fish oils (Ng et al. 2000). One of the difficulties in using plant proteins in aquafeeds
is the need for proper processing to destroy anti-nutritional compounds which may harm the fish
once fed. Researchers are looking at the possibilities of dealing with anti-nutritional factors by
producing feed enzymes to counteract them. Phytase is one example. This enzyme helps fish
make optimal use of the phosphorous available in plant-protein based feeds (Papatryphon and
Soares, 2001; Vielma et al., 2000; Van Weerd et al., 1999; Papatryphon et al., 1999; Storebakken
et al., 1998).

Dependable availability of quality fry to stock grow-out production systems has been one of the
most critical factors affecting commercial success of fish and shellfish production (Sorgeloos,
1995). Although nutritional and dietary requirements of most fish and shellfish species have
been identified, large-scale hatchery production of most aquatic species still depends on live
feeds, such as selected species of microalgae, the rotifer Brachionus and the brine shrimp Artemia.

More than 15 species of diatoms and green algae are used for first-feeding of hatchery produced
fish fry and shrimp larvae. Selection of these species has been done mainly by trial and error,
rather than on a nutritional scientific basis. The live feed production systems used in most
developing countries are still labour intensive. This lowers cost efficiency and poses many
problems for consistent mass production, including optimal nutritional quality and prevention
of microbial contamination. These problems have created a whole new area of biotechnological
research aimed at finding cost effective and efficient supplements to live microalgae, commercial
production of freeze-dried algae, microencapsulated diets, and manipulated yeasts. Results of
much of this work have shown significant success (Garcia-Ortega et al., 2001; Oliva-Teles and
Goncalves, 2001). This area requires further research and shows considerable potential for
reducing reliance on live microplankton in fish and shrimp hatchery production.

Artemia nauplii are the most widely used live feed in shrimp aquaculture (Sorgeloos and Leger,
1992). Considerable progress has been made in improving the dietary value of this planktonic
crustacean through selection of traits and batches, efficient cyst disinfection and decapsulation
(Garcia-Ortega et al. 2001), nauplius hatching, and enrichment and cold storage (Sorgeloos,
1995). Improvement of the nutritional quality of Artemia through bioencapsulation (enrichment),
especially with highly unsaturated fatty acids and vitamins, has improved larviculture outputs
in terms of quality, survival, growth, and stress resistance (Merchie et al., 1995). Bioencapsulation
has also been applied for oral delivery of vaccines, vitamins, and chemotherapeutants (Lavens
et al., 1995; Robles et al., 1998), notably for hatchery developmental stages of finfish (Majack et
al., 2000; Touraki et al., 1996, 1999) and shrimp ((Uma et al., 1999). Research into
bioencapsulation and use of live feed as a means of oral delivery for compounds to enhance
survival and fitness of larval stages of aquatic organisms merits further research priority.

Future aquaculture development ultimately depends on the ability of farmers and processors to
produce a product acceptable to consumers. Increasing consumer demands for quality and safe
products have to be recognised and addressed. Biotechnology also shows promise in this area,
especially for assessing and improving safety, freshness, colour, flavour, texture, taste, nutritional
characteristics, and shelf-life of cultured food products. Tools are already under development,
or commercially available, that can detect and assay toxins, contaminants, and residues in aquatic
products (Jellet et al., 1999; Quilliam, 1999; Marr et al., 1992, 1994; Pleasance et al. 1992).

Biotechnology tools can also be used to identify and characterize important aquatic germplasm
resources, including those of endangered species. The genetic make-up of aquatic species can
now be analysed, characterised and quantitative trait loci identified that code for phenotypic
characters that are beneficial for culture (e.g., fast growth, disease resistance and cold tolerance).
The study of biotechnology can also improve understanding of gene regulation and expression,
sex determination and definition of species, stocks, and populations (Alcivar-Warren, 2001;
Agresti et al., 2000; Davis and Hetzel, 2000; Ward et al., 2000; Moore et al., 1999; Sakamoto et
al., 1999; Liu et al., 1999Cross et al., 1998; Poompuang and Hallerman, 1997). This can be achieved

66
 by marker-assisted gene selection techniques, transgenic manipulations and improved
cryopreservation of gametes and embryos.

Progress in this arena will require sophisticated molecular biology technologies to be adapted to
aquatic organisms, in order to enhance understanding of their biological processes. For example,
approaches for gene transfer into eggs have been developed for many terrestrial organisms and
many freshwater species, but not for most marine species. This technology is needed for analyses
of gene regulatory systems and gene expression. In addition, methods need to be developed for
culturing tissues from marine organisms. Cultured cell lines will provide opportunities for gene
transfer and gene expression studies and enhance the usefulness of marine species as biomedical
research models.

