Original Paper
Title Brown nodules formed in the appendages of Penaeus monodon postlarvae cultured in a hatchery in Madagascar
Authers Motohiko SANO*1, Hideto IWAKAWA*2, Masanori SUEMITSU*2, Megumi MINAGAWA*1,4, Juliette RASOARINIRO*3, and Louisette RZAFINDRADOANY*3
Keywords Penaeus monodon, brown nodule formation, appendages necrosis
Citation Bull. Fish. Res. Agen. No.3, 1-22, 2002
Brown nodules, leading to shrimp mortality, frequently formed in the appendages of Penaeus monodon postlarvae cultured in a hatchery in Madagascar. The nodules resulted from a melanized hemocytic host reaction, which was activated by the necrosis of the cuticular epithelium of the appendages. In histopathological examinations, no evidence of bacterial invasion was detected in the postlarvae with brown nodules. The nodules were frequently formed in postlarvae that were starved for four days. The brown nodules in appendages disappeared after the postlarvae were transferred to a clean and less-densely reared aquarium. Furthermore, addition of formalin at 25ppm to the rearing water inhibited the formation of brown nodules, although the mechanism was not clear. These results suggest that the brown nodule formations in the appendages of the hatchery-reared postlarvae of P. monodon were probably caused by a combination of several factors related to water quality and feeding and not by bacterial infection.

Accepted on March 13, 2002
Contribution No.A 12 from Fisheries Research Agency
*1 Ishigaki Tropical Station, Seikai National Fisheries Research Institute, Ishigaki, Okinawa 907-0451, Japan
*2 Japan International Cooperation Agency, Yoyogi, Tokyo 151-8558, Japan
*3 Centre de Developpement de la Culture de Crevettes, Mahajanga B, P. 85, Madagascar
*4 Research Planning and Coordination Division, Seikai National Fisheries Research Institute, Nagasaki 850-0951, Japan

Title Annual variations in the conditions of fisheries for Euphausia pacifica in Sanriku and Joban waters, northeastern Japan
Authers Kenji TAKI*
Keywords Euphausia pacifica, fishery, Oyashio, CPUE
Citation Bull. Fish. Res. Agen. No.3, 2002
The relationship between annual variations in the ocean environment and the trends and conditions of the fisheries for Euphausia pacifica in Sanriku(Iwate and Miyagi prefectures) and in Joban(Fukushima and Ibaraki prefectures)waters were examined.
 The history of the fishery was classified into four time periods. During the first period, a small-scale fishery was conducted along the coastal waters off Miyagi Pref. beginning in the mid-1940's. During the second period, the fishing grounds were extended throughout the Sanriku and Joban waters and the annual total catch increased substantially beginning in the late-1960's. During the third period,the main fishing grounds shifted to the Sanriku waters beginning in the late-1980's. During the fourth period, the total catch has been regulated from the early-1990's.
 Along the coastal waters of Iwate Pref., the fisheries seem to be independent of the oceanographic conditions in this region and no apparent relationship between the CPUE(catch/boat/day)and annual variations in the oceanographic conditions was found. Along the coastal waters of Miyagi Pref., however, unfavorable conditions for the fishery are correlated with the northward anomaly of the southern limit of the first branch of the Oyashio Current(FOI). Along the Joban coastal waters, it was suggested that the southernmost extent of the FOI is an important factor in controlling the fishery conditions.

Accepted on March 13, 2002
Contribution No.A 13 from Fisheries Research Agency
* Tohoku National Fisheries Research Institute, 3-27-5 Shinhama, Shiogama, Miyagi 985-0001, Japan

Short Paper
Title Regeneration of Cd, P, and N on the continental shelf of the East China Sea in summer
Authers Kazuo ABE*
Keywords Regeneration, Cd, P, N, East China Sea
Citation Bull. Fish. Res. Agen. No.3, 2002
Regeneration of three biolimiting constituents, cadmium(Cd), phosphate(PO4), and nitrite plus nitrate(NO2+NO3), was examined on the continental shelf area of the East China Sea in the summer of 1999. Vertical profiles of these constituents showed increase of concentrations toward the sea bottom. The regenerated(molar)ratios to the consumed oxygen(O2)were estimated to be 0.00134×10-3:0.00684:0.0998:1 by plotting these constituents against the Apparent Oxygen Utilization(AOU), and these ratios generally agreed with a previous study near the Ryukyu Islands. The apparent increase of Cd, PO4, and NO2+NO3 in the bottom layer from winter to summer was around 0.079nM, 0.41μM, and 6.1μM, respectively, and this was attributable to the active supply caused by the decomposition of the organic matter in the bottom layer in this season.

