Original Paper
Title Changes in bioaccumulation of tributyltin chloride (TBTCl) in the tissues and organs of immature and maturating mummichogs (Fundulus heteroclitus)
Authers Hideo HORI*1, Akira KAKUNO*2, Kumiko IKEDA*2 and Hisashi YAMADA*2
Keywords BCF, tissue distribution, maturation, mummichog, tributyltin chloride
Citation Bull. Fish. Res. Agen. No.11, 1-10, 2004

Male and female mummichogs (Fundulus heteroclitus) were exposed to waterborne tributyltin chloride (TBTCl) for 56 or 77days at different maturation stages during the refractory and the maturating periods. Differences in TBT bioaccumulation in the tissues and organs as well as entire body by sex and degree of maturation were studied for a discussion on the influence of TBTCl on the reproduction processes.

The BCF during the refractory period was 9,100±640 for the males and 9,200±1,700 for the females, indicating that mummichogs bioaccumulated TBTCl in much higher concentration than other marine fish species. The TBT concentration was the highest in the blood and the lowest in the digestive tract of male and female mummichogs. The TBT concentrations in the liver and gonads were similar to one another. The differences between the males and females in terms of TBT concentration and BCF were neglegible during the refractory period. On the other hand, TBTCl had bioaccumulated significantly in the testes during the maturating period, and the BCF was 7 times larger in the testes than in the ovaries. The high bioaccumulation in the testes suggests that TBTCl inhibits the reproduction processes of male fish more seriously.

Received on February 18, 2004
Contribution No.A 49 from Fisheries Research Agency
*1 Japan Frozen Foods Inspection Corporation, 2-4-6, Shibadaimon, Minatoku, Tokyo, 105-0021, Japan
*2 National Research Institute of Fisheries and Environment of Inland Sea, 2-17-5, Maruishi, Ohno, Saeki, 739-0452, Japan

Title Bioconcentration and distribution of 4- tert -octylphenol in mummichogs (Fundulus heteroclitus) exposed during the maturation period
Authers Hideo HORI*1, Akira KAKUNO*2, Takatoshi MORITA*1,Kumiko IKEDA*2 and Hisashi YAMADA*2
Keywords mummichog, maturation, bioconcentration, tissue distribution, 4-tert-octylphenol
Citation Bull. Fish. Res. Agen. No.11, 11-18, 2004
Male and female mummichogs (Fundulus heteroclitus) were reared for 56 days in order to understand possible sexual differences in intake, elimination and tissue distribution of 4-tert-octylphenol (OP). The bioconcentration factors (BCF) for the entire body of the females and the males were 34±11 and 23±5.1, respectively. The difference was negligible. These BCFs were one order smaller than those reported previously for carp and Japanese medaka. The OP concentration was the highest in the digestive tract and the lowest in the blood of both the males and females. The BCF in the studied tissue and organs of the male fish was similar to that of the female fish. These results suggest that the bioconcentration of OP is independent of sex. OP was excluded rapidly from the fishes' tissues and organs in the elimination experiment, with the exception of the ovaries. The concentration of 0.08μg/g wet weight in the ovaries was observed on the 14th day of the elimination experiment. OP in the ovaries is retained for a long period compared to other fish tissues and organs. These results suggest that OP can inhibit developmental processes in the early life stage of the next generation of fish.

Received on February 18, 2004
Contribution No.A 50 from Fisheries Research Agency
*1 Japan Frozen Foods Inspection Corporation, 2-4-6, Shibadaimon, Minatoku, Tokyo, 105-0021, Japan
*2 National Research Institute of Fisheries and Environment of Inland Sea, 2-17-5, Maruishi, Ohno, Saeki, 739-0452, Japan

Technical Report
Title Characterization of the base collection strains preserved in the Marine Microorganism Division, a sub-bank of the Microbial Culture Collections, Fish Research Agency
Authers Motoharu UCHIDA*1, Yoko SATO*2, Masakazu MURATA*2 and Ryoji MATSUSHIMA*2
Keywords characterization, FRA culture collection, marine microorganism, microorganism Sub-Bank of FRA
Citation Bull. Fish. Res. Agen. No.11, 19-30, 2004
The Applied Microbiology Section in the National Research Institute of Fisheries Science is nominated as a Microbial Culture Collections Sub-Bank of the Gene Bank in the Fisheries Research Agency (FRA) and keeps a collection of fifty strains of bacteria and yeast as of May 2002, that are in preparation to be utilized as open cultures. Microbiological characterization was carried out with these collections to assist future culture utilization. The study was conducted using several commercial kits including NF- 18 test (for gram negative microorganisms), NF-20 test (for gram positive microorganisms), API50CH test, and API zym20 test in addition to conventional phenotypic characterization. The partial nucleotide sequences of 16S rRNA gene (for bacteria) and 18S rRNA gene (for yeast) were also determined and submitted to DDBJ. Based on all the obtained results, tentative identification was conducted for the current fifty FRA collections.

