Tetsuo Fujii

Japan Sea National Fisheries Research Institute
Fisheries Research Agency
1-5939-22, Suido-cho, Niigata 951-8121
JAPAN
Email: tefujii@affrc.go.jp

Key Words: Japanese flounder, migration, genetic diversity, mtDNA tag

Abstract

A method of tracking released Japanese flounder Paralichthys olivaceus was developed by using the extremely high sequence variability of the mitochondrial DNA, particularly in the control region. The determination of the mother of a released flounder is possible by sequencing the mitochondrial DNA control region because of its variability and maternal inheritance. In this method, sequences of hatchery-reared juveniles should be analyzed first. The sequence data are then registered in a database, so that when the flounder is later caught its origin can be determined. We can obtain information about the genetic variability of hatchery-reared stocks in the sea, as well as the migration of the released flounder. Although other methods have been used to track released flounder, mitochondrial DNA sequence appears to be less harmful for juveniles and potentially less expensive.

The Japanese flounder Paralichthys olivaceus is one of the most important species for the coastal fisheries in Japan. More than 30 million hatchery-reared juveniles have been released annually for the enhancement of stocks. It is well known that the wild flounder migrate widely as they grow (Minami, 1997). To elucidate the migration of the released flounder it is necessary to carefully manage the genetics as well as evaluate stocking effectiveness. Previous tagging studies have been performed using external tags or chemical markings. External tags may be harmful to juveniles (most juveniles are released less than 10 cm in total length) and difficult to place on large numbers of fish. On the other hand, chemical markings, such as fluorescent marking in otoliths, are comparably easy to apply, but the patterns of the marks are limited. To avoid these tagging obstacles, a method of tracking released Japanese flounder by mitochondrial DNA (mtDNA) sequence was developed. In this paper, the concept and advantages of the mtDNA tag are described. Potentials for use of the mtDNA tag and future programs are also introduced briefly.

Concept of mtDNA Tag

In this method, sequences of hatchery-produced juveniles must first be analyzed and registered in a database. The mtDNA of wild Japanese flounder is characterized by the extremely high sequence variability especially in the control region (D-loop region). The direct sequencing of the 350 base pairs (bp) in the first half of the control region showed that 126 sites (36.0%) were variable. The sequence differences between individuals were up to 8.3%, with an average of 4.3%, as 54 haplotypes were detected from 55 individuals (Fujii and Nishida, 1997). This means that almost all flounder have their own unique sequences and mothers of hatchery-reared juveniles can be determined because of its high variability and maternal inheritance. In Japan there is at least one hatchery in every prefecture, each having its own broodstock. This makes hatchery-rearing of juveniles localized. Since most of the hatcheries are supported by the prefectures, most juveniles are released in their home prefectures. When the released flounder are recaptured later, their origins of rearing and release can be determined by their mtDNA sequences.

Fortunately, most of hatchery-reared juveniles have abnormal pigmentation, called melanism, on their blind sides. Therefore it is easy to distinguish released flounder from wild ones. DNA analysis is also possible from a single fish scale preserved in 99.5% ethanol. All that would be necessary at the fish market is to pick one scale from each flounder having melanism on the blind side.

Advantages of the mtDNA Tag

The mitochondrial DNA tag has the following advantages: