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Fishes expressing a fluorescent protein in germ cells are useful to perform germ cell transfer experiments for conservation study. Nonetheless, no such fish has been generated in endangered endemic fishes. In this study, we tried to produce a fish expressing Venus fluorescent protein in germ cells using Honmoroko (Gnathopogon caerulescens), which is one of the threatened small cyprinid endemic to the ancient Lake Biwa in Japan. To achieve germ cell-specific expression of Venus, we used piwil1 (formally known as ziwi) promoter and Tol2 transposon system. Following the co-injection of the piwil1-Venus expression vector and the Tol2 transposase mRNA into fertilized eggs, presumptive transgenic fish were reared. At 7 months of post-fertilization, about 19% (10/52) of the examined larvae showed Venus fluorescence in their gonad specifically. Immunohistological staining and in vitro spermatogenesis using gonads of the juvenile founder fish revealed that Venus expression was detected in spermatogonia and spermatocyte in male, and oogonia and stage I and II oocytes in female. These results indicate that the Tol2 transposon and zebrafish piwil1 promoter enabled gene transfer and germ cell-specific expression of Venus in G. caerulescens. In addition, in vitro culture of juvenile spermatogonia enables the rapid validation of temporal expression of transgene during spermatogenesis.

Despite a recent study of Honmoroko, Gnathopogon caerulescens, reproduction in lagoons around Lake Biwa having demonstrated the importance of lagoon inlets as spawning habitats for the species, no quantitative studies have examined the conditions determining the environmental suitability of spawning habitats. A survey of Honmoroko egg distribution in the inlets indicated that mature Honmoroko selectively spawn in regions of greater current velocity, utilizing a greater abundance of submerged plants and larger substrate (gravel and pebble) size, not utilizing the emergent plants, habitat characteristics supported by a generalized linear mixed model. The biological information gained is essential for future conservation of Honmoroko spawning habitats.

Migration of wild and cultivated juvenile honmoroko Gnathopogon caerulescens of from the spawning and nursery areas in Lake Biwa were investigated, both in the Ibanaiko Lagoon and its outlet to Daido River, using beam-trawl surveys in 2013 and 2014. The study demonstrated migration of G. caerulescens from a nursery lagoon toward Lake Biwa after the juvenile stage. These findings appear to be the first direct evidence for migration of an exclusively pelagic cyprinid species from a littoral nursery to a pelagic adult habitat in a large deep lake.

Otolith growth increments in wild-caught alizarin complex one (ALC)-marked honmoroko Gnathopogon caerulescens were examined to verify the veracity of the age determination method in cyprinids. ALC-marked G. caerulescens recaptured from their natural environment had lapilli increment counts outside the ALC ring mark that had formed on a daily basis during the juvenile stage. This apparently being the first direct evidence of daily periodicity of otolith increment formation in wild-caught cyprinids.

Honmoroko Gnathopogon caerulescens is a critically endangered species and important for commercial fisheries; thus stock enhancement programs are being conducted to restore resources. We evaluated the genetic population structure of field collected samples including spawned eggs around spawning areas in Lake Biwa and the extent of genetic diversity in wild samples and hatchery stocks using sequences of mitochondrial cytochrome b gene. Pairwise Φst analysis and AMOVA clearly showed minimal population structure and haplotype network did not reveal any clear geographic pattern in Lake Biwa. It is probable that strays spawn in non-natal spawning areas, resulting in significant levels of gene flow among spawning areas. Genetic characteristics of hatchery stock F1, F2, and F3 were similar to those of wild samples in terms of haplotype diversity, nucleotide diversity and pairwise Φst values. These results indicate that the relatively high genetic diversity at its initiation was retained due to a lot of broodstock over two successive generations. Accordingly we propose that the current Honmoroko breeding method is appropriate for conserving the genetic diversity of Honmoroko and that the hatchery stock are genetically compatable for release and stock enhancement.

The influence of hatching date on the sex ratio of wild Gnathopogon caerulescens was examined. Cohorts reared from eggs collected in the early and middle parts of the spawning season showed almost balanced sex ratios, with female bias in some cohorts. Cohorts born later in the season mostly displayed male bias, and the mean proportion of males later in the season was significantly higher than in early- and mid-season cohorts. These results indicate that the sex ratio of G. caerulescens changes with the time of breeding, increasing along with the ambient water temperature of the lake.

