ADVANCES IN DIAGNOSIS AND MANAGEMENT OF AMYLOODINIOSIS IN
INTENSIVE FISH CULTURE
Sara M. Picón-Camacho, Ignacio Masson, Reginald Blaylock* & Jeffrey Lotz
Gulf Coast Research Laboratory
The University of Southern Mississippi
Ocean Springs, MS 39564 USA
Amyloodinium ocellatum is a parasitic dinoflagellate that infects a wide variety ofwarmwater marine and estuarine fishes and causes one of the most serious diseases inwarmwater marine fish culture. The life cycle of the parasite is direct and consists of threestages – the trophont which is parasitic primarily on the gills, the free-living reproductive
tomont, and the infective dinospore. The parasite is particularly troublesome for two reasons.
First, current diagnostic methods rely mainly on the microscopic identification of parasites on
the skin or gills; therefore, infections often go undetected until massive mortality occurs.
Second, the life cycle is direct and each tomont can produce up to 256 infective dinospores
resulting in the rapid buildup of heavy infections in recirculating systems. Although thegeneral course of infection is understood, no quantitative understanding of the survival andreproductive rates of the parasite that might lead to improved control is known.
Our program has focused on strengthening our ability to address these shortcomings
in diagnosis and management of amyloodiniosis. With respect to diagnosis, we developed anovel, highly sensitive and specific diagnostic tool based on the Loop Mediated Isothermal
Amplification (LAMP) reaction to detect free-swimming stages of A. ocellatum in water
samples. The advantages of LAMP reaction compared to the PCR are that is quicker and does
not require sophisticated equipment or skilled personnel. No amplification was detected using
DNA from related dinoflagellates species, demonstrating the specificity of the assay. The
LAMP assay also proved to be more sensitive than the PCR for the detection of A. ocellatumin water samples. The established LAMP assay provides a useful tool for the rapid andsensitive detection of A. ocellatum in water samples, which could assist in the early detection
and control of A. ocellatum infections in rearing systems.
With respect to management, we experimentally estimated the vital rates that
determine the parasite population’s growth and used that information to develop a population
model for evaluating the relative effects of changes in vital rates on the population growthrate. A schematic representation of the parasite’s life cycle was developed and a laboratory
challenge model was used to quantify each process in the life cycle in two species of hosts –the spotted seatrout (Cynoscion nebulosus) and the red snapper (Lutjanus campechanus). We
showed that although there were no significant differences in infection rates between the two
species, trophonts grew larger and remained attached longer to red snapper than spotted
seatrout and that spotted seatrout could tolerate a higher load of trophonts than red snapper.
The model demonstrated that the number of dinospores produced per tomont had the largest
effect on parasite population growth. Although the model clearly suggests that control

strategies should continue to focus on limiting the number of dinospores in a system, themodel provides a framework for evaluating the relative contribution of various factors to theparasite’s population growth rate under a variety of conditions.

PRE-SPAWNING CAROTENOID FORTIFIED DIETS IMPROVE REPRODUCTIVE
TRAITS OF CHANNEL CATFISH, ICTALURUS PUNCTATUS AND SUBSEQUENTPROGENY PERFORMANCE
Chatakondi, N. G., M. H. Li, B. C. Peterson and N. J. BoothUSDA ARS Catfish Genetics Research Unit
Thad Cochran National Warmwater Aquaculture Center
Stoneville, MS 38776, USA.
Availability of consistent number of ovulatory competent channel catfish females is a
pre-requisite for efficient production of channel catfish ♀ x blue catfish, I.furcatus ♂) hybridin hatcheries. Raising hybrid catfish in production ponds enables the catfish farmer to harness
improved growth rates, survival and feed conversion to improve production. Even thoughhybrid catfish production has been steadily increasing, fingerling production is stillinsufficient to meet the demand. Hybrid catfish embryo production involves hormone-induced
spawning of channel catfish, stripped eggs are fertilized with blue catfish sperm, and fertilized
eggs hatched in troughs in the same manner as channel catfish in hatcheries.
High quality broodstock maturation diets are an essential key for successful andsustainable production of hybrid catfish fry from hatcheries. Carotenoid fortification in broodfish diets has been suggested to improve maturation and egg quality in all aquatic animals.
However, fish and crustaceans are unable to produce astaxanthin de novo, only plants andprotists are capable of synthesizing carotenoids.
A 10-week pre-spawning broodfish nutrition study was conducted 2 months prior tospawning in twenty 1.5 m diameter, 760 L plastic tanks supplied with recirculated pond waterand continuous air. Each tank was stocked with 10 fully mature 4-year old ‘Delta’ strain offemale channel catfish per tank. This study was conducted to assess the effect of carotenoid
fortified diets on reproductive performance of channel catfish during the pre-spawning phase.
Four carotenoid fortified diets were prepared by mixing the required quantity of carotenoid inwater and sprayed on a 35% protein commercial catfish feed in a concrete blender, followed
by a coating of menhaden oil. The carotenoid treatments were: 1) 50 mg/kg Astaxanthin; 2)
100 mg/kg Astaxanthin; 3) 25 mg/kg of Lutein and 25 mg/kg of Zeaxanthin; 4) 50 mg/kg of
Lutein and 50 mg/kg of Zeaxanthin and 5) control diet that was sprayed with the same
quantity of water and oil. Four tanks were randomly allocated to a treatment.
Broodfish fed 100 ppm of Astaxanthin fortified feed had a higher (P < 0.05) percent
of gravid females suitable for hormone injection compared to other treatment groups. Gravid
females from all the 5 treatments were subjected to hormone-induced spawning procedures toproduce hybrid catfish fry in two spawning trials. Percent of females ovulated in response tohormone injection, fertility and hatching success did not differ (P > 0.05) among thetreatments. However, ovulatory index, fecundity, and fry produced per kg of female werehigher (P<0.05) in broodfish fed 100 mg/Kg of Astaxanthin fortified feed during the
pre-spawning phase.
The results of the present study suggest that supplemental 100 mg/kg astaxanthin inbroodfish diet not only improved fecundity, quality of eggs and fry production, but alsoenhanced the physiological response of broodfish to induced spawning. Further, progeny
derived by Astaxanthin fortification in brood fish diet had a positive effect on growth,
increased resistance to ESC disease challenge and had a reduced stress response to low
dissolved oxygen.

