Modulation of ecdysis in the moth Manduca sexta : the roles of the suboesophageal and thoracic ganglia
Department of Zoology, University of Washington, Seattle, WA
98195-38100, USA
*
Present address: Department of Biology, San Francisco State University, San
Francisco, CA 94132, USA
(e-mail: fuse{at}sfsu.edu )
Accepted 4 February 2002
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Summary |
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Key words: ecdysis-triggering hormone, eclosion hormone, crustacean cardioactive peptide, cyclic GMP, inhibition, eclosion, neuron, carbon dioxide, tobacco hornworm, Manduca sexta
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Introduction |
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Four key players in the regulation of the ecdysis sequence have been
identified. They include pre-ecdysis-triggering hormone (PETH)
(Zitnan et al., 1999),
ecdysis-triggering hormone (ETH) (Zitnan
et al., 1996
), eclosion hormone (EH)
(Truman and Riddiford, 1970
)
and crustacean cardioactive peptide (CCAP)
(Gammie and Truman, 1997b
).
These peptides probably play a role in the ecdysis behaviours of many, if not
all, insects (Adams and Zitnan,
1997
; Ewer and Truman,
1996
; Truman et al.,
1981
), but their roles have been especially well described in the
moth Manduca sexta (e.g. Ewer et
al., 1997
; Kingan et al.,
1997
).
In M. sexta, there is a working model for the organization of the
pre-ecdysis and ecdysis behaviours. Declining steroid titres and possibly an
early release of EH are thought to initiate the release of PETH and ETH from
epitracheal glands lining the body wall. PETH triggers pre-ecdysis behaviour
I, while ETH triggers pre-ecdysis behaviour II, both of which help the animal
to loosen the old digested cuticle (Zitnan
et al., 1999). Once ETH has been released, there is a positive
feedback cascade between ETH and EH in which ETH stimulates release of EH and
EH stimulates further release of ETH (Ewer
et al., 1997
; Gammie and
Truman, 1999
; Kingan et al.,
1997
). This feedback results in the massive release of both
hormones from their respective cells prior to the onset of ecdysis. EH then
stimulates the release of CCAP from a group of 50 cells in the ventral nerve
cord, called the cell 27/704 group (Davis
et al., 1993
; Ewer et al.,
1994
). Two major functions of CCAP are to release the ecdysis
motor pattern itself, enabling the animal to shed its old cuticle, and to
inhibit the ETH-induced pre-ecdysis behaviours
(Ewer and Truman, 1997
;
Gammie and Truman, 1997b
). The
action of EH on the 27/704 group of neurons involves an increase in cyclic GMP
(cGMP) levels, which increases cell excitability
(Ewer et al., 1994
;
Gammie and Truman, 1997a
;
Gammie and Truman, 1999
).
Measurements of changes in intracellular levels of cGMP have been
instrumental in determining the timing of many events associated with EH and
ETH activity (e.g. Ewer et al.,
1994). The timing of many of these events appears to depend on
descending inputs from the head (Baker et
al., 1999
; Ewer and Truman,
1997
; Zitnan and Adams,
2000
). In the case of larvae and pupae, there is a delay of 20-30
min between the release of EH and the onset of ecdysis behaviours, whereas
this delay is 2-3 h in adults. This paper examines the nature of this
inhibition through the use of surgical manipulations, ligatures and
pharmacological manipulations. It further characterizes other neurons
activated during ecdysis, of which little is currently known.
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Materials and methods |
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ETH was synthesized by the Howard Hughes Macromolecular Synthesis Unit at
the University of Washington. The peptide was stored as a 1 mmoll-1
stock in phosphate-buffered saline (PBS) and frozen until needed for
experiments. Experimental amounts were diluted in modified Weever's saline
(Trimmer and Weeks, 1989).
Larvae and pupae were injected in the terminal abdominal segment with 10 µl
of 2x10-5 moll-1 ETH (200 pmol of ETH per
animal).
