A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.

To address the dual needs for improved methods to assess potential health risks associated with chemical exposure in aquatic environments and for new models for in vivo mutagenesis studies, we developed transgenic fish that carry multiple copies of a bacteriophage vector that harbors the cII gene as a mutational target. We adapted a forward mutation assay, originally developed for transgenic rodents, to recover cII mutants efficiently from fish genomic DNA by in vitro packaging. After infecting and plating phage on a hfl؊ bacterial host, cII mutants were detected under selective conditions. We demonstrated that many fundamental features of mutation analyses based on transgenic rodents are shared by transgenic fish. Spontaneous mutant frequencies, ranging from 4.3 ؋ 10 ؊5 in liver, 2.9 ؋ 10 ؊5 in whole fish, to 1.8 ؋ 10 ؊5 in testes, were comparable to ranges in transgenic rodents. Treatment with ethylnitrosourea resulted in concentration-dependent, tissue-specific, and time-dependent mutation inductions consistent with known mechanisms of action. Frequencies of mutants in liver increased insignificantly 5 days after ethylnitrosourea exposure, but increased 3.5-, 5.7-and 6.7-fold above background at 15, 20, and 30 days, respectively. Mutants were induced 5-fold in testes at 5 days, attaining a peak 10-fold induction 15 days after treatment. Spontaneous and induced mutational spectra in the fish were also consistent with those of transgenic rodent models. Our results demonstrate the feasibility of in vivo mutation analyses using transgenic fish and illustrate the potential value of fish as important comparative animal models. medaka ͉ ethylnitrosourea A major challenge to the detection of spontaneous and induced mutations is the difficulty with which mutant genes can be efficiently recovered and accurately identified in vivo. Considering that mutations must be detected at low frequencies (e.g., Ϸ1 spontaneous mutation͞10 5 -10 7 loci), and that sufficient DNA sequence information must be available to distinguish mutant from nonmutant genes, the problem of efficiently detecting and quantifying mutations in whole animals can be formidable. Transgenic animals that carry specific genes for quantitation of spontaneous and induced mutations have been developed to assist in improving in vivo mutation analyses (1). In this approach, a transgenic animal carries a prokaryotic vector that harbors a gene that serves as a mutational target. After mutagen exposure, the vector is separated from the animal's genomic DNA and shuttled into indicator bacteria where mutant and nonmutant genes are readily quantified (2, 3). Transgenic mutation assays offer numerous benefits for in vivo mutation detection not available by using other approaches. Benefits include the ability to screen rapidly statistically meaningful numbers of genetically neutral mutational targets in a variety of tissues and the ability to characterize mutations to aid in disclosing possible mechanisms of mutagen action. † A significant additional attribute is the pot...

Telomerase plays a primary role in the maintenance of telomeres in immortal, germ, and tumor cells in humans but is lacking in most somatic cells and tissues. However, many species, including fish and inbred mice, express telomerase in most cells and tissues. Little is known about the expression of telomerase in aquatic species, although the importance of telomerase for longevity has been suggested. We compared telomerase activity and telomere lengths among a broad range of tissues from aquatic species and found telomerase at significant levels in both long- and short-lived aquatic species, suggesting constitutive telomerase expression has an alternative function. Telomere lengths in these aquatic species were comparable to those observed in normal human tissues and cell strains. Given that a host of aquatic species with short life spans have telomerase and a tremendous capacity to regenerate, we tested the hypothesis that telomerase upregulation is important for tissue regeneration. During regeneration, telomerase activity was upregulated and telomere lengths are maintained with the shortest telomeres being elongated, indicating the importance for maintaining telomere length and integrity during tissue regeneration. Thus, the expression of telomerase in aquatic animals is likely not related to longevity but to their ability to regenerate injured tissue.

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A novel sediment-contact assay using embryos of the transgenic medaka was developed to fully characterize the toxic effects induced by exposure to a mixture of organic pollutants in sediments. Embryos of the lambda transgenic medaka were exposed for 10 days to a clean reference sediment spiked with either the solvent alone, benzo[a]pyrene (B[a]P), or three concentrations (0.3x, 1x, and 2x) of an organic extract (OE) of sediments from the Seine estuary. The 1 x OE-spiked sediment contained concentrations of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls similar to those in field-collected sediment. Exposure to this sediment, but not to the B[a]P-spiked sediment, significantly increased embryo-larval mortality and prevalence of spinal deformities. Mutant frequency at the cII mutation target gene in the liver of 10-week-old medaka was significantly increased following exposure to either B[a]P or the three doses of OE. The predominant OE-induced liver mutations were G:C to T:A transversions, consistent with PAHs being the major contributors to the mutation induction. Liver and gonadal tumors were observed in 35-week-old medaka exposed to either B[a]P (1/25) or to the 1 x OE (1/24). The benefits of medaka as a fish model for toxicological assessment and the benefits of the cII mutation assay for mutation detection combine to provide comprehensive assessment of a wide range of genotoxic and nongenotoxic effects of aquatic pollutants.