gy and Ecology 354 (2008) 56–64www.elsevier.com/locate/jembe

Journal of Experimental Marine Biolo

Assessment of the nutritional status of field-caught larval Pacificbluefin tuna by RNA/DNA ratio based on a starvation

experiment of hatchery-reared fish

Yosuke Tanaka a,⁎, Keisuke Satoh b, Harumi Yamada b,Takayuki Takebe a, Hideki Nikaido a, Satoshi Shiozawa a

a Amami Station, National Center for Stock Enhancement, 955-5 Hyou-Sakiyama, Setouchi-cho, Ohshima, 894-2414,Kagoshima, Japan

b National Research Institute of Far Seas Fisheries, 5-7-1 Orido, Smizu, Shizuoka City, 424-8633, Shizuoka, Japan

Received 5 February 2007; received in revised form 15 October 2007; accepted 16 October 2007

Abstract

RNA/DNA ratio is a useful and reliable indicator of the nutritional status of fish larvae and juveniles. In order to assess thenutritional status of field-caught larval Pacific bluefin tuna Thunnus orientalis (Temminck et Schlegel), starvation experiments ofhatchery-reared larvae were conducted and changes in the RNA/DNA ratio of fed and starved larvae were analyzed. Starvationexperiments were conducted every 3 days after first feeding. The survival rate of Pacific bluefin tuna larvae ranged 10–50% after1 day of starved conditions and growth retardation was observed immediately. These results suggest that Pacific bluefin tuna larvaehave a very low tolerance to starvation. The RNA/DNA ratios of fed larvae were approximately 2.0–4.0. On the other hand, thevalue of starved larvae significantly decreased to 1.0–3.0. The nutritional status of 3 cohorts of field-caught tuna larvae collected inthe northwestern Pacific Ocean was examined based on the value of the RNA/DNA ratio of the 1 day starved larvae. 4.35–25.77%of the cohorts were regarded as the “starving condition”, which was negatively correlated to the ambient prey densities. Thesefindings suggest that the nutritional condition of larval Pacific bluefin tuna was influenced by the ambient prey density, andstarvation itself and starvation-induced predation could greatly contribute to mortality in the larval period of Pacific bluefin tuna.© 2007 Elsevier B.V. All rights reserved.

Keywords: Nutritional status; Pacific bluefin tuna; RNA/DNA ratio; Starvation

1. Introduction

Marine fish species generally produce a huge numberof small pelagic eggs, and mortality during the egg,larval and juvenile stages is considered to be the main

⁎ Corresponding author. Tel.: +81 997 75 0653; fax: +81 997 750637.

E-mail address: yosuket@affrc.go.jp (Y. Tanaka).

0022-0981/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.jembe.2007.10.007

cause of interannual variability in recruitment in manyspecies (Bailey and Houde, 1989). The nutritionalcondition of larvae and juveniles is one of the importantfactors for their survival. Larvae in a poor nutritionalcondition are not only more vulnerable to predation,disease and unfavorable environmental conditions butalso are less efficient at foraging (Amara and Galois,2004). Therefore, assessment of the nutritional status offield-caught larvae helps to predict larval survival andyear-class fluctuation (Richard et al., 1991).

Table 1Details of the starvation experiment. (WT: water temperature, DAH:days after hatching)

Mean WT(°C)±SD

Hatch date Trial DAH

Experiment 1 25.6±0.18 26 May 2006 3, 6, 9, 12, 15, 18 DAH

Experiment 2 28.6±0.29 20 July 2006 3, 6, 9, 12, 15 DAH

57Y. Tanaka et al. / Journal of Experimental Marine Biology and Ecology 354 (2008) 56–64

RNA/DNA ratio has been proven as a useful andsensitive indicator of the nutritional condition and recentgrowth of larval and juvenile fishes in relation to proteinsynthesis, which has been widely applied to both labora-tory-reared and field-caught fish (Buckley, 1980; Buckley,1984; Clemmesen, 1987; Rooker and Holt, 1996;Chicharo et al., 1998; Gwak and Tanaka, 2001; Chicharoet al., 2003; Islam and Tanaka, 2005). The quantity ofDNA in an animal cell is believed to be normally stable butthe quantity of RNA, primarily associatedwith ribosomes,is closely related to the rate of protein synthesis. Thenutritional condition is associated with the food supplyand feeding success of the fish and therefore, variability inthe trophic environment is reflected in the nutritionalcondition.