Bioremediation is another promising biotechnological approach for degradation of hazardous

waste to environmentally safe levels using aquatic microorganisms, or other filtering macro-
organisms (Srinivasa Rao and Sudha, 1996). Although this procedure has been used in various
situations, such as sewage treatment, application to shrimp and other aquaculture wastes is
fairly novel. There are a lot of commercial products on the market, mainly bacterial preparations,
but the mode of action and efficacy of many of these have yet to be scientifically measured. In
addition to microbes, bivalves, seaweeds, holothurians (sea cucumbers), etc., have been tested
to assess their ability to reduce organic loading, or reduce excess nutrients produced during
culture production. Various bioremediation preparations have also been developed with the
view to remove nitrogenous and other organic waste in water and bottom sludge, to reduce
chemically-induced physiological stress, e.g., in pond-reared shrimp. More products will
undoubtedly emerge with continued research in this field, however, controlled field trials are
urgently needed to determine the cost-benefit and effectiveness of these products under culture
conditions.

Concomitant with bioremediation is enhanced feed delivery. Aquaculture development in recent

years has, therefore, included investigation into methods for more efficient feeding. Underwater
closed circuit television is in use to record when fish are satiated (no longer feeding), so feeding
can be halted, and also to monitor the accumulation of wastes under moored cages. More recently,
research organisations, such as IFREMER1 , have been looking into the use of demand feeders,
where the fish trigger feeding by learning to push a lever. This method has shown some success
and may have potential for many farmed fish species. IFREMER reported a notable variation in
feed demand by European seabass on a daily and monthly scale (IFREMER, 2000). Training fish
to trigger feeding when hungry offers strong potential to lower feed costs, raise conversion
efficiency and reduce wastage and pollution. IFREMER are also looking to develop faecal
stabilisers for species such as turbot and seabass that have rather liquid waste. Feed additives
that would stabilise the faecal matter would benefit surrounding water quality in sea-cage rearing
situations.

Holding Systems
A notable technology-based development in the US freshwater farming sector has been a
significant expansion tilapia production using indoor closed-recirculation systems. This American
production, however, is still dwarfed by imports from countries such as China, Costa Rica,
Ecuador and Honduras, where production can be achieved using less capital investment.
Although this makes long-term sustainablility of tilapia farming in the US uncertain, there is
ongoing interest in diversifying to other species, such as carp, bass and perch that can take
advantage of lower ambient temperatures.

Recent technological advances in the salmon farming have been particularly in sea-cage design.
In the past, the industry had typically used steel-framed rectangular support structures for the
net cages, with walkways around them as work platforms. With the exception of the pond
rearing of some marine species practised in Asia, this general cage design has also typified the
1
Institut français de recherche pour l’exploitation de la mer
67
commercial culture methods used for most other sea fish, including Asian grouper and snapper,
and the Mediterranean seabass and bream. In recent years, however, there has been a move in
the salmon farming industry towards the use of circular cages with plastic support structures
and incorporating no walkway. Instead the cages are dependent on boats for maintenance. The
feeding of the fish, instead of being carried out by hand or by cannon/blower, is by automatic
cage-mounted machines with a capacity of up to 100 mt of feed. Visits by farm staff can thus be
reduced, lowering costs. As salmon prices have slipped lower, these technology shifts, and a
move towards amalgamation of companies, are allowing the industry to cut operating costs
and retain profitability. The change towards boat-maintained circular cages with plastic support
structures has not yet been seen in Europe’s seabream or seabass industry but it may be a trend
that will develop in these sectors in coming years.

If the commercial aquaculture of marine finfish is to continue expanding, it will likely take place
in more offshore locations than have traditionally been used. Atlantic salmon have been farmed
almost exclusively in sheltered nearshore waters, but this has been linked to production,
environmental and aesthetic problems. At offshore sites, removal/dilution of wastes is facilitated
by greater water exchange and volumes. In addition offshore sites offer greater salinity stability.
Cages developed specifically for offshore culture, such as the Ocean Spar® rectangular Sea
Cage design and the innovative double cone SeaStationTM Sea Cage, have been put into
commercial use in recent years. The Oceanic Institute of the US, in Hawaii, has developed a
similar bi-conical offshore cage design to that of Ocean Spar®, named the SeaStation 3000™
(Oceanic Institute 2001).

The double cone shaped net is suspended on a central floating vertical support column and can
be permanently submerged, with feed administered through a pipe from the surface. Access is
via zippered doors underwater and daily net cleaning is carried out by divers. In times of severe
storms, the structures can be sunk below the high-energy surface waves. An Oceanic Institute
cage 24m in diameter and 15m deep, moored 10m down and 3 km offshore in 30m of water,
has been used to grow batches of 70,000 Pacific threadfin or ‘moi’ (Polydactylus sexfilis) fingerlings
to 3-400g size in 4-5 months. In the USA, a recently formulated national aquaculture policy has
specifically identified open ocean aquaculture as one of two main areas for research and
development. The second being closed system (or “urban”) aquaculture, including research on
recirculating technologies for inland facilities (NOAA, 2001).