Establishing a cell line from endemic species facilitates the cell biological research of these species in the laboratory. In this study, an epithelium-like cell line RME1 was established from the blastula-stage embryos of the critically endangered cyprinid Honmoroko Gnathopogon caerulescens, which is endemic to ancient Lake Biwa in Japan. To the best of our knowledge, this is the first embryonic cell line from an endangered fish species. This cell line is well adapted to grow at 28°C in the culture medium, which was successfully used for establishing testicular and ovarian cell lines of G. caerulescens, and has displayed stable growth over 60 passages since its initiation in June 2011. Although RME1 did not express the genes detected in blastula-stage embryos, such as oct4, sox2, nanog, and klf4, it showed a high euploidy rate (2n = 50; 67.2%) with normal diploid karyotype morphology, suggesting that RME1 retains the genomic organization of G. caerulescens and can prove to be a useful tool to investigate the unique properties of endangered endemic fishes at cellular level.

SummarySperm cryopreservation is a valuable conservation method for endangered fish species. Here we report an easy and efficient cryopreservation method for juvenile whole testis by vitrification and successful sperm production from the vitrified whole testis viain vitrospermatogenesis in the critically endangered cyprinid honmoroko (Gnathopogon caerulescens). Juvenile testis (approximately 10 mm in length and 1 mm in width), consisting predominantly of spermatogonia, were aseptically dissected out and adherent fatty and non-testicular tissues were subsequently removed. Then, the testes were rapidly cooled on a nylon mesh by direct immersion in liquid nitrogen after serial exposures to pretreatment solution (PS), containing 2 M ethylene glycol (EG) and 1 M dimethyl sulfoxide (DMSO), for 20 or 30 min and vitrification solution (VS), containing 3 M EG, 2 M DMSO, and 0.5 M sucrose, for 5, 10, or 20 min. The highest survival rate of testicular cells (84.0%) was obtained from testes vitrified by immersion in PS for 20 min and in VS for 10 min. Spermatogonia were recovered from the vitrified testis by dissociation and cell culture produced many haploid sperm. Fertility and developmental competence were confirmed byin vitrofertilization assays. These results indicate that the vitrification of juvenile whole testis provides a new strategy to preserve the genetic resources of endangered fishes without affecting their reproductive population.

The age composition, and age- and size-dependent sex ratios of mass samples of “Honmoroko”, Gnathopogon caerulescens, were examined in Lake Biwa. The fish ranged from 0 to 2 years in age, but were mostly age 0 (97.9 %). The proportion of females was 48.6 % for age 0, 73.2 % for age 1, and 90.0 % for age 2 fish. Age 0 fish were significantly male biased, while ages 1 and 2 were significantly female biased, though the grand total showed a balanced sex ratio. The proportion of females was 48.0 % or less among age 0 fish in size classes of less than 9 cm in standard length (SL) and significantly different from a female:male ratio of 1:1 in such size classes, except for the 4–5.99 class. In size classes of 9 cm SL or greater, the proportion of females among age 0 fish was greater, more than 71.3 %, with significant differences from a balanced sex ratio for all such classes. These results suggest that G. caerulescens can be regarded as essentially an annual fish and that its spawning habits ought to result in a higher mortality for males than females, thus leading to female-biased sex ratios at ages 1 and 2.

Diel drift patterns of larvae of the endangered cyprinid Gnathopogon caerelescens in an inlet of the Ibanaiko Lagoon, connected to Lake Biwa in Japan, were assessed in April 2012. Peak occurrence of yolk-sac larvae was within a few hours after dark. Drift of newly hatched larvae is considered to be an important biological mechanism that ensures larval dispersal and recruitment from the inlets (spawning grounds) to the lagoon which functions as a nursery ground.

Egg size affects larval size, growth rate, survival and fecundity with maternal fitness being maximized by a trade-off between egg size and fecundity. Production of a small number of large eggs maximizes female fitness under poor food conditions, as does a large number of small eggs under rich food conditions. Gnathopogon caerulescens (Honmoroko) spawns over a wide range of water temperature from spring to summer. Thus, we determined whether or not egg size varied with water temperature and how egg size influenced hatchling size at different water temperature. Changes in egg size strongly correlated with seasonal changes in water temperature around the lake, regardless of time and area. An experiment using eggs from the lake indicated that hatchling size has a significant positive relationship with egg size in water temperatures of 24 °C. On the other hand, a lower incubation temperature, similar to that likely to be encountered at the beginning of the spawning season, resulted in a smaller hatchling size, eggs requiring a longer time to hatch. At the beginning of the spawning season, therefore, egg size may have a lesser impact on hatching size in the natural environment of Honmoroko because of the relatively lower temperatures. Although fish larvae below a certain threshold of effective body size are generally not expected to have a high early survival rate due to, for example, a lower competitive ability or a high susceptibility to predation, seasonal egg size variation in Honmoroko may be adaptive so as to enhance the likehood of early survival by establishing an effective body size under changing water temperature conditions.

