Worm Breeder's Gazette 15(4): 25 (October 1, 1998)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Biological Timing Lab, Institute of Cell, Animal and Population Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JT. E-mail: firstname.lastname@example.org
Defaecation in C. elegans is regulated in a periodic fashion. A previous study had suggested that this behaviour is controlled by a temperature-compensated ultradian clock (1) but we have recently shown that this is not the case (2,3). The period of this ultradian rhythm is highly dependent on several exogenous and endogenous factors. Age is one such factor and the choice of animal is therefore critical. A general problem is the high variability of the period both within and between animals which requires a large number of animals / cycles to be recorded in order to obtain reliable data. Because of the potential importance of this rhythm as a model oscillator, I have searched for an improved approach to the monitoring of this rhythm.
To avoid the complication of the effect of age on period, the defaecation rhythm in the four larval stages was investigated. The L4 stage was found to have, by far, the most regular rhythm. Whereas the coefficient of variation (CV) for the recordings of 30 young adults (for 15 cycles) was 8.5 % (3), that for 30 L4 larvae under identical conditions was only 4.9 %. The rhythm is also much more regular for a given animal. Whereas the CVs for the adults ranged between 2.7 and 22.8 % with a mean of 7.7 % (3), those for the L4 were between 1.5 and 6.4 % (mean 4.2 %). Although the rhythm in L4 is still not the output of an ultradian clock, the rhythm in many larvae showed a clock-like regularity.
During these studies, a further advantage of using L4 became apparent. On rich food, the larvae show very little locomotion. The protocol for recording was therefore altered as follows: NRO plates (4) were inoculated with 1-2 µl of bacterial solution and incubated overnight at 37°C (the size of the inoculum should be optimised such that the entire bacterial lawn can be seen in one field at the magnification chosen). Bacteria were killed by UV irradiation in order to ensure that the lawn did not increase. With this arrangement, more than 90 % of larvae remained within view during an 1 hour observation period. With L4 larvae the neutral red staining (4) is particularly effective and the defaecation of the animals can be observed under low magnification. As a result, the rhythm can now be recorded on video without any attendance.
The study of L4 offers dramatic advantages: i) precautionary measures to avoid effects of age are not necessary, ii) recording no longer requires attendance, iii) the increased regularity of the rhythm allows the total number of cycles to be reduced considerably. Whereas the recording of 30 adults over 15 cycles each does not provide completely satisfactory results, this can now be achieved with 15-20 L4 over 10 cycles each. This should prove particularly useful in future genetic analyses of oscillator mechanisms. Mutants with altered periods can be identified easily as has already been confirmed using strainds such as clk-1, gro-1 and lin-42.
(1) Liu DWC, Thomas JH (1995) J. Neurosci. 14:1953-1962. (2) Kowal S et al. (1998) European Worm Meeting, abstract 59. (3) Kowal S et al., submitted for publication. (4) Kowal S, Kippert F (1998) WBG 15(3):14.