Technology from the oil industry has supplied some of the background for the design of such
offshore cages. Another cross-over with the oil industry has been interest in conversion of disused
oil platforms for use as offshore fish farms. The high cost of decommissioning oil drilling and
pumping platforms at the end of their service life makes this an interesting proposition, though
to date, the costs and problems of conversion have proven to be formidable obstacles (Bugrov et
al., 1994; Osborn and Culbertson, 1998). In the USA, recently formulated national aquaculture
policy has specifically identified open ocean aquaculture as one of two main areas for research
and development. (The second being closed system (or “urban”) aquaculture, including research
on recirculating technologies for inland facilities (NOAA, 2001)). This emphasises the likely
future direction of aquaculture.

Another recent development in holding technology has been in closed-circulation systems. These
systems have shown great potential for reducing fishmeal consumption compared with open-
field farming. Although experiments rearing shrimp without water exchange date back to the
1970’s in Tahiti, and to the 1980s in Hawaii and South Carolina, USA, pilot projects have not
moved to commercial realisation. A commercial shrimp farming project in Belize in 1998 - initially
aiming to isolate the farm from the danger of disease introduction - took the technology to a
new level by keeping particulate matter aerobic and in suspension in the growout pond. This
facilitated nitrification of waste products (essential to a healthy rearing environment) by the
bacteria in the pond. As long as the system is aerated, pond conditions can be kept suitable for
shrimp to thrive and the flocculent bacteria and organic matter that form in the water contribute

68
directly to the food of the shrimp. As a result, protein and fishmeal contents of the feed can be
considerably reduced. Closed systems of this kind can also be housed inside buildings and there
are currently numerous projects at early stages of testing in the Americas and Asia to develop
this technology further.

The higher dissolved oxygen requirement of many finfish culture species makes it more difficult
to use similar systems to reduce protein demand in fish feeds but the production of catfish in
closed ponds is one example where closed systems can maximise the feed use and reduce the
need for outside protein inputs (Boyd and Tucker, 1995; Tucker et al., 1996).

Aquaculture Related Enhancement Technology

Sea ranching, where juveniles are produced in hatcheries and then released to the sea to grow,
dates back over one hundred years. There have been some notable successes, for instance with
the Japanese flounder (Paralichthys olivaceus), but there have also been failures where factors
affecting recruitment and loss from the fishery were not properly understood (Howell et al.,
1999). As understanding of the factors affecting the success of ranching programmes has
improved, interest in this technique has spread to new areas and targeted at new species.
Countries such as Norway, USA, Australia and China have all launched stock enhancement
projects. on a variety of species. To promote a more effective exchange of information, the First
International Symposium on Stock Enhancement and Sea Ranching was arranged in Norway
in 1997 (Howell et al., 1999) and a second symposium is to be held in Kobe, Japan in January
2002. Sea-ranching can be one useful approach to increasing overall landings, provided habitat
is adequate and that fishing is prudently regulated (Welcomme and Bartley, 1998)

Pre-Market Conditioning
An interesting sector which has opened up in recent years is the temporary holding of bluefin
tuna (Thunnus thunnus) to improve meat quality. The early development of this activity began in
Australia with the southern bluefin tuna (Thunnus maccoyii), in response to falling catches from
the South Australian wild fishery. Australian landings of this migratory species reached a peak
of 21,500 mt in 1982 but increasingly lower quotas had to be introduced, dropping to 5,265 mt
by 1989. The quality of the product being landed was poor which diminished export value,
thus, fishing/farming companies began holding 2-4 year old fish in cages for 3-5 months for
conditioning. This enhanced meat quality and enabled them to sell to high-value sushi markets
in Japan at prices of around US$18 per kg, or up to US$620 per fish. By 1997, tuna ‘fattening’
had become Australia’s most valuable single aquaculture sector (Brown et al., 1997).

Similar techniques were adapted by fishermen in the Mediterranean (Malta, Croatia and Turkey)
over the last few years, holding Atlantic bluefin tuna captured during the limited fishing season
(May-July). The fish are on a spawning migration at this time, thus flesh quality is poor, and
meat prices are depressed. The fish are held in floating cages until November or December and
fed on mackerel and herring. By then end of the holding period the fish improve in condition
and meet high market price quality for export to Japan. The cages used to hold and transport
the fish are large structures of up to 100 m in circumference and it can take a week or more to
tow them as much as 300 km from the fishing grounds to the holding area.

The capture of bluefin tuna from the declining, and possibly threatened, stocks in the Atlantic
has caused some controversy, thus, there interest in further development of this technology for
true farming – and reduce reliance on wild captured stocks. In addition, there is controversy
over the amount of fish required to feed the tuna during the ‘fattening’ process, especially since
these are species also used for human consumption in the Australian and Mediterranean (cf
discussion under “Feed Technologies” above). This presents a significant challenge, however
for this highly piscivorous migratory species.

