Worm Breeder's Gazette 15(5): 24 (February 1, 1999)
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.
Division of Molecular Biology Netherlands Cancer Institute. Plesmanlaan 121, 1066CX Amsterdam. The Netherlands.
Previously (European Worm Meeting, 1998), we have reported the use of a novel chemotaxis assay–based on the use of a four-quadrant Petri dish–in screens for soluble compound chemotaxis mutants. These screens have identified many spontaneous and EMS-induced mutations. The strategy in these screens has been to place worms at the intersection of four quadrants: two opposite quadrants are filled with agar that contains salt; the other quadrants contain no salt. Spacers between quadrants act as barriers to diffusion and thus the geometry of the assay produces very large, steep concentration gradients between adjacent quadrants. Thus, animals encountering a choice point are presented with a clear indication of gradient orientation. We have been using ammonium acetate as the attractant in these screens since it produces the most robust chemotaxis response that we have been able to characterise (a chemotaxis index in excess of 0.99 is not uncommon). One potential limitation of these screens is that the chemotaxis assay, when used as described above, is so robust that only very severe chemotaxis phenotypes are detected. Since our primary interest is in signal transduction, we are attempting to modify the assay such that more subtle phenotypes may be identified. We have been pursuing two ways to modify this assay.
First, we have been reducing the concentration of attractant dissolved in one pair of quadrants to a point where orientation within the gradient becomes more challenging, reflected as a decrease in the population chemotaxis index. This has allowed us to try to determine a perceptual threshold for a given attractant. To do this, the concentration of salt in the attractant-containing quadrants was reduced to a point where a population of worms failed to demonstrate a preference for that salt. A second approach has been to place the same attractant in both pairs of opposite quadrants at different concentrations and reduce the magnitude of the difference between the concentrations such that again worms fail to discriminate. Using this latter approach, we were able to demonstrate that concentration differences as low as 0.1 mM (i.e. 1mM NaCl vs. 1.1 mM NaCl) could be readily discriminated. Furthermore, across at least two orders of magnitude, the apportioning of populations of worms correlated with the ratio of attractant concentrations, rather than the absolute difference in attractant concentration. For example, a choice of 1.1 mM salt vs. 1 mM salt produced the same chemotaxis index as did a choice between 11 mM and 10 mM, or 55 mM and 50 mM salt. All of these choices reflect a 1.1 to 1 ratio; a 1.2 to 1 ratio produces a larger chemotaxis index that is not dependent on the absolute concentration of the salt. These studies are ongoing, but the trend suggests that worms obey a form of the matching law (Herrnstein, 1961) which hypothesises that animals apportion their responding between multiple behaviours according to the ratio of rewards presented by those behaviours.
The quadrant assay allows us to track the apportioning of populations of worms over attractants in the time domain. Any attractant in two opposite quadrants will typically produce a robust initial attraction that declines and asymptotes over the course of an hour or more. However, when worms are given a choice between two distinct attractants, characteristic kinetics of apportioning are revealed. For example, when Na+Cl- and Na+CH3COO- are used at equinormal concentrations, worms show a moderate preference for Cl- that lasts less than 10 minutes. After this time, the population shows a strong and long-lasting preference for acetate. Chemotaxis profiles have been obtained for several pairs of related and unrelated compounds. We have initiated a clonal screen using one of these characteristic profiles as a phenotype. We will identify lines in which the timecourse or preference for one class or the other of soluble attractant has changed. At the same time we will be able to bias the screen against detecting lines that are strongly chemotaxis defective; such lines can be easily identified as they will not show any preference for either attractant at any timepoint.