The biomechanical and neural control of hydrostatic limb movements in Manduca sexta
Department of Biology, Dana Laboratory, Tufts University, Medford, MA 02155, USA
* Author for correspondence (e-mail: barry.trimmer{at}tufts.edu)
Accepted 14 June 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: caterpillar, proleg adduction, muscle, nerve, planta, retractor muscle, motor inhibition, Manduca sexta, tobacco, hornworm
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Studies of hydrostatic locomotion have concentrated on swimming, burrowing
(Trueman, 1975) and telescopic
crawling in legless species such as worms (Quillin,
1998
,
1999
). Others have examined
the movements of myostatic tissues such as elephant trunks, vertebrate tongues
and octopus arms (Gutfreund et al.,
1998
; Matzner et al.,
2000
; Wilson et al.,
1991
). There is also considerable interest in the hydrostatic
components of spider leg joints (Sensenig
and Shultz, 2003
). However, legged crawling by terrestrial
soft-bodied animals such as caterpillars is particularly interesting and has
been the subject of both kinematic and energetic studies
(Barth, 1937
; Brackenbury,
1996
,
1997
,
1999
;
Casey, 1991
). These animals can
climb in a complex three-dimensional environment and exhibit remarkable static
and dynamic stability by virtue of their "swinging, discrete,
big-footed" gait (Yim,
1994
). Because of this stability, crawling insects do not need
widely spaced articulated legs and have a small frontal area to body size
ratio. Caterpillars, in particular, maintain a tight grip on the substrate
using cuticular hooks (crochets) at the tip (planta) of the abdominal prolegs.
In the tobacco hornworm, Manduca sexta, this grip is released at the
start of the proleg swing phase through the activation of a `retractor' muscle
attached to the planta (Belanger et al.,
2000
).
What is not known is how the prolegs re-extend and grip the substrate again. There are no specific extensor muscles, so the leg must extend passively by cuticle elasticity or hydrostatic pressure. The control must be local since proleg extension is not always accompanied by segment shortening, nor does segment compression always lead to proleg extension. The following studies examine the extension and adduction movements of the proleg in detail and show that biomechanical and neural mechanisms work in close coordination to correctly grasp the substrate. A noteworthy finding is that motor inhibition is a key element for active grasping rather than proleg eversion through an increase in hydrostatic pressure.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sensory-evoked adduction
Although the proleg withdrawal response is most easily evoked in isolated
abdomens (Weeks and Jacobs,
1987), the proleg extension and adduction reflex is more reliable
in intact larvae (S.M. and B.A.T., unpublished observations). Animals were
anesthetized by chilling on ice for at least 30 min and then restrained by
attaching their dorsal surface to a flat or slightly convex surface with
VetbondTM adhesive (3M Corp., St Paul, MN, USA). At least 30 min after
recovery from anesthesia, the different groups of sensory hairs (plant hairs,
PH; medial hairs, MH; and ventromedial hairs, VMH) were manually stimulated by
brief (1 s) deflection with a cactus spine or pin. Responses were videotaped
using an S-VHS-resolution camcorder (Canon ES-400) from both a lateral and
ventral view. The recorded behavioral responses were scored for at least five
animals. In some preparations, the MHs or VMHs were removed at their socket
using a razor blade while the larva was anesthetized. Animals were then
allowed to recover for 1 h before behavioral testing.
Kinematics
Proleg movements were tracked in three dimensions using a custom-built 3-D
motion capture system. Larvae were suspended dorsal surface uppermost attached
to a convex surface so that the prolegs were visible. Fluorescent polymer
microspheres of different sizes (22, 48 or 169 µm; Duke Scientific, Palo
Alto, CA, USA) were placed onto the cuticle at the attachment points of
specific muscles and at other landmarks on the proleg. The movements of these
points were recorded under ultraviolet illumination (Model B, 100 W, long
wavelength; Blak-Ray, Upland, CA, USA) using two digital camcorders (Canon
ZR10) fitted with Hoya green (X1) filters (Edmund Optics Inc., Barrington, NJ,
USA). The cameras were mounted on positioners placed at the same height,
angled approximately 90° to one another and 45° to the longitudinal
plane of the larva. The recordings on each camera were synchronized using a
green light-emitting diode (LED) in the field of view, manually activated to
flash (20 ms) at the start of an event. The two video recordings were
transferred to a Windows-based Pentium III PC through an IEE1394 (`Firewire')
interface. The positions of the microspheres were mapped using APAS software
(Advanced Performance Analysis System; Ariel Dynamics, Inc., San Diego, CA,
USA) with semi-automatic point tracking. Three-dimensional reconstructions
were calculated using a direct linear transform calibrated for each
preparation from at least 18 non-coplanar points. The maximum time resolution
was 16.7 ms (NTSC video field rate) but, for very slow movements, the points
were digitized every 5-10 video fields.