Received on January 19, 2004
Contribution No.A 47 from Fisheries Research Agency
*1 National Research Institute of Fisheries and Environment of Inland Sea, Maruishi, Ohno, Saeki, Hiroshima 739-0452, Japan
*2 National Research Institute of Fisheries Science, Fukuura, Kanazawa, Yokohama 236-8648, Japan

Doctoral Thesis
Title Studies on genetic management of hatchery broodstock in relation to the stock enhancement of Japanese flounder Paralichthys olivaceus
Authers Masashi SEKINO*
Keywords Japanese flounder, Genetic markers, Population genetics, Conservation genetics, Stock enhancement
Citation Bull. Fish. Res. Agen. No.11, 31-91, 2004

From the viewpoint of genetic conservation, this study presents application of molecular genetic markers to the population genetics studies of Japanese flounder Paralichthys olivaceus in order to propose several broodstock management options related to the stock enhancement.

A (CA)n-microsatellite enriched library of Japanese flounder was constructed using an efficient method to isolate microsatellite DNA regions, from which 16 PCR primer pairs to amplify microsatellites with a high amplification effectiveness were developed. Genetic variability of these loci varied depending on the locus, and all but one of the 16 loci conformed to Hardy-Weinberg's equilibrium (HWE).Mendelian inheritability of these loci was verified in five experimental families of Japanese flounder excepting one instance. The locus at which the significant HWE departure was observed had unstable and unreproducible PCR amplification of a particular allele in one family. These results suggest that 15 out of the 16 microsatellites are viable for further applications to the wide range of the Japanese flounder population genetics studies.

Genetic variability and structure maintained in wild Japanese flounder populations (seven populations) were assessed by using 11 microsatellites coupled with the nucleotide sequences of mitochondrial DNA (mtDNA) control region to outline the genetic stock management units. The 11 microsatellites examined showed considerable polymorphisms in all populations, and the section of mtDNA sequences examined was highly variable. Assessment of the genetic structure based on the 11 microsatellites revealed that 11 population pairs in 21 possible pairwise comparisons yielded significant genetic heterogeneities in terms of allele frequency distributions and/or pairwise FST values, and an overall FST value provided evidence of the genetic structuring among the seven populations, although these inter-populational genetic heterogeneities were not necessarily supported by the results of mtDNA sequencing analysis. According to the population relationships estimated from the phylogenetic trees based on the genetic distances (DA and DST) and the results of several hierarchical AMOVA analyses, which were calculated on the basis of the microsatellite data, the seven populations were possibly assigned into three groups consistent with the oceanogeographic positions of the populations. Although the level of the genetic differentiation between the groups was weak, the genetic management of wild flounder should be applied along with the group definitions so as to preserve the currently detectable genetic stock units.

Level of genetic variability maintained in currently available hatchery strains was compared with that of wild populations. Three Japanese flounder hatchery strains were screened by means of microsatellite and mtDNA sequencing analysis, and both the number of microsatellite alleles and mtDNA haplotypes, and mtDNA haplotype diversity showed marked reductions in the hatchery strains relative to the comparable three wild populations.

Pedigree structure of a hatchery strain was detailed in order to illustrate a skewed contribution of broodstock parents to the next generation, which may possibly cause a reduced effective population size, by using microsatellite profiling technique. The pedigrees of offspring including 113 individuals of larvae sampled within 24 hours after hatching 216 individuals of one-month-old, and 407 individuals of four-month-old were unambiguously identified. The contribution of candidate broodstock parents (18 individuals) to the next generation was highly skewed as one male of the six males used monopolized the contribution to the offspring pool, and a half of the females (6 of 12 females) did not produce any offspring. The contribution of one family to the released fish, which were culled for larger size in total length, was significantly higher, while those of other two families were lower, suggesting that culling operations for particular traits might cause a large family size variance of released fish through selection in favor of a few family lineages. Culling operations for particular traits thus should be avoided as much as possible to minimize the risk of unexpected pedigree structure to be a possible cause of the reduced effective size.

Possible way to retard loss of genetic variations in closed and non-pedigreed captive populations of Japanese flounder was explored by using a microsatellite-based kinship estimator (rxy statistic). The difference of pairwise rxy values among and between full-sib, half-sib, and unrelated categories in a hatchery strain was highly significant, suggesting that the rxy statistic would have a high utility to discriminate kinships. Minimal kinship (mk) selection approach based on the rxy statistic, which is a breeding strategy to minimize loss of genetic variability in captive populations, turned out to be effective in retention of both allelic diversity and gene diversity; this strategy however did not necessarily select unrelated fish as parents to create the next generation. It should thus be necessary to preclude the mating between highly related individuals in the selected fish by using the rxy statistic as a kinship indicator so as to minimize the risk of inbreeding.

From the results presented above, the present author discusses here several hatchery management options for further practical hatchery operations to minimize the genetic impact of stocking practice to the wild Japanese flounder populations.

Received on October 6, 2003
Contribution No.A 44 from the Fisheries Research Agency
* Tohoku National Fisheries Research Institute, 3-27-5, Shin-hama, Shiogama, Miyagi, 985-0001, Japan