Landlocking of salmon relaxes selective pressures on hypoosmoregulatory ability (seawater adaptability) and may lead to the abandonment of its physiological system. However, little is known about the mechanism and consequence of the process. Biwa salmon is a strain/subspecies of Oncorhynchus masou that has been landlocked in Lake Biwa for an exceptionally long period (about 500,000 years) and has low ability to adapt to seawater. We compared activity of gill Na+,K+-ATPase (NKA) of Biwa salmon with those of anadromous strains of the same species (masu and amago salmon) during downstream migration periods and after exogenous hormone treatment. Gill NKA activity in anadromous strains increased during their migration periods, while that in Biwa salmon remained low. However, treatments of Biwa salmon with growth hormone (GH) and cortisol increased gill NKA activity. Cortisol treatment also improved the whole body seawater adaptability of Biwa salmon. Receptors for GH and cortisol responded to hormonal treatments, whereas their mRNA levels during downstream migration period were essentially unchanged in Biwa salmon. Circulating levels of cortisol in masu salmon showed a peak during downstream migration period, while no such increase was seen in Biwa salmon. The present results indicate that Biwa salmon can improve its seawater adaptability by exogenous hormonal treatment, and hormone receptors are capable of responding to the signals. However, secretion of the endogenous hormone (cortisol) was not activated during the downstream migration period, which explains, at least in part, their low ability to adapt to seawater.

The sex ratios of the nigorobuna Carassius auratus grandoculis reared in paddy fields during the sex differentiation periods of larval and juvenile stages were examined, since the phenotypic expression of sex in the fish is thermolabile, so that sex determination is under the control of genetic factors and temperature. After 2 days from hatching, the larvae were introduced into five paddy fields at five different densities in late May. After 37 or 41 days from hatching the juveniles were collected when the water was drained from the paddy fields, and reared in laboratory tanks supplied with running freshwater pumped from the lake shore until the age of 144–164 days. The mean water temperatures in the paddy fields were 22.4–24.1 oC, and the maximum water temperature during the day frequently exceeded 30 oC during the supposed critical period of sex determination of the fish. The sex ratios in the five paddy fields did not show a male-bias. Instead, all fields produced either balanced or slightly female-bias sex ratios. These results suggest that temporary high and fluctuating temperatures in paddy fields with low mean temperatures have hardly any masculinization effects on genotypic females of C. auratus grandoculis during sex differentiation stages.

The honmoroko has been inferred to have an XX/XY sex determination system, but the parental genome can also affect the sex ratio of the offspring. The extent of parental effects on sex determination was examined by checking the sex ratios of F1 and F2 gynogenetic diploids and control diploids. Eleven gynogenetic broods from different females consisted of all or nearly all females, but eight broods showed a variable proportion of males (

Fish Sertoli cells play a critical role in spermatogenesis by mediating androgen and progestogen signaling. Their hormonal response, however, considerably differ among species. Therefore it would be ideal to use Sertoli cells originated from the fish of interest to investigate the effects of hormones as well as endocrine disrupting chemicals (EDCs). The aim of this study was to investigate the responses to reproductive hormones and EDCs of a Sertoli cell line that we established from an endemic cyprinid Gnathopogon caerulescens. As the Sertoli cell line expressed endogenous androgen and progestogen receptors, we were able to detect hormone responses by transfecting only a reporter vector (pGL4.36) expressing luciferase under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter into the cell line. Unlike previous reporter gene assays using fish steroid hormone receptors expressed in mammalian cell lines, luciferase activities were induced by the fish specific androgen (11-ketotestosterone) and progestogen (17α,20β-dihydroxy-4-pregnen-3-one), but not by testosterone and progesterone, at physiologically relevant concentrations. Furthermore, we found 4-nonylphenol (NP) but not bisphenol A showed strong anti-androgenic effects, implying that NP may have direct anti-androgenic effects on fish Sertoli cells in vivo. This is the first evidence, to the best of our knowledge, of anti-androgenic effects of NP in a fish Sertoli cell line. In addition, neither NP nor BPA showed anti-progestogenic effects. These results suggest that the Sertoli cell line established from the fish of interest can be a useful in vitro tool for investigating the mechanisms of reproductive hormones and EDCs in the specific fish.