Pacific bluefin tuna Thunnus orientalis is distributedwidely in the North Pacific Ocean (Collette, 1999) and isone of the most important fisheries species in Japan. Thespawning grounds of the Pacific bluefin tuna forms in thearea between the Philippines and the Ryukyu Islands inthe northwestern Pacific Ocean from April to June, andin the Sea of Japan inAugust (Yabe et al., 1966;Ueyanagi,1969; Okiyama, 1974, Kitagawa et al., 1995; Tanakaet al., 2007). It has been assumed that larvae hatched in thenorthwestern Pacific Ocean are carried by the KuroshioCurrent and juvenile bluefin tuna are transported to nearthe coast of Japan, where they are caught by troll fisheriesand some are used as fingerlings for aquaculture. Althougheco-physiological studies on the early life stages of Pacificbluefin tuna in the field are few, it has been suggested thatthe survival of larval Pacific bluefin tuna to recruitmentdepends largely on their growth in the very early stage oftheir life history (6–13 days old) in the spawning groundsoff the Ryukyu Islands, based on otolith microstructureanalysis (Tanaka et al., 2006). Since the RNA/DNA ratiohas been used as an indicator of recent growth (Bullow,1970; Buckley, 1984; Hovenkamp and Witte, 1991;Westerman and Holt, 1994), the RNA/DNA ratio can bean effective tool for more detailed examination of thesurvival processes in larval Pacific bluefin tuna.

Larval Pacific bluefin tuna shows a conspicuouspatch-like distribution (Nishikawa et al., 1985; Satoh,2006) similar to that reported for the southern bluefintuna Thunnus maccoyii (Davis et al., 1990; Jenkins andDavis, 1990; Jenkins et al., 1991). On the basis of thecharacteristics of the distribution of the present species,it is assumed that the survival of larval Pacific bluefintuna in the patches (cohorts) could be important for theirrecruitment. In this study, we could detect three cohortsof larval Pacific bluefin tuna and the nutritional status ofthese fish was assessed using the RNA/DNA ratio. Sincethe criteria of the RNA/DNA ratio representing the

“starving condition” is required in order to allow theassessment of nutritional status of field-caught fish,starvation experiments were carried out using hatchery-reared larvae to determine these criteria. The objectives ofthis study are (1) to determine the criteria of the starvingcondition based on the RNA/DNA ratio analysis bystarvation experiments, and (2) to assess the nutritionalstatus of the field-caught larval Pacific bluefin tuna.

2. Materials and methods

2.1. Rearing

Experimental fish were reared at the Amami Station,National Center for Stock Enhancement (NCSE),Fisheries Research Agency. Starvation experiments wereconducted at two rearing water temperatures, ca. 25 °C inMay and June (Experiment 1) and 28 °C in July andAugust 2006 (Experiment 2, Table 1). Fertilized eggs forexperiment 1 were obtained on 24 May 2006 from spon-taneous spawning of broodstock in a net pen in theAmamiStation. Fertilized eggs for experiment 2 were transferredon 19 July 2006 from Amami-Yougyo Co. Ltd and rearedat the Amami Station. The hatch dates of the experimentalfish of each experiment were 26 May and 20 July, res-pectively. Newly hatched larvae were transferred to a 50 trearing tank (fed group). Photoperiod was natural. Thelarvae in the experiment 1 were fed rotifers Branchionusrotundiformis, from 3 to 20 days after hatching (DAH),Artemia nauplii from 12 to 20 DAH. The larvae in theexperiment 2 were fed rotifers from 3 to 17DAH,Artemianauplii from 15 to 17 DAH. Twelve fish were taken everyday from 3 DAH, which were immediately preserved andstored at −80 °C.

