Channels constrain locomotor behavior to linear crawling
In this report we study two Drosophila larval locomotor behaviors: linear forward and linear reverse crawling. To reliably observe these behaviors we engineered narrow channels that force larvae to crawl in a straight line (). Compared with crawling on flat, open surfaces wild-type larvae in channels could crawl forward at the same average speeds, with bouts of linear crawling extending for longer durations, and larvae often crawling in reverse (data not shown). Channels can be used for both low-resolution recordings to observe organismal behavior (), and high-resolution recording to observe segmental and muscle movements during behavior (, ). In addition, in channels larvae can be viewed from any orientation—top, bottom or side. In conclusion, building channels was a key advance for our work, because it allows us to study extended bouts of linear crawling at multiple levels of detail.
Crawling is composed of periodic strides
Our ultimate goal is to use Drosophila
larval crawling as a model for studying rhythmic behavior. Thus, we examined the periodicity of the crawling behavior of larvae in channels. Previous studies of maggot (dipterian insect larvae) locomotion, including third-instar Drosophila melanogaster
larvae, showed that forward crawling is composed of repetitive cycles of motion called strides (Berrigan and Pepin, 1995
). We confirmed and extended these findings by showing that both forward and reverse crawls in first instar Drosophila melanogaster
larvae were composed of strides as well (). Specifically, we acquired bright-field movies of larvae crawling through channels, and measured the position of the larval head, tail and centroid in each frame of a movie (). We generated displacement plots to visualize the movement of head, centroid and tail over time, and see periodic cycles of motion in each trace (). To reliably recognize a cycle of motion in these data, we identified local maxima of centroid displacement, and used the interval between local maxima to calculate stride period (). We found that the average stride period in our bright-field recordings ranged from 0.6 to 2.1 s for forward crawls, and from 1.2 to 3.3 s for reverse crawls. In summary, both forward and reverse linear crawling in channels are rhythmic movements.
Crawling speed correlates with stride period rather than stride length
Next we wanted to understand the variability in behavior exhibited by wild-type larvae crawling through channels by measuring organism speed. We measured speed by taking the slope of the regression line describing centroid displacement of a crawl of five strides or more (). All regression models fit the data with an R2 value of at least 0.95 (data not shown). This method was based on the observation that, once initiated, larvae maintained a given speed until the crawl was terminated (data not shown). We find that larvae crawl at a range of speeds (forward 39.5–126.5 µm/s [n=14]; reverse 21.2–88.0 µm/s [n=17]), and, on average, forward crawls were significantly faster than reverse crawls (forward 88.7 ± 28.8 µm/s; reverse 62.3 ± 21.3 µm/s, p < 0.05). We noted that any given larva could crawl at multiple speeds in both forward and reverse directions (data not shown); furthermore an individual larvae always crawled faster traveling forward than traveling in reverse (data not shown). Thus, an individual larva is capable of producing both forward and reverse crawls at a range of speeds.
We wanted to understand the relationship between speed and stride cycle. In organisms such as earthworms, animals increase speed primarily by increasing the length of each stride (Quillin, 1999
). Other soft-bodied animals, such as caterpillars, increase speed primarily by increasing stride frequency (or decreasing stride period) (Casey, 1991
). Thus, we asked, as Drosophila
larvae crawl faster do they take larger strides or take faster strides? First, we analyzed our bright-field population data. We performed regression analysis between average stride period and crawling speed, as well as average stride length and crawling speed. For both forward and reverse crawls, crawling speed correlated much better with average stride period than average stride length (). Next, to control for non-neural factors, for example, staging, hormone level, genotype, or environment, we compared forward crawls produced by individual larvae that crawl at multiple speeds. For a given larva we did pairwise comparisons between every possible combination of crawls to determine the difference in average stride period, average stride length and crawling speed. We did regression analysis to examine the relationship between these three values, and find that 92% of the variation in forward crawling speed could be due to stride period (). Thus, Drosophila
larvae largely regulate crawling speed by regulating stride period as do many limbed organisms.
