United States-Japan
Natural Resources Panel on Aquaculture
40th Scientific Symposium“Hatchery Technology for High Quality Juvenile Production”
October 22-23, 2012
Honolulu, Hawai’i

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
reg.blaylock@usm.edu
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 three stages– the trophont which is parasitic primarily on the gills, the free-living reproductive tomont, andthe 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 isdirect and each tomont can produce up to 256 infective dinospores resulting in the rapid buildup
of heavy infections in recirculating systems. Although the general course of infection isunderstood, no quantitative understanding of the survival and reproductive rates of the parasitethat might lead to improved control is known.
Our program has focused on strengthening our ability to address these shortcomings indiagnosis and management of amyloodiniosis. With respect to diagnosis, we developed a novel,
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. ocellatum in water
samples. The established LAMP assay provides a useful tool for the rapid and sensitive detectionof 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 forevaluating the relative effects of changes in vital rates on the population growth rate. 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 thatspotted 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
Page | 2

parasite population growth. Although the model clearly suggests that control strategies should
continue to focus on limiting the number of dinospores in a system, the model provides aframework for evaluating the relative contribution of various factors to the parasite’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.
nagaraj.chatakondi@ars.usda.gov
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 ♂) hybrid in
hatcheries. Raising hybrid catfish in production ponds enables the catfish farmer to harness
improved growth rates, survival and feed conversion to improve production. Even though hybrid
catfish production has been steadily increasing, fingerling production is still insufficient to meetthe 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 troughsin 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 acoating 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 waterand oil. Four tanks were randomly allocated to a treatment.

Page | 3

Broodfish fed 100 ppm of Astaxanthin fortified feed had a higher (P < 0.05) percent ofgravid 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 the treatments.
However, ovulatory index, fecundity, and fry produced per kg of female were higher (P<0.05) inbroodfish 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, University ofWashington, 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
carolynf@uw.edu
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 othercountries, 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 have been
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 havedeveloped 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 byPage | 4

morphological changes that characterize this disease (e.g. pedal and digestive gland atrophy, anddigestive gland metaplasia). A real-time PCR (qPCR) assay has been developed 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 beused 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 withoxytetracycline. Avoidance is best accomplished by the establishment of a health history andmultiple health examinations prior to movement of animals. Although histology or in situ
hybridization are required to confirm infection, qPCR is able to detect small amounts ofpathogen DNA and is advised to be included as part of health 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
agarciao@hawaii.edu
Research on the larval rearing of giant grouper Epinephelus lanceaolatus was carried out
for the first time at the Pacific Aquaculture and Coastal Resources Center (PACRC), Universityof Hawaii at Hilo. Fertilized eggs from captive broodstock were obtained from Kampachi Farms,
Kailua-Kona, Hawaii. Approximately 36,000 viable eggs were stocked in three round tanks withcentral 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 anus closed, and half of the yolk was resorbed.
Microalgae Isochrysis galbana was supplied to all tanks from DPH 1 to 20 at a density from30,000 to 45,000/ml. Two feeding strategies were tested. In one tank, enriched trocophore larvaeof 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 consisted of 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 in larvae measuring 3.0 ± 0.0 mm was almost completely
resorbed, however, food was already present in the gut in the mixed copepod/rotifer feeding. At
6 DPH massive mortality occurred in the rotifer treatment as apparently the fish were not able toingest 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
Page | 5microscope. At 10 DPH a co-feeding treatment was initiated in one of the two remaining tankswith a microdiet of 200-300 µm particle size and shifted to 300-500 µm in DPH 14 at a fishlarvae length of 5.1 ± 0.3 mm. Despite being fed the same mixed rotifer/copepod ration, growthof larvae was higher in the co-fed treatment. Thus, at DPH 17 co-feeding was also applied in theother tank. Starting at 14 DPH instar I nauplii of Artemia was supplied to the larger fish tankfollowed by a mix of instar I nauplii and enriched metanauplii two days later. The same wasapplied 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 andat 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 was estimatedat 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 HATCHERYREARED PACIFIC BLUEFIN TUNA THUNNUS ORIENTALIS JUVENILES 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*Email: higuken@affrc.go.jp
Bluefin tuna has been cultured in many countries and regions, such as the MediterraneanSea, 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 theneed for a stable supply of artificially-reared tuna juveniles for aquaculture purposes. The
reasons for that is two-fold; first, to reduce the negative impacts on wild-stocks, and second, topromote the aquaculture industry. In 2002, Kinki University succeeded in the reproduction ofPacific bluefin tuna Thunnus orientalis (PBT) in captivity. Various studies to apply artificiallyreared juveniles to mass-culture system have been carried out actively since then, however a highmortality 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 cagenets at dawn. Techniques are being developed to prevent these collisions as the survival rate ismuch lower than that of other teleosts. In order to reduce the mortality in net cages, it isimportant to elucidate the developmental 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 causedby physical impact. In the present study, bone injuries of dead juveniles were investigated inPage | 6