69
Conclusion
Aquaculture biotechnology and other technological innovations are showing a positive impact
on aquaculture diversification success, investment potential, and international technology
exchange. The development of biotechnology in aquaculture should provide a means of producing
healthy and fast growing animals, through environmentally friendly means. However, this
development will largely depends on the desire and willingness of the producers to work hand-
in-hand with scientists and the international donor community to assist developing counties in
related research, capacity building and infrastructure development. Improved exchange of
information and discussion between scientists, researchers, and producers from different regions
on their problems and achievements will undoubtedly help this important sector to further
develop with the view to increasing sustainable aquatic animal production globally.

References
Alcivar-Warren, A. 2001. ShrimpMap: A genetic approach to understand immune response and
disease resistance in shrimp. In Aquaculture 2001: Book of Abstracts, World
Aquaculture Society, Baton Rouge, LA, USA, p. 11. (abstract only).
Agresti, J.J., S. Seki, A. Cnaani, S. Poompuang, E.M. Hallerman, N. Umiel, G. Hulata, G.A.E. Gall
and B. May. 2000. Breeding new strains of tilapia: development of an artificial
centre of origin and linkage map based on AFLP and microsatellite loci. Aquaculture,
185: 43-56.
Anderson, D.P. 1995. Novel techniques in fish disease diagnosis. In Diseases in Asian Aquaculture
II. M. Shariff, J.R. Arthur and R.P. Subasinghe, (eds.), Fish Health Section of the Asian
Fisheries Society, Manila, Philippines, pp. 27-42.
Ariel, E. and N.J. Olesen. 2001. Assessment of a commercial kit collection for the diagnosis of the
fish viruses: IHNV, IPNV, SVCV and VHSV. Bull. Euro. Assoc. Fish Pathol. 21(1):
6-11.
Austin, B. 1998. Biotechnology and diagnosis and control of fish pathogens. J. Mar. Biotech.
6: 1-2.
Bachère, E., E. Mialhe, D. Noel, V. Boulo, A. Morvan and J. Rodriguez. 1995. Knowledge and
research prospects in marine mollusc and crustacean immunology. Aquaculture,
132(1-2): 17-32.
Bedier, E., J.C. Cochard, G. Le Moullac, J. Patrois and Aquacop. 1998. Selective breeding and pathology
in penaeid shrimp culture: the genetic approach to pathogen resistance. World Aquacult.
29 (2): 46-51.
Benetti, D., J. Alarcón, O. Stevens, G. Banner-Stevens, F. Rotman, M. Feeley, W. Matzie,
R. Orbun and B. O’Hanlon. 2001. Marine fish culture prospects in Latin American and
Caribbean countries: review of candidate species and technological advances.
Proceedings of the Sixth Central American Symposium on Aquaculture, Tegucigalpa.
Benzie, J.A.H. 1998. Penaeid genetics and biotechnology. Aquaculture, 164: 23-47.
Browdy, C.L. 1998. Recent developments in penaeid broodstock and seed production technologies:
improving the outlook for superior captive stocks. Aquaculture, 164: 3-21.
Buchanan, J.T., J.F. LaPeyre, R.K. Cooper and T.R. Tiersch. 1999. Improved attachment and
spreading in primary cell cultures of the eastern oyster, Crassostrea virginica. In Vitro Cell.
Dev. Biol. Anim. 35(10): 593-598.
Buchanan, J.T., Y. Li and J.F. LaPeyre. 2001. The influence of substrates and culture media formula-
tions on the attachment and spreading of eastern oyster cells in primary
cultures. In Aquaculture 2001: Book of Abstracts, World Aquaculture Society, Baton Rouge,
LA, USA, p. 95.
Bugrov, L.Y., W.B. Murav’ev and O.M. Lapshin. 1994. Alternative using of petroleum-gas
structures in the Caspian and Black seas for fish-farming and fishing: real experience and
rigs conversion prospects. Bull. Mar. Sci. 55(2-3): 1331-1332.
Cheng, T.C., J.F. LaPeyre, J.T. Buchanan, T.R. Tiersch and R.K. Cooper. 2001. Cryopreservation of
heart cells from the eastern oyster. In Vitro Cell. Dev. Biol. Anim. 37(4): 237-244.

