Variation in responses to photoperiods and temperatures in Japanese medaka from different latitudes.

Seasonal changes are more robust and dynamic at higher latitudes than at lower latitudes, and animals sense seasonal changes in the environment and alter their physiology and behavior to better adapt to harsh winter conditions. However, the genetic basis for sensing seasonal changes, including the photoperiod and temperature, remains unclear. Medaka (Oryzias latipes species complex), widely distributed from subtropical to cool-temperate regions throughout the Japanese archipelago, provides an excellent model to tackle this subject. In this study, we examined the critical photoperiods and critical temperatures required for seasonal gonadal development in female medaka from local populations at various latitudes. Intraspecific differences in critical photoperiods and temperatures were detected, demonstrating that these differences were genetically controlled. Most medaka populations could perceive the difference between photoperiods for at least 1 h. Populations in the Northern Japanese group required 14 h of light in a 24 h photoperiod to develop their ovaries, whereas ovaries from the Southern Japanese group developed under 13 h of light. Additionally, Miyazaki and Ginoza populations from lower latitudes were able to spawn under short-day conditions of 11 and 10 h of light, respectively. Investigation of the critical temperature demonstrated that the Higashidori population, the population from the northernmost region of medaka habitats, had a critical temperature of over 18 °C, which was the highest critical temperature among the populations examined. The Miyazaki and the Ginoza populations, in contrast, were found to have critical temperatures under 14 °C. When we conducted a transplant experiment in a high-latitudinal environment using medaka populations with different seasonal responses, the population from higher latitudes, which had a longer critical photoperiod and a higher critical temperature, showed a slower reproductive onset but quickly reached a peak of ovarian size. The current findings show that low latitudinal populations are less responsive to photoperiodic and temperature changes, implying that variations in this responsiveness can alter seasonal timing of reproduction and change fitness to natural environments with varying harshnesses of seasonal changes. Local medaka populations will contribute to elucidating the genetic basis of seasonal time perception and adaptation to environmental changes.

Centromere localization in medaka fish based on half-tetrad analysis.

Gene-centromere (G-C) mapping provides insight into vertebrate genome composition, structure and evolution. Although medaka fish are important experimental animals, no genome-wide G-C map of medaka has been constructed. In this study, we used 112 interspecific triploid hybrids and 152 DNA markers to make G-C maps of all 24 linkage groups (LGs). Under the assumption of 50% interference, 24 centromeres were localized onto all corresponding medaka LGs. Comparison with 21 centromere positions deduced from putative centromeric repeats revealed that 19 were localized inside the centromeric regions of the G-C maps, whereas two were not. Based on the centromere positions indicated in the G-C maps and those of centromeric repeats on each LG, we classified chromosomes as either biarmed or monoarmed; n = 24 = 10 metacentrics/submetacentrics + 14 subtelocentrics/acrocentrics, which is consistent with the results of previous karyological reports. This study helps to elucidate genome evolution mechanisms, and integrates physical and genetic maps with karyological information of medaka.

Inter- and Intraspecific Variation in Sex Hormone-Induced Sex-Reversal in Medaka, Oryzias latipes and Oryzias sakaizumii.

We compared sex-reversal ratios induced by 17α-methyltestosterone (MT) and 17ß-estradiol (E2) exposure in two inbred medaka strains: Hd-rR derived from Oryzias latipes and HNI-II from O. sakaizumii. All MT exposures (0.2-25 ng mL-1) induced complete XX sex-reversal in HNI-II. Although MT exposure at 0.2 ng mL-1 induced XX sex-reversal at > 95% in Hd-rR, other concentrations tested caused XX sex-reversal at lower frequencies (<50%). MT exposure at 1, 5, and 25 ng mL-1 induced XY sex-reversal in Hd-rR, but not in HNI-II. In Hd-rR, E2 exposure induced XY sex-reversal at > 10 ng mL-1, and in all fish feminization occurred 500 ng mL-1. In HNI-II, E2 induced XY sex-reversal at 50 and 250 ng mL-1, but only at rates below 20%. To clarify whether the strain differences in sex hormone-induced sex-reversal are characteristic of each species, we examined the effects of MT and E2 exposure on sex differentiation in five and two additional strains or wild stocks/populations of O. latipes and O. sakaizumii, respectively. MT exposure induced low XX and high XY sex-reversal rates in O. latipes, except in the Shizuoka population, but the trend was reversed in O. sakaizumii. Furthermore, E2-induced XY sex-reversal rates varied intraspecifically in O. latipes. Our results demonstrated that sensitivity to MT and E2 varied within O. latipes species. To evaluate the ecological impacts of environmental chemicals using medaka, it is important to define not only the species, but the strains, stocks, and populations to obtain accurate results.

