**National Programs [Aquaculture]{.underline}**

**[FY2004]{.underline}**

**National Program Annual Report: FY 2004**

-   **Introduction**

-   **Genetic Improvement**

-   **Integrated Aquatic Animal Health Management**

-   **Reproduction and Early Development**

-   **Growth, Development, and Nutrition**

-   **Aquaculture Production Systems**

-   **Sustainability and Environmental Compatibility of Aquaculture**

-   **Quality, Safety, and Variety of Aquaculture Products for
    Consumers**

**Introduction**

The Aquaculture National Program enjoyed an exciting year with expanded
program, new hires, scientific productivity, and recognition of
scientific quality. The FY 2004 appropriations increased aquaculture
research in four of the seven research program component areas. ARS
implemented new program on Great Lakes Aquaculture at Milwaukee, WI.

The Aquaculture National Program was peer reviewed during 2004. The
program was organized into 26 projects that were submitted to two review
panels. I am proud to report that 70 percent of the projects were rated
needing only minor revision or needing no revision.

Congratulations to Brian Bosworth, Mid-South Area and Ken Overturf,
Pacific West Area for being named Early Career Scientists of the Year.

Welcome to 7 new permanent full-time scientists:

Shawn McNulty, microbiologist-Aquatic Animal Health, Auburn, AL

Sylie Quiniou, Molecular Immunologist, Catfish Genetics, Stoneville, MS

Peter Silverstein, Virologist, Catfish Genetics, Stoneville, MS

LaTonya Clay. Molecular Biologist, Catfish Genetics, Stoneville, MS

Cynthia Bower, Food Science/Microbiologist, Subarctic Agricultural
Research. Fairbanks, AK

Philip Pearson, Agricultural Engineer, Aquaculture Production Systems,
Pine Bluff, AR

Brett Dumbauld, Ecologist, Newport, OR

Two proposals for post-docs (Class of FY2005) were awarded to scientists
(Camara and Bilodeau) in the Aquaculture National Program.

ARS scientists delivered six invitational scientific presentations at
national and international conferences. The Agency helped support
symposia, workshops, field days or annual association meetings through
partial funding, participation, sponsorship or hosting on various
aquaculture research related topics.

Seven patents were filled or awarded in FY2004. Les Torrans received
Honorable Mention for Excellence in Technology Transfer from the Federal
Laboratory Consortium, Southeast Region for the "Sock Saver" method to
deliver oxygen to catfish. He also received a Mid-South Area Technology
Transfer Award.

**Genetic Improvement**

Since there has been limited genetic improvement of aquaculture stocks,
there are major opportunities for improvement through traditional animal
breeding, broodstock development, germplasm preservation, molecular
genetics, and allied technologies.  ARS research addresses improvement
of growth rates, feed efficiency, survival, disease resistance,
fecundity, yield, and product quality; genetic characterization and gene
mapping; and conservation and utilization of important aquatic
germplasm.

[Five North American Atlantic Salmon stocks evaluated]{.underline}.
Development of a genetically improved North American Atlantic Salmon is
important to survival of the Maine Salmon Industry. Atlantic salmon from
5 different sources was obtained from collaborators to initiate the
breeding program at the ARS National Cold Water Marine Aquaculture
Center, Franklin, ME. Eggs were incubated, hatched, and fish reared as
parr (juveniles) in indoor (greenhouse) tanks. Early growth evaluation
of the different genetic stocks indicate differences in growth between
stocks and individual families, an imperative result for genetic
improvement.

[Broodyear lines established and the top families selected.]{.underline}
Evaluation of germaplasm and development of broodstock lines is critical
to genetic improvement of economically important rainbow trout industry.
Scientist at the National Center for Cool and Cold Water Aquaculture in
Leetown, WV, conducted the first generation of selective breeding,
selecting the top 15% of 2002 broodyear families for breeding and
generating 100 full sib families for 2004 broodstock (F1). The 86
families from 2003 broodstock have been evaluated for growth and the top
10% of families are identified for breeding in Jan 2005.

[Stress hormone (costisol) heritable]{.underline}. Stress has a negative
impact on many traits in fish that are important to aquaculture
production, including growth rate, feed efficiency, disease resistance
and reproductive performance. Stress response, as measured by blood
levels of the hormone cortisol was found to be highly heritable in other
strains of rainbow trout, allowing lines of low and high responding fish
to be generated by ARS scientists at the National Center for Cool and
Cold Water Aquaculture, Leetown, WV. Investigations into the performance
benefits of altering stress response via breeding have been
inconclusive. Families of rainbow trout from 2002 broodstock development
program that differ by about 2X in cortisol response to stress have been
identified and increased stress response was correlated with increased
growth and feed efficiency, two selection criteria. High and low stress
broodstock families were bred to determine if the stress response is
heritable in fish. Positive correlation between stress response
indicators and performance traits will provide the information on which
to base selection to address the negative effects of stress in
aquaculture production,.