Regression analysis shows crawling speed is best correlated with stride period
During a stride the centroid and abdominal body wall move asynchronously
Because crawling speed can be understood in terms of strides, we focused the remaining analysis in this paper at the level of a stride cycle. In our bright-field data, we noted that in each stride there were two phases of movement. The centroid moved slowly (almost always) in a direction opposite to the crawl direction; and the centroid moved rapidly in the direction of the crawl. These two phases of movement were also easily detectable in the displacement plots of head and tail movements, and were seen for both forward and reverse crawls (). To understand in more detail how each segment of the Drosophila larval body moved during these two phases of movement we used wg-gal4 to express UAS-mCD8-GFP (wg>GFP) in a single row of cells in each segment of the abdominal and thoracic body wall, as well as the tail (). In addition, mouth hooks in the head could be tracked by following autofluorescence (, ). We placed wg>GFP larvae in channels and recorded crawling behavior. For this fluorescent dataset (wg>GFP), we used the tail movement rather than the centroid movement to determine the bounds of the stride cycle; we considered stride cycle to begin with movement of the tail in the direction of the crawl. We traced frames that represent the larval body at 0%, 25%, 50%, 75% and 100%/0% stride cycle completion (). For each frame we estimated which part of the larval body was in contact with the substrate (, horizontal red line), and calculated the position of the larval centroid (, red plus sign). Surprisingly, we found that that the centroid and abdominal body wall moved asynchronously. As larval centroid moved forward rapidly the abdominal segments remained stationary with respect to the substrate (, 0–25% of stride cycle). In contrast, when each abdominal segment was lifted off of the substrate, moved forward, and replaced back on the substrate in a more anterior position, the centroid remained stationary or moved slightly backward (, 25–0% of stride cycle). Thus, we conclude that a single stride is composed of two phases of movement: a phase of abdominal body wall advance and a phase of centroid advance.
Drosophila larvae use a visceral locomotor piston mechanism to move body mass
A crawling cycle consisting of two phases of movement, centroid advance and abdominal body wall advance has been recently documented in Manduca sexta
caterpillar forward crawling (Simon et al., 2010a
); this mechanism of movement is termed “visceral locomotor pistoning” (Simon et al., 2010a
). So we tested whether Drosophila
larvae also use a visceral piston mechanism for crawling. Specifically, we tested whether the centroid advance phase of the Drosophila
larval crawling stride cycle is a “visceral piston phase.” To be considered a visceral piston phase, we used Manduca
as a reference to lay out two criteria. First, the tail and gut should move together. Second, as the gut and tail move, abdominal segments should be stationary (Simon et al., 2010a
). We examined the movement of the Drosophila
larval gut relative to movement of the tail, and abdominal segment A4 (representing the center of the abdomen), using wg
larvae. In this genotype, autofluorescence allowed us to visualize a portion of the gut found near abdominal segment A4 when the larvae are at rest (). From movie data we constructed kymographs (see figure legend for details) (), and from kymographs we determined the position of each structure (). We found that the Drosophila
larval stride cycle contains a visceral piston phase. The gut and tail moved together, while abdominal segments remained stationary. First, this can be seen by examining the movements of gut, tail, and segment A4, on a displacement plot (gray shading, ). Second, we determined the speed of each structure over time, and calculated Pearson’s correlation coefficient for gut speed versus tail speed, as well as gut speed versus segment A4 speed. We found that on average the tail and gut speed has a strong positive correlation (forward r
= 0.67 ± 0.16[n
= 8], reverse r
= 0.56 ± 0.18 [n
= 3]), but gut and segment A4 speed were negatively correlated (forward r
=−0.39 ± 0.23 [n
= 8], reverse r
= −0.41 ± 0.15 [n
= 3]). Last, we found that gut movement and segment A4 movement were almost perfectly out of phase. This was seen on polar plots (see figure legend for details), which show high gut speeds occurred between 0 and 25% of the stride cycle, whereas high abdominal segment A4 speeds occurred between 50 and 75% of the stride cycle (). In contrast, movement of gut and tail showed a large amount of temporal overlap (). We conclude that the first part of the Drosophila
larval stride cycle—rapid movement of the head, gut, tail and centroid in the direction of the crawl, is a visceral piston phase.