WITHERING SYNDROME: DISTRIBUTION, IMPACTS, CURRENT DIAGNOSTIC
METHODS AND NEW FINDINGS
C.S. FRIEDMAN1, N.Wight1, L. Crosson1, G. R. VanBlaricom1,2, I. KIRYU3, J.D.
MOORE4,1

School of Aquatic and Fishery Sciences, University of Washington, Box 355020, Seattle,
WA 98195 USA
2
US Geological Survey, Washington Cooperative Fish and Wildlife Research Unit, Universityof Washington, Box 355020, Seattle, WA 98195USA
3
National Research Institute of Aquaculture, Fisheries Research Agency, Mie 516-0193,
Japan;
4
California Department of Fish and Game and UC Davis-Bodega Marine Laboratory, PO Box
247, Bodega Bay, CA 94923 USA
Withering Syndrome, WS, is a fatal disease of abalone caused by a Rickettsia-likeorganism (WS-RLO). The causative agent, “Candidatus Xenohaliotis californiensis” occurs
along the eastern Pacific margin of North America in California, USA and Baja California,
Mexico. However, as infected abalones have been transported to Chile, China (People’s Rep.
of), Taiwan, Iceland, Ireland, Israel, Spain, Thailand, and most recently Japan, and possibly
other countries, the geographical range of the etiological agent is suspected to be broad where
California red abalones, Haliotis rufescens, are cultured or areas where native species havebeen exposed to this species. Susceptibility varies among species with up to 99% losses of
black abalone, H. cracherodii, in lab and field studies in the USA to no losses among H.
diversicolor supertexta in Thailand. Some populations that have suffered catastrophic losses
to WS have developed an increased resistance to the disease. In addition, a newly identified
phage hyperparasite of the WS-RLO may reduce pathogenicity and dampen losses from the
WS-RLO. Diagnosis of WS requires the identification of infection with the pathogen(WS-RLO via in situ hybridization or via histology coupled with PCR and sequence analysis)

accompanied by morphological changes that characterize this disease (e.g. pedal and digestive
gland atrophy, and digestive gland metaplasia). A real-time PCR (qPCR) assay has beendeveloped and may be useful in quantifying amounts of pathogen DNA. Confirmation of
infection by the WS-RLO cannot be done by PCR analysis alone as this method only detects
pathogen DNA but can be used as a proxy for infection is areas where the agent is established.
Control measures include avoidance, culling infected animals and, as per federal regulations,oral or bath treatment with oxytetracycline. Avoidance is best accomplished by theestablishment of a health history and multiple health examinations prior to movement ofanimals. Although histology or in situ hybridization are required to confirm infection, qPCR
is able to detect small amounts of pathogen DNA and is advised to be included as part ofhealth examinations.

FEEDING HATCHERY-PRODUCED LARVAE FROM THE GIANT GROUPEREPINEPHELUS LANCEOLATUS
Armando García-Ortega, Adam Daw, Kevin Hopkins
Pacific Aquaculture & Coastal Resources CenterCollege of Agriculture, Forestry and Natural Resource Management
University of Hawaii at Hilo1079 Kalanianaole St.
Hilo, HI 96720, USA
Research on the larval rearing of giant grouper Epinephelus lanceaolatus was carriedout for the first time at the Pacific Aquaculture and Coastal Resources Center (PACRC),
University of Hawaii at Hilo. Fertilized eggs from captive broodstock were obtained from
Kampachi Farms, Kailua-Kona, Hawaii. Approximately 36,000 viable eggs were stocked inthree round tanks with central drain and conical bottom each with one m3 of seawater.
Fertilized eggs had a diameter of 0.89 ± 0.01 mm and hatched 30 h post fertilization. At 2
days post hatch (DPH) yolk sac larvae measured 2.4 ± 0.2 mm, had the mouth and anusclosed, and half of the yolk was resorbed. Microalgae Isochrysis galbana was supplied to alltanks from DPH 1 to 20 at a density from 30,000 to 45,000/ml. Two feeding strategies were
tested. In one tank, enriched trocophore larvae of the Pacific oyster (Crassostrea gigas) were
fed in DPH 2 and 3, shifting the feeding at DPH 4 to enriched S-type rotifers (Brachionus
rotundiformis) at a density of 20 per ml. In the second and third tank fish first food consistedof a mix of calanoid copepods (Parvocalanus crassirostris) (0.5 to 3.0/ml) and enriched
S-type rotifers (10/ml), both were fed to the fish twice every day. At 4 DPH the yolk sac inlarvae measuring 3.0 ± 0.0 mm was almost completely resorbed, however, food was alreadypresent in the gut in the mixed copepod/rotifer feeding. At 6 DPH massive mortality occurredin the rotifer treatment as apparently the fish were not able to ingest them. By 8 DPH
mortality was total in this tank. In contrast, fish in the mixed feeding regime showed constantfeeding behavior with gut fullness confirmed visually under the microscope. At 10 DPH a
co-feeding treatment was initiated in one of the two remaining tanks with a microdiet of200-300 µm particle size and shifted to 300-500 µm in DPH 14 at a fish larvae length of 5.1 ±0.3 mm. Despite being fed the same mixed rotifer/copepod ration, growth of larvae washigher in the co-fed treatment. Thus, at DPH 17 co-feeding was also applied in the other tank.
Starting at 14 DPH instar I nauplii of Artemia was supplied to the larger fish tank followed bya mix of instar I nauplii and enriched metanauplii two days later. The same was applied at
DPH 17 in the smaller fish. Artemia was fed two times per day at a density of 1-3/ml. At DPH
20 the supply of rotifers and copepods to the tanks was terminated. Grouper larvae presented
high growth rates at the Artemia feeding stage. Metamorphosis started at 25 DPH and at 35
DPH it was not yet completed with larvae remaining pelagic. Weaning was initiated at 35
DPH. Survival from hatched to pre-weaned larvae under the described conditions wasestimated at 2.1%. Photoperiod was maintained at 24L:0D from1 to 20 DPH, then changed to
12L:12D. Water temperature and salinity were 27.6 ± 0.2 °C and 29.5 ± 0.3 ppt respectively.

