Kinematic responses to changes in walking orientation and gravitational load in Drosophila melanogaster

Abstract

Walking behavior is context-dependent, resulting from the integration of internal and external influences by specialized motor and pre-motor centers. Neuronal programs must be sufficiently flexible to the locomotive challenges inherent in different environments. Although insect studies have contributed substantially to the identification of the components and rules that determine locomotion, we still lack an understanding of how multi-jointed walking insects respond to changes in walking orientation and direction and strength of the gravitational force. In order to answer these questions we measured with high temporal and spatial resolution the kinematic properties of untethered Drosophila during inverted and vertical walking. In addition, we also examined the kinematic responses to increases in gravitational load. We find that animals are capable of shifting their step, spatial and inter-leg parameters in order to cope with more challenging walking conditions. For example, flies walking in an inverted orientation decreased the duration of their swing phase leading to increased contact with the substrate and, as a result, greater stability. We also find that when flies carry additional weight, thereby increasing their gravitational load, some changes in step parameters vary over time, providing evidence for adaptation. However, above a threshold that is between 1 and 2 times their body weight flies display locomotion parameters that suggest they are no longer capable of walking in a coordinated manner. Finally, we find that functional chordotonal organs are required for flies to cope with additional weight, as animals deficient in these proprioceptors display increased sensitivity to load bearing as well as other locomotive defects.

Figure 1. Imaging setup and step parameters.

(A). Schematic of the recording setup. The fTIR apparatus was oriented in order to allow the flies to walk freely inside a walking chamber either horizontally (upright or inverted) or vertically (ascending or descending). A high-speed camera recorded body and tarsal contacts on the optical glass, see for details. Fly schematic adapted from . (B–G) Boxplots represent the median as the middle line, with the lower and upper edges of the boxes representing the 25% and 75% quartiles, respectively; the whiskers represent the range of the full data set, excluding outliers. Circles indicate outliers. Grey represents upright controls (n = 71, from [39]), green for inverted (n = 28), purple for ascending (n = 28) and brown for descending (n = 23). For C–G data was residual normalized and expressed as the difference to the upright control. Statistical analysis with one-way ANOVA followed by Tukey’s post hoc test (for normal distributions in panels B, C and E) or Dunn’s post hoc test (for non-normal distribution in panels D, F and G), ^* P<0.05; ^** P<0.01; ^*** P<0.001. Statistically significant increases or decreases are indicated in red and blue, respectively. (B) Non-upright animals display significantly reduced walking speed. (C) Swing speed was significantly increased in inverted walking animals. (D) All non-upright animals display increased step length. (E) Swing duration is increased only in ascending and descending animals while inverted animals show a small decrease. (F) Stance duration remains unchanged for all conditions. (G) Duty factor strongly increases in inverted walking animals while it is minimally reduced in ascending animals.

Figure 2. Spatial parameters.

Non-upright walking animals display more aligned footprints and less jitter during stance phases, while clustering remains unchanged. (A–D) Boxplots represent the median as the middle line, with the lower and upper edges of the boxes representing the 25% and 75% quartiles, respectively; the whiskers represent the range of the full data set, excluding outliers. Circles indicate outliers. Statistical analysis with one-way ANOVA followed by Tukey’s post hoc test, ^* P<0.05; ^** P<0.01; ^*** P<0.001. Statistically significant decreases are indicated in blue. In B, C and D data was residual normalized and expressed as the difference to the upright control. (A) Ascending animals show more aligned footprints. Data were grouped into slow (<20 mm/s) and medium (between 20 and 34 mm/s) speeds. (B). Non-upright animals display lower values for stance linearity indicating lower jitter during stance phases. (C–D) Footprint clustering remains unchained for Anterior Extreme Position (AEP) and Posterior Extreme Position (AEP). (b.u., body units).

Figure 3. Interleg coordination parameters and summary of kinematic effects in non-upright walking animals.

(A–D, F–G) Boxplots represent the median as the middle line, with the lower and upper edges of the boxes representing the 25% and 75% quartiles, respectively; the whiskers represent the range of the full data set, excluding outliers. Circles indicate outliers. Data was residual normalized and expressed as the difference to the upright control. Statistical analysis with one-way ANOVA followed by Tukey’s post hoc test, ^* P<0.05; ^** P<0.01; ^*** P<0.001. Statistically significant increases or decreases are indicated in red and blue, respectively. (A) Inverted walking animals display a significant decrease in tripod configurations while descending animals showed an increase. (B) Only ascending animals displayed a slight increase in tetrapod configurations. (C) Inverted walking animals display a significant increase in the use of wave conformations while ascending animals showed a slight decrease. (D) No variation was observed in the number of non-canonical combinations for all experimental groups. (E) Step patterns and tripod/transition phases. (E) and (E’) display two representative videos of animals walking upright and inverted at similar speeds, respectively. In the upper section, for each leg, swing phases are represented in black (from top to bottom: right hind (RH); right middle (RM); right front (RF); left hind (LH); left middle (LM); left front (LF)). Vertical dashed green lines represent the boundaries of a tripod stance phase. Lower section represents the periods associated with tripod and transition (or inter-Tripod time) phases depicted in green and grey, respectively. (F) Non-upright walking animals display a significant increase in inter-Tripod time. (G) Animals walking in a vertical plane display an increase in the average duration of each tripod stance phase. (H) Summary of kinematic affects under non-upright conditions. For simplicity, only the step cycle of the right foreleg is represented. Dashed green line represents the swing phase from PEP to AEP. Waved red line represents stance traces. Solid triangle represents the tripod conformation formed by RF, LM and RH. Dashed triangle represents the immediately subsequent tripod conformation. Purple dashed line represent the inter-Tripod time between the two tripod conformations. Brown and blue arrows represent the qualitative variations compared to upright walking animals observed for inverted and ascending/descending, respectively.

