Genetic dissection of behavior: modulation of locomotion by light in the Drosophila melanogaster larva requires genetically distinct visual system functions

Abstract

The Drosophila larva modulates its pattern of locomotion when exposed to light. Modulation of locomotion can be measured as a reduction in the distance traveled and by a sharp change of direction when the light is turned on. When the light is turned off this change of direction, albeit significantly smaller than when the light is turned on, is still significantly larger than in the absence of light transition. Mutations that disrupt adult phototransduction disrupt a subset of these responses. In larvae carrying these mutations the magnitude of change of direction when the light is turned on is reduced to levels indistinguishable from that recorded when the light is turned off, but it is still significantly higher than in the absence of any light transition. Similar results were obtained when these responses were measured in strains where the larval photoreceptor neurons were ablated by mutations in the glass (gl) gene or by the targeted expression of the cell death gene head involution defective (hid). A mutation in the homeobox gene sine oculis (so) that ablates the larval visual system, or the targeted expression of the reaper (rpr) cell death gene, abolishes all responses to light detected as a change of direction. We propose the existence of an extraocular light perception that does not use the same phototransduction cascade as the adult photoreceptors. Our results indicate that this novel visual function depends on the blue-absorbing rhodopsin Rh1 and is specified by the so gene.

Fig. 1.

Larval behavior during the ON/OFF assay.A, Videotape of a single CS larva tested in the ON/OFF assay was used to generate frame-by-frame photographs depicting 16 consecutive seconds. To the right of each panel is a schematic diagram of the larva representing the relative position of the head (arrowhead) and body (line). The first three frames (seconds00.08–00.10) show a larva immediately before a lightsOFF to ON transition. Lights are turned ON in the eleventh second, and head swinging is observed (00.12–00.14) followed by a change in direction (00.15–00.18). The final three framesshow a larva during lights OFF immediately after the lights ON to OFF transition (00.20–00.21). B, Line drawing of larval path shown in A. The solid lines represent the larval path during a portion of the dark pulse (00.08–00.10 and 00.21–00.23). The broken line represents larval path during the light pulse (00.11–00.20). The larval outlinedepicts the larval head swinging that occurs soon after the lights are turned on. During this time (00.12–00.15) the larva is stationary. This behavior is followed by a sharp change in the direction of the larval path.

Fig. 2.

Response in the ON/OFF assay of wild type and larvae with mutations in genes involved in phototransduction. A response index (R.I.) was derived per larva, and a genotype average was calculated. The RIs for the strains are significantly different (ANOVA F_(1,181) = 16.90, p < 0.001). Post hocanalysis of paired mean comparisons reveals no differences between the wild-type strains (OR, n = 30;CS, n = 30) and ninaE[ninaE¹⁷ (n = 20);ninaE⁸ (n = 20)], but a significant reduction in the larval response to light of thenorpA [norpA^P24(n = 30); norpA^P12(n = 20)] and ninaC[ninaC⁵ (n = 20);ninaC² (n = 19)] mutants.

Fig. 3.

Head swinging behavior of wild-type strains and larvae with mutations in genes involved in phototransduction during the ON/OFF assay. Head swings, defined as an abrupt movement of the anterior portion of the larva away from original path choice, were counted in light (stippled bar) and dark (gray bar) pulses on a per larva basis, and an average for each genotype was derived. There is a significant increase in head swinging by wild-type larvae (CS,n = 30; OR, n = 30) during light pulses, relative to that during dark (ANOVA:CS, F_(1,58) = 15.69,p < 0.001; OR,F_(1,58) = 20.51, p < 0.001). This difference is abolished in the phototransduction mutants norpA^P24 (n = 30), norpA^P12 (n = 20), and ninaC⁵ (n = 20) but not in theninaC² (n = 18),ninaE¹⁷ (n = 20), and ninaE⁸ (n = 20) mutants (ANOVA:norpA^P24,F_(1,58) = 0.09, NS;norpA^P12,F_(1,38) = 2.58, NS;ninaC⁵,F_(1,38) = 0.05, NS;ninaC²,F_(1,34) = 11.53, p < 0.001; ninaE¹⁷,F_(1,38) = 30.82, p < 0.001; ninaE⁸,F_(1,38) = 29.81, p < 0.001).

Fig. 4.