STUDY ON HIGH INCIDENCE OF DEATH DUE TO COLLISION OF
HATCHERY-REARED PACIFIC BLUEFIN TUNA THUNNUS ORIENTALISJUVENILES IN NET CAGES
Kentaro Higuchi*, Yosuke Tanaka, Takeshi Eba, Akefumi Nishi, Kazunori Kumon, Hideki
Nikaido, Satoshi Shiozawa
Research Center for Tuna Aquaculture, Seikai National Fisheries Research Institute, Fisheries
Research Agency
Bluefin tuna has been cultured in many countries and regions, such as theMediterranean Sea, Mexico, Australia and Japan, because of its high commercial value. The
decline of wild tuna stocks and the recent controversial issues of tuna capture-based
aquaculture catalyzed the need for a stable supply of artificially-reared tuna juveniles foraquaculture purposes. The reasons for that is two-fold; first, to reduce the negative impacts on
wild-stocks, and second, to promote the aquaculture industry. In 2002, Kinki University
succeeded in the reproduction of Pacific bluefin tuna Thunnus orientalis (PBT) in captivity.
Various studies to apply artificially-reared juveniles to mass-culture system have been carriedout actively since then, however a high mortality of hatchery-reared juveniles is frequently
observed after transfer from indoor tanks to net cages at around 30 days post hatch. The
mortality is likely due to collisions with the cage nets at dawn. Techniques are being
developed to prevent these collisions as the survival rate is much lower than that of otherteleosts. In order to reduce the mortality in net cages, it is important to elucidate thedevelopmental stages of reared PBT when the collisions occur in net cages.
Bone injuries can be considered an index for obvious collisions due to the damage
caused by physical impact. In the present study, bone injuries of dead juveniles wereinvestigated in order to examine the prevalence of net collisions. Juvenile PBT were reared in
3 net cages (20 m in diameter) for 90 days after being transferred from indoor tanks at Amami
Station, Seikai National Fisheries Research Institute, Fisheries Research Agency, Japan. Deadfish were sampled and examined by clearing and staining method or by dissection to detect
injury to the vertebral column and parasphenoid. Results showed that dislocation andfractures of the vertebral column usually occurred between the first and the 15th vertebrae. Up
to 30 days after being transferred, the prevalence of injuries of the vertebral column andparasphenoid was low (0.0-12.0%); with fish size being 5.5 to 15.2 cm in total length (TL).
From 31+ days after transfer, the injuries drastically increased to 17.8-78.0% (21.0 to 39.2 cm
in TL). These results suggest that the mortalities of juvenile PBT larger than 20 cm in TL incages were caused by net collision. Further studies are required to determine the reason forhigh incidence of collisions of juvenile PBT larger than 20 cm TL and to reduce collision
deaths for a consistent and stable supply of the seedlings for aquaculture.
SUCCESS OF SEED PRODUCTION OF HUMPHEAD WRASSE CHEILINUS
UNDULATUS WITH IMPROVEMENT OF SPAWNING INDUCTION, FEEDING,
AND REARING CONDITION.
Narisato Hirai1, Masahiko Koiso2, Kazuhisa Teruya3, Masato Kobayashi3, Takayuki Takebe3,
Taku Sato3, Koichi Okuzawa4, and Atsushi Hagiwara5
1

National Research Institute of Aquaculture, Aquaculture Systems Division
Japan Sea National Fisheries Research Institute, Stock Enhancement and Aquaculture
Division
3
Seikai National Fisheries Research Institute, Research Center for Subtropical Fisheries
4
National Research Institute of Aquaculture, Aquaculture Technology Division
5
Nagasaki University, Graduate School of Fisheries Science and Environmental Studies
2

Humphead wrasse, Cheilinus undulatus, is the largest labrid fish distributed intropical and sub-tropical regions of the Indo-Pacific and occur around coral reefs. Increasing
fishing pressure since the early 1990s for the live reef-fish trade, caused the number of thisspecies to drop in many countries, finally resulting in its listing in Appendix II of theConvention on International Trade in Endangered Species of Wild Fauna and Flora (CITES)
in 2004. Currently, international trade is government-controlled or the catch of humphead
wrasse banned in some of the countries. In Japan, the fishery of humphead wrasse is found in
Ryukyu Islands, and the annual catch was 6.4t in 1990, but decreased to approximately halfthat during the last two decades.
Attempts of aquaculture are becoming important for the sustainable utilization of
humphead wrasse. Successful seed production (120 juveniles) was achieved once in Indonesia
in 2003, however, reproducible production has not been established. Therefore, we examined
the method of spawning induction and initial food and rearing conditions of larvae.
Spontaneous spawning was found when the water temperature was greater than 28°C, andwithin 1 week of the new moon during June through September. Fertilization rate was 10 –25% with spontaneous spawning, but was successfully induced 100% with a drawdown ofseawater in rearing tanks of the broodstock. The mouth diameter of humphead wrasse larvae
is small (133 µm), therefore we also examined initial feeding. Two different types ofmonogonont, SS-type rotifer (Brachionus rotundiformis Thai-strain) and more minute Proalid
rotifer (Proales similis), were used as candidates of live food and boiled chicken yolk andpowdered milk were also examined. Larvae preferred Proales similis than any other dietduring the first 7days after initial feeding, and thereafter, preferred SS-type rotifer. The
survival rate of larvae was highest when fish were reared in 0.5 kL circle tanks with 20
ml/min aeration at 30 °C, with addition of a layer of surface oil to prevent surface death oflarvae. By using Proales similis as the initial live food, optimizing aeration rate and adding
feed oil, we produced 22 juveniles at 50 days post hatch [dph] (survival = 0.25%, TL= 9.0mm) on August 2011. In September 2011, we produced 537 juveniles at 50 dph (survival=
10.7%, TL= 9.1 mm) with the use of concentrated oxygen for aeration, indicating thatexamined rearing method was reproducible for the seed production of humphead wrasse.

WITHERING SYNDROME IN ABALONE IN JAPANI. Kiryu1, J. Kurita1, K. Yuasa1, T. Nishioka1, Y. Shimahara1, T. Kamaishi1, N. Tange2, N.
Oseko1 and C. S. Friedman3
1

National Research Institute of Aquaculture, Fisheries Research Agency, Mie 516-0193,
Japan
2
Fish Farming Center Department of Agriculture, Forestry and Fishery Tottori Prefectural
Government, Tottori 689-0602, Japan3
School of Aquatic and Fishery Sciences, University of Washington, Seattle, Washington
98195, USA
Withering syndrome (WS) is known as a chronic wasting disease in abalone caused
by Candidatus xenohaliotis californiensis, a Rickettsia-like organism (RLO) and has beenprevalent on the west coast of the United States since the mid-1980s. WS is listed as
notifiable disease in the International Aquatic Animal Health Code of the Office International
des Epizooties (OIE). In Japan, the WS-RLO was first detected in 2011 in farmed, juvenile
Japanese black abalone (Haliotis discus discus; 14 – 34mm in shell length). Those abalone
experienced monthly mortality rates from 3 to 10% with a cumulative mortality of 32.8%, andhistopathological changes and pathogens were not found except for basophilic, intracellularbacterial colonies within the epithelium of the digestive tract. Morphological characteristics
and target tissues containing the bacterial inclusions were consistent with those of the
WS-RLO. High intensity of infection by the RLO was observed in the epithelium of theposterior portion of the esophagus, the intestinal epithelium and, to a lesser extent, in theepithelium of transport ducts in the digestive gland. The RLOs observed were conclusively

identified as the agent of withering syndrome by the additional investigation of PCR andsequence analysis. Preventive measures against WS implemented in Japan will be shown.