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. Dead fish were
sampled and examined by clearing and staining method or by dissection to detect injury to thevertebral column and parasphenoid. Results showed that dislocation and fractures of thevertebral 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 and parasphenoid 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 in cages were caused by net
collision. Further studies are required to determine the reason for high incidence of collisions ofjuvenile PBT larger than 20 cm TL and to reduce collision deaths for a consistent and stablesupply 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
nrhirai@fra.affrc.go.jp
2

Humphead wrasse, Cheilinus undulatus, is the largest labrid fish distributed in tropicaland sub-tropical regions of the Indo-Pacific and occur around coral reefs. Increasing fishingpressure since the early 1990s for the live reef-fish trade, caused the number of this species todrop in many countries, finally resulting in its listing in Appendix II of the Convention 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 theannual catch was 6.4t in 1990, but decreased to approximately half that 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 themethod of spawning induction and initial food and rearing conditions of larvae. SpontaneousPage | 7

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 of seawater in rearing tanks ofthe broodstock. The mouth diameter of humphead wrasse larvae is small (133 µm), therefore wealso examined initial feeding. Two different types of monogonont, 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 and powdered milk were also examined. Larvae
preferred Proales similis than any other diet during the first 7days after initial feeding, andthereafter, preferred SS-type rotifer. The survival rate of larvae was highest when fish werereared 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 of larvae. 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= 9.0 mm) 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, indicatingthat examined 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
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
ikunari@affrc.go.jp
2Withering syndrome (WS) is known as a chronic wasting disease in abalone caused by
Candidatus xenohaliotis californiensis, a Rickettsia-like organism (RLO) and has been prevalenton 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%, and histopathologicalchanges and pathogens were not found except for basophilic, intracellular bacterial 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 the posterior portion of the esophagus,
the intestinal epithelium and, to a lesser extent, in the epithelium of transport ducts in thedigestive gland. The RLOs observed were conclusively identified as the agent of withering
Page | 8

syndrome by the additional investigation of PCR and sequence analysis. Preventive measuresagainst 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
leenrcc@gmail.com
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 adult production
systems, as well as, wet and dry laboratories for genetic, disease, nutrition and behavioral
research. In addition, several buildings are used for teaching and public educational purposes.
The primary objectives of the Center are to enhance Mississippi Gulf waters with commerciallyand recreationally important species and develop aquaculture technology for commercial
transfer. The immediate goals of the Center are to develop the methods to capture, maintain andspawn broodstock of marine fish in order to facilitate production, rearing, tagging, releasing andassessing of juveniles in a stocking program. The facility employs cutting-edge technology, peerreviewed research, and hands-on testing to grow fish in an environmentally sustainable andeconomically feasible manner. Research is funded through partnerships with state and federal
agencies, as well as by private companies. The facility is unique in that all hatchery andproduction tanks are closed, recirculating seawater systems filled with artificial seawater,
insuring biosecurity between tanks and the environment. The research and facilities can bedivided 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 been stocked into Gulf waters. Seatrout have beentagged and released and an assessment program is being developed in collaboration with theMississippi 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 of these expanding hatchery production projects. Additional species
have been the focus of the Demonstration 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