Role of head and tail during the visceral piston phase
Because it is a requisite step in understanding the cellular basis of behavior, we wanted to understand the muscle contractions powering the visceral piston phase.During the visceral piston phase we saw concurrent movement of tail and gut().Thus, we hypothesized that active movements of the tail could provide a source of power for larval translocation. We imaged muscles in the tail using MHC-GFP reporter larvae in which all .labeled (). During visceral piston phase we saw shortening of tail musculature as the tail moved forwards()(n = 3 crawls).Thus, contraction of muscles in the tail could power forward movement during the visceral piston phase.
We also noted that during the visceral piston phase the head moved forward ().This observation is consistent with the idea that the head moved passively forward;alternatively, this observation is consistent with the idea that active contraction of head muscles provide a source of power for larval translocation. The head musculature in our MHC-GFP
reporter line was not labeled distinctly enough to enable direct examination of muscle contraction (data not shown).Thus, to distinguish between these possibilities, we used an indirect measure, the movement of the head and mouth hooks which are controlled by head musculature (Bate, 1993
; Hans-lik et al., 2010
).We saw the mouth hooks move forward, exhibiting a distinct up and down motion before planting into the substrate (n
= 8 crawls), which is consistent with a prior report showing mouth hooks engage with the substrate (Wang et al., 1997
).In addition, the head region elongated in shape as segments A1, and A2 remained in place (, n
= 8 crawls).These observations support the idea that the movement of the head was not merely passive. Our data are consistent with the hypothesis that the forward visceral piston phase is powered by muscle contractions in both the head and tail.
Characterization of the wave phase of the stride cycle
We found that the stride cycle is composed of two phases: the visceral piston phase, described above, and the wave phase in which the abdominal body wall advances. A number of studies on Drosophila
larval forward crawling have documented peristaltic waves that travel from tail to head (Hughes and Thomas, 2007
; Song et al., 2007
; Dixit et al., 2008
; Lahiri et al., 2011
), and we characterize this wave phase in more detail here. We found that during forward crawling, there are sequential movements of abdominal segments. Moreover these movements were initiated in posterior segments and traveled toward the anterior (). These observations are in agreement with previous studies (Hughes and Thomas, 2007
; Song et al., 2007
; Dixit et al., 2008
; Lahiri et al., 2011
). In addition, we found that for reverse crawling, the wave phase was characterized by sequential movements of abdominal segments that travel from head to tail (data not shown). In both forward and reverse crawls the movements that occurred during the wave phase can be termed “direct,” because they traveled along the body axis in the same direction as the crawl (Elder, 1980
). Thus, consistent with previous work on Drosophila
larval behavior, we found that direct, sequential movements of external body segments occur during the wave phase of larval crawling.
Because we are interested in determining the neural basis of Drosophila
crawling we wanted to understand the intersegmental phase lag, which describes the phase relationship of segmental movements as a percentage of the stride cycle. Much experimental and theoretical work has gone into understanding the neural mechanisms contributing to intersegmental phase lag in preparations such as crayfish swimmerete beating (Friesen and Cang, 2001
).Using our wg
dataset, we measured the absolute latency between the onset of movement in segment A3 and onset of movement in segment A6, and divided by the 3, the number of segments traversed.This value gave us an approximation of the intersegmental travel time (ISTT) for segmental movements for each crawl. We found that ISTT varied linearly with stride duration (data not shown). Next, we normalized ISTT to stride period to determine the intersegmental phase lag. For forward crawls (n
= 20) we found an average intersegmental phase lag of 12 ±0.2% of the stride cycle/segment (). For reverse crawls (n
= 33) we found an average intersegmental phase lag of 10 ± 0.3% of the stride cycle/segment (). Notably, by examining displacement plots of our wg
data, we found that for both forward and reverse crawls up to three segments could be moving in the direction of the crawl at any time point (;data not shown).In conclusion, we have determined parameters of the wave phase that will be useful in further study of the neural basis of crawl behavior.