Many endemic fish species are threatened with extinction. Conservation strategies and the restoration of endemic fish after extinction must therefore be investigated. Although sperm cryopreservation is indispensable for the conservation of endangered fishes, the limited number of mature fish and limited availability (volume and period) of sperm from small endemic fish hinders the optimization and practical use of this material. In this report, we demonstrate the in vitro differentiation of fertile sperm from cryopreserved spermatogonia of juveniles of the endangered small cyprinid honmoroko (Gnathopogon caerulescens), which is endemic to Lake Biwa in Japan. The entire process of spermatogenesis was recapitulated in vitro using cryopreserved spermatogonia of non-spawning adult and juvenile fish. The differentiation of sperm from spermatogonia was captured as a time-lapse video and confirmed by 5-ethynyl-2′-deoxyuridine (EdU) incorporation into sperm. Fertility was demonstrated by artificial insemination. These results suggest that the combination of cryopreservation of spermatogonia and in vitro sperm differentiation will provide a new and promising strategy for the preservation of paternal genetic materials.

We investigated the feasibility of cryopreservation of spermatogonia and oogonia in the critically endangered cyprinid honmoroko Gnathopogon caerulescens using slow-cooling (freezing) and rapid-cooling (vitrification) methods. Initially, we examined the testicular cell toxicities and glass-forming properties of the five cryoprotectants: ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (BG), and we determined cryoprotectant concentrations that are suitable for freezing and vitrification solutions, respectively. Subsequently, we prepared the freezing solutions of EG, GC, DMSO, PG, and BG at 3, 2, 3, 2, and 2 M and vitrification solutions at 7, 6, 5, 5, and 4 M, respectively. Following the cryopreservation of the testicular cells mainly containing early-stage spermatogenic cells (e.g., spermatogonia and primary spermatocytes), cells were cultured for 7 days and immunochemically stained against germ cell marker protein Vasa. Areas occupied by Vasa-positive cells indicated that vitrification led to better survival of germ cells than the freezing method, and the best result was obtained with 5 M PG, about 50% recovery of germ cells following vitrification. In the case of ovarian cells containing oogonia and stage I, II, and IIIa oocytes, vitrification with 5 M DMSO resulted the best survival of oogonia, with equivalent cell numbers to those cultured without vitrification. The present data suggest that male and female gonial cells of the endangered species G. caerulescens can be efficiently cryopreserved using suitable cryoprotectants for spermatogonia and oogonia, respectively.

Size at maturity of fluvial white-spotted charr, Salvelinus leucomaenis, was studied in small headwater tributaries of nine rivers around the Lake Biwa water system, Japan. Threshold size at maturity in both sexes showed significant positive relationships with water discharge, indicating that smaller threshold sizes at maturity of fluvial white-spotted charr occurred in smaller habitats. These results provide a link between size at maturity and habitat size and have important implications for the management of both habitats and white-spotted charr populations.

The sex ratios of the offspring of males from broods of honmoroko Gnathopogon caerulescens that displayed thermolabile sex determination (TSD), and those from eggs collected from the wild, strongly suggest that the combination of parents plays an important supplementary role in TSD in this species. The proportion of females in all broods from eight pairings of fish captured in the field decreased significantly at 30°C compared to 20°C, and one brood was entirely female at the lower temperature. These results suggest that phenotypic males (XX-males) exist in nature, probably as a result of sex change from genetic females caused by TSD. Sex ratios in relation to water temperature fall into five patterns, two of which seem to result from normal pairings of XX-females and XY-males, and from pairings of XX-males and XX-females with non-thermosensitivity. Two other patterns are thought to result from the same types of pairing, but with thermosensitivity. The last pattern, which shows male bias at both low and high temperature, is not explained only by the combination of genotypic sex determination (XX/XY) and TSD. Fluctuating temperatures close to natural conditions showed little potential to masculinize broods laid in the field between April and early June. However, the sex ratios of fish spawned in early June showed male bias at low and high temperatures. The sex determination mechanism in G. caerulescens may involve the interaction between temperature and sex-determining genetic factors, and the relative importance of each component differs with breeding season.