Page | 9

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 SERIOLA DUMERILI
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.
matunari@affrc.go.jp
The amberjack Seriola dumerili is a commonly cultured species in southwest Japan andaquaculture production, together with yellowtail Seriola quinqueradiata, dominates that region.
Although a stable supply of amberjack juveniles artificially produced in Japan is urgently
needed, the current technology for mass seed production is still incomplete. Successful seedproduction of marine fish requires fortification of live foods that have insufficient essential
nutrients. The docosahexaenoic acid (DHA) and taurine contents of wild amberjack larvae werehigher than cultured larvae. These nutrients in Artemia nauplii and especially rotifers, which areused as live foods for amberjack larvae during seed production, were markedly lower comparedto wild zooplanktons. These findings suggest that DHA and taurine contents in rotifers used forseed production of amberjack are insufficient for the requirements 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 to improve growth,survival rate, and swim bladder inflation of amberjack larvae. Next, we investigated the effect ofalgae with different DHA contents on growth performance, survival, and swim bladder inflationof larval amberjack. The algae were used for rotifer enrichment and also supplemented to thelarval rearing tanks in static condition. Feeding trials were conducted from 1 to 10 dph. Rotifers
enriched with Nannochloropsis (EPA rich rotifers) were effective to enhance growth andsurvival, but DHA was essential to improve the swim bladder inflation in amberjack larvae.
Furthermore, the effect of feeding rotifers enriched with taurine on the growth performance andsurvival of larval amberjack was investigated. Amberjack larvae at 3 dph were fed rotifers
Page | 10

enriched with a commercial taurine supplement at 4 levels (0, 200, 400 and 800mg/l), and rearedfor 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 enrichmentof rotifers is effective to improve the survival of amberjack larvae.
The results of these studies indicate that enrichment of rotifers with DHA and taurine isessential 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 GENOMICS INFORMATIONIN YELLOWTAIL (SERIOLA QUINQUERADIATA)
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, Japan
aozaki at affrc.go.jp
The 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. Whenan efficient sex detection method becomes available for yellowtail, it will be possible to selectthe fastest-growing sex to increase food production. The sex-determining locus is located in
Squ12 Genetic linkage map, and the sex-linked alleles were inherited from the female parent.
This result suggests that yellowtail has a ZZ–ZW sex-determining system, and that it would bepossible to use these sex-linked markers to discriminate between sexes.
We also targeted disease resistance for important traits. Especially, Benedenia diseasecaused by the ectoparasite Benedenia seriolae and is difficult to prevent in marine aquaculture
systems. This is a serious parasitic disease in yellowtail aquaculture, leading to secondary viral
Page | 11

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 of removing 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 in yellowtail, a genome-wide andchromosome-wide linkage analysis was initiated using F1 yellowtail families. Two major
quantitative trait loci (QTL) regions on linkage groups Squ2 and Squ20 were identified and thenconfirmed 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 tothis 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 F2 family. F2
QTL congenic line had a significant phenotypic difference in the number of parasites, whencomparing 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
sarah.redmond@maine.edu
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 the lab and farmof Ocean Approved, LLC, of Portland, ME has allowed for the establishment of the firstcommercial kelp farm in the United States and an educational and research farm in Long Island
Sound. The technology was shared and further developed along the coast of Maine byintegrating 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 aresult 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