Immunohistochemistry
Animals were anesthetized on ice, and their nerve cords were dissected in
cold saline. Tissues were fixed in 4 % buffered paraformaldehyde for 1-2 h at
room temperature, then rinsed in PBS containing 1 % Triton-X 100 (PBST; Sigma
Chemical Co., St Louis, MO, USA. Tissues being processed for cGMP
immunoreactivity (cGMP-IR) were immediately transferred to sheep anti-cGMP
antibody (diluted to 1:10 000 in PBST; a generous gift from Dr Jan De Vente).
The other tissues were treated with collagenase (Type IV; Sigma) in PBST at
0.5 mg ml-1 for 60 min at room temperature, then rinsed in PBST and
blocked with 5 % normal donkey serum (Jackson ImmunoResearch Laboratories
Inc., West Grove, PA, USA) for 15 min at room temperature. Double-labeled
tissues were incubated with a combination of sheep anti-cGMP antibody (1:10
000) and one of various rabbit antibodies diluted to appropriate
concentrations with PBST; FMRFamide (1:1000, a gift from D. O. Willows),
Leucokinin IV (1:2500, a gift from J. Veenstra) and Manduca diuretic
hormone (1:2500, a gift from J. Veenstra). All tissues were incubated on a
shaker for 36-48 h at 4°C then rinsed in PBST and processed with secondary
antibodies (1:1000) on a shaker for 24 h at 4°C.
Tissues stained for cGMP-IR were transferred to peroxidase-conjugated donkey anti-sheep IgG (1:1000; Jackson Laboratories) for 24 h. After rinsing, the antibody complex was visualized using the chromagenic diaminobenzidene (DAB) reaction. Tissues were incubated with ammonium chloride (0.4 mg ml-1), beta-D-glucose (2 mg ml-1), DAB (50 µl ml-1) and glucose oxidase (6 µl ml-1) in PBST. Reactions generally took 5-30 min and were stopped by several rinses in PBST. The tissues were then dehydrated through an ethanol series, cleared in xylene and mounted in DPX (Fluka, Buchs, Switzerland) on poly-L-lysine-coated coverslips.
Double-labeled tissues were transferred to fluorescene isothiocyanate (FITC)-conjugated donkey anti-sheep IgG (1:1000; Jackson Laboratories) together with Texas-Red-conjugated donkey anti-rabbit IgG (1:1000; Jackson Laboratories) for 24 h. After rinsing, the tissues were dehydrated and mounted as described above. The samples were viewed on a confocal microscope (BioRad MRC 600; BioRad, Hercules, CA, USA).
Control tissues were treated as described above, with the omission of either the primary or the secondary antibody. No staining was noted in these preparations.
Signal quantification
The intensity of immunoreactivity based on the DAB colour reaction was
quantified subjectively using a scoring system from 0 (no stain) to 3 (maximal
staining of cell bodies and axons), as described by Ewer and Truman
(1997).
Surgical procedures
Animals were anesthetized on ice then placed on a wax mount or immersed in
cold saline. Debrained larvae and pupae had their brain removed through a
small incision in the head capsule. Sham-operated animals had forceps inserted
into the same opening but without removing the brain. After surgery, the
incision was sealed with wax and the animals were left to resume ecdysis. For
the ligations, a blood-tight ligature was placed at various segments, using
fine surgical thread, and the tissue anterior or posterior to the ligature was
then cut off using surgical scissors. Neck ligatures were placed between the
head and thorax to eliminate the brain and the suboesophageal ganglion (SOG).
Thoracic ligatures were placed between thorax and abdomen to eliminate all
thoracic ganglia. Terminal ligatures were placed between the last two
abdominal segments to eliminate the terminal abdominal ganglion (TAG). Animals
were dissected after the experiments to ensure that the ligatures had
eliminated the appropriate ganglia.
Sham-ligated animals had neck ligatures quickly removed after tightening to test for the effects of stress induced by the initial tightening. No incisions or visible injuries were produced.
Animals were treated with CO2 or N2 gas, for 1 min, at different times after ETH injection. The animals were placed in loosely sealed containers under a constant stream of gas for the 1 min interval.