Nerve recordings from isolated nerve cord/proleg preparations
An incision was made along the dorsal cuticle of the anesthetized larvae
and the gut was removed. The larvae were pinned dorsal side up to Sylgard
plates and bathed in cold Miyazaki saline
(Trimmer and Weeks, 1989). The
lateral branch of the ventral nerve (VNL) in A4 was dissected out
towards the proleg retractor muscles and cut distally. The remaining ventral
nerves of ganglion A4 were kept intact. The abdominal portion of the nerve
cord, along with the proleg in A4, was then removed and placed into a Sylgard
recording dish. A suction electrode was placed onto the cut end of
VNL to record spontaneous and evoked activity. Similarly, en
passant recordings were made from branches of the left A4 dorsal nerve
(DN). In some preparations, two suction electrodes were used to record from
pairs of dorsal nerves simultaneously.
In some experiments, active motoneurons were identified by severing connectives. In these preparations, only ganglia A4, A5 and A6 were isolated together with the proleg in A5. Recordings were made from DN in A5 before and after the A4-A5 connective was cut. All signals were amplified with cut-off filters of 10 Hz and 10 kHz (model 1700; A-M Systems Inc., Carlsborg, WA, USA).
Muscle recordings in reduced (`flatterpillar') preparations
After removing the gut, the larvae were pinned out in saline with the nerve
cord and muscles exposed dorsally. To gain access to MHs on the left-side
proleg, the muscles, cuticle and right-side proleg were removed from one
right-side body segment. The remaining ganglia, nerves and muscles were
intact. A suction electrode was used to record excitatory junction potentials
(EJPs) from muscles innervated by the posterior branch of the dorsal nerve
(DNP). These recordings were digitized (EGAA software; RC
Electronics, Santa Barbara, CA, USA) and viewed in Sigma Plot (SPSS Inc.,
Chicago, IL, USA). Periods of MH stimulation were recorded with a manually
activated event marker. EJPs were counted by threshold detection using
DataView software (W. Heitler, University of St Andrews, Scotland, UK).
Muscle and nerve ablation
Ventral muscles were severed in chilled, anesthetized animals using
micro-dissecting scissors on one side of the A4 segment (N=4), both
the ipsilateral and contralateral sides of the A4 segment (N=6) and
the ipsilateral sides on the A4 and A5 segments (N=4). In separate
experiments, micro-dissecting scissors were used to sever the dorsal nerve on
one side of the A4, A5 or A6 segment (N=4), the ipsilateral and
contralateral DNs in the A6 segment (N=3) or the ipsilateral DNs in
the A5 and A6 segment (N=3). Vetbond was used to seal the wounds.
After 24 h, the adduction response was tested by stimulation of the MHs with
fine forceps while mounted on their dorsal side. The location and extent of
muscle and nerve damage was assessed by dissection.