Change of direction in wild-type strains during the ON/OFF assay. Change of direction (in degrees) was measured at the dark to light (stippled bar), light to dark (gray bar), and in the absence of light transitions (solid bar). OR larvae display a significant difference between each of the light conditions (n = 30, F_(1,87) = 33.89, p < 0.001). CS larvae display a significant difference between the dark to light and light to dark transitions only (n = 30,F_(1,87) = 42.49, p < 0.001).

Fig. 5.

Change of direction in strains with mutations in genes involved in adult phototransduction during the ON/OFF assay. Change of direction (in degrees) was measured at the dark to light (stippled bar), light to dark (gray bar), and in the absence of light transitions (solid bar). norpA^P24 (n = 30, F_(2,87) = 10.12, p < 0.001),norpA^P12 (n = 20,F_(2,57) = 6.21, p < 0.005), and ninaC⁵ (n= 20, F_(2,57) = 5.17, p< 0.006) mutant larvae exhibit changes of direction at the dark to light and light to dark transitions that are not different from each other but are different from change of direction in the absence of light. ninaC² mutant larvae (n = 20, F_(2,57) = 5.64,p < 0.008) exhibit a significant difference at the dark to light and light to dark transition changes that in turn is not significantly different from that measured in the absence of light transition. ninaE¹⁷ mutant larvae also exhibit a significant difference between dark to light and light to dark, but the difference between the light to dark and absence of light transitions has been abolished (n = 20,F_(2,57) = 8.93, p < 0.001). The same is true of larvae that are heterozygous [ninaE¹⁷/ninaE⁸(n = 20, F_(2,57) = 12.2,p < 0.001)]. ninaE⁸(n = 20, F_(2,57) = 12.21, p < 0.001) larvae display a significant difference between each of the transitions.

Fig. 6.

RI in the ON/OFF assay of larvae with mutations in the so and gl genes. The RIs for the strains are significantly different (F_(7,178) = 15.55, p < 0.001). Post hoc analysis of paired means reveals no difference between the wild-type strains (OR,n = 30; CS, n = 30) and the pGMR-rpr (n = 20) andgl⁺ (n = 16). A significant reduction is observed in the larval response to light of the so^mda (n = 20),gl^60j (n = 20),gl¹ (n = 20), andpGMR-hid (n = 30) mutants.

Fig. 7.

Head swinging behavior in the ON/OFF assay of larvae with mutations in the so and glgenes. The increase in head swinging behavior seen during the light pulses (stippled bar) over that seen during the dark pulses (gray bar) is abolished in theso^mda mutant (n = 20,F_(1,38) = 0.76, p > 0.05) as well as in the gl mutantsgl^60j (n = 20,F_(1,38) = 0.03, p > 0.05) and gl¹ (n = 20, F_(1,38) = 0.03, p > 0.05) and in the pGMR-hid strain (n= 30, F_(1,58) = 0.03, p> 0.05), which lacks larval photoreceptor cells. A light pulse elicits differential head swinging behavior in pGMR-rpr(n = 20, F_(1,38) = 15.33, p < 0.001), which exhibits a less severe adult phenotype than pGMR-hid, and ingl⁺ (n = 16,F_(1,30) = 9.44, p < 0.005), which is the gl rescue line.

Fig. 8.

Change of direction in the ON/OFF assay of larvae with mutations in the so and gl genes. Change of direction (in degrees) was measured at the dark to light (stippled bar), light to dark (gray bar), and in the absence of light transitions (solid bar). Light has a significant effect on path direction in each of the strains tested, with the exception of pGMR-rpr(n = 20, F_(1,57) = 0.98,p > 0.05) and so^mda(n = 20, F_(1,57) = 1.79,p > 0.05), in which the presence or absence of light had no effect. The gl mutant strainsgl^60j (n = 20,F_(1,57) = 4.42, p < 0.02) and gl¹ (n = 20, F_(1,57) = 6.23, p < 0.005) and pGMR-hid (n = 30,F_(1,87) = 4.57, p < 0.01) show no difference between degree of direction change at the light transitions. However, change of direction in the absence of light transitions is significantly lower than either of the test conditions. The gl⁺ strain displays a degree of direction change in the dark to light transition that is significantly higher than the other test conditions (n = 16,F_(2,45) = 16.23, p < 0.001).