Accepted on March 13, 2002
Contribution No.A 14 from Fisheries Research Agency
* Ishigaki Tropical Station, Seikai National Fisheries Research Institute, 148-446 Fukai-Ota, Ishigaki Okinawa 907-0451, Japan

Doctoral thesis
Title Studies on a Novel Bitter Amino Acid, Pulcherrimine in the Green Sea Urchin Gonads*1
Authers Yuko MURATA*2
Keywords sea urchin, bitterness, amino acid, Hemicentrotus pulcherrimus, gonad
Citation Bull. Fish. Res. Agen. No.3, 2002
The present study was undertaken to clarify the bitter substance in the green sea urchin gonads. The substance was elucidated to be a novel sulfur-containing amino acid. It was named pulcherrimine after the scientific name of the green sea urchin, Hemicentrotus pulcherrimus. The results of the investigation were described in five Chapters.
 ‘Chapter Ⅰ’described the results of the preliminary examination of this study. The frequency of occurrence of bitter-tasting sea urchins collected from the sea off Iwaki in Fukushima Prefecture was examined. All of the individuals of which the gonads tasted bitter were found to be mature females. This evidence indicates that bitter-tasting gonads are specific to mature ovaries. Free amino acid contents were not significantly different between ovaries and testes. Thus, the possibility of the participation of the bitter tasting amino acids, i.e. Val, Leu and Ile in the bitterness of the sea urchin ovaries was ruled out.
 The relation between the tri-monthly changes in the gonad index(GI)and the frequency of bitter gonads of the green sea urchin at the sea off Iwaki was investigated from November 1998 to November 1999. The mean GI value of mature male and female individuals was lowest in May 1999. However, the GI values showed a large variation among mature specimens in each season, therefore, the maturation process of the sea urchins in the sea off Iwaki may vary among individuals. Immature individuals were found in May, August and November 1999, and their percentages were 20%, 60% and 4%, respectively. However, many mature sea urchins were found in all months when examined. These facts indicate that the reproductive cycle seems to be unclear and the occurrence of the mature stage extends over a long term in this area. More than 95% of the mature ovaries had a bitter taste in November 1998, February 1999 and November 1999. In May and August 1999, 60% of the mature ovaries had a bitter taste. In August 1999, 20% of the immature gonads tasted bitter. From these results, the seasonal change in the frequency of bitter ovaries agreed well with that in the distribution of mature ovaries.
 ‘Chapter Ⅱ’described the isolation of a novel sulfur-containing amino acid from the green sea urchin ovaries, and the elucidation of its structure. This amino acid was named pulcherrimine(Pul).
 Ovaries were extracted with 80% aq EtOH and then 20% aq MeOH. The 80% EtOH extract was partitioned between water and diethyl ether. The aqueous layer and the 20% aq MeOH extract were combined and subjected successively to ODS flash chromatography, gel filtration on Sephadex G-10, MPLC on ODS and RP-HPLC. Thirty milligrams of the bitter principle was obtained from 628g of the starting material.
 Molecular weight and the molecular formula of the bitter compound was determined to be 249 and C9H15O5NS, respectively, by HRFAB-MS. The structure was elucidated as 4- (2'-carboxy -2'-hydroxyethylthio)-2-piperidinecarboxylic acid by 1H and 13C NMR, 1H-1H COSY, HMBC, and HMQC experiments.
 The absolute stereochemistry was determined by NOE experiments, chiral HPLC analysis and the modified Mosher method. Accordingly, the absolute stereochemistry of Pul was 2’S, 2R, and 4S .
 In ‘Chapter Ⅲ’, a method for determination of Pul in sea urchin gonads was devised. This method consists of the formation of dimethylaminoazobenzenesulphonyl chloride(Dabs-Cl)derivatives of Pul(dabsylation), and separation of the dabsylate using RP-HPLC. The dabsylated Pul was analyzed by RP-HPLC using a linear gradient elution consisting of methanolaquous/acetic acid and methanol/acetic acid mixture after dabsylation. Linearity of the caribration curve for Pul was extremely high(r=0.994)in the tested range from 0μg/ml to 4μg /ml.
 The Pul content in the ovary and testis of the green sea urchin collected from the sea off Iwaki in November 1998 was analyzed by this method. The mean Pul content in the ovary was 1.37mg /100g. On the other hand, no Pul was detected in all testes examined. Correlation between the bitterness and the Pul content was statistically significant. 
 Individuals with a low pulcherrimine content in their ovaries increased from February to August 1999 and decreased from August to November 1999. These results suggest that the amount of pulcherrimine is related to a seasonal change, probably the maturity of ovaries of the green sea urchin.
 ‘Chapter Ⅳ’ described the result of sensory tests for Pul in relation to the thresholds for detection and recognition.
 The detection and recognition thresholds determined by a triangle test were 0.17mM and 0.34mM, respectively. Furthermore, by the triangle tests, the thresholds for Pul added to the extracts from the green sea urchin gonads were estimated to be 0.25mM and 0.52mM(immature gonads), 0.22mM and 0.46mM(ovaries), and 0.27mM and 0.46mM(testes), respectively. These findings suggested that the compound exerts remarkable effects of bitter taste on the extracts from both immature and mature green sea urchin gonads.
 In ‘Chapter V’, in order to examine the possibility of differences in receptor mechanisms among bitter substances, behavioral responses to Pul using a conditioned taste aversion(CTA) paradigm in C57BL/6 and BALB/c mice were compared. The behavioral aversion threshold to Pul was determined to be 1.0mM in C57BL/6 mice and 0.1mM in BALB/c mice after aversions were conditioned in mice to 4mM Pul. This fact indicated that taste sensitivity to Pul is higher in BALB/c than in C57BL/6 mice.
 Generalization patterns across various bitter and other taste stimuli in the two strains of mice suggested that Pul may taste similar to sulfur-containing bitter compounds, but different from any other bitter amino acids and even L-methionine. A hierarchical cluster analysis showed that Pul was grouped with sulfur-containing bitter substances, and SOA, Den and Str.