Functional sex reversal of all-female nigorobuna Carassius carassius grandoculis to phenotypic males was examined by immersion exposure of fry to 17-methyltestosterone (17-MT) and controlled water temperature during early development. Fry were reared in water containing differ- ent concentrations of 17-MT at 24 and 30°C for 80 days starting 20 days after hatching. Although the fish exposed to 0.1 and 1.0 mg/L 17-MT at 24°C were all male, treatment with 10.0 mg/L 17-MT resulted in 43% females. Twenty-two percent males appeared in the control treatment at 30°C but the control at 24°C was entirely female. The proportion of males in treatments exposed to 0.01- 1.0 mg/L 17-MT at 30°C was slightly lower than in the respective treatments at 24°C. These results indicate that the phenotypic expression of sex in nigorobuna is thermolabile and that sex determi- nation is under the control of genetic factors and temperature. Also, control of temperature during early development has been shown to be important for the production of all-female offspring for use as breeding stock for pond culture of fish suitable for preparation as 'funazushi'.

We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz’s L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and β-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 α promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function.

Changes in lipid content, lipid class composition, and fatty acid composition in wild and cultur-ed biwa and amago salmon underyearlings were examined during the period of body silvering. Cultured biwa salmon showed slight changes in these components of the muscle during body sil-vering. Although cultured amago salmon parr showed seasonal fluctuations in the components, the smolts did not show definite difference as compared with the parr in early smolting season. On the other hand, wild biwa salmon displayed a marked difference in the lipid class composition and fatty acid composition between the parr in the river and the silvery fish in the lake. In silvering biwa salmon, depletion of 16:1, 18:2n6, 18:3n3 and increase in 22:6n3 of nonpolar lipids were observed, the pattern being similar to the marine fish lipid. Since no change was seen in cultured biwa salmon, differences in the lipid class and fatty acid compositions between the parr in the river and the silvery fish in the lake in wild biwa salmon seem to be related to differences in their feeding habit and/or in swimming activity.

Downstream migratory behavior, plasma T4 concentrations and histological changes in the thyroid gland of biwa salmon underyearlings were examined during late spring and early summer for three consecutive years. The downstream migration was observed specifically when the flux of the river increased following rainfall. A close correlation was seen between the number of migrants and the rate of flux increase of the river. Plasma T4 levels of the fry caught in the river were variable, and no correlation was seen between the plasma T4 levels in the fish remaining in the river and lunar phase, rainfall, water flux or water temperature. However, significantly higher T4 concentrations were observed in the fish during downstream migration when compared with those in the fish remaining in the river and postmigrants in the lake. The mean epithelial height of the thyroid folliciles was greatest in the underyearling in May, shortly before the onset of the migratory season. The thyroidal activity of the migrants in late May and June tended to decrease and postmigrants in the lake had inactive thyroid. These results suggest that the rainfall or the associated changes in turbidity and flux is one of the initiators of the migratory behavior in biwa salmon, and also that thyroid hormone is involved directly or indirectly in downstream movement.

Parr-smolt transformation, growth rate, maturity and appearance or disappearance of red spots on the body were observed in juvenile biwa salmon Oncorhynchus rhodurus indigenous to Lake Biwa, reared in pond for eighteen months, and compared with those of amago salmon reared under the same conditions. Biwa salmon transformed from juvenile parr to silvery smolt in underyearling June to July (standard length 7.5-8.0cm), and 60-80% remained as smolt till yearling autumn, Smolts of amago salmon appeared mainly in underyearling September to yearling March, and all of them transformed back to parr or silvery parr by yearling May. Most of the smolt of biwa salmon, unlike those of amago and masu salmon, did not show complete silvering of body color or intense blackening at outer extremities of dosal fin. Growth after smolt transformation in biwa salmon was slower than that of amago salmon, though there was no significant difference between the juveniles of biwa and amago salmon in underyearling April to May. Almost all of the amago salmon matured in yearling autumn, wherease only a part of the biwa salmon males matured at the same time. Red spots appeared on body of almost all of the amago salmon, and the number and density increased with growth. On the contrary, those of biwa salmon decreased during smolt transformation, though a few red spots existed at parr stage. Thus biwa salmon differed from amago salmon in several characteristics during smoltification.