Phosphodiesterase inhibitors
The cGMP-specific phosphodiesterase inhibitor Zaprinast (Sigma) was
dissolved in 100% dimethyl sulfoxide (DMSO) and injected into the animals in a
similar manner to ETH injections. Animals were injected with 10 µl of
Zaprinast 10 min before ETH injection. Control animals were injected with 10
µl of 100% DMSO. Ganglia from some Zaprinast-injected animals were also
fixed and processed for cGMP-IR, as described above.
Adult behaviours and videography
Adult eclosion was monitored and timed by eye and was videotaped using a
Sony DCR-TRV11 digital camcorder. Images were captured on a Macintosh iMac
using iMovie software.
Results are presented as means ± S.E.M.
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Results |
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Fig. 1 also shows the time
course of cGMP-IR in the two other pairs of neurons that are not
CCAP-containing cells (Zitnan and Adams,
2000). Similar cells have been described during ecdysis of locusts
(Truman et al., 1996
). As
shown in Fig. 1, cGMP was
detected in these cells approximately 10-15 min after it appeared in the cell
27/704 group. Peak levels of cGMP-IR were evident at ecdysis, with a rapid
decay thereafter. The intensity of cGMP staining in these cells did not reach
the levels seen in the cell 27/704 group. These ventrolateral cells were
located posterior to cells 27 and 704, and their axons projected ipsilaterally
through the ventral nerve. Hence, these cells would be in the L cell group on
the basis of the nomenclature of Davis et al.
(1993
) (see Discussion).
Homologous pairs of cells were noted in each abdominal ganglion, with the
greatest intensity of staining occurring in abdominal ganglia 1 and 2 and
progressively weaker staining occurring in cells of the more posterior
segments.
Double labeling with anti-cGMP and anti-FMRFamide
(Fig. 2B), anti-cGMP and
anti-leucokinin IV (Fig. 2C) or
anti-cGMP and anti-Manduca diuretic hormone
(Fig. 2D) revealed that these
neurons did not contain FMRFamide-, leucokinin- or diuretic-hormone-like
peptides. There was no apparent overlap between peptide immunoreactivity and
cGMP-IR. The immunohistochemical data suggested that cGMP-positive cells were
not the L3,4 neurons described in the month central nervous system
(Chen et al., 1994). We will
therefore refer to them as the L2 and L5 cells.
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Effects of surgery and decapitation on the timing of ecdysis
Larvae were decapitated at various times after ETH injection, and the time
to ecdysis was determined (Fig.
3). In controls, larval ecdysis began on average 37 min after ETH
injection. If animals were decapitated 10 min after ETH injection, after the
onset of pre-ecdysis behaviours but prior to detectable levels of cGMP, the
animals did not ecdyse. In contrast, larvae decapitated at 15, 20 or 25 min
after ETH injection all began ecdysis significantly sooner than controls
(P<0.001). The onset of ecdysis occurred approximately 28 min
after ETH injection, irrespective of when the animals were decapitated.
Similarly, for pupal ecdysis, decapitation at 10 min resulted in no ecdysis
whereas the same treatment at 15, 20 or 30 min after ETH injection resulted in
animals ecdysing at least 15 min earlier than controls.
|
As with previous studies (Baker et al.,
1999; Ewer and Truman,
1997
; Zitnan and Adams,
2000
), these results indicated that the head has a dual role in
the control of ecdysis. It is necessary to activate the behaviour, presumably
through the release of EH (Reynolds et
al., 1979
), but it also has an inhibitory function. The locus of
this inhibition was examined by assessing the effects of removing various
ganglia on the timing of ecdysis. The influence of the brain, SOG, thoracic
ganglia or terminal abdominal ganglion (TAG) was assessed by direct removal
(debraining) or by ligature. Larvae did not ecdyse when the brain was removed
10 min after ETH injection (data not shown). When animals were debrained 20
min after ETH injection (Fig.
4; -Brain), they ecdysed at the same time as the controls, whereas
the application of a neck ligature accelerated ecdysis
(Fig. 4; -Br/SOG).