[Transgenesis used to demonstrate disease resistance]{.underline}.
Aquaculture production suffers large and variable losses to disease.
Conveying disease resistance by selection is slow. Scientists at the
University of Connecticut, Storrs, CN in collaboration with NCCCWA
developed the P1 transgenic fish in 1999 (Rbt-99)and a total of 6
families of F2 transgenic fish. This is the first time in this project
that F2 transgenic fish are ready for disease challenge studies. Animals
from these six families were subjected to challenge studies with A.
salmonicida, a potent bacterial pathogen of trout. Results of the
challenge studies showed animals from two families exhibited a
significant protection from infection by A. salmonicida. More F2 animals
will be established from 8 additional families of F1 transgenic fish for
further disease challenge studies and improve understanding of gene
action.

[Characterized a rainbow trout BAC library]{.underline}. Genome research
requires the development of species-specific molecular genetic resources
for organisms of interest. Unfortunately, these resources are very
expensive to construct and maintain, thus they are not available for
every species. ARS scientists at the National Cool and Cold Water
Aquaculture Center at Leetown, WV, (NCCCWA) characterized a rainbow
trout bacterial artificial chromosome library for use in genetic and
physical mapping and sequencing DNA segments of interest. The library,
made up of \~135,000 base pair DNA segments retained in bacterial cells,
has the equivalent of 10 times the amount of DNA of the rainbow trout
genome. This means that for any DNA segment of interest, there is a 95%
probability of identifying at least one clone retaining that segment. On
average, each DNA segment is retained \~10 times as expected. As rainbow
trout have undergone an evolutionarily recent genome duplication, some
DNA segments were retained 20 times. Further DNA fingerprinting analyses
revealed that these were actually two sets of clones retaining recently
diverged duplicates of a single gene. Mapping these copies has revealed
homeologous segments of the genome expected to harbor similar genes.
These results have had an impact on strategies for constructing physical
maps and the eventual sequencing of the rainbow trout genome. The NCCCWA
library is publicly available to the research community.

[Genes identified for early development of rainbow trout]{.underline}.
Reproductive fecundity and early life forms survival are important
traits for an aquaculture species. A high-quality cDNA library from
rainbow trout oocyte has been successfully constructed and normalized by
scientists at West Virginia University, Morgantown, WV in collaboration
with NCCCWA. A total of 20,160 EST sequences from the normalized library
was generated. The information of these sequences provides a global
picture of genes expressed in trout oocyte and is important for
identification of key genes essential for oocyte development,
maturation, and early embryogenesis.

[Calpain proteins characterized for rainbow trout.]{.underline} Calpain
proteins from rainbow trout muscle have been isolated and characterized
by scientists at West Virginia University, Morgantown, WV in
collaboration NCCCWA. These proteins are important in muscle texture
development. The cDNAs for calpain regulatory subunits of the two
calpains were successfully cloned and characterized. The expression of
these genes in muscle during starvation has been determined. These
determinations will improve the accuracy and reduce time in selective
genetic improvement of rainbow trout.

[Performance rank order similar for trout in different
systems]{.underline}. Domesticated strains of rainbow trout from
flow-through culture environments were used as the founding population
for the selective breeding program by ARS scientists at the National
Center for Cool and Cold Water Aquaculture in Leetown, WV. Individuals
from each strain were reared in tanks receiving recirculated or single
use water to examine the impact of culture environment on growth
characteristics. Surprisingly, fish reared in tanks receiving
recirculated culture water gained more weight than fish reared in the
flow-through tanks, even though water quality in the recirculating
aquaculture system was poorer, with respect to ammonia and organic
concentrations. Additionally, the results indicated that the strains
maintained the same rank order in terms of growth while being reared in
either environment. The implications of this work suggest that a single
selective breeding program may be sufficient to improve performance
characteristics of rainbow trout when reared in a recirculating or
flow-through environment.

[State-of-the-art controlled facilities set-up for culture of
oysters]{.underline}. Improved oyster germplasma with superior traits
for growth, quality, and disease resistance is important for oyster
farming. Molecular technology is needed to enhance selective breeding.
The ARS scientist at Newport, OR constructed a large greenhouse with
temperature controlled oyster rearing facilities and large algae-rearing
containers in cooperation with the Oregon State University Molluscan
Breeding Program. This accomplishment permits initiation of a
large-scale genetic analyses. The new infrastructure for nursery and
field stages of oyster culture will provide critical data and generate
experimental materials for genetic mapping. Microsatellites will be the
workhorse technology for oyster breeding efforts due to their high
levels of polymorphism, ease of scoring, and transferability among
laboratories, populations, and because very high throughput genotyping
will be required. The ARS scientist optimized over 50 microsatellite
loci for Pacific oysters and automated PCR setup using robotic liquid
handling. PCR primer pairs published in the literature or provided by
colleagues were tested using a matrix of reaction conditions and the
best set of conditions was determined using a diverse panel of oysters.
This accomplishment has permitted progress on the high throughput
demanded by our research and will accelerate our progress and the
transfer of our results to industry

[Unique trait established for oyster breeding]{.underline}. Ongoing
international negotiations that limit cadmium content in seafood may
impact the marketability of Pacific oysters, and there is need to know
whether selective breeding can modify this character. The ARS scientist
at Newport, OR collected oyster tissue samples for preliminary study of
the heritability of cadmium concentration in Pacific oysters from an
ongoing experiment setup by collaborators at OSU. The research was
accomplished by consultation and collaboration with OSU and other ARS
researchers in Corvallis, OR. This accomplishment will permit a critical
evaluation of the feasibility of breeding to select for reduced cadmium
accumulation, an issue of great concern to the shellfish industry.