MARINE FISH HATCHERY TECHNOLOGY AND RESEARCH AT THE THAD
COCHRAN MARINE AQUACULTURE CENTER
Phillip G. Lee, Reginald Blaylock, Eric Saillant and Jeffery Lotz
Thad Cochran Marine Aquaculture Center
Gulf Coast Research Laboratory-University of Southern Mississippi
703 E. Beach Road, Ocean Springs, MS 39564
The Thad Cochran Marine Aquaculture Center is composed of 7 new laboratorybuildings dedicated to the development of Gulf of Mexico marine fish aquaculture. The
buildings include live feed production systems, hatchery, broodstock, juvenile and adultproduction systems, as well as, wet and dry laboratories for genetic, disease, nutrition andbehavioral research. In addition, several buildings are used for teaching and public
educational purposes. The primary objectives of the Center are to enhance Mississippi Gulfwaters with commercially and recreationally important species and develop aquaculture
technology for commercial transfer. The immediate goals of the Center are to develop the
methods to capture, maintain and spawn broodstock of marine fish in order to facilitate
production, rearing, tagging, releasing and assessing of juveniles in a stocking program. The
facility employs cutting-edge technology, peer-reviewed research, and hands-on testing togrow fish in an environmentally sustainable and economically feasible manner. Research isfunded through partnerships with state and federal agencies, as well as by private companies.
The facility is unique in that all hatchery and production tanks are closed, recirculatingseawater systems filled with artificial seawater, insuring biosecurity between tanks and theenvironment. The research and facilities can be divided into three programs, Spotted Seatrout
Program, Red Snapper Program and Demonstration Program for new species. Over the last
few years, more than half a million Spotted Seatrout (Cynoscion nebulosus) have beenstocked into Gulf waters. Seatrout have been tagged and released and an assessment programis being developed in collaboration with the Mississippi Department of Marine Resources.
The second major project focuses on Red Snapper (Lutjanus campechanus); several thousand
have also been released in recent years. The Red Snapper Program has been significantly
expanded during the last 2 years with the construction of the new Red Snapper Hatchery and
Grow-out buildings. Improvements in live feed production have been key to the success ofthese expanding hatchery production projects. Additional species have been the focus of theDemonstration Project research, including Cobia (Rachycentron canadum), Tripletail(Lobotes surinamensis) and Atlantic Croaker (Micropogonias undulatus). The Center has led
an effort to establish a Gulf of Mexico Marine Fish Hatchery Consortium that would link a
number of research and private scientific institutions on the Gulf coast, improving

communication and coordinating joint research.
EFFECTS OF DOCOSAHEXAENOIC ACID AND TAURINE LEVELS IN
ROTIFERS ON GROWTH AND SURVIVAL OF LARVAL AMBERJACK SERIOLADUMERILI
Hiroyuki Matsunari1*, Hiroshi Hashimoto2, Takashi Iwasaki3, Kentaro Oda2, Yoshitsugu
Masuda2, Hitoshi Imaizumi2, Kazuhisa Teruya2, Hirofumi Furuita1, Takeshi Yamamoto1,
Kazuhisa Hamada4 and Keiichi Mushiake1
1

National Research Institute of Aquaculture, Fisheries Research Agency, Minami-ise, Mie,
Japan.
2
Shibushi Station, National Center for Stock Enhancement, Fisheries Research Agency,
Shibushi, Kagoshima, Japan.
3
Stock Enhancement Technology Development Center, National Research Institute of
Aquaculture, Fisheries Research Agency, Saiki, Oita, Japan
4
Komame Branch, Stock Enhancement Technology Development Center, National ResearchInstitute of Aquaculture, Fisheries Research Agency, Otsuki, Kochi, Japan.
The amberjack Seriola dumerili is a commonly cultured species in southwest Japanand aquaculture production, together with yellowtail Seriola quinqueradiata, dominates that
region. Although a stable supply of amberjack juveniles artificially produced in Japan isurgently needed, the current technology for mass seed production is still incomplete.
Successful seed production of marine fish requires fortification of live foods that haveinsufficient essential nutrients. The docosahexaenoic acid (DHA) and taurine contents of wild
amberjack larvae were higher than cultured larvae. These nutrients in Artemia nauplii andespecially rotifers, which are used as live foods for amberjack larvae during seed production,
were markedly lower compared to wild zooplanktons. These findings suggest that DHA andtaurine contents in rotifers used for seed production of amberjack are insufficient for therequirements for this species.
First, we investigated the effect of DHA on growth and survival of larval amberjack.
Larvae at 3 days post-hatch (dph) were fed rotifers enriched with commercial DHA
supplements at 4 levels, and reared for 7 days. DHA enrichment of rotifers was effective toimprove growth, survival rate, and swim bladder inflation of amberjack larvae. Next, weinvestigated the effect of algae with different DHA contents on growth performance, survival,
and swim bladder inflation of larval amberjack. The algae were used for rotifer enrichment
and also supplemented to the larval rearing tanks in static condition. Feeding trials wereconducted from 1 to 10 dph. Rotifers enriched with Nannochloropsis (EPA rich rotifers) wereeffective to enhance growth and survival, but DHA was essential to improve the swim bladderinflation in amberjack larvae. Furthermore, the effect of feeding rotifers enriched with taurineon the growth performance and survival of larval amberjack was investigated. Amberjack
larvae at 3 dph were fed rotifers enriched with a commercial taurine supplement at 4 levels (0,200, 400 and 800mg/l), and reared for 7 days. Growth and survival of fish fed rotifers
enriched with taurine supplement at 800mg/l were improved compared to fish fed the rotifers
without taurine enrichment. Taurine enrichment of rotifers is effective to improve the survivalof amberjack larvae.
The results of these studies indicate that enrichment of rotifers with DHA and taurine
is essential for the growth and survival of larval amberjack, and the requirements of DHA andtaurine in rotifers are estimated at 1.5 and 5.3 mg/g on a dry matter basis.

APPLICATION TO THE GENETIC BREEDING USING
INFORMATION IN YELLOWTAIL (SERIOLA QUINQUERADIATA)GENOMICS

Akiyuki Ozaki1*, Kazunori Yoshida2, Kai Wataru1, Jun-ya Aoki1, Yumi Kawabata1, Kanako
Fuji3, Satoshi Kubota3, Kazuki Akita3, Takashi Koyama3, Masahiro Nakagawa2, Takurou
Hotta2, Tatsuo Tsuzaki2, Nobuaki Okamoto3, Takashi Sakamoto3, and Kazuo Araki1
1

