Production of juvenile masu salmon (Oncorhynchus masou) from spermatogonia-derived sperm and oogonia-derived eggs via intraperitoneal transplantation of immature germ cells.

No effective cryopreservation technique exists for fish eggs and embryos; thus, the cryopreservation of germ cells (spermatogonia or oogonia) and subsequent generation of eggs and sperm would be an alternative solution for the long-term preservation of piscine genetic resources. Nevertheless, in our previous study using rainbow trout, we showed that recipients transplanted with XY spermatogonia or XX oogonia produced unnatural sex-biased F1 offspring. To overcome these obstacles, we transplanted immature germ cells (XX oogonia or XY spermatogonia; frozen for 33 days) into the body cavities of triploid hatchlings, and the transplanted germ cells possessed a high capacity for differentiating into eggs and sperm in the ovaries and testes of recipients. Approximately 30% of triploid recipients receiving frozen germ cells generated normal salmon that displayed the donor-derived black body color phenotype, although all triploid salmon not receiving transplants were functionally sterile. Furthermore, F1 offspring obtained from insemination of the oogonia-derived eggs and spermatogonia-derived sperm show a normal sex ratio of 1:1 (female:male). Thus, this method presented a critical technique for practical conservation projects for other teleost fish species and masu salmon.

Proteomic analysis of inviable salmonid hybrids between female masu salmon Oncorhynchus masou masou and male rainbow trout Oncorhynchus mykiss during early embryogenesis.

Early embryos of inviable hybrids between female masu salmon Oncorhynchus masou masou and male rainbow trout Oncorhynchus mykiss at 9, 12, 15 and 20 days after fertilization were examined for protein expression profiles. A total of 44 proteins, mostly down-regulated products of house-keeping genes and those involved in nucleic acid metabolism or chromatin replication, were identified in hybrid embryos by mass spectrometry analysis and protein database searching. The identified down-regulated proteins may be responsible for the inviability in the hybrids.

Modeling predicts that redd trampling by cattle may contribute to population declines of native trout.

Unrestricted livestock grazing can degrade aquatic ecosystems, and its effects on native vertebrate species are generally mediated by changes to physical habitat. Recently, high estimated rates of cattle trampling on artificial redds within federal grazing allotments in southwestern Montana, USA, has raised concern that direct mortality from trampling may contribute to imperilment of native westslope cutthroat trout (Oncorhynchus clarkii lewisi). To explore the implications of cattle trampling, we built two mathematical models. First we used a temperature-driven model of egg-to-fry mortality representative of the developmental stages during which embryos would be vulnerable to trampling. Cattle trampling was an additional source of mortality (beyond natural mortality), and we modeled egg-to-fry mortality across a range of trampling rates (25-125% per month) for scenarios assuming low (0.60), moderate (0.81), and high (0.95) natural mortality. We then used a matrix model to determine how trampling affected population growth (lambda), assuming initially stable (lambda = 1.008) or slow-growing populations (lambda = 1.025 and 1.05). Cattle trampling concentrated over a few days when the embryos were most sensitive caused greater egg-to-fry mortality than when the same amount of trampling occurred over one month. Trampling caused a large increase in egg-to-fry mortality when that natural mortality was low, but the overall population-level effect was far less than might have been anticipated from the rate of trampling itself. Nonetheless, small reductions in population growth rate could be biologically significant for populations with little or no demographic resilience, and trampling rates as low as 25% could lead to negative population growth. The rapid reduction in resilience with increased trampling rates (>50%) means that even growing populations are less likely to recover from periodic fluctuations. The overall risk posed by trampling will depend on whether cutthroat trout populations face concurrent threats that have already reduced their abundance and resilience. Biologists can potentially use the egg-to-fry model and thermograph data to identify dates when limiting cattle presence in or near stream habitats would likely reduce mortality from trampling. Evaluation of grazing policies on federal lands may be needed to ensure that species conservation and land use concerns are equitably balanced.

Biotechnology: surrogate broodstock produces salmonids.

A worldwide decline in the number of wild salmonids calls for strategies to restore endangered populations. Here we show that germ cells can be transplanted between two different salmonid species, with the subsequent production of xenogenic, donor-derived offspring. This pioneering xenotransplantation technology may eventually find applications in facilitating the production of commercially valuable fish, as well as in species conservation.

Expression of a cysteine-rich protein (CRP) encoding gene during early development of the trout.

Members of the cysteine-rich protein (CRP) define a subclass of LIM-only proteins implicated mainly in muscle differentiation. Until now, very little is known concerning the expression of CRP encoding genes during vertebrate development. We describe here the isolation of a trout (Oncorhynchus mykiss) gene encoding a cysteine-rich protein (TCRP) and the pattern of its mRNA accumulation during embryogenesis, focusing on somitogenesis. TCRP encodes a putative protein with two LIM domains linked to a short glycine-rich region that displays 86%, 76%, 67% identity with chicken CRP2, CRP1 and MLP/CRP3 proteins, respectively. Whole-mount in situ hybridisation showed that TCRP transcript is first detected just before somitogenesis in the paraxial mesoderm, while it is absent in the axial structures. During somitogenesis, the expression of TCRP was observed caudally in the elongating presomitic mesoderm and in the last formed somites. The labelling for TCRP was found to fade as the somites mature. At the end of the somitogenesis, TCRP transcripts accumulation was restricted to pronephros and bronchial arches.

Vertebrate floor plate transiently expresses a compound recognized by antisera raised against subcommissural organ secretion.