2.2. Starvation experiment

Experimental trials were conducted every 3 days from3 DAH (Experiment 1: 3, 6, 9, 12, 15, 18 DAH;Experiment 2: 3, 6, 9, 12, 15 DAH). The initial meanstandard length (SL) in each trial is summarized in

Table 2Mean standard length (mm±S.D.) of fish at the onset of each trial (DAH: days after hatching)

3 DAH 6 DAH 9 DAH 12 DAH 15 DAH 18 DAH

Experiment 1 (25 °C) 3.36±0.19 4.05±0.20 4.69±0.31 5.36±0.31 6.51±0.44 7.61±0.54Experiment 2 (28 °C) 3.31±0.16 3.91±0.22 4.97±0.19 6.12±0.41 7.60±0.53

58 Y. Tanaka et al. / Journal of Experimental Marine Biology and Ecology 354 (2008) 56–64

Table 2. To determine the survival rate without a foodsupply, 60–120 fish were carefully transferred using alarge glass pipette from the 50 t tank to aerated 10 Ltanks (n=3) for each trial. Food was withheld. Deadlarvae within 3 h after transfer were removed becausemortality was assumed to be associated with handlingstress. Thereafter, dead larvae were counted and re-moved using a pipette from each tank every day afterthe transfer.

600–1200 experimental fish for the RNA/DNAanalysis were carefully transferred using a large glasspipette from the 50 t to a 100 L tank and kept incirculated sea water with aeration without food (starvedgroup). Samples of starved groups were taken from the100 L tank every day after the transfer. Twelve fish were

Fig. 1. Temporal changes in the survival rate (%) of Pacific bluefin tuna l

taken every day from the onset of each trial, which wereimmediately preserved and stored at −80 °C.

2.3. Field collection

We detected three patches (cohorts) in the north-western Pacific Ocean. Wild Pacific bluefin tuna larvaewere collected by surface trawl in the northwesternPacific Ocean aboard RV ‘Shunyo-Maru’ in 2004 and2005 (Table 2). Sampling was done at nighttime using a2 m diameter net with a 335 μm mesh. The net wastowed at the sea surface beside the hull for 10 min in2004 and 5 minutes in 2005 at a speed of approximately1.5 knots. The volume of water filtered was calculatedfrom the mouth area of the net multiplied by the distance

arvae under unfed conditions. Error bars show standard deviations.

Fig. 2. Changes in mean standard length (SL) of fed and unfed groupsreared at 25 (experiment 1) and 28 °C (experiment 2), shown with eachfeeding scheme. Error bars show standard deviations. ⁎⁎: pb0.01 (t-test).

59Y. Tanaka et al. / Journal of Experimental Marine Biology and Ecology 354 (2008) 56–64

traveled, which was measured by a calibrated flowmeterattached to the net mouth. Collected tuna larvae werefrozen (−70 °C) onboard immediately after beingsorted. Copepods and their nauplii as prey organismswere simultaneously collected by filtering surface seawater sampled by a bucket (1 L in 2004, 10 L in 2005).

At each station, the sea surface temperature wasobserved by direct measurement (0 m depth) and CTDmeasurement (N3 m depth with 1 m pitch); mean watertemperature ≤10 m depth was used to represent theambient water temperature for larvae collected at thestation, because water temperature at depths shallowerthan 2 m (ring diameter) may be influenced by weatherconditions such as rain and wind.

2.4. Determination of RNA and DNA

SL of sampled fish was measured to the nearest0.1 mm on an ice-cold Petri-dish immediately beforemeasurements of RNA and DNA contents. Measure-ments of RNA and DNA contents were individuallycarried out. Nucleic acids were extracted from the wholebody transferred to a mixture of Tris–EDTA buffer(0.05 M Tris, 0.1 M NaCl, 0.01 M EDTA, pH 8.0),proteinase-K (pro-K) and sodium dodecyl sulfate(SDS). The quantity of RNA and DNA in the wholebody was determined by the fluorescence techniqueusing the nucleic acid specific fluorescent dye ethidiumbromide, as described by Clemmesen (1993) and slight-ly modified by Sato et al. (1995).In order to measure theDNA content of a sample, RNA was enzymaticallydigested with RNAase and the remaining quantity ofDNAwas determined with ethidium bromide. The fluo-rescence due to total RNA was calculated as the dif-ference between the total fluorescence (RNA and DNA)and the fluorescence after RNAase treatment, which isassumed to be due to the presence of only DNA.