Pressure measurements
Most recordings reported here were carried out with a saline-filled
polyethylene catheter (0.28 mmx0.061 mmx0.011 mm, 42 cm long)
inserted at the subcoxa and body wall junction in A4 of an anesthetized animal
and held in place with Vetbond. The catheter was connected to a mineral
oil-filled polyethylene tube (0.83 mmx1.6 mmx0.2 mm, 35 cm long)
and a solid-state pressure sensor (PX170 or PX40; Omega Engineering Inc.,
Stamford, CT, USA). In an attempt to increase the resolution of local pressure
changes, we also used an implantable solid-state Mikro-Tip sensor (tip
diameter 0.47 mm; model SPR 671; Millar Instruments, Houston, TX, USA). This
was inserted through a small incision in the cuticle and allowed to seal in
place by hemolymph coagulation. Although less sensitive to movement artifacts
(`catheter whip'), recordings from the solid-state sensor were similar to
those of the remote catheter sensors. When both sensors were implanted at the
same location and the caterpillar squeezed repeatedly, the remote sensor had a
negligible response lag (28 ms by cross-correlation). Signals were amplified
(Brownlee Precision Co., San Jose, CA, USA) and digitized at 1-10 kHz using
WinDaq Software (Dataq Instruments Inc., Akron, OH, USA). The dorsal side of
the caterpillar was glued to a flat surface, and proleg movements were
captured on videotape using an SVHS-resolution camcorder (Canon ES-400). The
video and pressure recordings were synchronized using a voltage pulse that
also triggered an LED in the camera's field of view. The timing and amount of
proleg movement were monitored by measuring the distance between the left and
right crochets on each body segment in a single two-dimensonal view using
APAS. The phase relationships of proleg movements and pressure changes were
estimated from the peak lag calculated by cross-correlation using MatLab
(Mathworks, Inc., Natick, MA, USA). Because the sampling frequency of the
movement data and pressure records was different, correlations were carried
out using a discrete cross-correlation function that employs binning and a `z'
transform. This function was kindly provided by Dr E. Ofek, School of Physics
& Astronomy, Tel-Aviv University, Israel. Both sensors were calibrated
with a head of water, and the values converted to Pa.
Electromyography
Bipolar electrodes were made from 50 µm-diameter Formvar-insulated
Nichrome wire (AM Systems). The electrode was inserted through the cuticle
into the origin of the principal planta retractor muscle (PPRM) or the ventral
internal lateral muscle (VIL) in anesthetized animals on a chilled metal
block. A silver ground electrode, 75 µm in diameter, was inserted into the
body cavity in the terminal segment. The animal was epoxyed (Pacer Technology,
Rancho Cucamonga, CA, USA) to a flat surface and videotaped using a digital
video camcorder (Canon ZR10). The EMG signal was amplified (AM Systems, model
1700) and digitized at 10 kHz using WinDaq software. After 30 min of recovery,
adduction and retraction were stimulated via pin manipulation of the
PHs and MHs.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Two main muscles insert into the proleg: the principal planta retractor
(PPRM), with its insertion point at the lateral edge of the planta, and the
accessory planta retractor (APRM), which inserted more proximally and
laterally on the wall of the coxa-planta boundary. Both muscles originate from
apodemes high up on the lateral body wall posterior to the spiracle
(Weeks and Truman, 1984).
During crawling, contraction of PPRM disengages the crochets from the
substrate (Belanger et al.,
2000
). Stronger contraction of PPRM during the proleg withdrawal
reflex retracts the crochets further into the inverted planta region
(Weeks and Jacobs, 1987
). APRM
is also active during crawling and complete proleg withdrawal. A number of
small muscles are attached close to the rim of the subcoxa (see
Weeks and Ernst-Utzschneider,
1989
).
Kinematics of adduction
When the prolegs grasp an object at the ventral midline, the movement
consists of a relatively smooth eversion of the leg and its simultaneous
adduction. Most of the initial eversion is generated by an increase in the
size of the coxal segment, with smaller contributions from the proximal part
of the planta. This increase in length is accomplished primarily through an
unfolding of the crease between the subcoxa and coxa and a smaller extension
of the coxal cuticle itself (Fig
2A). The relative contribution of these mechanisms is variable and
may depend on the hydrated state of the caterpillar and its resting body
pressure. Adduction occurs because the lateral coxal wall expands more than
the medial wall, thereby rotating the planta medially
(Fig. 2B). The final grasping
movement involves an eversion of the planta, which inflates medially and fans
out into a broad lobe along the rostral-caudal axis. At this point, the
crochets appear to erect as they meet at the midline. The whole movement is
variable in duration but typically takes between 0.3 and 0.4 s.
|
Stimulation of adduction
It has been proposed that adduction is elicited when the large identified
ventromedial hairs (VMHs) are bent (Levine
et al., 1985). We found that careful stimulation of VMHs alone in
one body segment [avoiding the nearby medial hairs (MHs)] rarely initiated
adduction. Furthermore, removing the VMHs had no detectable effect on
crawling, grasping or adduction. Instead, adduction could be evoked more
reliably by stroking the MHs on the inside surface of the proleg (see also
Peterson and Weeks, 1988
).