National Research Institute of Aquaculture, Fisheries Research Agency, 422-1,
Nakatsuhamaura, Minamiise-cho, Watarai-gun, Mie, 516-0193, Japan
2
Goto Branch of Seikai National Fisheries Research Institute, Fisheries Research Agency,
122-7, Nunoura, Tamanoura-machi, Goto-shi, Nagasaki, 853-0508, Japan
3
Faculty of Marine Science, Tokyo University of Marine Science and Technology, 4-5-7,
Konan, Minato-ku, Tokyo, 108-8477, JapanThe capture marine products industry has developed as the majority of the fisheryindustry, directly using aquatic resources. Only recently has the breeding of marine fish been
considered important research, due to the decrease of available aquatic resources. Beyond thedepletion of aquatic resources, the expectation of aquaculture research is getting higher.
Genetic improvement of economic traits is needed to improve phenotypes suitable for captive
breeding, improving with every generation. We are researching practical application for
selection of economically important traits from natural genetic resources of yellow tail
(Seriola quinqueradiata).
In our study, we identified sex-linked markers for the genetic-sex of yellowtail as arepresentative binary trait, which can be useful for controlling sex in yellowtail breeding.
When an efficient sex detection method becomes available for yellowtail, it will be possible
to select the fastest-growing sex to increase food production. The sex-determining locus islocated in Squ12 Genetic linkage map, and the sex-linked alleles were inherited from thefemale parent. This result suggests that yellowtail has a ZZ–ZW sex-determining system, andthat it would be possible to use these sex-linked markers to discriminate between sexes.
We also targeted disease resistance for important traits. Especially, Benedenia
disease caused by the ectoparasite Benedenia seriolae and is difficult to prevent in marine
aquaculture systems. This is a serious parasitic disease in yellowtail aquaculture, leading tosecondary viral or bacterial infections. Because fish rub their bodies against the fish cage toremove the parasite, mortality is quite high especially juveniles. The common way ofremoving the parasite is to soak the fish in a freshwater bath. However, this method requires a
lot of time, cost, and effort. To evaluate the genetic basis of Benedenia disease resistance inyellowtail, a genome-wide and chromosome-wide linkage analysis was initiated using F1yellowtail families. Two major quantitative trait loci (QTL) regions on linkage groups Squ2and Squ20 were identified and then confirmed in F1 families. These QTL regions explained
32.9– 35.5% of the phenotypic variance. This is the first genetic evidence discovered that
contributes to detailing the phenotypic resistance to Benedenia disease, and the results will
help resolve the mechanism of resistance to this important disease of yellowtail.
Advanced research is progressing. DNA marker-assisted selection “MAS” program
were performed for linkage groups Squ2 and Squ20 QTL significant region using the F2family. F2 QTL congenic line had a significant phenotypic difference in the number ofparasites, when comparing resistant-parents with susceptible-parents in parental genotype
combinations.
The next step is to apply MAS breeding to other target aquatic species. The effective
utilization of natural genetic marine resources in aquaculture has more advantages comparedbecause wild species are not selected and still maintain high genetic diversity.

THE COOPERATIVE CULTURE OF SEAWEED IN NEW ENGLAND—HOW
RESEARCH, INDUSTRY, AND EXTENSION ARE CULTIVATING A NEW FIELD
IN AQUACULTURE
Sarah Redmond*, Dana Morse Charles Yarish, Jang Kim, Paul Dobbins, Tollef Olson
Maine Sea Grant & University of Maine Cooperative Extension
University of Maine
Orono, ME 04469
A partnership of research, industry, and extension in the Northeast has resulted insuccessful commercial culture of native seaweed species in Maine and Long Island Sound.
Development of nursery culture and grow-out technologies for sugar kelp (Saccharina
latissima) in the Seaweed Biotechnology Laboratory at the University of Connecticut and thelab and farm of Ocean Approved, LLC, of Portland, ME has allowed for the establishment of
the first commercial kelp farm in the United States and an educational and research farm inLong Island Sound. The technology was shared and further developed along the coast ofMaine by integrating kelp lines on six different shellfish farms through a Maine Sea Grant
extension-led collaborative research project. The first regional seaweed aquaculture workshop
was held as a result of this work, bringing together members of the seaweed industry,
potential new farmers, researchers, extension, students, and entrepreneurs. This emerging new
field continues to grow in the Northeast with the sharing of ideas, technologies andinformation through the open and collaborative relationships of extension, research, andindustry.

INTENSIVE JUVENILE PRODUCTION OF YELLOWTAIL AMBERJACK (Seriolalalandi) IN SOUTHERN CALIFORNIA
Federico Rotman*, Kevin Stuart, and Mark Drawbridge
Hubbs-SeaWorld Research Institute2595 Ingraham St.
San Diego, CA 92109, USA
Hubbs-SeaWorld Research Institute (HSWRI) has been culturing yellowtail
amberjack (Seriola lalandi) at its laboratory in San Diego, CA since 2003. Beginning in 2007
intensive-level production began at the Mission Bay research facility in order to refine
methodologies at a commercial scale. Initial results yielded survival levels to 1 gram juvenile
between 0.2 to 5.0% with varying degrees of skeletal malformation (often greater than 40%).
Moreover, larval swim bladder inflation rates have been highly variable; an issue that poses
potential problems for the cage-based production of the species. In 2012 live feed quality andlarval rearing microbial management improvements were made to production protocols andsystems in order to increase juvenile quantity and quality. Many of these changes were basedon observations made during HSWRI larval rearing trials coupled with advancements in the
global aquaculture industry.
Live feed quality was enhanced through the improvement of the water quality
supplying both the rotifers (Brachionus plactilis) and Artemia (Artemia franciscana), as wellas limitation of bacterial loading in the live feeds culture systems. The water quality
supplying live feeds systems was improved by adding high-capacity 1 µm pleated filters andhigh-intensity ultraviolet sterilizers. Furthermore, Artemia protocols were modified todecrease Vibrio spp loading through the use of INVE Separt systems, INVE Hatch Controller,
improved sanitation and automated cold storage. Rotifer systems were modified to promote
more stable microbial communities through the use of recirculation technology and improved
cold storage.
Larval rearing systems were improved through the addition of a dedicated,
high-capacity 1 µm pleated filter, ultraviolet sterilizer and heaters. In order to remove
accumulated bio-film from tanks, plumbing and equipment, a routine total-system disinfection
protocol was established in between production runs. Moreover, quantitative bacterial
screening methods were put into place to confirm cleaning and disinfection protocols. With
these systems in place, various larval rearing strategies were trialed at intensive levels in“commercial-scale” larval tanks (volumes between 1.6 – 8.0 m3) based on previous
experimental results. In most trials, rotifers and turbidity (algae paste or clay) wereadministered to larval tanks continuously via an automated temperature controlled
cold-storage unit. Although clay-based turbidity protocols were trialed with relatively poor

success, algae-paste turbidity protocols coupled with larval passive-transfer methods yielded
the most productive results.
Ultimately, utilizing improved systems and protocols, two Seriola lalandi production
runs were completed in the summer of 2012 and one is currently underway. The first run
yielded more than 200,000 weaned 0.75 gram juveniles (30% survival from egg); the second
produced more than 45,000 weaned 1.3 gram juveniles (7% survival from egg) and the thirdis currently still in its late larval stage but appears to be following a similar successful trend.
Skeletal malformation rates have been relatively very low (< 5.0%) compared to previous
years production. Although these results have been encouraging, larval swim bladder inflation
rates have been very problematic, even compared to previous years, with the first two runs
demonstrating inflation rates of < 1.0% and 23.0%, respectively.
Current research and development efforts include further refining systems andprotocols, reducing variability in larval survival and improving swim bladder inflation.