3. Results

3.1. Survival rate and growth under starved conditions

Survival rate of the larvae 1 day after starved con-ditions ranged from 10.9 to 57.1% in both experiments(Fig. 1). Particularly, the survival rates of the trials 3 and6 DAH were low and ranged 10.9 to 29.6%. The numberof days to 100% mortality in experiment 1 was 4 days inthe trials 3, 6 and 9 DAH, and 3 days in the trials 12, 15and 18 DAH. In experiment 2, the number of days to100% mortality was 3 days in the trials 3, 6 and 9 DAH,and 2 days in the trials 12 and 15 DAH. Fed groups inboth experiments showed a constant growth in SL up to

the end of the experiments (Fig. 2). The mean SL of thefed groups in experiment 1 and 2 reached 8.37 mm at20 DAH, and 9.03 mm at 17 DAH, respectively. Theeffects of starvation on growth were recognized in bothexperiments except for 1 day after starvation in the trial3 DAH in experiment 2. Significant growth retardationof the starved group in both experiments was observedimmediately after starvation (t-test, pb0.01).

3.2. Changes in the RNA/DNA ratio of fed and starvedlarvae

RNA and DNA contents of 216 fish of the fed and170 fish of the starved group in experiment 1 and 184fish of the fed and 107 fish of the starved group inexperiment 2 were measured.

Changes in the RNA/DNA ratio of the fed and starvedgroups in both experiments are shown in Fig. 3. Inexperiment 1, the mean value of the RNA/DNA ratio ofthe fed group initially ranged from 3.0 to 4.0, and thengradually decreased to 3.0. The mean value of the RNA/DNA ratio of the fed group in experiment 2 was ap-proximately 3.0 at 3 DAH and increased to 4.0 at 6 DAH.

Fig. 4. Relationship between standard length (SL) and the RNA/DNAratio of fed and 1 day starved groups.

Fig. 3. Changes in mean RNA/DNA ratio of fed and unfed groupsreared at 25 (experiment 1) and 28 °C (experiment 2). Error bars showstandard deviations. n.s.: not significant, ⁎⁎: pb0.01 (t-test).

60 Y. Tanaka et al. / Journal of Experimental Marine Biology and Ecology 354 (2008) 56–64

Subsequently, these values decreased to 2.0–3.0. TheRNA/DNA ratio of the starved group showed a sig-nificant decrease 1 day after starvation in the later part oftrials (9, 12, 15 and 18 DAH) compared to that of the fedgroup although that of the starved group did not sig-nificantly decrease at the trials 3 and 6 DAH in bothexperiments (Fig. 3).

The RNA/DNA ratio of the fed groupwas compared tothat of the group of 1 day after starvation (1 d starvedgroup) in terms of SL (Fig. 4). The RNA/DNA ratios ofthe fed group smaller than 5 mm SL showed a markedvariation. However, those of the fed group larger than5 mm SL were less variable relative to the fish smallerthan 5 mm SL. The RNA/DNA ratio of the 1 d starvedgroup ranged from2.0 to 3.0 in the size class ofb5mmSLand from 1.5 to 2.0 in the size class of ≥5 mm SL. Thedistribution of the RNA/DNA ratio of the fed and 1 dstarved groups were significantly different (ANCOVA,df=1 278, F=88.1, pb0.01 in experiment 1; df=1 236,F=49.8, pb0.01 in experiment 2). The RNA/DNA ratiosof the 1 d starved group were lower than those of the fedgroup. However, some of the fed group smaller than 5mmSL showed a lower RNA/DNA ratio than that of the 1 dstarved group.

Tanaka et al. (2006) suggested that growth retarda-tion in larval stage of Pacific bluefin tuna could reducetheir survival to the juvenile stage. In the present study,

growth retardation in SL and decrease in the RNA/DNAratio were observed in the 1 d starved group. In addition,the survival rates of the 1 d starved group in the trials 3and 6 DAH were very low (less than 50%) although thesurvival rate of them may be underestimated (see Dis-cussion). From these findings, we regarded the RNA/DNA ratios of the 1 d starved group as the criteriarepresenting the crucial starved condition of larvae,which were used for the comparison to the field-caughtPacific bluefin tuna larvae.

3.3. Nutritional status of field-caught larvae

Three cohorts of Pacific bluefin tuna larvae weredetected in the northwestern Pacific Ocean in 2004 and2005. The ambient water temperature and the mean SL(±standard deviation) are shown in Table 3. Maximumdensity observed in the cohorts 1, 2 and 3 were 123.1,645.4 and 126.7 individual/1000 m3, respectively. Themean RNA/DNA ratios (±standard deviation) of thecohorts 1, 2 and 3 were 3.17 (±1.07), 3.61 (±1.02) and3.20 (±0.61), respectively (Fig. 5)