70
Chi, S.C., W.W. Hu and B.J. Lo. 1999. Establishment and characterisation of a continuous
cell-line (GF-1) derived from grouper, Epinephelus coicoides (Hamilton): a cell line susceptible
to grouper nervous necrosis virus. J. Fish Dis. 22(3): 173-182.
Crawford, S.A., I.A. Gardner and R.P. Hedrick. 1999. An enzyme-linked immunosorbent assay
(ELISA) for detection of antibodies to channel catfish virus (CCV) in channel catfish.
J. Aquat. Anim. Health, 11(2): 148-153.
Cross, T.F., P. Galvin and E. Dillane. 1998. The use of molecular markers in aquaculture
genetics. ICES Theme Session on the use of Genetics in Aquaculture. International Council
for the Exploration of the Sea, Copenhagen, Denmark, 11 pp.
Davis, G.P. and D.J.S. Hetzel. 2000. Integrating molecular genetic technology with traditional
approaches for genetic improvement in aquaculture species. Aquacult. Res. 31(1): 3-10.
Durand,-S.; D.V. Lightner, L.M. Nunan, R.M. Redman, , J Mari.and ,J.-R. Bonami. 1996.
Application of gene probes as diagnostic tools for white spot baculovirus (WSBV) of penaeid
shrimp. Dis. Aquat. Org. 27(1): 59-66.
FAO. 1997. Review of the state of world aquaculture. FAO Fish. Circ. No. 886, Rev. 1, 163 pp.
FAO. 2000. DNA-based molecular diagnostic techniques. In P. Walkerand R.P. Subasinghe, (eds.),
Research needs for standardization and validation of the detection of aquatic animal patho-
gens and diseases. Report and proceedings of the Expert Workshop on DNA-based Molecu-
lar Diagnostic Techniques, Bangkok, Thailand, 7-9 February 1999, FAO Fish. Tech. Pap.
No. 395, 93 pp.
FAO and NACA. 2001. Asia Diagnostic Guide to Aquatic Animal Diseases. FAO Fish.
Tech. Pap. No. 402/2, 237 pp.
Flegel, T.W. 1996. A turning point for sustainable aquaculture: the white spot virus crisis in Asian
shrimp culture. Aquaculture Asia, July-September 1996. NACA, Bangkok,
Thailand, pp 29-34.
Forster, J. and R. Hardy. 2001. Measuring efficiency in intensive aquaculture. World Aquacult. 32(2):
41-45.
Garcia, D.K., A.K. Dhar, and A. Alciva-Warren. 1996. Molecular analysis of PAPD marker (B20)
reveals two microsatellite and differential mRNA expression in Penaeus vannamei. Mol. Mar.
Biol. Biotech. 5: 71- 83.
Garcia-Oretga, A., E.A. Huisman, P. Sorgeloos and J. Verreth. 2001. Evaluation of protein
quality in microbound starter diets made with decapsulated cysts in Artemia and fishmeal
for fish larvae. J. World Aquacult. Soc. 32(3): 317-329.
Ghosh, D., A.K. Dasmahaptra and A.K. Ray. 1995. Primary culture of prawn hepatocytes in serum
free media. In Vitro Cell. Dev. Biol. Anim. 31(11): 811-813.
Gjedrem, T. 1997. Selective breeding to improve aquaculture production. World Aquacult.
28: 33 – 45.
Gjerde. B. and M. Rye. 1998. Design of breeding programmes in aquaculture species:
possibilities and constraints. In D. Bartley and B. Basurco, (eds), Genetics and breeding of
Mediterranean aquaculture species. Cahiers OPTIONS Méditerranéennes Vol 34. Zaragoza,
Spain. pp. 181–192.
Groff, J.M. and S.E. La Patra. 2000. Infectious diseases impacting the commercial culture
of salmonids. J. Appl. Aquacult. 10(4): 17-90.
Handy, R.D. and M.H. Depledge. 1999. Physiological responses: their measurement and use as
environmental biomarkers in ecotoxicology. Ecotoxicoloy, 8(5): 329-349.
Howell, B.R., E. Moksness and T. Svåsand. (eds.) 1999. Stock Enhancement and Sea Ranching: Pro-
ceedings of the First International Symposium on Stock Enhancement and Sea
Ranching, Norway. Fishing News Books, Oxford, United Kingdom, 672 pp.
IFREMER. 2000. Self-service sea bass. Press release, October 2000. http://www.ifremer.fr/anglais/
actual/presse/medias.htm#sea bass (updated 04-10-01).
Jellett, J.F., E.R. Belland, L.I. Doucette, A.J. Windust, S. Gallagher and M.A. Quilliam. 1999.
Validation of the Maritime In Vitro Shellfish Test (MISTTM) kits for marine biotoxins. Can.
Tech. Rep. Fish. Aquat. Sci. 2261: 120. (abstract only).
LaPeyre, J.F. and Y. Li. 2000. Isolation and primary culture of eastern oyster haemocytes.
J. Shellfish Res. 19(1): 646. (abstract only)
Lavens, P., P. Sorgeloos, P. Dhert and B. Devresse. 1995. Larval foods. In N.R. Bromage, and
J.R. Roberts, (eds.), Broodstock Management and Egg and Larval Quality. Blackwell Science
Limited. Oxford, United Kingdom, p. 373.