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in the Northeast with the sharing of ideas, technologies and information through the open andcollaborative 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
kstuart@hswri.org
Hubbs-SeaWorld Research Institute (HSWRI) has been culturing yellowtail amberjack(Seriola lalandi) at its laboratory in San Diego, CA since 2003. Beginning in 2007 intensivelevel production began at the Mission Bay research facility in order to refine methodologies at acommercial 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 and larval rearing microbial
management improvements were made to production protocols and systems in order to increasejuvenile quantity and quality. Many of these changes were based on 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 well as limitationof 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 and high-intensity ultraviolet
sterilizers. Furthermore, Artemia protocols were modified to decrease Vibrio spp loading throughthe use of INVE Separt systems, INVE Hatch Controller, improved sanitation and automatedcold 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-filmfrom tanks, plumbing and equipment, a routine total-system disinfection protocol wasestablished 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” larvaltanks (volumes between 1.6 – 8.0 m3) based on previous experimental results. In most trials,
rotifers and turbidity (algae paste or clay) were administered to larval tanks continuously via an
automated temperature controlled cold-storage unit. Although clay-based turbidity protocols
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were trialed with relatively poor success, algae-paste turbidity protocols coupled with larvalpassive-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 third is 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 and protocols,
reducing variability in larval survival and improving swim bladder inflation.

IN SITU SWIMMING AND SETTLEMENT BEHAVIOR OF CULTURED SERRANIDLARVAE, 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
shibunot@affrc.go.jp
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 and 6.1 cm/s for E. malabaricus. Whenreleased at shallower sites (7 m) close to the reef edge (3 m), 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 and15.4m, respectively. However, there were no significant differences between the two species insettlement depth, time to settlement, swimming speed and swimming distance. When released
3m from the slope and 3m above the rubble bottom at sites 400 m offshore and 16 m deep, 73 %of P. leopardus larvae settled underneath prominent live or dead coral on the slope, and 23 %settled underneath rocks on the bottom.

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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 Agency
dshimizu@affrc.go.jp
2

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 cycle inbroodstock management. On the other hand, in hatcheries for seed production, light environment
can significantly affect the first-feeding success in various fish species. Rearing techniques thatmatch the optimal light environment conditions of the target species is essential to efficient andhealthy 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 flounder thatlive in the deep sea, feeding status was better and growth and survival rate was been improved indark 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 theresults 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% from50%).

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LARVAL REARING ADVANCEMENTS FOR YELLOWTAIL AMBERJACK (Seriolalalandi) IN SOUTHERN CALIFORNIA
Kevin Stuart*, Federico Rotman, and Mark Drawbridge
Hubbs-SeaWorld Research Institute2595 Ingraham St.
San Diego, CA 92109, USA
kstuart@hswri.org
Hubbs-SeaWorld Research Institute (HSWRI) has been culturing yellowtail amberjack(Seriola lalandi) experimentally at its laboratory in San Diego, CA since 2003. In 2007, webegan to intensively rear juveniles, and between 2007 and 2011, we achieved survival rates ashigh 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 the bacterial 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 well as
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 5dph (4.6 mm notochord length). The second trial attempted to remove 1st instar Artemia from thecurrent feeding regime because 1st instar cannot be enriched. This trial consisted of threetreatments: 1) rotifers to 2nd instar offered at 5 dph, 2) rotifers to 2nd instar offered at 7 dph, and3) rotifers to 1st instar to 2nd instar (i.e. traditional HSWRI protocol). Results showed nosignificant differences in growth or survival between any of the treatments, 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 other factors likewater treatment and temperature. We conducted two trials to evaluate methods to minimize thebacterial load (Vibrio spp.) in the water, while maintaining good larval performance. The firsttrial 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 water column (377 ± 120 CFU) than thealgae paste treatment (5,692 ± 2,396 CFU) after 14 days of culture. Growth was not
significantly different among the treatments, however survival was significantly higher in the
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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 results from this trial showed that the water in thetransfer 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 tojuvenile 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 of variability
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.
sshige@affrc.go.jp
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 stock enhancement programwith annual release of approximately 100,000 seedlings has been promoted since 1987. This
program is inferred to be effective because released seedlings are recaptured every year.
However, the risk of losing genetic diversity from the base population is ever-present: theproportion 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 broodstock individuals
using microsatellite DNA markers. Moreover, individual identification and artificial
insemination techniques were developed to support the breeding plan. The broodstock consistedof 37 wild and 41 hatchery-reared barfin flounders. Each individual was identified using a PIT
Page | 17

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 that of the broodstock’s, showingthat the concept of minimum kinship selection is useful for genetic conservation in barfinflounder 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, andHarry Ako1.
1