Accepted on January 21, 2002
Contribution No.A 15 from Fisheries Research Agency
* 1 Ph.D. Thesis, Kyoto University
* 2 National Research Institute of Fisheries Science, 2-12-4 Fukuura, kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan.

Title Studies on the early life ecology of skipjack tuna, Katsuwonus pelamis, in the tropical western-north Pacific
Authers Toshiyuki TANABE*
Citation Bull. Fish. Res. Agen. No.3, 2002
The objective of this study is to provide new information on the early life ecology of skipjack tuna. It is necessary to clarify the recruitment process of the skipjack tuna population and to establish a basis for stock management in the western Pacific.
 In order to collect large number of juvenile skipjack tuna, a new sampling protocol and associated gear were developed. The midwater trawl net TANSYU had a estimated mouth opening of 20m×20m, 72m total length, 1000-57mm mesh size at the body, 8mm mesh size at the codend, and 5 knots maximum towing speed. A total of 497 tows was conducted in the tropical western Pacific(0-20°N, 130-160°E), from October to December during the years 1992-1996, resulting in the collection of 6724 skipjack, 6-172mm standard length(SL), and 0-1163 individuals(inds)/1 hr tow. These results indicate the establishment of a new sampling method for capturing much larger numbers of juvenile skipjack tuna than was possible using the sampling gear of previous studies. In addition, samples of 1373 other tunas, 8-140mm SL, 0-128 inds/1 hr tow, were collected. These results verify that the TANSYU is an effective sampling gear for juveniles of not only skipjack tuna but also of other tunas and large oceanic fishes.
 Juvenile skipjack tuna are widely distributed in the north equatorial current area(NEC), the north equatorial counter current area(NECC), and the boundary area of these two currents. Vertically, juvenile skipjack tuna appeared mainly from the lower portion of the mixed layer to the upper portion of the thermocline. The abundance indices, frequency of occurrence(%)and density(inds/1 hr tow)were calculated as 61% and 17inds/1 hr tow at the NECC, 35% and 6 inds/1 hr tow at the boundary, and 32% and 15 inds/1 hr tow at the NEC. The distributional pattern of juvenile skipjack tuna was different by year. The characteristics of the horizontal distribution of juvenile skipjack were as follows. During 1992 and 1994, juvenile skipjack tuna were relatively abundant in the southern area but poor in the northern area. During 1995, high concentrations of juveniles in the eastern area appeared. During 1993 and 1996, wider distributions with no clear differences between localities were observed. Young skipjack were collected only from the NECC at night, and were distributed mainly in the lower portion of the mixed layer.
 The vertical distribution of skipjack changed with growth stages. For daytime in the NECC, larvae at the metamorphosis stage of around 10mm SL are distributed through depths of 0-200m, and juveniles after metamorphosis of 10-40mm SL are distributed through depths of 0-220m. Juveniles of 50-60mm SL are distributed through depths of 60-140m. Juveniles of 70-80mm SL are concentrated at about 90m depth. The vertical distribution of juvenile skipjack is related to the vertical temperature profile, with a shallower thermocline corresponding to a shallower distribution of juveniles and a deeper thermocline resulting in a deeper distribution of juveniles. The vertical distribution of juvenile skipjack is also related to the distribution of other organisms sampled. This result indicates that juvenile skipjack are preferentially distributed in the abundant layers of other organisms collected by the midwater trawl net.
 In contrast, other juvenile tuna, Thunnus spp. including yellowfin tuna Thunnus albacares and bigeye tuna Thunnus obesus are mainly distributed in the mixed layer shallower than about 80m depth in the NECC, indicating a clear difference with juvenile skipjack. The physical charasteristics of distribution area of juvenile skipjack is mainly from 20 to 29℃ temperature and from 33.6 to 35.5PSU salinity. Those of other juvenile tuna ranged from 26 to 29℃ temperature and from 33.6 to 33.7PSU salinity. These results indicate that skipjack and other tuna juveniles have habitat segregation in the pelagic ocean of the tropical western Pacific.