[Marker assisted selection of catfish]{.underline}. Catfish families
demonstrate variation in growth rate, but the mechanisms underlying
genetic differences between families are unclear. Levels of mRNA and
protein of insulin-like growth factors (IGF) I and II were measured in
slow and fast growing families of catfish by ARS scientists at the
Catfish Genetics Unit, Stoneville, MS. Fish in fast growing families
demonstrated higher levels of plasma IGF-I and higher IGF-II mRNA
expression compared to slower growing families. The changes in protein
levels of IGF-I supported IGF-I\'s role in growth regulation of channel
catfish and provide evidence for a role of IGF-II in growth of channel
catfish. These gene products could be used as markers for selection of
broodstock with superior genetic potential to increase the rate of
improvement.

[Performance evaluation of several types of catfish shows advantage of
blue catfish]{.underline}, Mortalities and reduction in growth due to
diseases are an increasing problem for producers growing channel
catfish, but blue catfish and channel x blue catfish hybrids are less
susceptible than channel catfish for enteric septicemia and
proliferative gill disease, two of the most prevalent diseases. ARS
scientists at the Catfish Genetics Unit, Stoneville, MS conducted a
comparison of production traits of blue catfish, channel x blue catfish
hybrids, and two channel catfish strains (Norris and NWAC103).
Fingerling growth and survival were better for blue catfish and channel
x blue catfish hybrids than for the channel catfish strains. Feed
records indicate hybrids consumed the most feed, followed by blue
catfish, then NWAC103s, and then Norris strain channel catfish during
the fingerling to market weight portion of the study. Results
demonstrated that use of blue or channel x blue catfish hybrids could
benefit producers through improved growth and survival.

**Integrated Aquatic Animal Heath Management**

Despite progress in aquatic animal health, significant losses to
diseases still occur.  ARS research addresses improvement of survival,
growth, vigor, and wellbeing of cultivated aquatic animal stocks through
integrated aquatic animal health research, improved technologies and
practices, such as population health management; and development of
health management products, including vaccines and therapeutics, and
disease detection/diagnostic techniques.

[Rainbow trout pathogen sequenced]{.underline}. Sequence analysis of
Flavobacterium psychrophilum genes will be useful for developing
diagnostic assays, identifying putative virulence factors and prototype
vaccine candidates. A low coverage (\~3.4X) genome sequence of
Flavobacterium psychrophilum strain CFS 259-93 was generated by ARS
scientists at the National Center for Cool and Cold Water Aquaculture in
Leetown, WV. This strain was chosen as it is representative of disease
causing isolates from Idaho and a challenge protocol has already been
established by an industry collaborator. A single virulent clone was
isolated, genomic DNA extracted, and clones from a small-insert library
sequenced. The sequence has been assembled into 1,160 contigs ranging in
size from 0.8-13.6 kb. Computer analysis identified 2,420 potential open
reading frames. Of these, 53% could be assigned putative functions.
Based on this sequence analysis, Flavobacterium psychrophilum is
predicted to have a relatively small genome compared to other microbial
pathogens.

[A disease challenge model for a rainbow trout pathogen.]{.underline} A
standardized disease challenge model for the salmonid pathogen Yersinia
ruckeri has been established by ARS scientists at the National Center
for Cool Cold Water Aquaculture in Leetown, WV. This accomplishment was
a required first step for further studies of the pathogenesis of this
bacterium and for the assessment of Yersinia ruckeri mutants. Methods
for the genetic manipulation of gram negative bacteria have been adapted
for use in Yersinia ruckeri with the purpose of defining virulence
factors.

[Rainbow trout immune gene identified]{.underline}. Immune system
characterization has focused on identifying chemokine ligands/receptors
and tumor necrosis superfamily ligands/receptors. These families of
genes were targeted by ARS scientists at the National Center for Cool
and Cold Water Aquaculture in Leetown, WV because of their known roles
in immune system signaling, activation, and memory. They identified a
number of putative family members in the Institute for Genomic Research
(TIGR) rainbow trout, expressed sequence tag (EST) database. Fifty-four
unique cDNA clones were isolated for further characterization.

[Virus found for a fish pathogen]{.underline}. ARS scientists at the
National Center for Cool and Cold Water Aqauculture in Leetown, WV
discovered a bacteriophage that specifically infects and kills the
salmonid pathogen Yersinia ruckeri. Methods for purifying this
bacteriophage and assays for measuring its viability and antibacterial
activity have been developed. This discovery could lead to new
biocontrol technology to protect fish from pathogens.

[A patent issued for Bacterial Kidney Disease diagnostic
test]{.underline}. This test will be useful to evaluate the health of
salmonids and document pathogen presence upon administration of a known
dosage of a pathogenic bacterium. A quantitative real-time PCR assay was
devised by the ARS scientist at Aberdeen, ID to detect the presence of
the bacterial genome and quantify its absolute copy number in infected
tissue. With this test there now exists a method to assay dietary effect
on fish health status by administration and monitoring of precise
pathogen numbers.