Table 3Details of the field sampling and field-caught fish analyzed

Sampling year Sampling date Lat. (N) Long. (E) WT (°C) No. of tows Maximum density(No./1000 m3)

No. of fishanalyzed

Mean SL(mm) ±SD

Cohort 1 2004 16–17 May 21°57–22°12 124°40–124°43 28.7 3 123.1 33 6.26±0.41Cohort 2 2004 1–2 June 26°07–26°12 129°07–129°23 26.1 8 645.4 92 5.78±0.47Cohort 3 2005 2–5 June 24°35–25°09 126°00–126°38 27.7 21 126.7 97 4.87±0.54

61Y. Tanaka et al. / Journal of Experimental Marine Biology and Ecology 354 (2008) 56–64

The RNA/DNA ratios of the three cohorts werecompared to those of 1 d starved group (Fig. 5). Basedon the ambient water temperature of each cohort, theRNA/DNA ratios of cohorts 1 and 3 were compared tothose of 1 d starved group reared at 28 °C, and those ofcohort 2 to those reared at 25 °C. The data sets of 1 dstarved group were divided into two size classes, b5 mmSL and ≥5 mm SL. Thereafter, the regression line and95% confidence limits were independently calculatedfor each data set of the two size classes because therelationship between the SL and the RNA/DNA ratiowas not linear.

Experiment 1 (25 °C)b5 mm SL: R/D=−0.29SL+3.73, r2 =0.17, n=33≥5 mm SL: R/D=−0.14SL+2.83, r2 =0.21, n=33

Fig. 5. Frequency distribution of RNA/DNA ratio of 3 field-caught cohorts an3 field-caught cohorts, compared to those of 1 day starved group.

Experiment 2 (28°C)b5 mm SL: R/D=−0.70SL+5.39, r2 =0.63, n=27≥5 mm SL: R/D=−0.99SL 10−2 +1.64, r2 =0.0014,

n=32where R/D indicates the RNA/DNA ratio of 1 d

starved group. SL indicates the standard length (mm).The field-caught larvae with the RNA/DNA ratio

below the 95% confidence upper limit were judged as“starving fish”. The size, 5 mm SL corresponds to mor-phological changes in the relative growth (Uotani et al.,1990;Miyashita et al., 2001) and a shift of feeding habit ofwild larvae (Uotani et al., 1990).

Starving larvae occurred in all cohorts. Percentages ofthe starving larvae in cohorts 1, 2 and 3 were 15.15, 4.35and 25.77%, respectively. The mean densities of ambient

d relationship between standard length (SL) and the RNA/DNA ratio of

Fig. 6. Relationship between prey density (copepod adults and nauplii)and percentages of starving fish in each cohort.

62 Y. Tanaka et al. / Journal of Experimental Marine Biology and Ecology 354 (2008) 56–64

prey (Copepod adults and nauplii) of the cohorts 1, 2 and3 were 2.5, 5.1 and 1.4 individuals/L, respectively, andnegatively correlated to the percentages of starving lar-vae (Fig. 6).

4. Discussion

4.1. Tolerance to starvation in relation to the changesin the RNA/DNA ratio

The survival rates of the 1 d starved group ranged from10.9 to 57.1% and the duration for 100%mortality was 3–4 days at 25 °C and 2–3 days at 28 °C through thesampling period. In addition, the significant growth re-tardation in SL was observed immediately. For wild Pa-cific bluefin tuna, growth retardation in the larval stagecould seriously influence their survival to the juvenilestage (Tanaka et al. 2006). Accordingly, these findingsindicate that larval Pacific bluefin tuna have a very lowtolerance for starvation.

In general, hatchery-reared Pacific bluefin tuna larvaeshow a high level of mortality during the early larvalstages until 10 DAH due to unsuitable environmentalconditions, mismatch of the live food size and insufficientnutritional contents of the food as the possible factors(Sawada et al., 2005). In the present study, the survivalrates of the trials 3 and 6 DAH were very low 1 day afterthe commencement of starvation (Fig. 1). In this period,differences in the RNA/DNA ratio between the fed andstarved groups were not always sufficiently large enoughto be significant (Fig. 3). On the basis of SL, some of thefed group smaller than 5mmSL showed lowerRNA/DNAratios than that of the 1 d starved group (Fig. 4). Theseresults suggest that even the fed group had low nutritional

status which is crucial for their survival. Accordingly, thesurvival rate of the starved group in the early periods (3 and6 DAH) may be underestimated.

