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Distribution and movement of Caenorhabditis elegans on a thermal gradient

Yohko Yamada^* and Yasumi Ohshima

Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Hakozaki, Fukuoka 812-8581, Japan

View larger version (17K): [in a new window]   Fig. 1. (A) Illustration of the apparatus for the temperature gradient and set-up of an agar plate for behavior assays. (B) Temperature gradients on an aluminum slab and an agar plate. A temperature gradient was produced on an aluminum slab, and temperatures of surfaces of the slab (open circles, broken line) and an agar plate placed on it (crosses, solid line) were monitored at various positions, as described in the Materials and methods section. The means (± S.D.) of temperatures obtained in 19 experiments are shown.

View larger version (39K): [in a new window]   Fig. 2. Distribution and movement of wild-type, fed worms on a temperature gradient. Wild-type N2 adults either (A) maintained in our laboratory or (B) freshly obtained from the Caenorhabditis Genetics Center (CGC) fed at 15°C (open circles), 20°C (filled circles) or 25°C (open triangles) were placed around the center of an agar plate (which corresponds to a temperature of approximately 19°C) and incubated on a temperature gradient, as described in Materials and methods. After 1 h of incubation, distribution of worms along the gradient was determined. Approximately 30 worms were tested in one experiment and the results of several experiments were combined. Total numbers of worms (N) and experiments (in parentheses) were as follows: A: 25°C, N=130 (5); 20°C, N=175 (6); 15°C, N=163 (6); B: 25°C, N=79 (3); 20°C, N=109 (4); 15°C, N=117 (4). (C) N2 worms (CGC line) fed at 20°C were put on a temperature gradient and movement was observed for approximately 10 min. Instantaneous velocities (upper panel) and their elements in directions of the gradient (lower panels) are shown (dots) with their means in each area of the gradient (line). Positive values of velocity elements indicate movement up the gradient, and negative values indicate movement down the gradient. Results of 3743 points are shown. (D) Examples of traces observed in C are shown with starting positions (closed circles). Broken vertical lines show positions on the temperature gradient. (E) Movement of N2 (CGC) worms fed at 15°C was observed for approximately 10 min on a temperature gradient. Means of instantaneous velocities (filled circles) and their elements in directions of the gradient (open circles; positive values indicate upward movement; negative values indicate downwards movement) were obtained from 1141 intervals in total. (F) Examples of traces observed in E are shown with starting points (filled circles) and positions on the temperature gradient (broken lines).

View larger version (21K): [in a new window]   Fig. 3. Distribution and movement of starved animals on a temperature gradient. (A) N2 worms were starved for 6 h (upper and middle panels) or overnight (lower panel) and examined for distribution on the temperature gradient. Upper panel, N2 (lab line) starved at 20°C (filled circles; N=83 worms, 3 experiments) or 15°C (open circles; N=84 worms, 3 experiments); middle panel, N2 (CGC line) starved at 20°C (filled circles; N=48 worms, 2 experiments) or 15°C (open circles; N=58 worms, 2 experiments); lower panel, N2 (CGC line) starved at 20°C (N=77 worms, 3 experiments). (B) N2 worms (CGC line) starved at 20°C for 6 h were put on a temperature gradient and movement was observed for about 10 min. Instantaneous velocities and their elements in gradient directions (dots; positive values indicate upward movement; negative values indicate downwards movement) are plotted with their means in each area of the gradient (lines). Results of 2759 points are shown. (C) Examples of traces observed in B are shown with starting points (filled circles), and positions on the temperature gradient (broken lines).

View larger version (17K): [in a new window]   Fig. 4. Effects of serotonin treatment on starving worms. N2 adults were starved on an agar plate with or without serotonin for 6-7 h at 20°C and were then tested for distribution on a temperature gradient. Blue, without serotonin; black, with 8 mmol l-1 serotonin; red, 8 mmol l-1 serotonin with 5 mmol l-1 serotonin added 1 h before the test. Results of two experiments were combined.

