We believe that Nothobranchius rachovvi, a killifish, is a better vertebrate model for longevity genetics because their maximum lifespan is about ten months (Herrera and Jagadeeswaran, 2004). Nothobranchius rachovvi are tele-ostei like zebrafish and have already been used in aging studies. They have been shown to begin senescent changes at approximately four months of age. They are relatively easy to breed and are approximately 2 inches in size. The females lay approximately 20 eggs per day. Nothobranchius rachovvi can breed even at an old age until the very end of their lives. This occurs because the natural habitat of these fish dries out seasonally. In order to preserve their species, these annual fish developed schemes to lay eggs up to the time of death. In the laboratory, eggs are collected in peat moss and then stored in a dry container for four months. The eggs hatch when placed in water, and they grow at 28-30°C. The larvae mature by the age of 3-4 weeks when males develop colors and are easy to distinguish from females. Since they belong to the cyprinodontiformes form of fish, the information on the zebrafish and fugu fish gene sequences should be useful in obtaining polymorphic EST markers for Nothobranchius. There are about 40 species of Nothobranchius, and most Nothobranchius species have a diploid number of chromosomes ranging from 2N = 16 to 28. Recently, another group claimed that Nothobranchius furzeri has the shortest lifespan of 2 months under circulating water conditions, which is similar to what we observed for Nothobranchius rachovvi under these same conditions. We do not know the normal lifespan of Nothobranchius furzeri under stagnant water conditions. Furthermore, different strains of Nothobranchius rachovvi are available, whereas for furzeri we do not know whether multiple strains are available. A prerequisite for genetic mapping is the availability of strains. Thus, Nothobranchius rachovvi is an ideal vertebrate organism that has a short lifespan if we could adapt the mutagenesis methods that are well established in zebrafish and medaka. These protocols for mutagenesis are standard and should be easily adaptable for the annual fish. In addition, several genetic markers for Nothobranchius have already been isolated.
From our published work, Nothobranchius would be a good vertebrate model for studying the genetics of longevity due to its shorter lifespan. However, the survivorship curves as presented appear to be less rectangular than the one we obtained for zebrafish (Austad, 2004). Furthermore, in the work of Walford on Cynolebias, the curves are rectangular (Liu and Walford, 1969). The difference between their work and our work is the difference in temperature at which the fish were maintained. Thus, optimization of survivorship curves is required using different temperatures. In addition, another possible cause for a nonrectangular curve is the existence of pathogens. We believe the alternation of one or two degrees in temperature maintenance of Nothobranchius as well as a clean water system will make a significant impact on the survivorship curves. Before using this fish, conditions for optimal growth and maintenance should be established. Since these fish have been used in commercial sales and the conditions for growth and maintenance are available from vendors, they should be helpful. For example, water quality, temperature, presence of good amounts of denitrifying bacteria, elimination of bacterial contamination, and appropriate monitoring of pH and ammonia are some of the conditions which might provide us with the desired rectangular shape of the survivorship curves. Since bubbles generated in the water circulation system may be causing the early deaths, the efficiency could be improved by using tanks that are connected at the bottom, whose water is recirculated slowly.
It is well established that heterosis or hybrid vigor exists in organisms. Thus, the effects of heterosis on longevity using progeny derived from the crossing of two strains should be determined. Studies on several parameters such as the body weight, growth, their physical activity measurable by analyzing images of locomotion captured by a video monitor system, the gill movements per minute, and lateral line responses observable by movements in response to a pendulum should help in determining any differences among the strains and their progeny. Additional important information on differences between survivorship curves among the parental strains and their hybrid progeny is also needed. A compilation of these curves will assist us in forming an appropriate baseline for the survivorship curves. Any noticeable increase in longevity among these hybrids would help determine an accurate time point for the selection of longevity mutants. In Nothobranchius, embryos must be kept dormant for four to twelve months before they can be hatched. The effect of dormancy over the lifespan needs to be investigated by keeping the eggs under dry conditions for different periods of time. We still do not know whether there is a decline in reproductive activity with age, so measurement of the egg-laying activity of Nothobranchius as a function of age is important. Due to the fact that fish embryos can be diploidized from haploid eggs, we can increase the speed of generating homozygous mutant fish (one generation is needed rather than two) for recessive mutations. The principle of generating the gynogenetic diploids is described above. This should be easily adaptable for this annual fish. The haploid eggs of females of the F1 progeny are fertilized in vitro with UV irradiated sperm and subjected to high pressure to rapidly generate diploid F2 progeny. The embryos can then be kept under dry conditions as described above. The lifespan of the F2 progeny can be analyzed for their similarities to normal survivorship curves.
This information would provide us with two facts: first, the diploidization is feasible; and second, the gynogenetic diploids behave as wild-type normal fish as far as their survivorship curves are concerned. Important information that is also needed is the histology to look for any observable pathology. Since zebrafish histology and other fish histology and pathology are available, again it should be relatively easy to extend this information for the annual fish.
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