Preliminary investigation of the circadian rhythms of wild-collected Neurospora strains.
Louis W. Morgan and Jerry F. Feldman - Department of Biology, University of California, Santa Cruz, Santa Cruz, CA 95064.
A medium that allows for measurement of circadian rhythms in wild-collected strains of Neurospora is reported. Preliminary results with N. intermedia strains from four different latitudes suggest natural variation in clock-affecting loci.
Gall and Lysek used 60 mM RbCl in their medium, with fructose as a carbon source, which caused ~50% reduction in growth rate and affected viability of the csp-2 strain at temperatures differing significantly much from 20C. In order to reduce the amount of rubidium chloride required to induce circadian banding of conidiation, we used an acetate-based medium. This acetate/casamino acids medium causes thinner bands of conidiation and a slower linear growth rate (~60%) as compared to standard glucose-arginine medium (Sargent and Kaltenborn 1972 Plant Physiol. 50: 171-175).
Race tube medium containing 1.2% sodium acetate, 0.5% casamino acids, and 2% agar (Feldman and Hoyle 1973 Genetics 75:605-613) was tested at 5 mM, 15 mM, and 25 mM RbCl concentrations with four wild-collected N. intermedia strains and an N. crassa strain (band). The race tube assay was performed at 25C as described in Feldman and Hoyle. Standard (12 inch) race tubes allowed 6 to 7 days of growth after transfer to darkness. The results from the 15 mM RbCl medium, which gave the clearest banding, are presented in Table 1.
The linear growth rate of the band strain was reduced by only about 25% at 25C by the addition of 15 mM rubidium chloride to the medium. In this assay, three of the wild N. intermedia strains had a circadian rhythm similar to the N. crassa wild-type (band) strain with a period length of about 22 hours. However, the P2264 strain collected in New Zealand had a long period length. This long period length may reflect naturally occurring allelic variation at a clock-affecting locus, since there are several known alleles of N. crassa clock genes which lengthen period length. These results show that the circadian rhythm of wild-collected Neurospora strains can be easily seen without the need to introduce any mutations.
These results extend possibilities for looking into natural variability in circadian behavior, within and between populations and species, in different ecological settings, and at different latitudes. For example, Pittendrigh and Takamura (1989 J. Biol. Rhythms 4: 217-235) showed a latitudinal cline in the properties of the Drosophila clock using wild-collected strains. However, our experiments with wild-type strains were exploratory and are peripheral to our current research. We do not expect to have an opportunity to do further work using the rubidium medium, but we have been encouraged by Dr. David Perkins (who generously gave us the wild-collected strains) to report these preliminary results. We thank Dr. Perkins and Ken Langdon (both of Stanford University) for their enthusiasm and assistance with this study.
Table 1. Period lengths and linear growth rates of Neurospora strains on acetate/casamino acids medium containing 15 mM RbCl at 25C.
| Strain | n | Period length (hours) | Growth rate (mm/day) |
| band (no RbCl) | 6 | 22.0 ± 0.5 | 24.3 ± 1.8 |
| band | 5 | 21.4 ± 0.7 | 17.7 ± 1.9 |
| P9 (Unzen, Japan) | 5 | 22.6 ± 0.3 | 33.2 ± 0.5 |
| P277 (Singapore) | 3 | 22.7 ± 1.1 | 34.2 ± 3.6 |
| P146 (Bogor, Java) | 6 | 22.6 ± 0.6 | 33.4 ± 1.9 |
| P2264 (Ahipara, N.Z.) | 3 | 25.7 ± 0.8 | 34.0 ± 0.9 |