View larger version (21K): [in a new window]   Fig. 5. Behavior of mutant worms on a temperature gradient. Wild-type N2 (CGC) or mutant worms were fed at 20°C and transferred onto a temperature gradient. (A) Population distribution after 1 h or (B) means of instantaneous velocities (filled circles) and their elements in gradient directions (open circles; positive values indicate upward movement; negative values indicate downwards movement) at each position of the gradient observed during 10 min were determined. In A, the following alleles were used, and results of 2-4 experiments were combined: tax-2(ks10), tax-4(p678) (solid line); tax-4(ks11) (broken line); egl-4(ks61), eat-4(ad572) (solid line); eat-4(ky5) (dashed line); ttx-3(ks5) (solid line); ttx-3(tm268) (dashed line); and tax-6(p675) (solid line). In B, the total numbers of analysed intervals were as follows: N2, 3743; tax-2(ks10), 723; tax-4(p678), 2607; eat-4(ad572), 984; ttx-3(ks5), 2337; tax-6(p675), 1724; lin-11(n389);him-5(e1467), 731.

View larger version (23K): [in a new window]   Fig. 6. AFD-killed worms avoid warm temperature. (A) AFD neurons of N2 (CGC) were irradiated during the L1 stage. After worms were grown to adults at 20°C, population distribution on a temperature gradient was examined. Results with 48 AFD-killed worms (combined from four experiments; filled circles) are shown with 51 control N2 animals (simultaneously assayed in three experiments; open circles). (B) AFD neurons of N2 carrying an AFD marker, the gcy-8::gfp fusion gene, were irradiated in the L1 stage and, after the worms had grown to adults, movement on a temperature gradient was observed. Means of instantaneous velocities (filled circles) and their elements in gradient directions (open circles; positive values indicate upward movement; negative values indicate downwards movement) are shown in the left-hand panel (N=1178 intervals). Examples of traces are shown in the right-hand panel.

View larger version (36K): [in a new window]   Fig. 7. Rescue of tax-4 defect by cell-specific expression. The tax-4(p678) worms expressing the tax-4 cDNA::gfp gene under indicated promoters were examined for their behavior. (A) Population distribution was determined with worms carrying the tax-4 expression construct and kin-8::gfp as an injection marker. Control worms were injected solely with kin-8::gfp. Combined results of 2-3 experiments are shown. Total worm numbers analysed and experiment numbers (in parentheses) were as follows: control; 74 (3), 49 (2), 80 (3); tax-4p, 76 (3), 71 (3), 54 (2); nhr-38p, 74 (3), 69 (3), 78 (3); gcy-8p, 56 (2), 45 (2), 39 (2); gpa-3p, 63 (3), 80 (3), 82 (3). (B,C) Movement was examined with worms transformed by a tax-4 cDNA expression construct without an injection marker. Control represents the results of the parent tax-4(p678). (B) Means of instantaneous velocities (open symbols) and their elements in gradient directions (closed symbols; positive values indicate upward movement; negative values indicate downwards movement) are shown. Total numbers of analysed intervals of each line were as follows: no transgene, 1109; tax-4p, 249 and 448; nhr-38p, 439, 494 and 303; gcy-8p, 271 and 754; gpa-3p, 575 and 518. In both distribution and movement analysis, two or three lines were tested for each transformation and are shown with different symbols. (C) Examples of traces.

View larger version (21K): [in a new window]   Fig. 8. Modulation of distribution of ttx-3 mutant worms by growth temperature or starvation. ttx-3(ks5) mutant worms were (A) fed or (B) starved for 6 h at 15°C (open circles), 20°C (filled circles) or 25°C (open triangles) and examined for distribution on a temperature gradient. Combined results of two separate experiments are shown.

View larger version (23K): [in a new window]   Fig. 9. Movement of egl-4 mutant worms. N2 and the egl-4(ks61) worms were placed in an area at about 25°C on the gradient and observed for movement for 20 min. (A) Instantaneous velocities (filled circles) and their elements in gradient directions (open circles; positive values indicate upward movement; negative values indicate downwards movement) observed during the indicated period after the placement were averaged. After 10 min of the placement, N2 worms were excluded from a warm area above 24°C and no data was obtained above 24°C. Numbers of analysed intervals were as follows: N2: 0-5 min, 994; 5-10 min, 480; 10-20 min, 261; egl-4: 0-5 min, 1610; 5-10 min, 1148; 10-20 min, 890. (B) Examples of traces of the egl-4 worms. Time is indicated by the color scale.