Touching MHs on one proleg generally evoked adduction of both prolegs in a
body segment, although a single proleg could be extended on its own. Most
commonly, a single touch to MHs in one segment evoked both bilateral and
multisegmental adduction that spread anteriorly and posteriorly. These
responses could be elicited in segments with the VMH removed but not when the
MHs were cut (although adduction in these segments was normal when evoked by
stimulating MHs in other segments). Adduction can also be stimulated by
lightly blowing air over the intact insect, although we have not determined
which sensory hairs are involved in this response.
Adduction cannot be elicited reliably in isolated abdomens or in larvae with the abdominal connectives severed anterior to the stimulated proleg. This implies either that essential adduction circuitry is present in anterior ganglia or that adduction is `gated' by descending information. Cutting the connectives between the subesophageal and thoracic ganglia does not prevent adduction (although it eliminates crawling), but cuts between A2 and A3 eliminate adduction in all the prolegs.
Motoneurons involved in adduction
Although adduction requires most of the abdominal and thoracic nerve cord
to be intact, MH stimulation will stimulate motoneuron activity in chains of
isolated abdominal ganglia and in flatterpillar preparations. In both of these
preparations, recordings were made from the ventral nerve (VN) and branches of
the dorsal nerve (DN) in different body segments while stimulating MHs on the
left proleg in segment A4. MH stimulation had no effect on the anterior and
lateral branches of the dorsal nerve, but activity in the ipsilateral VN and
the DNP were both increased by MH stimulation
(Fig. 3; P=0.0017,
N=4; P=0.019, N=6, respectively). The effect on VN
activity was weak and not closely time-locked to the stimulus. By contrast,
the increase in DNP activity was robust and coincident with the
duration of the stimulus. Spike analysis (separated by amplitude alone) of
DNP activity suggested that all the MH-evoked activity was
accounted for by one or two large amplitude units.
|
The DNP in A5 contains the axons of nine motoneurons [VIL
neurons 1 and 2, ventral external oblique (VEO) neurons 1 and 2, the ventral
internal oblique (VIO) neuron, the ventral internal medial (VIM) neuron,
neuron 28, and two unidentified neurons, all with their cell bodies in A4] and
one ventral unpaired midline neuron (with its cell body in A5)
(Levine and Truman, 1985;
Taylor and Truman, 1974
).
Severing the connective between A4 and A5 abolished MH-evoked activity in the
DN of segment A5. This implies that MH sensory input activates one of the
descending motoneurons in A4.
Using a flaterpillar preparation, recordings from each of the muscles VIM, VEO, VIO and VIL showed that only VIL is reliably excited by MH stimulation (Fig. 4). The activation of VIL was bilateral, persisted throughout the MH stimulation (mean EJP frequency: spontaneous, 1.10±0.25 Hz; evoked, 5.81±0.54 Hz, N=27) and could be detected in other segments (A3 mean EJP frequency: spontaneous, 3.93±0.55 Hz; evoked, 6.29±0.40 Hz, N=24) (A5 mean EJP frequency: spontaneous, 3.59±0.53 Hz; evoked, 4.67±0.35 Hz; N=14) (A6 mean EJP frequency: spontaneous, 0.94±0.42 Hz; evoked, 4.81±1.29 Hz; N=7). In 22 out of 35 recordings from VIL, the EJPs could be sorted into two amplitude groups that might correspond to the activity of the two VIL motoneurons. The EJP activity was increased by MH stimulation regardless of its amplitude. Although the timing of A4 MH deflection could not be controlled precisely, the responses of VIL in body segments A3-A6 were initiated within 0.5-1.2 s of one another.
|
Electromyography
During cycles of proleg retraction and adduction, the activity of PPRM was
strongly correlated with the prolegs separating and moving away from the
midline. Withdrawal movements began immediately at the onset of EMG activity
in PPRM, and adduction began precisely when EMG burst activity ceased
(Fig. 5). This relationship was
consistent in both spontaneous and evoked movements. The activity of ventral
muscles (recordings were made close to VIL) was usually coincident with proleg
activity (Fig. 6) but there
were periods during which EMG bursts in VIL did not correspond to proleg
movements and some retractions during which VIL was not active.
|
|
Muscle ablation and nerve section
Activity in VIL and other large segmental ventral muscles is implicated in
proleg movements, but damage to these muscles in one body segment did not
block adduction or retraction (Table
1). The section of the dorsal nerve through which the axon of VIL
projects also failed to block adduction responses. Although it would be
informative to cut selected branches of the ventral nerve (e.g.