In many rhythmic behaviors such as leech and lamprey swimming, intersegmental phase lags are constant over different cycle periods; it is thought that this helps maintain the body form (Skinner and Mulloney, 1998
). It has been postulated that for terrestrial forms of locomotion such as leech crawling, that it is less important to maintain a specific body form to move efficiently (Cacciatore et al., 2000
). Thus, we wanted to determine whether the intersegmental phase lag was constant over a range of stride periods in Drosophila
larvae. We plotted intersegmental phase lag for both forward and reverse crawling against stride period, and found it did not vary over a range of stride periods (). An additional observation that supports the idea that Drosophila
larvae maintain intersegmental phase relationships over differing stride periods comes from examine composite polar plots (). To generate these plots, speed of each segment was normalized to stride period, and three crawls performed at different stride periods were overlaid. The composite data well recapitulates data from a single crawl; for example, on composite plots up to three segments can be moving at any point in the crawl cycle. Together these data suggest that for both forward and reverse crawling Drosophila
larvae maintain their body form even as they crawl at different speeds.
Visceral piston phase and wave phases can be uncoupled in embryo
During forward larval crawling both the visceral piston and wave phase began as the tail moved forward (, ). This observation caused us to ask whether these two phases were causally linked. We decided to look for evidence of decoupling between wave phase and visceral piston phase in embryos. If we could see such uncoupling, this would suggest that the visceral piston phases may not be causally linked, but rather distinct movements.
We looked at late stage embryos just before hatching, because previous studies have shown fully mature waves of segmental contraction occur by this stage (Suster and Bate, 2002
; Pereanu et al., 2007
; Crisp et al., 2008
), but to our knowledge embryonic visceral piston movements not have been reported. One of the advantages of our wg
assay used to monitor segmental movements is that this assay allows for a quantitative comparison between segmental movements in newly hatched larval and segmental movements in the embryo ) To ensure that we were examining healthy embryos, we recorded embryos until they hatched, and sampled segmental movements that occurred within 15 min of initiation of hatching.
First, we asked whether we could find an embryonic wave phase, equivalent to larval wave phase. Many studies have documented wave-like movements in the embryo (Suster and Bate, 2002
; Pereanu et al., 2007
; Crisp et al., 2008
). Consistent with previous work, in wg
embryos we two found patterns of movement that were similar to forward and reverse wave phases observed in larvae. In one pattern, we found each abdominal and thoracic body wall segment moved toward the head; these movements were initiated in posterior segments, and moved sequentially with up to three segments moving at one time (). This pattern was similar to the pattern of segmental movement seen in forward larval wave phases, and we term it an embryonic forward wave phase. The other pattern was similar to the pattern seen in reverse larval wave phases (), and we term it an embryonic reverse wave phase. To further compare the embryonic wave phases and larval wave phases, we measured how long it took for a single segment to travel toward the head during each type of wave phase; we saw the ranges overlap with a wider range seen in the embryo (). Thus, we found patterns of movement in the embryo similar to larval wave phases.
Next, we asked whether equivalents of larval visceral piston phases were present in embryos.We observed patterns of segmental movement in the embryo that bear similarity to larval visceral piston phase. First, both larval and embryonic visceral piston phases showed a movement of the viscera relative to the abdominal body wall (data not shown). Notably, we did not expect abdominal segments to be stationary during an embryonic visceral piston phase because embryos are encased in a series of membranes and cannot make contact with the substrate. Second, as in larvae, we observed two types of embryonic visceral piston phases, those that simultaneously moved segments toward the tail and those that simultaneously moved segments toward the head (). Third, embryonic visceral piston phases had significantly shorter duration than the duration of embryonic wave phases ().Thus, we find likely embryonic equivalents to both forward and reverse larval visceral piston phases.