71
Le Groumellec, M., C. Martin, P. Haffner, B, Martin and Aquacop. 1995. Cell culture from tropical
shrimp. J. Aquacult. Trop. 10: 277-286.
Liao, I.C. and N.H. Chao. 1997. Developments in aquaculture biotechnology in Taiwan. J. Mar.
Biotechnol. 5: 16-23.
Liu, Z., P. Li, H. Kucuktas, A. Nicols, G. Tan, X. Zheng, B.J. Argue and R.A. Dunham. 1999.
Development of amplified fragment length polymorphism (AFLP) markers suitable
for genetic linkage mapping of catfish. Trans. Am. Fish. Soc. 128(2): 317-327.
Lorenzen, E., B. Carstensen and N.J. Olesen. 1999. Inter-laboratory comparison of cell-lines for
susceptibility to three viruses: VHSV, IHNV and IPNV. Dis. Aquat. Org. 37(2): 81-88.
Majack, T.J., M.B. Rust, K.C. Massee, G.W. Kissil, R.W. Hardy and M.E. Peterson. 2000.
Bioencapsulation of erythromycin using brine shrimp, Artemia franciscana (Latreille).
J. Fish Dis. 23(1): 71-76.
Marr, J.C., T. Hu, S. Pleasance, M.A. Quilliam and J.L.C. Wright. 1992. Detection of new
7-0-acyl derivatives of diarrhetic shellfish poisoning toxins by liquid chromatography – mass
spectrometry. Toxicon, 30(12): 1621-1630.
Marr, J.C., L.M. McDowell and M.A. Quilliam. 1994. Investigation of derivitisation reagents for the
analysis of diarrhetic shellfish poisoning toxins by liquid chromatography with
fluorescence detection. Nat. Toxins, 2(5): 302-311.
Meloni, S. and G. Scapigliati. 2000. Evaluation of immunoglobulins produced in vitro by
head-kidney leucocytes of sea bass Dicentrarchus labrax by immunoenzymatic assay. Fish Shell-
fish Immunol. 10(1): 95-99.
Mendoza, R., A. De Dios, C. Vasquez, E. Cruz, D. Ricque, C. Aguilera and J. Montemayor. 2001.
Fishmeal replacement with feather-enzymatic hydrolyzates co-extruded with
soya-bean meal in practical diets for the Pacific white shrimp (Litopenaeus vannamei). Aquacult.
Nutr. 7(3): 143-151.
Merchie, G., P. Lavens, P. Dhert, M. Dehasque, H. Nelis, A. DeLeenheer and P. Sorgeloos. 1995.
Variation in ascorbic acid content in different live food organisms. Aquaculture,
134(3-4): 325-337.
Mialhe, E., E. Bachere, V. Boulo, J.P. Cadoret, C. Rousseau, V. Cedeno, E. Saraiva, L. Carrera and
R.R. Colwell. 1995. Future of biotechnology-based control of disease in marine invertebrates.
Mol. Mar. Biotechnol. 4: 275-283.
Mishra, S.S. 1998. Use of immunoassay for rapid detection of pathogenic bacteria Vibrio alginolyticus
and Aeromonas hydrophila from shrimp and fishes. Indian J. Mar. Sci. 27(2): 222-226.
Moore, S.S., V. Whan, G.P. Davis, K. Byrne, D.J. S Hetzel and N. Preston. 1999. The development
and application of genetic markers for the Kuruma prawn Penaeus japonicus. Aquaculture,
173(1-4): 19-32.
Munoz, M., R. Cedeno, J. Rodriguez, W.P.W. van der Knapp and E. Mialhe. 2000.
Measurement of reactive oxygen intermediate production in haemocytes of the penaeid shrimp,
Penaeus vannamei. Aquaculture, 191(1-3): 89-107.
Nadala, E.C.B. Jr. and P.C. Loh. 2000. Dot-blot nitrocellulose enzyme immunoassays for the detec-
tion of white spot virus and yellow head virus of penaeid shrimp. J. Virol. Methods, 84:
175-179.
Naylor, R.L., R.J. Goldburg, J.H. Primavera, N. Kautsky, M.C.M. Beveridge, J. Clay, C. Folke,
J. Lubchenko, H. Mooney and C. Troell. 2000. Effect of aquaculture on world fish
supplies. Nature, 405(6790): 1017-1024.
Ng, W., M. Tee and P. Boey. 2000. Evaluation of crude palm oil and refined palm olein as dietary
lipids in pelleted feeds for a tropical bagrid catfish Mytsus nemurus (Cuvier
& Valenciennes). Aquacult. Res. 31(4): 337-347.
NOAA 2001. NOAA’s Aquaculture Policy. http://swr.ucsd.edu/fmd/bill/aquapol.htm
Noel, T., J.L. Nicolas, V. Boulo, E. Mialhe and P. Roch. 1996. Development of a colony-blot ELISA
assay using monoclonal antibodies to identify Vibrio P1 responsible for ‘brown ring disease’
in the clam Tapes philippinarum. Aquaculture, 146(3-4): 171-178.
Oceanic Institute News. 2000. Oceanic Institute Achieves Breakthrough by Spawning Red Snapper.
http://www.oceanicinstitute.org
Ogunji, J.O. and M. Wirth. 2001. Alternative protein sources as substitutes for fishmeal in the diet of
young tilapia Oreochromis niloticus (Linn.). Isr. J. Aquacult. 53(1): 34-43.