Hyperosmotic tolerance of adult fish and early embryos are determined by discrete, single loci in the genus Oryzias.

The acquisition of environmental osmolality tolerance traits in individuals and gametes is an important event in the evolution and diversification of organisms. Although teleost fish exhibit considerable intra- and interspecific variation in salinity tolerance, the genetic mechanisms underlying this trait remain unclear. Oryzias celebensis survives in sea and fresh water during both the embryonic and adult stages, whereas its close relative Oryzias woworae cannot survive in sea water at either stage. A linkage analysis using backcross progeny identified a single locus responsible for adult hyperosmotic tolerance on a fused chromosome that corresponds to O. latipes linkage groups (LGs) 6 and 23. Conversely, O. woworae eggs fertilised with O. celebensis sperm died in sea water at the cleavage stages, whereas O. celebensis eggs fertilised with O. woworae sperm developed normally, demonstrating that maternal factor(s) from O. celebensis are responsible for hyperosmotic tolerance during early development. A further linkage analysis using backcrossed females revealed a discrete single locus relating to the maternal hyperosmotic tolerance factor in a fused chromosomal region homologous to O. latipes LGs 17 and 19. These results indicate that a maternal factor governs embryonic hyperosmotic tolerance and maps to a locus distinct from that associated with adult hyperosmotic tolerance.

Estrogen alters gonadal soma-derived factor (Gsdf)/Foxl2 expression levels in the testes associated with testis-ova differentiation in adult medaka, Oryzias latipes.

Testis-ova differentiation in sexually mature male medaka (Oryzias latipes) is easily induced by estrogenic chemicals, indicating that spermatogonia persist in sexual bipotentiality, even in mature testes in medaka. By contrast, the effects of estrogen on testicular somatic cells associated with testis-ova differentiation in medaka remain unclear. In this study, we focused on the dynamics of sex-related genes (Gsdf, Dmrt1, and Foxl2) expressed in Sertoli cells in the mature testes of adult medaka during estrogen-induced testis-ova differentiation. When mature male medaka were exposed to estradiol benzoate (EB; 800ng/L), testis-ova first appeared after EB treatment for 14days (observed as the first oocytes of the leptotene-zygotene stage). However, the testis remained structurally unchanged, even after EB treatment for 28days. Although Foxl2 is a female-specific sex gene, EB treatment for 7days induced Foxl2/FOXL2 expression in all Sertoli cell-enclosed spermatogonia before testis-ova first appeared; however, Foxl2 was not detected in somatic cells in control testes. Conversely, Sertoli-cell-specific Gsdf mRNA expression levels significantly decreased after EB treatment for 14days, and no changes were observed in DMRT1 localization following EB treatment, whereas Dmrt1 mRNA levels increased significantly. Furthermore, after EB exposure, FOXl2 and DMRT1 were co-localized in Sertoli cells during testis-ova differentiation, although FOXL2 localization was undetectable in Sertoli-cell-enclosed apoptotic testis-ova, whereas DMRT1 remained localized in Sertoli cells. These results indicated for the first time that based on the expression of female-specific sex genes, feminization of Sertoli cells precedes testis-ova differentiation induced by estrogen in mature testes in medaka; however, complete feminization of Sertoli cells was not induced in this study. Additionally, it is suggested strongly that Foxl2 and Gsdf expression constitute potential molecular markers for evaluating the effects of estrogenic chemicals on testicular somatic cells associated with estrogen-induced testis-ova differentiation in mature male medaka.

Androgen induces gonadal soma-derived factor, Gsdf, in XX gonads correlated to sex-reversal but not Dmrt1 directly, in the teleost fish, northern medaka (Oryzias sakaizumii).

In the inbred HNI-II strain of Oryzias sakaizumii, Dmy and Gsdf are expressed in XY gonads from Stages 35 and 36, respectively, similarly to the inbred Hd-rR strain of Oryzias latipes. However, Dmrt1 respectively becomes detectable at Stage 36 and 5 days post hatching (dph) in the two strains. In XX HNI-II embryos, 17α-methyltestosterone (MT) induces Gsdf mRNA from Stage 36, accompanied by complete sex-reversal in all treated individuals (MT, 10 ng/mL), while Dmrt1 mRNA was first detectable at 5 dph. In XX d-rR, MT induced Gsdf mRNA expression and sex-reversal in only some of the treated individuals. Together, these results suggest the testis differentiation cascade in XY individuals differs between the HNI-II and Hd-rR strains. In addition, it is suggested that androgen-induced XX sex-reversal proceeds via an androgen-Gsdf-Dmrt1 cascade and that Gsdf plays an important role in sex-reversal in medaka.

Origin of Boundary Populations in Medaka (Oryzias latipes Species Complex).

The Japanese wild population of the medaka fish (Oryzias latipes species complex) comprises two genetically distinct groups, the Northern and the Southern Populations, with boundary populations having a unique genotype. It is thought that the boundary populations have been formed through introgressive hybridization between the two groups, because they are fixed with the Northern alleles at two allozymic loci, with the Southern alleles at two other loci, and have a unique allele at one locus. In this study, we examined the genetic population structure of the boundary populations using genome-wide single nucleotide polymorphism (SNP) data. Most SNPs of the Toyooka population, a typical boundary population, were shared with the Northern Population, some were shared with the Southern Population, and the remaining SNPs were unique to this population, suggesting that the boundary populations originated and diverged from the Northern Population. Further analyses of different populations using SNPs at eight genomic loci indicated that the boundary populations at different locations share similar genomic constitutions, and can be genetically distinguished from typical Northern Populations by unique SNPs. In addition, the boundary populations in the Maruyama River Basin had Northern mitochondrial DNA (mtDNA), while others, from the Fukuda and Kishida River Basins and from the Kumihama Bay area, had Southern mtDNA. These findings suggested that the boundary populations originated from the Northern Population, and then their genomes diverged as a result of geographical isolation, followed by mtDNA introgression from the Southern Population that occurred independently in some populations.

High levels of plasma cortisol and impaired hypoosmoregulation in a mutant medaka deficient in P450c17I.

scl is a spontaneous medaka mutant deficient in P450c17I, which is required for production of sex steroids, but not of cortisol, the major role of which is osmoregulation in teleost fish. The scl mutant provides a new model to study the functions of these hormones. We first found that fish homozygous for this mutation have plasma cortisol constitutively at a high physiological level (1000 nM). Since we previously showed that this level reversed the seawater-type differentiation of the medaka gastrointestinal tract, hypoosmoregulation of the scl mutant was analyzed. Muscle water contents in freshwater were normal in scl homozygotes, but the contents were lower than those of the wild type (WT) after seawater transfer. There were no differences in gill mRNA levels of corticosteroid receptors or ion transporters between scl homozygotes and WT. In the intestine, expression of glucocorticoid receptors and Na(+)/K(+)/2Cl(-) cotransporter were induced in WT during seawater acclimation, but not in scl homozygotes. The high plasma cortisol may prevent hypoosmoregulation by inhibition of increased intestinal water absorption, essentially by the Na(+)/K(+)/2Cl(-) cotransporter, in seawater.

Evolution of the sex-determining gene in the teleostean genus Oryzias.

In the genetic sex determination of vertebrates, the gonadal sex depends on the combination of sex chromosomes that a zygote possesses. Despite the discovery of the sex-determining gene (SRY/Sry) in mammals in 1990s, the sex-determining gene in non-mammalian vertebrates remained an enigma for over a decade. In most mammals, the male-inducing master sex-determining gene is located on the Y chromosome and is therefore absent from XX females. A second sex-determining gene, Dmy, was described in the Oryzias latipes in 2002 and has a DNA-binding motif that is different from the motif in the mammalian sex-determining gene SRY or Sry. Dmy is also located on the Y chromosome and is therefore absent in XX females. Seven other sex-determining genes, including candidate genes, are now known in birds, a frog species, and 5 fish species. These findings over the past twenty years have increased our knowledge of sex-determining genes and sex chromosomes among vertebrates. Here, we review recent advances in our understanding of sex-determining genes and genetic sex determination systems in fish, especially those of the Oryzias species, which are described in detail. The facts suggest some patterns of how new sex-determining genes emerged and evolved. We believe that these facts are common not only in Oryzias but also in other fish species. This knowledge will help to elucidate the conserved mechanisms from which various sex-determining mechanisms have evolved.

Turnover of Sex Chromosomes in Celebensis Group Medaka Fishes.

Sex chromosomes and the sex-determining (SD) gene are variable in vertebrates. In particular, medaka fishes in the genus Oryzias show an extremely large diversity in sex chromosomes and the SD gene, providing a good model to study the evolutionary process by which they turnover. Here, we investigated the sex determination system and sex chromosomes in six celebensis group species. Our sex-linkage analysis demonstrated that all species had an XX-XY sex determination system, and that the Oryzias marmoratus and O. profundicola sex chromosomes were homologous to O. latipes linkage group (LG) 10, while those of the other four species, O. celebensis, O. matanensis, O. wolasi, and O. woworae, were homologous to O. latipes LG 24. The phylogenetic relationship suggested a turnover of the sex chromosomes from O. latipes LG 24 to LG 10 within this group. Six sex-linkage maps showed that the former two and the latter four species shared a common SD locus, respectively, suggesting that the LG 24 acquired the SD function in a common ancestor of the celebensis group, and that the LG 10 SD function appeared in a common ancestor of O. marmoratus and O. profundicola after the divergence of O. matanensis. Additionally, fine mapping and association analysis in the former two species revealed that Sox3 on the Y chromosome is a prime candidate for the SD gene, and that the Y-specific 430-bp insertion might be involved in its SD function.

Co-option of Sox3 as the male-determining factor on the Y chromosome in the fish Oryzias dancena.

Sex chromosomes harbour a primary sex-determining signal that triggers sexual development of the organism. However, diverse sex chromosome systems have been evolved in vertebrates. Here we use positional cloning to identify the sex-determining locus of a medaka-related fish, Oryzias dancena, and find that the locus on the Y chromosome contains a cis-regulatory element that upregulates neighbouring Sox3 expression in developing gonad. Sex-reversed phenotypes in Sox3(Y) transgenic fish, and Sox3(Y) loss-of-function mutants all point to its critical role in sex determination. Furthermore, we demonstrate that Sox3 initiates testicular differentiation by upregulating expression of downstream Gsdf, which is highly conserved in fish sex differentiation pathways. Our results not only provide strong evidence for the independent recruitment of Sox3 to male determination in distantly related vertebrates, but also provide direct evidence that a novel sex determination pathway has evolved through co-option of a transcriptional regulator potentially interacted with a conserved downstream component.

The complete mitochondrial genome of Dugesia japonica (Platyhelminthes; order Tricladida).

We used two sequencing methods, namely long polymerase chain reaction (PCR) and primer walking, to determine the complete mitochondrial DNA (mtDNA) sequence of Dugesia japonica and most of the mtDNA sequence of Dugesia ryukyuensis. The genome of D. japonica contained 36 genes including 12 of the 13 protein-coding genes characteristic of metazoan mitochondrial genomes, two ribosomal RNA genes, and 22 transfer RNA genes. The genome of D. ryukyuensis contained 33 genes, including 12 protein-coding genes, two ribosomal RNA genes, and 19 transfer RNA genes. The gene order of the mitochondrial genome from the Dugesia species showed no clear homology with either the Neodermata or other free-living Rhabditophora. This indicates that the platyhelminths exhibit great variability in mitochondrial gene order. This is the first complete sequence analysis of the mitochondrial genome of a free-living member of Rhabditophora, which will facilitate further studies on the population genetics and genomic evolution of the Platyhelminthes.

Molecular cytogenetic identification and characterization of Robertsonian chromosomes in the large Japanese field mouse (Apodemus speciosus) using FISH.

Robertsonian (Rb) karyotypic polymorphism in Apodemus speciosus has interested many researchers with particular referece to the genetic divergence between Rb and non-Rb populations. Failure to find morphologic, biochemical, or genetic differences in previous studies reveals the necessity of focusing on loci on Rb chromosomes, which can be characterized by FISH mapping with DNA probes. In an Rb heterozygote, DNA probes from laboratory mouse chromosomes (MMUs) 1 and 10 were simultaneously hybridized to the long arm of a metacentric and a medium-sized acrocentric chromosome and to the short arm of the metacentric and a small acrocentric chromosome, respectively. Four additional probes derived from each of MMUs 1 and 10 were mapped to the long and short arms, respectively, of the Rb chromosome identified by the above markers. Homologies between the long arm of the Rb chromosome and MMU 1 and between the short arm and MMU 10 were supported by all ten markers, which were dispersed along nearly the entire lengths of the Rb chromosomes. These results indicate that the long and short arms of the Rb chromosomes are homologous to Apodemus speciosus chromosomes 12 and 19 (defined in a previous study), respectively. This ten-marker series can be useful for detecting chromosome-specific divergence between the two karyotypic populations at the gene level.

Tracing the emergence of a novel sex-determining gene in medaka, Oryzias luzonensis.

Three sex-determining (SD) genes, SRY (mammals), Dmy (medaka), and DM-W (Xenopus laevis), have been identified to date in vertebrates. However, how and why a new sex-determining gene appears remains unknown, as do the switching mechanisms of the master sex-determining gene. Here, we used positional cloning to search for the sex-determining gene in Oryzias luzonensis and found that GsdfY (gonadal soma derived growth factor on the Y chromosome) has replaced Dmy as the master sex-determining gene in this species. We found that GsdfY showed high expression specifically in males during sex differentiation. Furthermore, the presence of a genomic fragment that included GsdfY converts XX individuals into fertile XX males. Luciferase assays demonstrated that the upstream sequence of GsdfY contributes to the male-specific high expression. Gsdf is downstream of Dmy in the sex-determining cascade of O. latipes, suggesting that emergence of the Dmy-independent Gsdf allele led to the appearance of this novel sex-determining gene in O. luzonensis.

Molecular cloning and characterization of the repetitive DNA sequences that comprise the constitutive heterochromatin of the W chromosomes of medaka fishes.

Among the medaka fishes of the genus Oryzias, most species have homomorphic sex chromosomes, while some species, such as Oryzias hubbsi and Oryzias javanicus, have heteromorphic ZW sex chromosomes. In this study, a novel family of repetitive sequence was molecularly cloned from O. hubbsi and characterized by chromosome in situ and filter hybridization, respectively. This repetitive element, which we designated as a BstNI family element, localized at heterochromatin regions on the W chromosome, as well as on two pairs of autosomes. Homologous sequences to this element were found only in O. javanicus, which is a sister species of O. hubbsi, suggesting that this repeated element originated in the common ancestor of these two species. However, the intensity of the hybridization signals was lower in O. javanicus than in O. hubbsi, and the chromosomal location of this element in O. javanicus was confined to heterochromatin regions on one pair of autosomes. Thus, we hypothesize that this repetitive element was extensively amplified in the O. hubbsi lineage, especially on its W chromosome, after the separation of the O. javanicus lineage. In addition, we also found the W chromosomal location of the 18S-28S ribosomal RNA genes in both O. hubbsi and O. javanicus. Our previous studies showed no linkage homology of the sex chromosomes in these species, indicating that the RNA genes were shared between W chromosomes of different origins. This situation may be explained by a translocation of the sex-determining region with the ribosomal RNA genes in either species or an independent accumulation of the RNA genes as a convergent process during W chromosome degeneration.

Occurrence of a short variant of the Tol2 transposable element in natural populations of the medaka fish.

Tol2 is a member of the hAT (hobo/Activator/Tam3) transposable element family, residing as 10-30 copies per diploid genome in the medaka fish. We previously reported that this element is highly homogeneous in structure at both the restriction map level and the nucleotide sequence level. It was, however, possible that there is variation of such a low frequency as not to have been detected in our previous surveys, in which samples from 12 geographical locations were used. In the present study, we first conducted searches of genome sequence databases of medaka, and found a 119-bp-long internal deletion. We then conducted a survey of samples from 58 locations for this deletion by performing PCR preceded by restriction enzyme digestion to increase the sensitivity to this deletion. We found that copies suffering this deletion have spread, or have been generated by multiple origins, in the northern-to-central part of mainland Japan. Thus, although the high homogeneity in structure is a distinct feature of Tol2, variation does exist at low frequencies in natural populations of medaka. The current status of Tol2 is expected to provide information with which results of future surveys can be compared for clarification of determinants of population dynamics of this DNA-based element.

The National BioResource Project Medaka (NBRP Medaka): an integrated bioresource for biological and biomedical sciences.