[Efficacy of amoxicillin demonstrated for hybrid striped
bass]{.underline}. Streptococcal infections can cause devastating
mortalities in hybrid striped bass production. The efficacy of
amoxicillin for controlling Streptococcus iniae infection in tilapia was
demonstrated using a laboratory disease model developed by ARS
scientists at the HKD Stuttgart National Aquaculture Research Center,
Stuttgart, AR. This data could support the future approval of
amoxicillin as an antibiotic to treat S. iniae infection in tilapia,
which can be a devastating disease. Trials were run to show that
amoxicillin is effective in decreasing the mortalities in infected fish
from 96% to 6%. The immediate impact of this accomplishment has been for
the FDA to allow the continued temporary use of this compound for
disease control of S. iniae in hybrid striped bass production.

[Need to understand stress and disease susceptibility]{.underline}. The
bacterial pathogen Edwardsiella ictaluri is a prevalent source of
substantial economic loss to catfish farmers, and stress has been shown
to increase E. ictaluri susceptibility. ARS scientist at the Catfish
Genetics Unit, Stoneville, MS conducted trials to determine the effect
of cortisol in the presence and absence of stress on mortality during an
E. ictaluri challenge. Increased plasma cortisol levels due to stress
correlated to increased mortality; however, stress-free cortisol
administration had no effect on mortality. These results suggest that
although cortisol is considered the primary stress hormone in fish, it
in itself is not immunosuppressive. Understanding the relationship
between stress and disease resistance is crucial to the development of
biomarkers and management strategies for improving disease resistance.

[Increased expression of toll-like receptors for increased disease
resistance]{.underline}, Little is known of host-response mechanisms
that are activated when catfish are infected by the bacteria
Edwardsiella ictaluri, which causes enteric septicemia of catfish (ESC).
The genes encoding toll-like receptors 3 and 5 (TLR3, TLR5) were
identified by DNA sequencing and changes in the expression levels (using
real-time PCR) were measured in liver, kidney, and spleen tissue in
non-exposed fish and fish challenged with virulent Edwardsiella ictaluri
by ARS scientists at the Catfish Genetics Unit, Stoneville, MS.
Expression of TLR5 was higher than TLR3 in all tissue types sampled and
was elevated in liver tissue compared to non-exposed fish throughout
experimental challenge. This indicates TLR5 is involved in the innate
immune response to E. ictaluri. Understanding factors influencing the
expression of toll-like receptors that are related to disease resistance
in catfish could benefit catfish producers and consumers by leading to
the development of catfish with higher levels of innate resistance to
ESC.

[Intestinal shedding may be a factor in ESC transmission,]{.underline}
Factors influencing transmission of enteric septicemia of catfish (ESC)
are not well understood. Researchers at Mississippi State University, in
collaboration with ARS scientists at the Catfish Genetics Research Unit,
Stoneville, MS measured changes in pathogen levels in gut and intestinal
contents of catfish infected with ESC to quantify levels of bacterial
shedding during ESC infection. Evidence of bacteria in intestinal
samples indicated bacterial shedding may be one mechanism of disease
transmission. Ongoing research will address the issue of bacterial
shedding and improve understanding of factors influencing disease
transmission.

[Fish vaccines being commercialized]{.underline}. Vaccines against
Flavobacterium columnare, Edwardsiella tarda and Streptococcus
agalactiae, all fish pathogens were developed and U.S. patent
applications filed by ARS scientists at the Aquatic Animal Health
Research Unit, Auburn, AL. Intervet, Inc. is currently licensing a
previous developed F. columnare vaccine that may be used in 7-10 day old
fry and a U.S. patent was approved, and a divisional application has
been filed. Schering-Plough is licensing a previously patented S. iniae
vaccine. The availability of these vaccines is expected to increase fish
farming profits by \$80-\$100 million annually.

**Reproduction and Early Development**

Reproduction and early development are important aspects of species
domestication and sustainability.  ARS research on this component
interacts with several of the other components of the Aquaculture
National Program.  The selection below exemplifies the
multi-disciplinary character of husbandry research. 

[Technology to produce sterile fish refined]{.underline}. Sterile fish
are important to aquaculture for production efficiency, risk reduction
of escapes and protection of germplasm. In order to effectively make
rainbow trout strains with four sets of chromosomes (tetraploids) to
cross with normal diploids and produce sterile triploids, it is
necessary to define the timing of the first cell division in fertilized
eggs. Scientists at the National Center for Cool and Cold Water
Aquaculture in Leetown, WV.determined the time to first cleavage in
embryos from individual females of a number of rainbow trout strains.
Results from the analyses revealed that at a given incubation
temperature the time until first cleavage varied by as much as 40 min.
among the strains. Knowledge of this variation allows the development of
a treatment protocol that will increase the production of tetraploids
from different strains and may be an indicator of genetic differences in
embryonic development rate that can be utilized for selection.

[A first step toward domestication of pompano]{.underline}. Florida
pompano can be subjected to broodstock maturation procedures and
successfully induced to spawn. Growth and expansion of the US
mariculture industry depends on development of methods for sustainable
reproduction and larviculture of target species such as Florida pompano.
ARS scientists at the Harbor Brabch Oceanographic Institute, Ft Pierce,
FL collected pompano from the wild, acclimated them to indoor culture
conditions, transferred them to a commercial diet, and successfully
spawned them over a four-month period. Results suggest the potential for
year-round production of pompano seedstock for commercial farmers.