VNAP, which innervates the MHs and VMHs;
Trimmer and Weeks, 1993), we
have not yet managed to perform this surgery without damaging the cuticle and
muscles at the base of the proleg.
|
Pressure recordings
In unrestrained larvae, the internal pressure changes were complex and
dominated by large peaks corresponding to gross body movements. Because cycles
of proleg retraction and adduction were synchronized to these crawling
movements, specific pressure changes related to proleg control could not
always be detected. However, in restrained larvae, some pressure changes
closely matched overall proleg movements
(Fig. 7A). These changes varied
between 1500 and 7900 Pa depending on the number of prolegs moving
simultaneously. A cross-correlation analysis of these pressure changes with
proleg movements showed that proleg retraction was preceded by an increase in
pressure with a lead time of 0.1-1 s (Fig.
7B). A drop in pressure usually preceded adduction. In some
recordings, the prolegs at the recording site (A4) were glued together to
prevent them retracting. When a single pair of prolegs moved in another
segment, a small pressure pulse could often be detected preceding the
movement.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Movements of the proleg
Because caterpillars do not have a hard skeleton, they cannot use levers to
extend or direct the positions of their limbs. It has generally been assumed
that prolegs are extended by pressure, but movements of the crochets towards
the midline have not been directly examined. The kinematics of adduction in
intact caterpillars reveal that extension and adduction are not distinct from
one another but proceed in a smooth movement with no discontinuity in any of
the three planes. Adduction occurs through a larger increase in the length of
the lateral margin relative to the medial surface, which causes a rotation
around the anterior-posterior axis. Most of this extension involves an
unfolding of the membrane between the coxa and the subcoxa, with a smaller
contribution from expansion of the subcoxal cuticle. This differential
stretching probably results from differences in the local cuticle stiffness.
However, it is unlikely that the physical properties of the proleg cuticle are
sufficient to direct adduction. When pairs of prolegs are removed and mounted
on a syringe barrel, they inflate and deflate with changes in saline pressure
but they do not adduct in a natural way (N.P. and A.T., unpublished
observations). This highlights the importance of maintaining the appropriate
cuticle geometry and suggests that either passive tension provided by
retractor muscles or active tension in the ventral muscles (see below) is
essential for normal gripping.
Neural control
Previous research has shown that activation of the mechanosensory planta
hairs can stimulate proleg withdrawal through the activation of PPRM and APRM
(Weeks and Jacobs, 1987). This
reflex is context-sensitive; it is inhibited during the stance phase of
crawling, habituated by repeated stimuli
(Weil and Weeks, 1996
) and
sensitized by noxious stimuli (Walters et
al., 2001
). Unlike retraction, adduction is not reliably evoked in
isolated abdomens or in reduced preparations. Furthermore, because proleg
extension is the `default' state (the prolegs are extended in anesthetized or
resting larvae), adduction could be viewed as the cessation of retraction.
However, from a behavioral and experimental perspective, adduction is a
distinct process. For example, during the swing phase of crawling, the prolegs
do not truly retract but instead shorten from a stretched state back to their
resting length (Belanger et al.,
2000
; Belanger and Trimmer,
2000
). Hence, adduction during the stance phase cannot be defined
as the end of retraction.
Because adduction is very hard to see in freely moving animals, we have
used restrained larvae with their ventral surface uppermost. In this
situation, gripping can be initiated by placing a small probe along the
midline. This stimulus causes the proleg to extend and adduct whether it is
fully or partially retracted. As described previously, adduction is stimulated
mainly by proleg MHs (Peterson and Weeks,
1988) and we have found that it does not require the VMHs. To try
and identify changes in neural activity that accompany adduction, we examined
the effects of MH stimulation on different nerves in semi-intact preparations.