IN SITU SWIMMING AND SETTLEMENT BEHAVIOR OF CULTUREDSERRANID LARVAE, PLECTROPOMUS LEOPARDUS AND EPINEPHELUS
MALABARICUS
Shibuno, T.1, O. Abe2, Y. Takada3, K. Hashimoto4
1

National Research Institute of Aquaculture, Stock Enhancement and Aquaculture Division
National Research Institute of Far Seas Fisheries, Bluefin Tuna Resources Division3
Japan Sea National Fisheries Research Institute, Stock Enhancement and Aquaculture
Division
4
Seikai National Fisheries Research Institute, Research Center for Fisheries and Environment
in the Ariake and Yatsushiro Bay
2

Cultured serranid larvae, Plectropomus leopardus (n=81) and Epinephelus
malabaricus (n=61) were released during day time in June and July of 1997 and 1998 at
various locations along a fringing reef at Ishigaki Island, southern Japan, and their behavior
observed by divers. At offshore (1 km) deeper sites (30 m), released larvae swam at an
average depth and swimming speed of 10.2 m and 4.9 cm/s for P. leopardus and 7.5 m and6.1 cm/s for E. malabaricus. When released at shallower sites (7 m) close to the reef edge (3m), both species of larvae swam directionally to open water to avoid predators. At sites 400 m
offshore and 18 m deep, released P. leopardus and E. malabaricus larvae settled on the slope
at average depths of 16.4 m and 15.4m, respectively. However, there were no significant
differences between the two species in settlement depth, time to settlement, swimming speed
and swimming distance. When released 3m from the slope and 3m above the rubble bottom atsites 400 m offshore and 16 m deep, 73 % of P. leopardus larvae settled underneathprominent live or dead coral on the slope, and 23 % settled underneath rocks on the bottom.

EFFECTS ON GROWTH OF FLATFISH JUVENILE BY ARTIFICIAL
LIGHTENING CONDITIONS: INTENSITY, PHOTOPERIOD AND WAVELENGTH
Daisuke Shimizu*1 and Yuichiro Fujinami2
1

Tohoku National Fisheries Research Institute, Fisheries Research Agency
Miyako Laboratory, Tohoku National Fisheries Research Institute, Fisheries Research
Agency2

Mechanisms that respond to light are deeply involved in life history of fish and arewidely known. Light control technology has been essential for control of the spawning cyclein broodstock management. On the other hand, in hatcheries for seed production, lightenvironment can significantly affect the first-feeding success in various fish species. Rearing
techniques that match the optimal light environment conditions of the target species isessential to efficient and healthy seed production.
Therefore, in the present study, three flatfish species of importance for fisheries innorthern Japan, spotted halibut Verasper variegatus, slime flounder MIicrostomus achne, andJapanese flounder Paralichthys olivaceus juveniles were studied to elucidate the necessary
artificial lighting conditions: intensity, photoperiod and wavelength. We conducted rearing
experiments for each developmental stage, growth, survival, and feeding status (feeding
incidence, average number of rotifer in digestive tract) as an indicator of the optimal lightenvironment.
Light intensity: In spotted halibut that live in very shallow seas, feeding status was
better, and growth and survival rate was improved in light conditions (＞12 µmol･m-2 s-1). In
addition, they fed even in the dark conditions, showing increased growth. In slime flounderthat live in the deep sea, feeding status was better and growth and survival rate was been
improved in dark conditions (<0.2 µmol･m-2 s-1).
Photoperiod: In spotted halibut and Japanese flounder, feeding status was better andgrowth and survival rate was improved in continuous lightening (24 h). Photoperiod had noeffect on slime flounder.
Wavelength: As a result of rearing juveniles under various wavelengths in spottedhalibut and flounder, green light (518nm) feeding status was better compared with white light.
Result of electro-intraretinal-retinogram in adult fish of these flatfish, the peaks of spectral
sensitivities of spotted halibut were 521 nm, Japanese flounder were 511nm, thus supporting
the results of rearing experiments.
As a result of incorporating optimal light environment obtained in the above
experiments, seed production technology of spotted halibut has been improved dramatically(average survival rate: improved to 50% from 20%, average normal rate: improved to 80%
from 50%).

LARVAL REARING ADVANCEMENTS FOR YELLOWTAIL AMBERJACK (Seriola

lalandi) IN SOUTHERN CALIFORNIA
Kevin Stuart*, Federico Rotman, and Mark Drawbridge
Hubbs-SeaWorld Research Institute2595 Ingraham St.
San Diego, CA 92109, USA
Hubbs-SeaWorld Research Institute (HSWRI) has been culturing yellowtail
amberjack (Seriola lalandi) experimentally at its laboratory in San Diego, CA since 2003. In
2007, we began to intensively rear juveniles, and between 2007 and 2011, we achieved
survival rates as high as 5.0% from egg to approximately 50 days post hatch (dph). In 2012,
we continued to conduct manipulative larval rearing studies to improve overall larval
performance. These studies targeted optimizing the live prey feeding regime and reducing thebacterial load (Vibrio spp.) in the larval culture tanks.
First, we attempted to further optimize our larval live prey feeding procedures. The
traditional HSWRI feeding regime for yellowtail amberjack began with offering rotifers(Brachionus plicatilis) at 2 dph through 7 dph, 1st instar Artemia (Artemia franciscana) were
offered from 6 through 8 dph, and 2nd instar Artemia are were offered from 7 through 35 dph.
In the first trial, larvae were co-fed rotifers, 1st instar Artemia, and 2nd instar Artemia from 3
through 20 dph in order to gain a better understanding of appropriate weaning times, as wellas ingestion levels of each feed over time. Results from this trial showed that the larvae
selected 1st instar Artemia as early as 3 dph (4.5 mm notochord length) and 2nd instar
Artemia as early as 5 dph (4.6 mm notochord length). The second trial attempted to remove
1st instar Artemia from the current feeding regime because 1st instar cannot be enriched. This
trial consisted of three treatments: 1) rotifers to 2nd instar offered at 5 dph, 2) rotifers to 2nd
instar offered at 7 dph, and 3) rotifers to 1st instar to 2nd instar (i.e. traditional HSWRI
protocol). Results showed no significant differences in growth or survival between any of thetreatments, indicating that larvae can be weaned from rotifers directly onto 2nd instar Artemia
at 5 dph.
Second, we wanted to reduce bacterial load levels, specifically Vibrio species, in thelarval culture tanks. The bacterial load in larval rearing environment is heavily influenced by
the inputs introduced to the rearing tanks (e.g. algae cells and live prey), as well as otherfactors like water treatment and temperature. We conducted two trials to evaluate methods tominimize the bacterial load (Vibrio spp.) in the water, while maintaining good larval
performance. The first trial examined the use of Bentonite clay as a turbidity agent to replace
algae in a greenwater-type environment. This study consisted of three treatments: 1) clay with
continuous feeding, 2) clay with batch feedings, and 3) algae paste with batch feedings. The
results showed that both clay treatments had significantly fewer Vibrio colonies in the watercolumn (377 ± 120 CFU) than the algae paste treatment (5,692 ± 2,396 CFU) after 14 days ofculture. Growth was not significantly different among the treatments, however survival was
significantly higher in the clay treatment with continuous feeding (14.1 ± 2.6%) than either

the batch fed clay (2.3 ± 0.5%) or algae paste treatments (2.8 ± 1.5%). The second study
attempted to limit bacterial loading in the larval rearing culture tank by siphoning the larvae
into adjacent, clean tanks at 1, 5 and 9 dph during the first two weeks of culture. The resultsfrom this trial showed that the water in the transfer tank had fewer Vibrio colonies (1,025 ±541 CFU) than the water in the control tanks where larvae were not moved (1,962 ± 1,415
CFU). Also, survival was significantly higher among larvae that were transferred (43.9 ±
13.5%) than in the control tanks (23.1 ± 6.3%).
Based on these results, we applied these same experimental methods on threecommercial-scale production runs in 2012 that yielded survival rates as high as 30% from egg
to juvenile fish. This work demonstrates the value and transferability of laboratory-scale
experimental results to a production scale. Additional experimental work is required to
improve the overall consistency of larval survival, including an improved understanding ofvariability among batches of eggs.