[Artifical insemination-spawning of flounder and pompano]{.underline}.
Procedures were developed for the collection, handling, and refrigerated
storage of sperm from two marine fish species, Southern flounder and
Florida pompano by ARS and Harbor Branch Oceanographic Institute
scientists, Ft. Pierce. FL. Utilization of refrigerated sperm improves
hatchery techniques, by allowing efforts to be focused on maintaining
female broodstock, monitoring ovarian development, and increasing
efficiency during the strip-spawning process. Sperm that were collected
from domesticated southern flounder and wild Florida pompano retained
motility for 10 d at 4 C when the sperm were suspended in diluted
calcium-free Hanks\' balanced salt solution (200 and 300 mOsmol/kg).
This study demonstrated that southern flounder and Florida pompano sperm
can be stored for short-term repeated use in the hatchery needed to
support an industry.

[Additional nitrogen improves fry survival]{.underline}. Catfish fry
survival to fingerling stage averages around 60%, and is affected by the
availability of natural food resources in the ponds - particularly
zooplankton. ARS scientists at the Catfish Genetic Research Unit
determined the effect of catfish fry pond fertilization to optimize
plankton composition in collaboration with Mississippi State University
researchers. Industry practices do not provide sufficient nitrogen for
growth of desirable algae, thereby limiting zooplankton population size
in ponds. Optimal fertilization practices have been assessed on
commercial ponds and management recommendations adopted by fingerling
producers.

[Large zooplankton important in catfish fry]{.underline}. The objective
of zooplankton research is to help catfish farmers adopt management
practices that maximize profitability of fry culture through utilization
of natural pond productivity and reduced feed costs. Zooplankton are
more important to catfish fry culture than previously thought. Catfish
fry actively forage on large zooplankton taxa (i.e., copepods and
cladocerans); large bodied zooplankton from catfish nursery ponds meet
or exceed all nutritional requirements for channel catfish fry (65%
protein, 9% fat). Mississippi State scientists at the National Center
for Warm Water Aquaculture, Stoneville,MS conducted preliminary
laboratory studies that indicate zooplankton, in conjunction with
commercial feed, have a positive effect on fry growth, survival, and
general fish health. In several trials, vaccinated fry fed zooplankton
performed better when challenged with ESC than vaccinated fry not fed
zooplankton and non-vaccinated fry. This research may have major
implications regarding management decisions. Ponds should be managed to
increase the numbers of large sized zooplankton.

**Growth, Development, and Nutrition**

There are substantial opportunities to improve the growth, development,
and nutrition of cultivated aquatic organisms.  ARS research addresses
improving survival, growth rates, feed conversion, environmental
tolerances, and feed formulations and feeding strategies to reduce
dependence on marine fish-based protein in aquaculture diets.

[New hormone involved in growth]{.underline}. ARS scientist at the
National Center for Cool and Cold Water Aquaculture in Leetown, WV
identified a new regulatory path controlling secretion of growth
hormone, and an important hormone controlling growth in fish. They
determined that a new compound stimulated growth hormone release in
tilapia and channel catfish. This provided the first evidence for the
presence of \'ghrelin\', a recently discovered molecule and growth
hormone regulator, in these species. Their continued research in rainbow
trout found that ghrelin stimulates growth hormone release and increases
feed intake in this species. This research may result in novel ways to
promote growth, feed efficiency and immunity in commercially important
fish.

[Large Variation in nutrient utilization among groups.]{.underline}
Nutrient utilization not only influences the cost of production, but
nutrient retention has become very important for reducing phosphorus and
nitrogen excretion to the environment. ARS scientists at the National
Center for Cool and Cold Water Aquaculture in Leestown, WV are
conducting research to identify fish with superior nutrient utilization
efficiency and measure the genetic component of this trait. Significant
strain variation for this trait was identified. Significant variation
between families within strains was also detected. Importantly, a
significant correlation between efficiency measured on individually
reared fish and efficiency of groups of their siblings was detected.
This result suggests that feed efficiency measured on groups may be an
adequate selection criterion to improve performance of the population.

[Technique developed to measure palatability of shrimp
feed]{.underline}. There is a need for a simple efficient test for
quantifying the effectiveness of shrimp feed attractants and
palatability aids. Attractants can enhance the value and use of nutrient
sources otherwise unpalatable to animals A testing system was developed
by scientists at the Ocean Institute in collaboration with NOVUS, Inc.
consisting of a series of tanks each equipped with two feeding bowls in
which shrimp were allowed to choose between a neutral control diet, and
a control diet spiked with an attractant. After vigorous testing several
putative attractants were ranked according to their effectiveness. This
methodology will be useful to reliably screen attractants and
palatability aids for shrimp, and can be used to develop nutritious,
inexpensive feeds containing high levels of protein from plant sources
in place of fish meal.

[Differential gene expression related to source of
nutrients]{.underline}. Understanding the genetic components in trout
involved in diet utilization is critical for US aquaculture industry to
remain competitive particularly in the face of higher feed costs,
limited marine protein resources, and stricter environmental standards.
ARS scientists at Aberdeen, ID measured differential expression relating
to trout growth, health and metabolism of trout reared on diets
containing cereal grain and commercial feeds. Rainbow trout of distinct
lineage were separated and reared on either a commercial diet or a diet
containing cereal grain for one year after which, tissue samples were
taken and the changes in gene expression measured. This work will be the
basis to evaluate selected broodstock for improved performance on diets
containing different sources of nutrients.

