Positive geotactic behaviors induced by geomagnetic field in Drosophila

Abstract

Background: Appropriate vertical movement is critical for the survival of flying animals. Although negative geotaxis (moving away from Earth) driven by gravity has been extensively studied, much less is understood concerning a static regulatory mechanism for inducing positive geotaxis (moving toward Earth).

Results: Using Drosophila melanogaster as a model organism, we showed that geomagnetic field (GMF) induces positive geotaxis and antagonizes negative gravitaxis. Remarkably, GMF acts as a sensory cue for an appetite-driven associative learning behavior through the GMF-induced positive geotaxis. This GMF-induced positive geotaxis requires the three geotaxis genes, such as cry, pyx and pdf, and the corresponding neurons residing in Johnston's organ of the fly's antennae.

Conclusions: These findings provide a novel concept with the neurogenetic basis on the regulation of vertical movement by GMF in the flying animals.

Keywords: Cryptochrome; Drosophila melanogaster; Geomagnetic field; Gravity; Johnston’s organ; Negative geotaxis; Positive geotaxis; Vertical movement.

Fig. 1

Near-zero GMF potentiates the negative geotactic behaviors in flies. a Schematic drawing of the rectangular Helmholtz coil system used to regulate intensity or direction of three GMF vectors by active cancellation. b Left: Photo of the test cube used for the tube-positioning assay. Right: Imaginary drawing of geotactic positioning by the flies under the sham and shield (−) condition in the assay. The geotactic positioning score was calculated at the end point of the test using the following equation: (number of flies at the S2–S5 sections of the test tube equally divided into five imaginary sections/total number of flies) × 100 % (details in Methods). S, section; Sham, ambient GMF; Shield, near-zero GMF. Scale bar: 2 cm. c Negative geotactic positioning of fly strains in the shield (−) condition (n = 10 trials). Note the significance in all the strains. Error bars: SEM. **, P < 0.01; ***, P < 0.005; ****, P < 0.001 by Student’s t-test. d Time-course measurements of the positioning score in Canton-S flies under the sham and the shield (−) condition. ***, P < 0.005; ****, P < 0.001 by Student’s t-test. e Photo of the six-exit Y-maze used in the assay. For each experiment, 25 ± 2 flies were allowed to enter the maze through the entrance (details in Methods). f Exit profiles of the vertical choice Y-maze assay under the sham and shield (−) conditions (n = 12 trials). Note the significantly higher scores at the upper exits (1 and 3) under the shield condition compared to the sham condition. Error bars: SEM. *, P < 0.05 by Student’s t-test. g Schematic drawing of the cubic arena used for the free-flight assay (details in Methods). The geotactic flying score was calculated as the percentage of flies that flew to the upper section of the front plane. h Geotactic flying scores for the sham and shield (−) in the free-flight assay. Error bars: SEM. *, P < 0.05 by Student’s t-test (n = 15 trials)

Fig. 2

Modulated GMF with increased intensity induces positive geotaxis. a Left: Comparisons of the geotactic positioning under the GMF conditions with modulated intensities. A positive geotaxis was induced in conditions b and c. Error bars: SEM. n.s.: not significant. ***, P < 0.005; ****, P < 0.001 by ANOVA Tukey’s test. (n = 10 trials). Right: A representative image of geotactic positioning under the sham and b, respectively. b, c Comparisons of the geotactic positioning of the wild-type flies between light (500 lx) versus dark (0 lx) conditions under the near-zero GMF condition and the GMF condition b in Fig. 2a, respectively. Note the increase of positioning score of the sham samples under the dark condition. −; near-zero GMF, +; GMF condition b. Error bars: SEM. n.s.: not significant. ***, P < 0.005; ****, P < 0.001 by Student’s t-test. d Schematic drawing of the associative learning assay using food as an unconditioned stimulus and the non-geotactic GMF as a conditioned stimulus. The GMF denotes the non-geotactic GMF a. Note that the test tubes were inverted during the rest and the GMF was provided from the bottom side of the tubes. The homogeneous space for the GMF is marked as dashed rectangles. e Comparisons of the geotactic positioning induced by the control, trained, and test conditions in the presence of the ambient GMF condition (Sham) or the GMF stimulus, or either one associated with food. GMF, the non-geotactic GMF condition a in Fig. 2a. Food, the rearing diet. Error bars: SEM. n.s.: not significant. ****, P < 0.001; *****, P < 0.0001; ***, P < 0.005 compared to the sample of the middle group, associated with the GMF alone and then tested under the same GMF, by ANOVA or Student’s t-test (n = 10 trials)