Downstream migration and body silvering of landlocked biwa salmon Oncorhynchus rhodurus indigenous to Lake Biwa were observed in Lake Biwa and the rivers which flow to the lake in central Japan from 1983 to 1986. Underyearling biwa salmon inhabited the gently flowing shallows of the river shore in April, and moved into the riffle in May, when they began to migrate downstream into the lake. Downstream migration of the juvenile fish (5 to 7cm in standard length) was mainly observed from the middle of May to the end of June. The downstream migration seems to be related with rainfall, as the number of the fish moved into the lake increased on the day and/or the next day of rain, Although the sex ratio of the juvenile fish in the river in May was equal, the ratio of male became greater from June to July. Juvenile fish were scarcely observed in the river after July; in autumn only a small number of residual precocious mature males inhabited the river. After descending into the lake, they inhabited the offshore zone of the lake deeper than 20m. There was a significant difference in the slopes in the growth lines between the juvenile fish in the river and in the lake; the fish in the river grew faster than those in the lake. Body silvering of the juvenile fish began from the end of May in the river, and parr marks were scarcely observed in some of the fishes just after descending into the lake. Although parr marks completely disappeared in the fish greater than 10cm in standard length after September, even those fish did not show in-tense darkening at outer extremities of dorsal fin. The size and age of biwa salmon migrating downstream were similar to those of chum and pink salmon, and different from those of closely related amago and masu salmon. The process of body silvering in biwa salmon seems to be related to the downstream migratory behavior, since they are known not to acquire seawater adapt-ability as underyearlings

Morphological changes associated with growth and parr-smolt transformation of biwa salmon, reared in pond from underyearling May 1983 to yearling September 1984, were examined by follow-ing relative growth of various body parts, body weight-standard length ratios and changes of condi-tion factor, compared with those of amago salmon reared under the same conditions. Changes of body shapes were observed in association with parr-smolt transformation in biwa salmon; relative growth lines differed between each phase, and also from amago salmon. Parr in amago salmon showed inflections in all the relative growth lines at about 10cm in standard length, and relative growth of body depth and body width changed from negative allometry to isometry or positive allometry. On the other hand, inflection in relative growth line was recognized only in eye dia-meter of biwa salmon smolt. Although similar changes of the body shapes in biwa and amago salmon as in the other salmonids were observed in association with smoltification, such as relative decrease of body depth, body weight and condition facter, degree of these changes was smaller in biwa salmon than in amago salmon, reflecting incomplete smoltification in biwa salmon. Significant differences were recognized in almost all of the relative growth lines between biwa and amago salmon in each phase, most remarkable differences being lower body depth and larger eye diameter in biwa salmon than in amago salmon.

Most of the positional isomers of mono-, di-, tri- and tetranitrobiphenyls were synthesized and assayed for their mutagenicity in Salmonella typhimurium strains TA98, TA98NR and TA98/1,8DNP 6 in the absence of S9 mix. In mono- and dinitrobiphenyls, the structure requirements favoring mutagenic activity are the presence of a nitro group at the 4-position and its absence at the 2-position. TA98 and TA98/1,8DNP 6 were reverted by 2-position-free 4-nitro analogues, but TA98NR was not reverted. The results suggest that direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes. Some of the tri- and tetranitrobiphenyls e.g. 3,4,3′, 3,4,4′-, 3,4,3′,4′- and 3,4,2′,4′-derivatives reverted not only TA98 and TA98/1,8DNP 6 but also TA98NR. Those derivatives commonly have 2 nitro groups at an adjoining position (3,4-dinitro group), whereas 2,4,2′,4′-tetranitrobiphenyl, which has strong potency not only in TA98 and TA98/1,8DNP 6 but also in TA98NR, possesses 2 nitro groups at the 2-position of each benzene ring.

Seasonal changes in hypoosmoregulatory ability of hatchery-reared biwa salmon Oncorhynchus rhodurus and amago salmon O. rhodurus were examined by following changes in plasma Na+ concentration after transfer to 70% seawater. The landlocked biwa salmon lose parr marks and become silvery bodied (smolt) underyearling during early summer. They showed the maximum hypoosmoregulatory ability from underyearling May to July, when they transformed from parr to smolt and migrated to the lake. However, the smolt did not show increased seawater adaptability as compared with parr in underyearling May, and the ability decreased markedly in autumn and winter. There was no essential difference in seawater adaptability between smolt and dark parr or yearling parr. On the other hand, amago salmon, which smoltify and go down stream to the sea in late autumn and early winter, showed better seawater adaptability than biwa salmon throughout all developmental stages examined. Especially, the smolts during the period from November to January showed good osmoregulatory ability. The poor hypoosmoregulatory ability of biwa salmon as compared with amago salmon may be related to their lacustrine nature and a long period passed since this species was landlocked in Lake Biwa.