EXPERIMENTAL STUDY ON BROODSTOCK MANAGEMENT OF BARFIN
FLOUNDER UNDER THE CONCEPT OF MINIMUM KINSHIP SELECTION
Shigenori Suzuki1*, Naoto Murakami2, Takashi Ichikawa2
1

Minami-Izu Laboratory, National Institute of Aquaculture, Fisheries Research Agency(FRA), Irouzaki, Minami-Izu, Shizuoka 415-0156, Japan.
2
Akkeshi Laboratory, Hokkaido National Fisheries Research Institute, Fisheries Research
Agency (FRA), 2-1 Tsukushikoi, Akkeshi, Hokkaido 088-1108, Japan.
A large flatfish, the barfin flounder Verasper moseri, is distributed from the southern
Sea of Okhotsk to northern Japan. This fish is the highest-priced righteye flounder in Japan,
but fishery yields have been endangered since the 1970s by its severe depletion. This
reduction has rendered barfin flounder a rare species. To correct this situation, a stockenhancement program with annual release of approximately 100,000 seedlings has beenpromoted since 1987. This program is inferred to be effective because released seedlings arerecaptured every year. However, the risk of losing genetic diversity from the base population
is ever-present: the proportion of hatchery born fish in broodstock has been increasing
because obtaining wild barfin flounder is almost impossible. For this reason, optimal
broodstock management is necessary to avoid inbreeding and loss of genetic variation in
future generations.
In this study, we applied the concept of minimum kinship selection to broodstock
management of barfin flounder using three microsatellite DNA markers. The breeding plan
was designed based on the kinship value, which was calculated among the broodstockindividuals using microsatellite DNA markers. Moreover, individual identification andartificial insemination techniques were developed to support the breeding plan. The
broodstock consisted of 37 wild and 41 hatchery-reared barfin flounders. Each individual wasidentified using a PIT tag. The sex was recognized according to the gonad shape, as analyzed
by sonography. Artificial fertilization was performed based on the factorial mating design,
excluding closely related individuals. Furthermore, the numbers of larvae used for seedling
production were equalized among the pairs to maximize the effective population size. Using
this procedure, we produced seedlings with high genetic variation that was comparable to thatof the broodstock’s, showing that the concept of minimum kinship selection is useful for
genetic conservation in barfin-flounder stock enhancement.

IMPROVING THE HATCHERY OUTPUT OF THE HAWAIIAN PINK SNAPPER,PRISTIPOMOIDES FILAMENTOSUS
Clyde S. Tamaru1, Karen Brittain1, Benjamin Alexander1, Petra H. Lenz2, James Jackson2,and Harry Ako1.
1
2

College of Tropical Agriculture and Human Resources/Hawaii Institute of Marine Biology,
Pacific Biosciences Research Center

The opakapaka, one of the highly prized “Deep Seven” bottom fish species, has beenbranded with an “over fishing” status. This situation has mandated that Hawaii develop a
fishery management plan for the bottom fish fishery in the Main Hawaiian Islands. Part of thisplan was the development of hatchery protocols where initial efforts have resulted in the only
spawning broodstock maintained in captivity. This has offered the opportunity to develop
hatchery techniques for their artificial propagation.
To insure that the nutritional requirements of opakapaka larvae are being met, fatty
acid profiles of spawned opakapaka eggs were determined. Total fatty acid content in the
opakapaka eggs matched that of wild caught copepods. While the total fat content may berelatively low the percent composition of the essential fatty acids, DHA and EPA (26.9% ±
4.3% and 2.7% ± 0.4%, respectively) are consistent with other reports indicating that bothessential fatty acids are critical for survival and growth of the opakapaka larvae. In addition,
the composition of essential fatty acids for the various live food organisms were obtained andwith the exception of Nanno-fed rotifers, the live feeds employed, have similar profiles from apercent composition standpoint and indicates that protocols that were developed can be
adjusted to meet the nutritional requirements of the opakapaka larvae.
Using changes in total length between fed and unfed larvae and monitoring temporalchanges in gape size, it is clearly evident that introduction of live food organisms must takeplace no later than the third day post-hatch. When larvae were presented with a variety of live
food organisms either alone or in combination, the results indicate that a copepod
nauplius-only diet is a superior feeding regime over the all others tested.
Since adult copepod and nauplius production is dependent on cell density of thephytoplankton being used, the team used these findings to consistently time production of
Parvocalanus sp. nauplii at densities ranging between 5-10 individuals/ml in a 3,000-Lrearing tank by the third day post-hatching. This was achieved by “conditioning” copepod
cultures using Isochrysis galbana (Tahitian Strain) at cell densities of 4 x 105 cells/ml prior tostocking into the larval rearing tank. Employing this strategy resulted in unprecedented larval
survival with a mean of 80% passing the first feeding stages up until 10-12 day post-hatching.
Utilizing video recordings of the foraging behavior of opakapaka larvae revealed thatthe evasive abilities of adult and copepodite stages of calanoid copepods allowed them toavoid being captured by the larvae. While rotifers could be easily captured the growing bodyof evidence suggests that the ubiquitous rotifer is not a suitable live food for opakapaka larvae
and another transitional live food is needed.