Finally, we wanted to determine whether the embryonic visceral piston phase could be uncoupled from embryonic wave phase.We scored the sequence of embryonic wave phases and embryonic visceral piston phases that occurred in the 15 min before embryo hatching ().We found clear examples where embryonic wave phases and embryonic visceral piston phases did not occur within five seconds of other segmental movements (15/30 “forward”embryonic wave phases, 7/20 forward embryo visceral piston phases, 6/11 “reverse” embryonic wave phases, 1/6 reverse embryo visceral piston phases) (). For example, two wave phases without an intervening visceral piston phase, or a piston phase with out an associated wave phase (>5 s apart).In conclusion we found evidence to suggest that visceral piston phases could be considered distinct from the wave phase.
Intrasegmental phase relationship of dorsal and ventral muscles
A relatively well accepted idea of how insect larvae, such as caterpillars, use muscles to crawl was proposed first in 1937 by Barth (Barth, 1937
; Wigglesworth, 1950
; Champman, 1998
). In insects, muscles fall into the three groups: dorsal muscles (D), ventral muscles (V) and lateral muscles (L) (Snodgrass, 1935
); these three muscle groups are found in Drosophila
larvae (Bate, 1993
). Barth’s model suggests that within a segment, D muscles contract before L muscles and L muscles contract before V muscles. Thus a defining feature of Barth’s model is that D muscles and V muscles within a segment contract asynchronously. This motor pattern was thought to help lift each segment off the substrate and then replace it. Recently, however, a study in Manduca sexta
caterpillars has challenged Barth’s model (Simon et al., 2010b
). As Manduca sexta
caterpillars crawl forward, D and V muscles contract simultaneously (Simon et al., 2010b
). Thus, we wanted to understand the relationship of D muscle contractions versus V muscle contractions in Drosophila
larvae. In Drosophila
larvae, do D and V muscles contract simultaneously, as seen during Manduca
crawling, or with a phase shift as suggested by Barth?
MHC-GFPlarvae that express GFP in all somatic muscles were imaged as they crawled forward or in reverse through channels (). We selected three muscles for detailed comparison: from the D group—DO1 (Dorsal Oblique 1 [muscle 9], dorsal-most), from the L group—LT1 (Lateral Transverse 1 [muscle 21] posterior-most), and from the V group, VL1 (Ventral Longitudinal 1 [muscle 6]; ). First, we analyzed coordination of D and V muscle contractions during forward crawling. We examined movies of MHC-GFP larvae crawling, frame-by-frame. We noted that within a given segment D and V muscles contracted simultaneously (). Next, we plotted VL1 and DO1 muscle length over time (), and we found that D and V muscles contracted in synchrony (n = 7 crawls, 4 segments each). For each segment examined there was no significant difference in timing of muscle contraction or muscle relaxation (data not shown). In addition, we measured the average latency between peak V and D contractions. To compute V-D latency we took the difference between peak contraction time of VL1 and peak contraction time of DO1 for three stride cycles on three consecutive segments. We found a V-D latency of 0.05±0.07 s. (),and this latency did not vary with stride period (). In summary, we find that a feature of the muscle contraction pattern for forward crawling in Drosophila larvae is that D and V muscles within the same segment contract simultaneously.
Next we asked whether during reverse crawling in Drosophila larvae D and V muscles also contracted simultaneously. We found that VL1 muscles contracted slightly, but significantly, before the DO1 muscles of the same segment (). We found an average latency V-D latency of 0.35 ± 0.08 s (), and this latency was constant over a range of stride periods. In summary, we find the phase-shifted relationship between V and D muscle contractions during reverse crawling in Drosophila larvae, which is slightly different from that seen for forward crawling.