Medaka (Oryzias latipes) is a small freshwater teleost fish that serves as a model vertebrate organism in various fields of biology including development, genetics, toxicology and evolution. The recent completion of the medaka genome sequencing project has promoted the use of medaka as a comparative and complementary material for research on other vertebrates such as zebrafish, sticklebacks, mice, and humans. The Japanese government has supported the development of Medaka Bioresources since 2002. The second term of the Medaka Bioresource Project started in 2007. The National Institute for Basic Biology and Niigata University were selected as the core organizations for this project. More than 400 strains including more than 300 spontaneous and induced mutants, 8 inbred lines, 21 transgenic lines, 20 medaka-related species and 66 wild stock lines of medaka are now being provided to the scientific community and educational non-profit organizations. In addition to these live fish, NBRP Medaka is also able to provide cDNA/EST clones such as full-length cDNA and BAC/fosmid clones covering 90% of the medaka genome. All these resources can be found on the NBRP Medaka website (http://shigen.lab.nig.ac.jp/medaka/), and users can order any resource using the shopping cart system. We believe these resources will facilitate the further use of medaka and help to promote new findings for this vertebrate species.

Dermal morphogenesis controls lateral line patterning during postembryonic development of teleost fish.

The lateral line system displays highly divergent patterns in adult teleost fish. The mechanisms underlying this variability are poorly understood. Here, we demonstrate that the lateral line mechanoreceptor, the neuromast, gives rise to a series of accessory neuromasts by a serial budding process during postembryonic development in zebrafish. We also show that accessory neuromast formation is highly correlated to the development of underlying dermal structures such as bones and scales. Abnormalities in opercular bone morphogenesis, in endothelin 1-knockdown embryos, are accompanied by stereotypic errors in neuromast budding and positioning, further demonstrating the tight correlation between the patterning of neuromasts and of the underlying dermal bones. In medaka, where scales form between peridermis and opercular bones, the lateral line displays a scale-specific pattern which is never observed in zebrafish. These results strongly suggest a control of postembryonic neuromast patterns by underlying dermal structures. This dermal control may explain some aspects of the evolution of lateral line patterns.

Dichotomous haplotypic lineages of the immunoproteasome subunit genes, PSMB8 and PSMB10, in the MHC class I region of a Teleost Medaka, Oryzias latipes.

Sequence comparison of the medaka, Oryzias latipes, major histocompatibility complex (MHC) class I region between two inbred strains, the HNI (derived from the Northern Population) and the Hd-rR (from the Southern Population), revealed a approximately 100 kb highly divergent segment encompassing two MHC class IA genes, Orla-UAA and Orla-UBA, and two immunoproteasome beta subunit genes, PSMB8 and PSMB10. To elucidate the genetic diversity of this region, we analyzed polymorphisms of the PSMB8 and PSMB10 genes using wild populations of medaka from three genetically different groups: the Northern Population, the Southern Population, and the China-West Korean Population. A total of 1,245 specimens from 10 localities were analyzed, and all the PSMB8 and PSMB10 alleles were classified into the N (fixed in the HNI strain) or the d (fixed in the Hd-rR strain) lineage. Polymerase chain reaction analysis of the region from PSMB8 to PSMB10 indicated that the two allelic lineages of these genes are segregating together constituting dichotomous haplotypic lineages. Both haplotypic lineages were identified in all three groups, although the frequency of d haplotypic lineage (73-100%) was much higher than that of N haplotypic lineage (0-27%) in all analyzed populations. The two allelic lineages of the PSMB8 gene showed curious substitutions at the 31st and 53rd residues of the mature peptide, which are likely involved in formation of the S1 pocket, suggesting that these alleles have a functional difference in cleavage specificity. These results indicate that the two medaka MHC haplotypic lineages encompassing the PSMB8 and PSMB10 genes are maintained in wild populations by a balancing selection.

Distribution of complete and defective copies of the Tol1 transposable element in natural populations of the medaka fish Oryzias latipes.

DNA-based transposable elements are present in the genomes of various organisms, and generally occur in autonomous and nonautonomous forms, with a good correspondence to complete and defective copies, respectively. In vertebrates, however, the vast majority of DNA-based elements occur only in the nonautonomous form. Until now, the only clear exception known has been the Tol2 element of the medaka fish, which still causes mutations in genes of the host species. Here, we report another exception: the Tol1 element of the same species. This element was thought likely to be a "dead" element like the vast majority of vertebrate elements, but recent identification of an autonomous Tol1 copy in a laboratory medaka strain gave rise to the possibility that the element is still "alive" in medaka natural populations. We examined variation in the structure of Tol1 copies through genomic Southern blot analysis, and revealed that 10 of the 32 fish samples examined contained full-length Tol1 copies in their genomes. The frequency at which these copies occur among Tol1 copies is at most 0.5%, yet some of them still have the ability to produce a functional transposase. The medaka fish thus harbors two active DNA-based elements in its genome, and is in this respect unique among vertebrates.