[Poultry byproduct meal replaces fishmeal in hybrid striped bass
diets]{.underline}. A major success toward replacing fishmeal and
reducing costs in hybrid striped bass production was achieved by ARS
scientists at the HKD Stuttgart National Aquaculture Research Center,
Stuttgart, AR when hybrid striped bass were observed to grow equally
well on test diets in which poultry byproduct meal (PBM) or fishmeal was
the major protein source. Fishmeal is an expensive ingredient in
aquaculture feeds because natural supplies are limited and it is in high
demand. Because potential replacements for fishmeal are often deficient
in essential nutrients and availability of amino acids, several animal
byproducts were evaluated by feeding them to hybrid striped bass. The
Ideal Protein Model was used to estimate which amino acids were most
limiting in PBM. Minimal supplementation of two amino acids in the test
diets ameliorated potential deficiencies when PBM totally replaced
fishmeal.

[Hormone found useful to optimize culture conditions]{.underline}.
Insulin-like growth factor-I (IGF-I) is an indicator of optimum culture
conditions. It appears that IGF-I levels in the blood can be used, after
a shorter period of treatment than previously thought, to identify diets
and culture conditions that optimize growth. IGF-I is a hormone produced
in the liver and is thought to be the most important regulator of fish
growth. ARS scientists at the HKD Stuttgart National Aquaculture
Research Center, Stuttgart, AR used plasma (blood) concentrations of
IGF-I to help identify optimum temperature and minimum dietary
tryptophan (amino acid) levels for sunshine bass. Stress and withholding
feed also resulted in a decrease of IGF-I.

**Aquaculture Production Systems**

There are opportunities to improve the performance of aquaculture
production systems through development and application of innovative
engineering approaches and technologies.  ARS research addresses
development and successful application to aquaculture of new
technologies as well as relevant existing technologies and engineering
presently employed in other sectors of the economy.

[Recirculation system in place]{.underline}. While rainbow trout
production is based on single use water strategies, effluents discharge
standards and limited freshwater supplies suggest that efforts to
increase production will have to employ water recirculation
technologies. ARS scientist at the National Center for Cool and Cold
Water Aquaculture at Leetown, WV, completed a 34-tank recirculation
system for to rear rainbow trout that will be included in the
facility\'s selective breeding program. The recirculating aquaculture
system uses biological and mechanical filtration to remove ammonia and
suspended solids. Additionally, carbon dioxide is stripped and dissolved
oxygen levels increased prior to returning the water to the culture
tanks. This system has enabled the NCCCWA to maintain its genetic stock,
while conserving water resources. Additionally, this system allows
evaluation of the various genetic strains of rainbow trout under
different culture environments and to determine if alternative selection
criteria are required for each type of culture environment.

[Alternative live feed not successful]{.underline}. Threadfin shad
stocked as a forage fish did not substitute for formulated feed in
multiple-batch catfish production ponds when feed application was
restricted. Formulated feed represents the largest component of variable
costs, and improved feed management could result in lower feed
expenditures for channel catfish production. ARS scientists at the
Aquaculture Systems Research Unit, Pine Bluff, AR determined if channel
catfish feeding frequency could be reduced to once every three days,
compared with daily in ponds co-stocked with threadfin shad as a forage
species. The presence of threadfin shad did not substitute for feed when
feed was offered every third day. Feeding channel catfish every third
day resulted in significantly lower yield and significantly smaller
fish, but did not affect feed conversion ratio compared with daily
feeding. Thus, additional grow-out time would be needed for fish to
attain market size in ponds where threadfin shad was stocked to
substitute for formulated feed.

[Stocking rates measured for stocker catfish.]{.underline} Stocker
channel catfish growth and yield were related linearly to stocking rate
over the range of 3,500 to 5,500 fish/acre in single-batch culture.
There is increasing interest in using stocker channel catfish (Ictalurus
punctatus) instead of fingerlings in grow out ponds. There is little
data available on the effect of stocking rate on stocker catfish growth
during grow-out. ARS scientists at the Aquaculture Systems Research
Unit, Pine Bluff, AR quantified the effect of stocking rate on
production characteristics of stocker channel catfish in single-batch
culture. Net yield increased linearly with stocking rate and averaged
4,591, 4,802, and 5,533 lb/ac for the 3,500, 4,500, and 5,500 fish/ac
stocking rates, respectively. Average fish weight at harvest decreased
linearly as stocking rate increased.

Farmers can select the appropriate stocking rate to ensure that
harvested fish meet processing plant fish size requirements

**Sustainability and Environmental Compatibility of Aquaculture**

The overall goal of ARS research in this area is to protect and conserve
the nation\'s water resources and natural environments by conducting
research and technology transfer to improve the sustainability and
environmental compatibility of aquaculture production systems. 