The critical value of the RNA/DNA ratio for survivalis species specific. The critical value is 1.3 for sardineSardina pilchardus (Chicharo, 1998), 4.1 for cod andhaddock (Lough et al., 1996), 2.0 for striped bassMorone saxatilis (Martin et al. 1985) and 1.08–5.36 forJapanese flounder Paralichthys olivaceus (Gwak andTanaka, 2001). The critical value of the RNA/DNA ratioestimated for the 1 d starved group ranged from 1.5 to3.0 (Fig. 4), which seems to be in a range common toother fishes.

4.2. Assessment of nutritional status of field-caughtlarvae

Food availability (e.g., Clemmesen, 1996) and watertemperature (Goolish et al., 1984; Ferguson andDanzmann, 1990; Mathers et al., 1992) are the mainfactors affecting the RNA/DNA ratio of fish larvae. Ithas been found that a temperature difference of around2 °C is necessary to produce a significant effect on theRNA/DNA ratio (Buckley et al., 1999). In the presentstudy, the water temperature of the cohorts 1, 2 and 3was 28.7, 26.1 and 27.7 °C, respectively (Table 3). Thecritical value of the RNA/DNA ratio at 25 °C was usedfor comparison to the cohort 2 and that at 28 °C to thecohorts 1 and 3. The differences between the ambientwater temperature of the cohorts and the rearing tem-perature were less than 2 °C, suggesting that tempera-ture effects on the RNA/DNA ratio for assessment wereminimal.

In the present study, starving larvae collected in thefield occurred in all of the three cohorts and the percen-tages of starving larvae ranged from 4.35 to 25.77%. Ithas been reported that the assessment of the nutritionalstatus of field-caught larvae in various fish species bythe RNA/DNA ratio. Occurrence of starving larvae wasvariable depending on the species. Percentage of starvinglarvae in the field was estimated as 5.5% for Japaneseflounder (Gwak and Tanaka, 2001), 0.0–4.8% for sardineS. pilchardus (Chicharo et al., 1998; Chicharo et al.,2003). In anchovy Engraulis anchoita, although thepercentage starving fish was high (52.4%) only in the sizeclass of 5–6 mm, the percentage was 0.0–1.75% in othersize classes (Clemmesen, 1996). In larval Japanese sar-dine Sardinops melanostictus at first feeding, thepercentage of starving larvae was zero (Kimura et al.,1996). Thus, the percentage of starving larvae in Pacificbluefin tuna appears to be relatively higher than those ofother species although we analyzed only three cohorts.

63Y. Tanaka et al. / Journal of Experimental Marine Biology and Ecology 354 (2008) 56–64

The percentage of starving field-caught larvae wasnegatively correlated to the ambient prey density (Fig. 6).This result indicates that the nutritional condition of larvalPacific bluefin tuna was influenced by the ambient preydensity as also observed in other species (e.g. Martin et al.,1985; Islam and Tanaka, 2005). The target species in thestudies mentioned above are distributed in more neriticwaters relative to the distribution of Pacific bluefin tuna.Generally, oceanic sub-tropical waters where larval Pacificbluefin tuna are distributed are oligotrophic and theproductivity of zooplankton is low relative to more neriticwaters. In such an environment, the prey density could be acritical factor for the nutritional condition, consequentgrowth and survival of larval Pacific bluefin tuna becausethe present study revealed that larval Pacific bluefin tunashowed a very low tolerance to starvation. Therefore,starvation itself and starvation-induced predation couldgreatly contribute tomortality in the larval period of Pacificbluefin tuna, whereas predation is generally considered tobe most important factor which controls larval mortality inmarine fishes (Houde, 1987; Bailey and Houde, 1989).

Acknowledgements

We are grateful to Captain M. Onoda and the crew ofthe RV ‘Shunyo-Maru’ for their assistance in the fieldsampling of larval bluefin tuna. The authors expresstheir sincere thanks to H. Imaizumi and K. Ide and themembers of Amami Stations, NCSE, FRA for produc-ing the larvae for the experiment, Mr. K. Noda and T.Naiki, students of Tokyo University of Marine Scienceand Technology for their helpful assistance in the ex-periment. This study was supported by the FisheriesAgency of Japan and the Research Fellowships of theJapanese Society for the Promotion of Science (JSPS)for Young Scientists (No. 03810). [SS]

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