The strongest and most consistent effect of MH stimulation was the bilateral
activation of VIL in all the proleg-bearing segments, which closely matches
the recruitment of prolegs during normal adduction. Analysis of VIL EJPs
suggests that both motoneurons are activated by MH stimulation. Because
neither the MH projections nor the VIL dendritic arbors cross the midline, the
activation of contralateral prolegs must involve interneurons. VIL is a large,
wide muscle extending from the anterior ventral apodeme to a similar position
at the posterior margin of the same segment
(Levine and Truman, 1985
). The
contraction of VIL would be expected to shorten or stiffen a large region of
the body wall ventral to the spiracle.
In intact larvae, EMG recordings showed that both evoked and spontaneous
adduction occur at the end of a burst of activity in PPRM. As discussed below,
the prolegs cannot be extended during strong contractions of PPRM. Because
movements of the MHs can initiate adduction, it was expected that MH
stimulation would inhibit the proleg retractor motoneurons. However, in
semi-intact flaterpillar preparations, activity in the lateral branch of the
ventral nerve, which carries these axons, was not reduced but sometimes
increased by MH stimulation. This result is consistent with previous findings
using isolated proleg/ganglion preparations in which activation of the sensory
branches of the ventral nerve (including VNAP, which innervates the
MHs) excited the retractor motoneuron principal planta retractor
(Trimmer and Weeks, 1993).
These apparently contradictory findings probably reflect major differences in the way information is processed in intact insects and reduced preparations and they highlight the need for high-resolution EMG recordings in freely moving larvae. Unlike other model systems such as locusts and cockroaches, Manduca muscles are not innervated by fast and slow motoneurons, nor do they have common inhibitors. This simplicity should help in the interpretation of EMG activity and in relating it to the role of individual muscles in normal movement.
The role of pressure and muscle activation
Anatomically, Manduca differs from both classical hydrostats and
muscular hydrostats. Classical hydrostats such as the mollusks Lingula
anatina and Donax serra use fluid-filled appendages to burrow
and provide muscular antagonism (Trueman
and Brown, 1985; Trueman and
Wong, 1987
). Likewise, sea anemones are able to modify their size
by exploiting the inherent elasticity of their hydrostatic skeleton
(Truman, 1992
). In muscular
hydrostats, the pressure tissues are largely compartmentalized and packed with
muscles (Trueman and Clarke,
1988
) that can be selectively activated to control local pressure
changes (Nishikawa et al.,
1999
). This type of skeletal system provides support for
appendages ranging from squid tentacles to frog tongues and can be used for
precise movements (Kier and Curtin,
2002
; Nishikawa et al.,
1999
). Manduca can be viewed as a combination of these
two hydrostatic systems, with segments functionally partitioned by muscle and
with hemolymph that can move between compartments.
The results reported here show that proleg extension occurs when the
retractors are inactive and that adduction results from a differential
unfolding of the intersegmental membrane on the lateral and medial surfaces.
Although this arrangement is simple, there are important aspects that have not
previously been noted. First, although it is possible that weak MH excitation
of PPR (Trimmer and Weeks,
1993) could stiffen the medial plane of the proleg and force the
leg to adduct, this activity is not evident in restrained larvae. Second,
pressure pulses are not used to help extend the proleg. Instead, hemolymph
pressure often rises before retraction and usually falls before extension.
These fluctuations are probably caused by motor activity in muscles that
stiffen the body wall and assist in directing movements. Our physiology
recordings suggest that VIL is one of these muscles because it is activated by
MH stimulation. However, damage to VIL and other body wall muscles does not
prevent adduction so it is clear that local stiffening is not essential for
movements of the proleg. By bracing the ventral-lateral wall, the contraction
of PPRM and APRM retracts the proleg instead of buckling the upper attachment
point.