[Biosecure aquaculture production]{.underline}. Recirulating aquaculture
systems offer higher biosecurity than other systems of production.
Internal disinfection processes should control and reduce risk of
amplification of pathogens and other microbial populations. Scientists
at the Conservation Fund\'s Freshwater Institute in Shepherdstown, WV
are working to evaluate the applicability of advanced oxidation
processes for controlling and mitigating microbial populations and
certain water quality parameters within intensive fish farming systems.
Research determined the ozone dose required to inactivate total
heterotrophic and total coliform bacterial populations using a
side-stream of water flow from an operating high-density aquaculture
recirculating system. The study results are significant in that it
identified conditions required to achieve nearly complete ozone
disinfection of the recirculating flow, i.e., maintaining a dissolved
ozone concentration of 0.1-0.2 mg/L at the outlet of a contact basin
that provides 8 to 16 min of hydraulic residence time. This finding is
importance for biosecure production and ensuring product quality.

[Settling tank design important for solids removal]{.underline}.
Dual-drain circular culture tanks can rapidly and effectively
concentrate settleable biosolids into a relatively small bottom-center
drain flow, which can then be treated using a relatively small settling
device. Scientists at the Conservation Fund\'s Freshwater Institute in
Shepherdstown, WV evaluated two settling tank designs, a radial flow
settler and a more conventional swirl separator, that were both operated
at a surface loading rate of 0.00086 m3/s of flow per square meter of
settling area (i.e., 1.3 gpm/ft2). Results indicate a highly significant
difference (P \< 0.001) in mean TSS removal efficiency of the swirl
separator and radial-flow settler, (± se) 37.1 ± 3.3% and 77.9 ± 1.6%,
respectively. These findings will help fish farmers improve
recirculating system water quality, increase the particle removal
efficiency from recirculating systems containing large \`Cornell-type\'
dual-drain circular culture tanks, and decrease the solids loading on
the recirculating system's more costly and complex microscreen drum
filter.

[Fine sand fluidized biofilters achieve desired control]{.underline}.
Biofilters are used to remove ammonia and nitrite from recirculating
aquaculture systems and the steady-state removal efficiency of the
biofilter controls the concentrations of ammonia and nitrite that
accumulate within the recirculating system. Scientists at the
Conservation Fund's Freshwater Institute in Shepherdstown, WV completed
an evaluation of sand bed expansion and long-term nitrification within a
full-scale fluidized-sand CycloBio(tm) biofilter. The finest (D10 0.18
mm) sand tested within the full-scale fluidized-sand CycloBio(tm)
biofilter, which expanded approximately 60% (i.e., the clean sand
expansion) at a hydraulic loading rate of 0.77 cm/s (11 gpm/ft2),
maintained a mean 92.2±0.7% total ammonia nitrogen(TAN) removal
efficiency and a mean nitrite nitrogen concentration of 0.06±0.00 mg/L
when makeup water flow rates ranged from 4-8% of total recirculating
flow. The results show that exceptionally low TAN and nitrite nitrogen
concentrations, similar to that obtained in a single-pass system, can be
achieved within large commercial recirculating salmonid systems that use
fine sand fluidized-sand biofilters.

[Integrated pest management of burrowing shrimp]{.underline}. Burrowing
shrimp are serious pests on west coast oyster culture farms and formerly
used chemical methods for controlling these shrimp are being phased out
by legislation. The Forage Seed and Cereal Research Unit, Corvallis, OR
recruited a new research ecologist who entered on duty in May, 2004 and
established laboratory and field operation capabilities that interface
with the ARS oyster genetics program at Newport, OR. This accomplishment
integrated an ARS research team with other state and federal
collaborators at the Hatfield Marine Science Center in Newport, OR with
a mission to develop sustainable approaches to enhance west coast
shellfish production. New integrated pest management strategies to
control burrowing shrimp will permit continued and profitable production
of oysters on west coast aquaculture farms.

[Reduced costs to protect ponds from birds]{.underline}. A modified
technique was devised to non-lethally exclude fish-eating birds from
aquaculture ponds. Using two privately owned, commercial catfish farms
within a few miles of the largest roosting colony of double-crested
cormorants, ARS scientists at the HKD Stuttgart National Aquaculture
Research Center, Stuttgart, AR evaluated the effectiveness of modifying
a low-cost, physical barrier system for deterring cormorants. The
modification limited the materials, setup time, and overall cost
significantly and yielded similar significant results for limiting
cormorant access to aquaculture ponds by 4 to 10 fold, prevented other
fish-eating birds from landing at similar rates to complete exclusion at
some ponds, and limited the duration cormorants stayed on ponds. The
modification involved changing a previously researched technique using a
simple barrier of twine, flagging and t-posts. This economical,
nonlethal method is used by the industry.

.

[An alternative to saline water for shrimp culture]{.underline}. ARS
scientists at the Aquaculture Systems Research Unit, Pine Bluff, AR
amended the ionic composition of freshwater to achieve successful
production of the Pacific white shrimp Litopenaeus vannamei in inland
ponds. Culture of the Pacific white shrimp in inland ponds filled with
low-salinity ground water is attracting attention as a means for farmers
to diversify production and increase profits. However, the availability
of low-salinity ground water may not necessarily be a requirement for
channel catfish farmers considering shrimp culture as an alternative. L.
vannamei was produced successfully in freshwater amended with potassium,
magnesium, sulfate, sodium, and chloride ions. Shrimp yield was 3,078
lb/acre and individuals averaged 19.3 g each for the125-d study. Inland
culture of marine shrimp could expand to areas that lack saline ground
water.