Intrasegmental phase relationship of ventral and lateral muscle groups
Having defined the phase relationships of D and V muscle contractions during forward and reverse larval crawling in Drosophila larvae, we next we looked at the lateral (L) muscle contractions. We found that V/D muscles contract before L muscles of the same segment (). We measured V-L latency by determining the time between peak VL1 contraction and peak LT1 contraction, and found and average of 0.85 ± 0.11 s (). Similarly, we measured D–L latency by determining the time between peak DO1 and peak LT1 muscle contractions, and found an average of 0.87 ± 0.02 s (). These data are consistent with the finding that D and V muscle groups contract simultaneously. We find that V-L latency varied with stride period: with longer stride period we found longer V–L latencies (). In summary, we find that in Drosophila larvae during forward crawls the muscles contraction pattern has three distinguishing features: D and V muscles contract simultaneously; D/V muscle contract before L muscles of the same segment; V-L latency changes with changing stride period.
We next assayed reverse crawls. Here too we found the L muscles contract after V and D muscles within the same segment. We found a V-L latency of 0.51 ± 0.18 s and a D–L latency of 0.21 ± 0.1 s (). The V-L latency was significantly longer than the D–L latency (t test, p < 0.05), consistent with the idea that V and D muscle contractions are phase shifted relative to each other. In contrast to forward crawls (), during reverse crawls the V–L latency did not vary with changing stride period. In summary, we observe the pattern of muscle contractions within a given segment during reverse crawls in Drosophila larvae to have two distinct features. First, the sequence of muscle contraction was V muscles, followed by D muscles, and finally contraction of L muscles. Second, the latency between contractions of different muscle groups did not depend on stride period.
Intersegmental phase relationship between ventral and lateral muscle groups
Crawling in soft-bodied organisms such as leech and earthworm is driven by waves of muscle contractions in functionally antagonistic groups of muscles— circumferential and longitudinal muscles. Contraction waves pass from head to tail sequentially; as the circumferential wave passes through a segment causing elongation, the longitudinal wave is passing through a more anterior segment causing shortening (Gray and Lissmann, 1938
; Baader and Kristan, 1995
). Thus, for Drosophila
larval crawling we wanted to define the intersegmental relationship between functionally antagonistic muscle groups. We chose to examine contraction of the V muscle, VL1, which is oriented along the larval anteroposterior axis, and contraction of the L muscle, LT1, which is oriented along the dorsoventral axis (). We found that during forward crawling, V muscles contract at nearly the same time as L muscles in the adjacent posterior segment (n
= 7 crawls, 4 segments). For example VL1 in segment A4 contracted as LT1 in segment A5 contracted (). To quantify this relationship we calculated the V-L(+1) latency by determining the time difference between peak contraction of VL1 in segment n
, and peak contraction of LT1 in segment n
+1, and found an average latency 0.04 ±0.06 s. (). This latency did not vary with stride period (data not shown). In summary, a defining feature of forward crawling in Drosophila
larvae is that V muscles and L muscles in adjacent posterior segments contract in phase, nearly simultaneously.
We analyzed the relationship between contractions of V and L muscle groups during reverse crawls. We saw V muscles contract at nearly the same time as L muscles in the adjacent anterior segment (n = 6 crawls, 4 segments).For example, VL1 in segment A4 contracted while LT1 in segment A3 contracted (). In addition, we calculated the V-L( −1) latency by determining the time difference between peak contraction of VL1 in segment n and LT1 in segment n-1. We found the average V-L( −1) latency to 0.04 ± 0.08 s (), and this did not vary with stride periods (data not shown). In conclusion, a defining feature of reverse crawling in Drosophila larvae is that the contractions of V muscles and L muscles in adjacent anterior segments are coordinated to temporally coincide.