UNDERSTANDING ARTEMIA BIOGEOGRAPHY RELATED TO HATCHERYPRODUCTION AND JUVENILE QUALITY
Laura Torrentera
NOAA Visitor Scientist
Kirkland, WA 98034
The culture of fish, mollusk, and crustacean larvae is generally carried out undercontrolled hatchery and nursery conditions, but the failure and high cost to garner high yields
is partly due to the lack of ecological studies into live food resources such as the brine shrimp
Artemia. Developing larvae are usually very small, extremely fragile, and generally notphysiologically developed. Proper larval nutrition is one of the major bottlenecks incommercial aquaculture. The best sources of nutrition continue being the development anduse of a succession of live food organisms as feed for the developing larvae. Artemia is one of
the most important live foods in commercial aquaculture. Artemia can ingest small foodparticles ranging from 1 to 50 µm in size including diverse species of live microalgae, baker’s
yeast, dried microalgae, probiotics, and waste products from the food industry. The
non-selective consumption of particulate food by the brine shrimp enables its use as a vector
or “biocapsule” for delivering specific nutrients and biological protectors to marine and fresh
water larvae, guaranteeing healthy juveniles. These nutrients and immune protectors are notstored in appreciable amounts in the larvae body, so signs of deficiency and sickness usuallyappear within weeks in young, rapidly growing fish. In many countries products from theprocessing of plant and animal products from human foods are the primary ingredientsavailable for aquatic animal feeds (rice bran, corn bran, soybeans, whey powder, etc.). Mostof these ingredients have limited nutrient levels, or even introduced anti-nutritional factors.
Even dough these inert foods could be enriched the active predator larvae cannot take it easily.
There is no artificial replacement for natural food sources, and such synthetic feed canincrease water eutrophication and stunt larvae development. Artemia is considered now morethan ever an important source of nutrients not just for its natural nutritional properties but also
as an important “Biocapsule” of essential nutrients, vitamins, pigments, antioxidants, active
enzymes, probiotics, antimicrobial agents, and vaccines. Considering the costs and difficulties

in the formulation of non-live food with the adequate amount of key nutrients, Artemia has
several advantages. For all these reasons further studies of Artemia biogeography and its
nutritional qualities are increasing in recent years. These field and laboratory studies of brine
shrimp strains are demonstrating Artemia’s nutritional value, as well as an indicator of natural
ecological condition. In brief, Artemia’s use as live aquaculture feed is advantageous overexisting inert food sources; as a result several new studies of Artemia biogeography andecology are emerging to evaluate the quality and the production of cysts of different Artemia
population’s worldwide.

CONDITIONING TECHNOLOGIES FOR FLATFISH STOCK ENHANCEMENT:GLOBAL PROGRESS AND PITFALLS
Michelle L. Walsh
Office of Sustainable Fisheries, National Marine Fisheries Service, NOAA
Silver Spring, MD 20910
A successful stocking program requires survival of released fish, and to achieve this,fish must be able to adjust to their new environment, feed successfully, and avoid predation.
However, hatchery-reared flatfishes (e.g., flounders, halibuts, soles) often exhibit irregularswimming, feeding, and cryptic (burying and color change) behavioral patterns comparedwith wild conspecifics, and these behavioral "deficits" are assumed to lead to increased
predation risk once fish are released. Conditioning flatfish to natural stimuli before release
may offer fish an opportunity to refine these behaviors, which may increase survival in natureand subsequent recruitment to the fishery.
Examples of conditioning technologies that have been applied to flatfish in thehatchery include providing sediments in rearing tanks, live (or life-like) feeds, or predator
cues. Marbled flounder, Pseudopleuronectes yokohamae, reared for stock enhancement at
Hyogo Prefecture Hatchery in Japan, are fed a mixture of minced frozen mysids with theaddition of formulated, pellet feed to boost nutritional content. However, pellet feeding issuspended approximately 2 wk before release to focus fish on the more natural feed. In theUS, providing hatchery-reared winter flounder, Pseudopleuronectes americanus, with live
feeds (oligochaete worms and amphipods) during rearing conformed post-release feeding
performance closer and more quickly to that of wild fish1. Cost and effort, however, are still
the biggest impediments to implementing a prolonged, live-feed strategy in the hatchery.
Technologies that can be applied to ease the transition to the wild at, or near, therelease site include, conducting “operant conditioning” on fish to respond to light or soundcues for supplemental food provision during the first few days or weeks post release, or
short-term release into predator-exclusion cages before full release. Cage conditioning allows
hatchery fish to experience natural substrates and sediments, wild (live) food sources, and“safe” predator exposure before actual release. Since 2004, Danish scientists have been cage

conditioning reared Atlantic turbot, Psetta maxima, before release, and this practice has
resulted in a much lower post-release mortality.2 Obama Laboratory, National Center for
Stock Enhancement, Japan, has been conducting pre-release, experimental cage conditioning
for Japanese flounder, Paralichthys olivaceus, since 2008 and found that conditioned fish
have significantly higher recapture rates in the fishery as well as enhanced burying andfeeding skills3. In both Danish and Japanese studies, evidence has been detected that intensive
researcher recollection efforts at, or near, the release site may disproportionately sample
weaker (i.e., non-feeding or non-moving) fish. At the University of New Hampshire in the US,
protocols for releasing winter flounder have included cage conditioning since 2004, however,evidence arose that cages themselves attract 'structure-philic' predators, mostly crabs4 ,so cage
design has been modified in recent years.
Successful conditioning of stocked fish before release can increase post-releasesurvival and recapture. However, choosing a location that can be monitored adequately may
be just as important if the success of a stocking effort will influence future efforts (i.e.,
funding, resources, support).

CHARACTERISTIC EVALUATION METHOD RELATING TO BENEDENIADISEASE OF YELLOWTAIL (SERIOLA QUINQUERADIATA)
Kazunori Yoshida1*, Akiyuki Ozaki2, Masahiro Nakagawa1, Takurou Hotta1, Jun-ya Aoki2,
Takashi Koyama3, Kazuo Araki2, Nobuaki Okamoto3, Takashi Sakamoto3, Tatsuo Tsuzaki1
1

Goto Branch of Seikai National Fisheries Research Institute, Fisheries Research Agency,
122-7, Nunoura, Tamanoura-machi, Goto-shi, Nagasaki, 853-0508, Japan
2
National Research Institute of Aquaculture, Fisheries Research Agency, 422-1,
Nakatsuhamaura, Minamiise-cho, Watarai-gun, Mie, 516-0193, Japan
3
Faculty of Marine Science, Tokyo University of Marine Science and Technology, 4-5-7,
Konan, Minato-ku, Tokyo, 108-8477, Japan
Yellowtail (Seriola quinqueradiata) is one of the most important species in marine
fishery resources and aquaculture in Japan. The production of yellowtails occupies more than
50% in marine finfish aquaculture and reaches about 15,000 tons/year. At present, mostyellowtail aquaculture production uses captured wild juveniles, but artificial rearing ofjuveniles is needed to sustain aquaculture production.
Benedenia is a parasitic disease caused in Seriola species by Benedenia seriolae.
This parasite can cause growth reduction and external injuries in yellowtail, increasing therisk of secondary viral or bacterial infection. The major way of removing the parasite is tosoak the fish into a freshwater bath. However, this method requires a lot of time, cost andeffort.
Under this situation, we have been studying DNA maker-assisted selection "MAS"breeding, in order to use artificial seeds holding resistance to Benedenia disease inaquaculture production. Three components ("Reproduction technology", "Character
evaluation", and "DNA analysis") are critically important to promote "MAS" breedingcertainly.
Here I would like to introduce one of the key components, "Characteristic evaluation
method relating to Benedenia disease in the present species".