The increased hemolymph pressure caused by tension in body wall muscles is unlikely to influence retraction. We have found that the prolegs can be retracted fully even when the hemolymph pressure is increased to 10 times its normal level by saline injection. It is also clear that basal body pressure is sufficient to extend the proleg. From Pascal's principal relating force, displacement and area in connected fluid-filled compartments, a shortening of the body will extend the smaller-diameter proleg by a much larger distance. Assuming a body radius of 0.5 cm with the gut occupying 36% of the cross-sectional area (both measurements from magnetic resonance imaging of a 5th instar larva), a proleg radius of 0.125 cm and a constant pressure of 2 kPa, a proleg could be extended by 0.5 cm for a 500 µm shortening of the body. This calculation does not take into account other geometric or pressure changes but it demonstrates that a very small amount of body shortening can account for full proleg extension. Because the proleg radius is about a quarter of that of the body, tension in the limb walls will be about a quarter of that in the body at the same pressure. This probably explains why extension occurs mainly through unfolding of intersegmental membranes. Unless the proleg cuticle is considerably less stiff than the body wall, the normal hydrostatic pressure that maintains turgor will not be sufficient to expand the proleg cuticle itself.
An important aspect that has not been addressed in these studies is the force of adduction and gripping by the prolegs. It is quite likely that restrained and supported larvae do not grip in the same way that freely moving larvae do. Despite the strong MH activation of VIL, the nerve sectioning and muscle ablation experiments imply that adduction does not require active contraction of ventral muscles in restrained larvae. However, we have not yet been able to measure the proleg grip force in normal and surgically altered animals. It is possible that muscles such as VIL are more important when the larva needs to support itself and that they are recruited to generate the normal adduction force. We are currently exploring these possibilities using custom-designed force sensors and multi-site EMG recordings in freely moving larvae.
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barth, R. (1937). Muskulatur und Bewegungsart der Raupen. Zool. Jb. Physiol. 62,507 -566.
Belanger, J. H., Bender, K., J. and Trimmer, B. A. (2000). Context-dependency of a limb-withdrawal reflex in the caterpillar Manduca sexta. J. Comp. Physiol. A 186,1041 -1048.[Medline]
Belanger, J. H. and Trimmer, B. A. (2000). Combined kinematic and electromyographic analyses of proleg function during crawling by the caterpillar Manduca sexta. J. Comp. Physiol. A 186,1031 -1039.[Medline]
Bell, R. A. and Joachim, F. A. (1978). Techniques for rearing laboratory colonies of tobacco hornworms and pink bollworms. Annu. Entomol. Soc. Am. 69,365 -373.
Brackenbury, J. (1996). Novel locomotory mechanism in caterpillars: life-line climbing in Epinotia abbreviana (Tortricidae) and Yponomeuta padella (Ypononmeutidae). Physiol. Entomol. 21,7 -14.
Brackenbury, J. (1997). Caterpillar kinematics. Nature 390,453 .[CrossRef]
Brackenbury, J. (1999). Fast locomotion in caterpillars. J. Insect Physiol. 45,525 -533.[CrossRef][Medline]
Casey, T. M. (1991). Energetics of caterpillar locomotion: biomechanical constraints of a hydraulic skeleton. Science 252,112 -114.
Gutfreund, Y., Flash, T., Fiorito, G. and Hochner, B.
(1998). Patterns of arm muscle activation involved in octopus
reaching movements. J. Neurosci.
18,5976
-5987.
Hinton, H. E. (1955). On the structure, function and distribution of the prolegs of the panorpoidea, with a criticism of the Berlese-Imms theory. Trans. R. Ent. Soc. Lond. B 106,455 -541.
Kier, W. M. and Curtin, N. A. (2002). Fast
muscle in squid (Loligo pealei): contractile properties of a specialized
muscle fibre type. J. Exp. Biol.
205,1907
-1916.
Levine, R. B., Pak, C. and Linn, D. (1985). The structure, function and metameric reorganization of somatotopically projecting sensory neurons in Manduca sexta larvae. J. Comp. Physiol. A 157,1 -13.
Levine, R. B. and Truman, J. W. (1985). Dendritic reorganization of abdominal motoneurons during metamorphosis of the moth, Manduca sexta. J. Neurosci. 5,2424 -2431.[Abstract]
Matzner, H., Gutfreund, Y. and Hochner, B.
(2000). Neuromuscular system of the flexible arm of the octopus:
physiological characterization. J. Neurophysiol.
83,1315
-1328.
Nishikawa, K. C., Kier, W. M. and Smith, K. K.
(1999). Morphology and mechanics of tongue movement in the
African pig-nosed frog Hemisus marmoratum: a muscular hydrostatic
model. J. Exp. Biol.
202,771
-780.