72
OIE. 2000. Diagnostic Manual for Aquatic Animal Diseases. 3 rd edn. Office International des
Epizooties, Paris, France, 237 pp.
OIE. 2001. International Aquatic Animal Health Code. 4th ed. Office International des Epizooties,
Paris, France, 155 pp.
Oliva-Teles, A. and P. Goncalves. 2001. Partial replacement of fishmeal by brewers yeast (Saccaromyces
cerevisae) in diets for sea bass (Dicentrarchus labrax) juveniles. Aquaculture, 202(3-4): 269-278.
Osborn, H.R. and J.C. Culbertson. 1998. Mariculture options with Texas rigs to reefs. Sea Grant
College Program Publication. Texas A&M University, pp. 47-51.
Papatryphon, E. and J.H. Soares. 2001. The effects of phytase on apparent digestibility of four
practical plant feedstuffs fed to striped bass, Morone saxatilis. Aquacult. Nutr. 7(3): 161-167.
Papatryphon, E., R.A. Howell and J.H. Soares Jr. 1999. Growth and mineral absorption by striped
bass Morone saxatilis fed a plant feedstuff based diet supplemented with phytase. J. World
Aquacult. Soc. 30(2): 161-173.
Pernas, M., B. Novoa, C. Tafalla and A. Figueras. 2000. Efficiency of different monoclonal antibod-
ies in immunological assays developed for the detection of Marteilia sp. isolated from Mytilus
galloprovincialis. Bull. Euro. Assoc. Fish Pathol. 20(5): 193-198.
Pleasance, S., J. Kelly, M.D. LeBlanc, M.A. Quilliam, R.K. Boyd, D.D. Kitts, K. McErlane, M.R. Bailey
and D.H. North. 1992. Determination of erythromycin A in salmon tissue by liquid
chromatography with ionspray mass spectrometry. Biol. Mass Spectrom. 21(12): 675-687.
Quilliam, M.A. 1999. Recent developments in analytical chemistry of marine toxins. Can. Tech. Rep.
Fish. Aquat. Sci. 2261, p. 120 . (abstract only)
Robles, R., P. Sorgeloos, H. Duffel and H. Nelis. 1998. Progress in biomedication using live foods.
J. Appl. Ichthol. 14(3-4): 207-212.
Romalde, J.L. 1999. Assessment of a magnetic bead-EIA based kit for rapid diagnosis of fish
pasteurellosis. J. Microbiol. Methods, 38(1-2): 147-154.
Saakamoto, T., R.G. Danzmann, N. Okamoto, M.M. Ferguson and P.E. Ihssen. 1999. Linkage
analysis of quantitative trait loci associated with spawning time in rainbow trout
(Oncorhynchus mykiss). Aquaculture, 173(1-4): 33-43.
Shelby, R.A., C.A. Shoemaker, J.J. Evans and P.H. Klesius. 2001. Development of an indirect ELISA
to detect humoral response to Streptococcus iniae of Nile tilapia, Oreochromis niloticus. J. Appl.
Aquacult. 11(3): 35-44.
Shimizu, C., H. Shike, K.R. Klimpel and J.C. Burns. 2001. Hemolymph analysis and evaluation of
newly formulated media for culture of shrimp cells (Penaeus stylirostris). In Vitro Cell. Dev.
Biol. Anim. 37(6): 322-329.
Shipton, T.A. and P.J. Britz. 2000. Partial and total substitution of fishmeal with plant protein
concentrates in formulated diets for the South African abalone, Haliotis midae. J. Shellfish
Res. 19(1): 534. (abstract only)
Sorgeloos, P. 1995. Bioengineering of hatcheries for marine fish and shellfish. J. Marine Biotechnol.
3: 42-45.
Sorgeloos, P. and P . Leger. 1992. Improved larviculture outputs of marine fish, shrimp, and prawn.
J. World Aquacult. Soc. 23 (4): 251-264.
Sriniwasa Rao, P.S. and P.M. Sudha. 1996. Emerging trends in shrimp farming. Fishing Chimes,
16(3): 25-26.
Storebakken, T., K.D. Shearer and A.J. Roem. 1998. Availability of protein, phosphorus and other
elements in fish meal, soy-protein concentrate and phytase-treated soy-protein-concentrate-
based diets to Atlantic salmon, Salmo salar. Aquaculture, 161(1-4): 363-377.
Subasinghe, R.P., U. Barg, M.J. Phillips, D. Bartley and A. Tacon. 1998. Aquatic animal health
management: investment opportunities within developing countries. J. Appl. Ichthyol. 14
(3-4): 123-129.
Toullec, J.Y. 1995. Crustacean cell cultures: state of the art. In Biology of Protozoa, Invertebrates
and Fishes. In vitro experimental models and applications. Actes Colloq. IFREMER No. 18,
pp.43-47
Touraki, M., S. Mourelatos, G. Karamanlidou, S. Kalaitzopoulou and C. Kastritsis. 1996.
Bioencapsulation of chemotherapeutics in Artemia as a means of prevention and treatment of
infectious diseases of marine fish fry. Aquacult. Eng. 15(2): 133-147.
Touraki, M., I. Niopas and C. Kastritsis. 1999. Bioaccumulation of trimethorpim, sulfamethoxazole
and N-aceytl-sulfamethoxazole in Artemia nauplii and residual kinetics in seabass
larvae after repeated oral dosing of medicated nauplii. Aquaculture, 175(1-2): 15-30.