Located along the ventral midline of the neural tube, the floor plate (FP) performs an essential role in central nervous system development, especially in the patterning of the ventral region of the neural tube and axonal guidance. Several studies have been directed to the identification of molecules mediating some of the functions of the FP. Most of the models proposed for floor plate actions involve contact-mediated- and/or gradients of diffusible-signals acting throughout the nervous tissue. This report presents and discusses findings showing that the FP cells secrete a novel compound, which is recognized by antisera raised against secretory products of the subcommissural organ (SCO). This immunoreactive compound appears to be very similar to one of the glycoproteins secreted by the SCO. This immunoreactivity is expressed transiently during central nervous system development, and its rostro-caudal extension along the anterior-posterior axis of the FP displays some species variations. However, a constant feature in all species investigated is that this immunoreactive compound is highly expressed in the FP located in the mesencephalic-metencephalic boundary. The distribution of this compound is compatible with basal and apical pathways of release from FP cells. The former might participate in the formation of some brain commissures. The latter might involve the use of the cerebrospinal fluid as a route for performing actions on distant targets, a pathway somehow disregarded by most models accounting for morphogen actions.

Developmental toxicity of selenium to Dolly Varden char (Salvelinus malma).

Gametes were collected from Dolly Varden char (Salvelinus malma) from waterbodies in a region exposed to mining-related selenium (Se) releases in British Columbia, Canada. Fertilized eggs were incubated in a laboratory and deformities were assessed on newly-hatched alevins using a graduated severity index. No effects were observed on egg or alevin survival or larval weight across the studied exposure range of 5.4 to 66 mg/kg dry weight in egg. Length of some larvae was reduced at the highest egg Se concentrations and a clear residue-response relationship was observed for larval deformity. The egg concentration corresponding to a 10% increase in the frequency of deformity (EC10) was 54 mg/kg dry weight, which is substantially higher than reported for other cold-water fish species.

Production of trout offspring from triploid salmon parents.

Many salmonids have become at risk of extinction. For teleosts whose eggs cannot be cryopreserved, developing techniques other than egg cryopreservation to save genetic resources is imperative. In this study, spermatogonia from rainbow trout were intraperitoneally transplanted into newly hatched sterile triploid masu salmon. Transplanted trout spermatogonia underwent spermatogenesis and oogenesis in male and female recipients, respectively. At 2 years after transplantation, triploid salmon recipients only produced trout sperm and eggs. With use of these salmon as parents, we successfully produced only donor-derived trout offspring. Thus, by transplanting cryopreserved spermatogonia into sterile xenogeneic recipients, we can generate individuals of a threatened species.

Synchronous gene expressions during embryogenesis of Oncorhynchus masou (Yamame).

In order to detect periods of synchronous gene expressions during embryogenesis of Oncorhynchus masou (Yamame), we analyzed the peptide constituents of the embryos by means of two-dimensional electrophoresis. New kinds of peptides were mainly detected in three periods, i.e. between the 12th and 13th day, between the 20th and 21st day, and between the 30th and 31st day after fertilization. Hatching occurred between the 34th and 37th day. These indicate that these three periods, which corespond to (1) the shifting stage from maternal to nuclear events in gene expression, (2) the development of external organs such as fins, and (3) the preparative stage for hatching, respectively, may be critical stages in gene expression.

The effect of bioaccumulated selenium on mortalities and deformities in the eggs, larvae, and fry of a wild population of cutthroat trout (Oncorhynchus clarki lewisi).

To determine if elevated concentrations of waterborne selenium (Se), caused by coal mining, in the Elk River in southeastern British Columbia, may be causing reproductive or teratogenic effects in wild cutthroat trout (Oncorhynchus clarki lewisi), fertilized eggs from exposed and reference fish were raised in the laboratory. Eggs from each female were reared separately and the percent mortalities and deformities were related to the selenium content of the eggs. Selenium concentrations in females from the exposed site were highest in the liver (36.6 +/- 22.5 microg/g dry weight, range: 18.3 to 114), followed by the eggs (21.0 +/- 18.3 microg/g, range: 8.7 to 81.3) and the muscle (12.5 +/- 7.7 microg/g, range: 6.7 to 41). Despite these elevated egg Se concentrations, there was no significant effect on fertilization; time to hatch; percent hatch; or egg, larvae, and fry deformities or mortalities. Reproductive failure and embryonic terata have been reported at much lower egg Se concentrations in other fish species. The lack of any toxic response in this study may be due to an evolved tolerance to higher tissue Se concentrations in a population of fish living in a seleniferous river system.

Proteome analysis of Oncorhynchus species during embryogenesis.

To understand the molecular mechanisms underlying normal and abnormal development of two salmonids, masu salmon (Oncorhynchus masou) and rainbow trout (O. mykiss), we used two-dimensional (2-D) electrophoresis to construct a series of 2-D maps during the embryonic period. We identified all visible protein spots on the 2-D map by assigning numbers for masu salmon and rainbow trout, and we determined N-terminal sequences of proteins for one hundred of the spots, that appear at very high concentrations in the whole embryos of masu salmon and rainbow trout. We also characterized embryonic stages according to the periods of appearance of spots. Most of the N-terminal sequences were identical or at least highly similar to partial sequences reported for vitellogenin (Vtg) of O. mykiss. A potential proteolytic processing of Vtg for rainbow trout is discussed in relation to the time of appearance and relative position of Vtg fragments within the complete protein sequence.