 The stomach contents of skipjack and other tuna juveniles collected from the NEC and NECC areas were analyzed. The importance of each organism as a prey of skipjack or other tuna juveniles was evaluated by the index of relative importance of prey taxa(IRI). The IRI of fish larvae for juvenile skipjack was remarkably high, 14107 in the NEC and 10852 in the NECC. These data indicate that fish larvae are the principal diet of juvenile skipjack. In the NEC, Euphausiacea(IRI=162), Amphipoda(9.2), and Copepoda(0.3)appeared. In the NECC, Copepoda(IRI=158.4), Cephalopoda(66.8), Euphausuiacea(24.8), and others including Saggitoidea, Isopoda, and fish eggs(11.6), and Amphipoda(2.1)appeared. Unidentified organisms comprised the second-highest score of the IRI, 214.4 in the NEC and 346.5 in the NECC. These organisms were consisted mainly of digested fish larvae.
 Diel periodicity of feeding activities of juvenile skipjack was studied, based on the temporal change in the stomach content index(SCI), stomach fullness, and digestion index. The percentage of empty stomachs was 100% at 22-02 hours, 80% at 02-06 hours, and decreased rapidly after sunrise, reaching the minimum score of 5.3% at 14-18 hours(before sunset). In contrast, the percentage of full stomachs reached the maximum score of 60.6% at 14-18 hours, and decreased to 0% from 22 to 06 hours. The SCI and digestion index also indicated the maximum score at 14-18 hours in contrast with the minimum score at before sunrise. Therefore, juvenile skipjack are actively feeding during daytime from morning to sunset, but they do not feed at night.
 The stomach contents of other juvenile tuna were characterized by extremely high scores of fish larvae with no clear difference between the NEC and NECC ; the only occurrences of other prey organisms were Euphausiacea and Cephalopoda. These data show that other tuna are stronger piscivourous feeders than skipjack during the juvenile stage. Feeding activities of other juvenile tuna occur during daytime, similar to skipjack juvenile.
 In order to clarify the growth of skipjack during the early life stages, daily otolith increments were analyzed. Samples from skipjack larvae and juveniles were collected during 1994 to 1997. The otolith measurement system(Ratoc System Engineering Inc., Tokyo)was used for counts of growth increments and measurements of increment width. Daily growth increments were validated by marginal increment analysis for juvenile skipjack.
 Growth rate from larva to juvenile stages of skipjack was studied using the relationship between body length and the number of daily increments of 548 individuals(3.3 to 57.7mm SL). In 15 samples from 1996 and 1997(3.3 to 7.8mm SL), daily growth rate was slow, 0.55mm/day at 3 to 9 days old. In 285 samples from 1994(9 to 24 days old), daily growth rate rapidly increased to 3.3mm/day, and for 248 samples from 1995(9 to 29 days old)growth was 2.5mm/day. The estimated length at 30 days after hatching was about 60mm SL. After metamorphosis from larva to juvenile at 10 to 12 days after hatching, a period of extremely rapid growth with large individual variation was observed. Juvenile skipjack growth rates depend on hatching year and nursery area. The analyses of distance from core to margin at each otolith increment show that a larger individual had a faster growth period after 5 days after hatching. A rapid growth in the early life stage enables the utilization of larger prey organisms, resulting in increased survival rates in the nursery ground that are nutritionally poor. Consequently, these characteristics play an important role in maintaining the population level of skipjack in the pelagic ocean of the tropical western Pacific.

Accepted on March 13, 2002
Contribution No.A 16 from Fisheries Research Agency
*National Research Institute of Far Seas Fisheries, 5-7-1, Orido, Shimizu,Shizuoka 424-8633, Japan