73
Uma, A., T.J. Abraham and V. Sundararaj. 1999. Effect of a probiotic bacterium, Lactobacillus plantarum
on disease resistance of Penaeus indicus larvae. Indian J. Fish. 46(4): 367-373.
Van Weerd, J.H., K.A. Khalaf, F.J. Aartsen and P.A.T. Tijssen. 1999. Balance trials with African
catfish Clarias gariepinus fed phytase-treated soybean meal-based diets. Aquacult. Nutr.
5(2): 135-142.
Vielma, J., T. Makinen, P. Ekholm and J. Koskela. 2000. Influence of dietary soy and phytase levels
on performance and body composition of large rainbow trout (Oncorhynchus mykiss) and
algal availability of phosphorus load. Aquaculture, 183(3-4): 349-362.
Walker, P. and R. Subasinghe. (eds.) 2000. DNA-based molecular diagnostic techniques: research
needs for standardisation and validation of the detection of aquatic animal pathogens and
diseases. Report and proceedings of the Expert Workshop on DNA-based Molecular
Diagnostic Techniques: Research Needs for Standardization and Validation of the Detection
of Aquatic Animal Pathogens and Diseases. Bangkok, Thailand, 7-9 February 1999, FAO
Fish. Techn. Pap. No. 395, 93 pp.
Walton, A. and V.J. Smith. 1999. Primary culture of the hyaline haemocytes from marine decapods.
Fish Shellfish Immunol. 9(3): 181-194.
Wang, C.H., H.N. Yang, C.Y. Tang, C.H. Lu, G.H. Kou and C.F. Lo. 2000. Ultrastructure of white
spot syndrome virus development in primary lymphoid organ cell cultures. Dis. Aquat. Org.
41(2): 91-104.
Wang, Y. 1998. Utilization of genetic resources in aquaculture: a farmer’s view for sustainable
development. Paper presented at the Bellagio Conference, Towards Policies for
Conservation and Sustainable Use of Aquatic Genetic Resources. FAO/ICLARM, Bellagio,
Italy, 14–18 April, 1998.
Ward, R.D., L.J. English. D.J. McGoldrick, G.B. Maguire, J.A. Nell and P.A. Thompson. 2000.
Genetic improvement of the Pacific oyster Crassostrea gigas (Thunberg) in Australia. Aquacult.
Res. 31(1): 35-44.
Weirich, C.R. and R.C. Reigh. 2001. Dietary lipids and stress tolerance of larval fish. In C. Lim and
C.D. Webster, (eds.), Nutrition and Fish Health, Food Products Press, 10 Alice Street
Binghamton NY 13904-1580, USA, pp. 301-312.
Welcomme, R.L. and D.M. Bartley. 1998. An evaluation of present techniques for the enhancement
of fisheries. In T. Petr, (ed.), Inland Fishery Enhancements. FAO/ODA Expert Consultation
on Inland Fishery Enhancement, April 7 - 11, 1997, Dhaka, Bangladesh. FAO Fish. Tech. Pap.
No, 374, pp.1-35.
Wongteerasupaya, C, S. Wongwisansri, V. Boonsaeng, S.,Panyim, P. Pratanpipat, G.L. Nash, B.
Withyachumnarnkul and T.W. Flegel. 1996. DNA fragment of Penaeus monodon baculovirus
PmNOBII gives positive in situ hybridization with white spot viral infections in six penaeid
shrimp species. Aquaculture, 143: 23-32.
Wright, J. Jr., A. Bonen, J.M. Conlon and B. Pohajdak. 2000. Glucose homeostasis in the teleost fish
Tilapia: Insights from Brockman body xenotransplantation studies. Am. Zool. 40(2): 234-245.

74