College of Tropical Agriculture and Human Resources/Hawaii Institute of Marine Biology,
Pacific Biosciences Research Center
clydetamaru@gmail.com
2

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 this plan 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 acidprofiles 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 be relatively low thepercent 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 both essential fatty acids are criticalfor survival and growth of the opakapaka larvae. In addition, the composition of essential fatty
acids for the various live food organisms were obtained and with the exception of Nanno-fed
rotifers, the live feeds employed, have similar profiles from a percent 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 take placeno later than the third day post-hatch. When larvae were presented with a variety of live foodorganisms either alone or in combination, the results indicate that a copepod nauplius-only diet isa 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 ofPage | 18

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 culturesusing Isochrysis galbana (Tahitian Strain) at cell densities of 4 x 105 cells/ml prior to stocking
into the larval rearing tank. Employing this strategy resulted in unprecedented larval survivalwith 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 that theevasive abilities of adult and copepodite stages of calanoid copepods allowed them to avoid
being captured by the larvae. While rotifers could be easily captured the growing body ofevidence suggests that the ubiquitous rotifer is not a suitable live food for opakapaka larvae andanother transitional live food is needed.

UNDERSTANDING ARTEMIA BIOGEOGRAPHYPRODUCTION AND JUVENILE QUALITY

RELATEDTO

HATCHERY

Laura Torrentera
NOAA Visitor Scientist
Kirkland, WA 98034
ltorrentera@yahoo.com
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 ispartly 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 in commercial
aquaculture. The best sources of nutrition continue being the development and use of a
succession of live food organisms as feed for the developing larvae. Artemia is one of the mostimportant live foods in commercial aquaculture. Artemia can ingest small food particles ranging
from 1 to 50 μm in size including diverse species of live microalgae, baker’s yeast, driedmicroalgae, 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 not stored inappreciable amounts in the larvae body, so signs of deficiency and sickness usually appear
within weeks in young, rapidly growing fish. In many countries products from the processing ofplant and animal products from human foods are the primary ingredients available for aquatic
animal feeds (rice bran, corn bran, soybeans, whey powder, etc.). Most of 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 can increase water eutrophicationand stunt larvae development. Artemia is considered now more than ever an important source ofPage | 19

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 foodwith 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 over existing inert food sources; as a result several newstudies of Artemia biogeography and ecology 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
michelle.walsh@noaa.gov
A successful stocking program requires survival of released fish, and to achieve this, fishmust 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 compared withwild 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 nature and subsequent
recruitment to the fishery.
Examples of conditioning technologies that have been applied to flatfish in the hatcheryinclude 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 the addition of formulated,pellet feed to boost nutritional content. However, pellet feeding is suspended approximately 2 wk
before release to focus fish on the more natural feed. In the US, providing hatchery-reared winterflounder, Pseudopleuronectes americanus, with live feeds (oligochaete worms and amphipods)
during rearing conformed post-release feeding performance closer and more quickly to that ofwild 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, the releasesite include, conducting “operant conditioning” on fish to respond to light or sound cues for
supplemental food provision during the first few days or weeks post release, or short-term
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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, hasbeen 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 and feeding skills3. In both Danish and Japanese studies,
evidence has been detected that intensive researcher recollection efforts at, or near, the releasesite 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 'structurephilic' predators, mostly crabs4 ,so cage design has been modified in recent years.
Successful conditioning of stocked fish before release can increase post-release survivaland 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 RELATINGDISEASE OF YELLOWTAIL (SERIOLA QUINQUERADIATA)TO

BENEDENIA

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, 1227, 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, Japankyosida at affrc.go.jp
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 of juveniles
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 the risk of
secondary viral or bacterial infection. The major way of removing the parasite is to soak the fish
into a freshwater bath. However, this method requires a lot of time, cost and effort.
Page | 21

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 "DNAanalysis") are critically important to promote "MAS" breeding certainly.
Here I would like to introduce one of the key components, "Characteristic evaluation
method relating to Benedenia disease in the present species".

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