**Quality, Safety, and Variety of Aquaculture Products for Consumers**

The overall goal of ARS research in this area is to improve the quality,
safety, and variety of aquaculture products through research and
technology transfer.  ARS research addresses improvement of the safety,
freshness, flavor, texture, taste, nutritional characteristics, and
shelf life of cultivated fish and shellfish, and development of new and
improved value-added products and processes.

[Liquids from fish processing contain valuable nutrients]{.underline}.
Over one million metric tons of fish processing by-products is produced
each year from fish harvested in Alaska; however, much of this protein
is not utilized and during the manufacturing of fish meal the soluble
protein fraction, called stickwater, is often discarded. ARS scientists
in the Subarctic Agriculture research Unit, Fairbanks, AK. determined
the composition and properties of soluble protein generated during the
production of fish meal and chemically and nutritionally characterized
the stick water obtained from a commercial fish meal plant. Samples had
high protein contents on a dry-basis of which approximately 25 % was
connective tissue protein. Proteins in stick water were shown to have
chemical and nutrient contents useful for development of food and
non-food products.

[Liver separated from waste stream]{.underline}. Viscera, a major part
of fish waste, contains substantial quantities of liver, and in Alaska
most fish liver is made into fish meal and oil or discarded. Efforts to
separate liver from other by-products during fish processing and
utilizing them in high value products are lacking. ARS scientists at the
Subarctic Agricultural Research Unit, Fairbanks, Alaska and
collaborators examined the chemical content of liver protein isolated
from six different species of cold water marine fish including
Arrowtooth Flounder, Pacific Halibut , Alaska Pollock, Pink Salmon,
Flathead Sole and Spiny Head Rock Fish. Liver protein was of high
nutritional quality and ranged from 18 to 7% of the fish weight. Liver
protein from the six different species of fish was of similar chemical
composition with useful nutrient values that can be exploited for
commercialization.

[Alaskan fish meal may be better than commercial source]{.underline}.
Replacement of traditional fish meals, which are often imported, with
fish meals made from by products of the Alaska fish processing industry,
will enhance the Alaskan economy. ARS scientists in the Subarctic
Agricultural Research Unit, Fairbanks, AK and collaborators at the
University of Alaska found that the nutritional quality of Alaska fish
meal for Longfin amberjack was high in two growth trials. The growth and
survival of the fish fed the diet containing the Alaska meal was similar
to those fed the commercial feed and the liver lipid levels were lower
in the Alaska fish meal feed than in those fed the commercial feed. The
information will be used to persuade aquatic feed manufacturers to
utilize fish waste meals from Alaska in fish diets.

[Previously thought harmless alga cause fish kills]{.underline}. An
alga, previously thought to be benign, has been associated with a series
of fish kills in Mississippi, North Carolina, Texas, and Arkansas. The
causative organism has been identified and cultured by ARS scientists at
the Catfish Genetics Unit, Stoneville, MS, in collaboration with NOAA\'s
Harmful Algal Bloom Chemistry Staff who have identified a small
molecular weight toxic fraction. Identification of the mode of action,
toxin identity, and resulting management recommendations during toxic
bloom episodes is ongoing from this work. This alga is easily controlled
by low copper sulfate additions, providing a simple management remedy
for commercial fish farmers.

[An alga that produces an anatoxin correlated with fish
kills]{.underline}. VTC, visceral toxicosis of catfish, has killed
30-40% of brood fish during fall and winter in the four-state region.
This condition was studied by ARS scientists at the Catfish Genetics
Unit, Stoneville, MS in collaboration with scientists from the MSU
College of Veterinary Medicine. An alga capable of anatoxin-a production
has been correlated with these events. Ongoing research includes
exposure of fish to sub-acute doses to reproduce the fish responses;
management alternatives will result from this research.

[Natural algicide tested]{.underline} . Currently there are only two
algicides, copper sulfate and diuron, registered for managing
algae-related off-flavors in pond-raised catfish. Mississippi State
scientists at the National Warm Water Aquaculture Center, Stoneville, MS
cooperated with ARS scientists at the Natural Products Research Unit,
Oxford, Mississippi, to identify and evaluate new algicides for use in
managing algae-related off-flavors. One promising compound - a modified
anthroquinone - identified in laboratory screening was tested in outdoor
mesocosms and found to be selectively algicidal to odor-producing
blue-green algae, thereby reducing levels of odorous compounds in water.
This compound is undergoing patent application.

[Conditions established to observe the presence of viruses]{.underline}.
With \"normal,\" single-celled bacteria, viruses lyse the cells when
plated at high density and form a clearing called a plaque. The
cyanobacteria producing off-flavor compounds are not single cells, but
rather they grow in long filaments that don\'t allow plaques to form.
ARS scientists at the Food Processing and Sensory Quality Unit, New
Orleans, LA established methods to shear the filaments into smaller
pieces. The plaque-forming capacity of environmental water samples can
now be tested for viruses.. Viruses against cyanobacteria could be used
as a strategy to control their growth and production of off-flavors
compounds in pond water.

.