Figure 4. Imaging setup and step parameters.

(A) Recording setup. Flies were allowed to walk freely inside a walking chamber on an optical glass with a metal ball bearing attached to the notum. A high-speed camera recorded body and tarsal contacts on the optical glass. (A’) Representative image of a fly carrying a metal ball bearing. (B–F) Box plots represent the median as the middle line, with the lower and upper edges of the boxes representing the 25% and 75% quartiles, respectively; the whiskers represent the range of the full data set, excluding outliers. Circles indicate outliers. Statistical significance was determined using 2-way-ANOVA with post-hoc t-tests, where ^* p<0.05; ^** p<0.01; ^*** p<0.001. Statistically significant increases or decreases are indicated in red and blue, respectively. (B) Average Speed. Flies bearing 0.66× and 1.14× show a recovery in average speed by one week, while flies bearing 2.02× do not. (C) Swing speed. The immediate effect of weight is an increase in swing speed, although it decreased to that of controls for flies bearing 0.66× and 1.14× by 1 week. (D) Step length. Step length shows weight-dependence only, where intermediate weights increases step length at 2 hours and the heaviest weight decreases step length at 1 week. (E) Swing duration. Swing duration is significantly decreased for flies bearing 2.02×. (F) Stance duration. Stance duration is increased only for the heaviest weight.

Figure 5. Spatial parameters.

(A–C) Footprint positions relative to the body center. AEP and PEP values for each leg are represented on the left and right sections of the plot, respectively. Values are normalized for body size. Line size denotes standard deviations, while intersection indicates mean value. Statistical significance was determined using 2-way-ANOVA with Tukey’s post-hoc tests and post-hoc t-tests, where ^* p<0.05; ^** p<0.01; ^*** p<0.001. Statistically significant increases are indicated in red. (D–E) AEP and PEP Clustering. Box plots represent the median as the middle line, with the lower and upper edges of the boxes representing the 25% and 75% quartiles, respectively; the whiskers represent the range of the full data set, excluding outliers. Circles indicate outliers. Values show a significant footprint dispersal in 2.02× bearing flies compared to controls.

Figure 6. Stance linearity, interleg coordination parameters and summary of kinematic effects under different load conditions.

(A–D) Box plots represent the median as the middle line, with the lower and upper edges of the boxes representing the 25% and 75% quartiles, respectively; the whiskers represent the range of the full data set, excluding outliers. Circles indicate outliers. Statistical significance was determined using 2-way-ANOVA with post-hoc t-tests, where ^* p<0.05; ^** p<0.01; ^*** p<0.001. Statistically significant increases or decreases are indicated in red and blue, respectively. (A) Stance Linearity Index. Flies bearing intermediate weights show less wobble during stance phases compared to controls at 2 hours, while flies bearing the heaviest loads display more wobble at all time points. (B) Tripod Index. Flies bearing 2.02× weights show significantly reduced tripod index at all three time points. (C) Non-canonical Index is markedly increased for flies bearing 2.02× weights. (D) Full Stance Index is increased for 2.02× bearing flies at all time points. (E) Summary of kinematic affects due to load bearing. For simplicity, only the step cycle of the right foreleg is represented. Dashed green line represents the swing phase from PEP to AEP. Waved red line represents stance traces. Solid triangle represents the tripod conformation formed by RF, LM and RH. Dashed triangle represents the immediately subsequent tripod conformation. Purple dashed line represents the transition between the two tripod conformations. Blue points represent the qualitative effects transiently observed for animals carrying 1.14× or lighter weights, while brown points represent the effects observed for walking animals carrying 2.02× weights.

Figure 7. Effects of chordotonal organ deprivation while weight bearing.

Box plots represent the median as the middle line, with the lower and upper edges of the boxes representing the 25% and 75% quartiles, respectively; the whiskers represent the range of the full data set, excluding outliers. Circles indicate outliers. Statistical significance was determined using 3-way-ANOVA with post-hoc t-tests, where ^* p<0.05; ^** p<0.01; ^*** p<0.001. Statistically significant increases or decreases are indicated in red and blue, respectively. (A) Stance Duration. Only weighted nan^36a flies show increased stance duration, and this effect occurs independently of time. (B) Step period only increases significantly for weighted nan^36a flies in a time-independent manner. (C) Stance linearity index. Animals carrying intermediate weights display a less wobbly stance phase 2 hours after weight bearing, this effect is absent in nan^36a flies. (D) Tripod index. Sensory deprived nan^36a flies display a significant reduction in the use of tripod configurations during weight bearing. (E) Non-canonical index. nan^36a flies weight bearing show an increase in non-canonical configurations in a time-independent way. (F) Inter-Tripod time. Weight bearing nan^36a flies display an increased transition time between tripod configurations. This effect is only visible after 2 hours of weight bearing.