Fig. 3

CRY, PDF, and Pyx pathways are required for GMF-modulated geotaxis. a, e, i Comparisons of the geotactic positioning of wild-type and the CRY-, PDF- and Pyx-deficient flies, respectively, under the negative geotactic GMF condition. Error bars: SEM. n.s.: not significant. **, P < 0.01; ****, P < 0.001 by Student’s t-test. b, f, j Comparisons of the geotactic positioning of the wild-type and null mutant flies for cry, pdf, and pyx, respectively, under the positive geotactic GMF condition. Error bars: SEM. n.s.: not significant. **, P < 0.01 by Student’s t-test. c, g, k Comparisons of the geotactic positioning of the flies mutant for cry, pdf, and pyx, and the flies in which these genes were genetically restored in the mutant background, respectively. Error bars: SEM. n.s.: not significant. **, P < 0.01; ***, P < 0.005 by Student’s t-test. d, h, l Comparisons of the geotactic positioning of the wild-type flies, control flies (GAL4 transgene alone, UAS-RNAi alone), and flies with knockdown of cry, pdf, and pyx transcripts using the gene-specific GAL4 driver, respectively. Error bars: SEM. n.s.: not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001 by Student’s t-test. For all the data, n = 10 trials

Fig. 4

cry-, pdf- and pyx-GAL4 expressing neurons are necessary for the GMF-induced geotactic positioning. a Schematic representation of JO in the second antennal segment. b, c Expression of GFP in cry-GAL4/UAS-mCD8::GFP and pdf-GAL4/UAS-mCD8::GFP, respectively. Scale bar: 10 μm. d The geotactic positioning of JO-injured flies. The second antennal segments of flies were pinched with fine forceps under CO₂ anesthesia 24 h before the tube-positioning assay. Anesthetized flies without JO injury were controls. Error bars: SEM. n.s.: not significant. **, P < 0.01; ***, P < 0.005 by ANOVA (n = 10 trials). e, f, g The geotactic positioning of the flies with targeted inhibition of the neurons expressing CRY, PDF, and Pyx, respectively, by expressing TNT under the positive geotactic GMF condition. Control flies expressed impTNT. Error bars: SEM. n.s.: not significant. **, P < 0.01; ***, P < 0.005; ****, P < 0.001 by Student’s t-test. For all the data, n = 10 trials

Fig. 5

CRY and Pyx function in Johnston’s organ for GMF-modulated positive geotaxis. a, c, e Comparisons of the geotactic positioning of wild-type flies, the fly mutants for cry, pyx and pdf, and the mutant flies in which these genes were genetically restored using nan-GAL4 driver, a JO-specific GAL4 driver, respectively. Error bars: SEM. n.s.: not significant. **, P < 0.01; ***, P < 0.005; ****, P < 0.001 by Student’s t-test. b, d, f Comparisons of the geotactic positioning of wild-type flies, the fly mutants for cry, pyx and pdf, and the mutant flies in which these genes were genetically restored using iav-GAL4 driver, another JO-specific GAL4 driver, respectively. Error bars: SEM. n.s.: not significant. **, P < 0.01; ***, P < 0.005 by Student’s t-test. g, h Comparisons of the geotactic positioning of wild-type flies, control flies (GAL4 driver alone, UAS-pdf RNAi alone), and the flies with RNAi knockdown of pdf using cry-GAL4 or pyx-GAL4 driver, respectively. Error bars: SEM. n.s.: not significant. **, P < 0.01; ***, P < 0.005; ****, P < 0.001 by Student’s t-test. For all the data, n = 10 trials