Peterson, B. A. and Weeks, J. C. (1988). Somatotopic mapping of sensory neurons innervating mechanosensory hairs on the larval prolegs of Manduca sexta. J. Comp. Neurol. 275,128 -144.[Medline]
Quillin, K. J. (1998). Ontogenetic scaling of
hydrostatic skeletons: geometric, static stress and dynamic stress scaling of
the earthworm Lumbricus terrestris. J. Exp.
Biol. 201,1871
-1883.
Quillin, K. J. (1999). Kinematic scaling of
locomotion by hydrostatic animals: ontogeny of peristaltic crawling by the
earthworm lumbricus terrestris. J. Exp. Biol.
202,661
-674.
Sensenig, A. T. and Shultz, J. W. (2003).
Mechanics of cuticular elastic energy storage in leg joints lacking extensor
muscles in arachnids. J. Exp. Biol.
206,771
-784.
Snodgrass, R. E. (1952). A Textbook of Arthropod Anatomy. Ithaca, NY: Comstock Pub. Associates.
Suzuki, Y. and Palopoli, M. F. (2001). Evolution of insect abdominal appendages: are prolegs homologous or convergent traits? Dev. Genes Evol. 211,486 -492.[CrossRef][Medline]
Taylor, H. M. and Truman, J. W. (1974). Metamorphosis of the abdominal ganglia of the tobacco hornworm, Manduca sexta. J. Comp. Physiol. 90,367 -388.
Trimmer, B. A. and Weeks, J. C. (1989). Effects of nicotinic and muscarinic agents on an identified motoneurone and its direct afferent inputs in larval Manduca sexta. J. Exp. Biol. 144,303 -337.
Trimmer, B. A. and Weeks, J. C. (1993).
Muscarinic acetylcholine receptors modulate the excitability of an identified
insect motoneuron. J. Neurophysiol.
69,1821
-1836.
Trueman, E. R. (1975). The Locomotion of Soft-Bodied Animals. London: Edward Arnold.
Trueman, E. R. and Brown, A. C. (1985). Dynamics of burrowing and pedal extension in Donax serra Mollusca Bivalvia. J. Zool. 207,345 -356.
Trueman, E. R. and Clarke, M. R. (1988). Form and function. In The Mollusca, vol.11 (ed. K. M. Wilbur), pp.211 -247.San Diego: Academic Press.
Trueman, E. R. and Wong, T. M. (1987). The role of the coelom as a hydrostatic skeleton in Lingulid brachiopods. J. Zool. 213,221 -232.
Truman, J. W. (1992). Developmental neuroethology of insect metamorphosis. J. Neurobiol. 23,1404 -1422.[Medline]
Walters, E., Illich, P., Weeks, J. and Lewin, M.
(2001). Defensive responses of larval Manduca sexta and their
sensitization by noxious stimuli in the laboratory and field. J.
Exp. Biol. 204,457
-469.
Weeks, J. and Ernst-Utzschneider, K. (1989). Respecification of larval proleg motoneurons during metamorphosis of the tobacco hornworm, Manduca sexta: segmental dependence and hormonal regulation. J. Neurobiol. 20,569 -592.[Medline]
Weeks, J. C. and Jacobs, G. A. (1987). A reflex behavior mediated by monosynaptic connections between hair afferents and motoneurons in the larval tobacco hornworm, Manduca sexta. J. Comp. Physiol. A 160,315 -329.[Medline]
Weeks, J. C. and Truman, J. W. (1984). Neural organization of peptide-activated ecdysis behaviors during the metamorphosis of Manduca sexta: II. Retention of the proleg motor pattern despite loss of the prolegs at pupation. J. Comp. Physiol. A 155,423 -433.
Weil, D. E. and Weeks, J. C. (1996). Habituation and dishabituation of the proleg withdrawal reflex in larvae of the sphinx moth, Manduca sexta. Behav. Neurosci. 110,1133 -1147.[CrossRef][Medline]
Wilson, J. F., Mahajan, U., Wainwright, S. A. and Croner, L. J. (1991). A continuum model of elephant trunks. J. Biomech. Eng. 113,79 -84.[Medline]
Yim, M. (1994). Locomotion with a unit modular reconfigurable robot. PhD Thesis, Stanford University, Palo Alto, CA, USA.