Interestingly, ligation between the third thoracic ganglion (T3) and the first
abdominal ganglion (A1) to remove the brain, SOG and thoracic ganglia
(Fig. 4; Br-TG) resulted
in a strikingly faster onset of ecdysis compared with the neck ligatures
(P<0.001). No other treatments affected the timing of ecdysis,
including sham operations (Fig.
4; ShamBr) and stresses such as sham ligatures
(Fig. 4; ShamLig), sharp
pinching of the cuticle (data not shown) or ligation of the terminal abdominal
segments (Fig. 4;
Terminal).
|
As shown in Fig. 4, a similar relationship is evident for pupal ecdysis. Sham ligations, however, were omitted in pupae because these inevitably ruptured the new pupal cuticle. The high internal pressure in pupae led to excessive haemolymph loss during the course of ecdysis and usually to the eventual cessation of the ecdysis behaviours before the cuticle was shed. If the ligatures were not removed, as was the case for brain/SOG removal, haemolymph loss was minimized and ecodysis occurred prematurely, without noticeable differences in the quality of the visualized behaviours. Because terminal ligatures were followed by a normally timed, robust ecdysis, the stress of ligation did not seem to be sufficient to trigger premature ecdysis.
Thus, it appears that, after the release of EH, the brain can be removed without affecting the subsequent onset of the ecdysis behaviour. The removal of the SOG and thoracic ganglia, in contrast, results in a progressive advance in the onset of ecdysis in both larvae and pupae.
Effects of ligation on the duration of the ecdysis motor program
To determine whether there were differences in the roles of the SOG and
thoracic ganglia during ecdysis, the duration of the ecdysis motor program was
measured in both neck-ligated and thorax-ligated larvae. Duration was defined
as the persistence of peristaltic waves in the abdomen, once ecdysis had
started, irrespective of whether the cuticle was shed. This definition was
necessary, since ligatures artificially held the cuticle in place during
ecdysis in most animals. As can be seen in
Fig. 5, thoracic ligatures
significantly decreased the duration of the ecdysis motor program compared
with that of animals ligated at the neck. Therefore, the thoracic ganglia may
have dual roles, to aid in delaying the onset of ecdysis and to maintain the
motor program.
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Effects of altered cGMP levels on the timing of ecdysis
The importance of cGMP to the timing of ecdysis was examined by injecting
larvae with Zaprinast, a specific cGMP phosphodiesterase inhibitor
(Burns et al., 1992). Animals
were injected with Zaprinast 10 min before
(Fig. 6A) or after (data not
shown) injection of ETH, and the time to the onset of ecdysis was determined.
When larvae were injected with the solvent DMSO prior to ETH injection, they
showed an ecdysis latency of 38 min, which is not significantly different from
that of controls injected with ETH without prior DMSO treatment
(P>0.5). Treatment with Zaprinast, in contrast, produced a
dose-dependent delay in the time of ecdysis onset
(Fig. 6A).
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Zaprinast has been used in Drosophila melanogaster as a specific
cGMP phosphodiesterase inhibitor (Chyb et
al., 1999). To assess its effectiveness in Manduca sexta
in maintaining cGMP levels, we used immunocytochemistry to monitor the levels
of cGMP in the ventral ganglia 60 min after ETH injection. Using the scoring
system illustrated in Fig. 1,
the intensity of cGMP-IR in the 704 cells of ganglion A3 of controls and of
animals treated with 500 µg of Zaprinast was 1.0±0.4 (N=6)
and 2.8±0.2 (N=6), respectively. The controls showed the
expected waning of the cGMP response, whereas Zaprinast-treated animals still
showed high levels of this second messenger in the cell bodies and processes
of these neurons (P<0.001; Fig.
6B), showing that Zaprinast was, in fact, affecting cGMP levels.
Preliminary results in naturally ecdysing larvae also showed that injection of
Zaprinast after the start of the pre-ecdysis behaviour delays the onset of
ecdysis and increases cGMP staining in CCAP-containing neurons (J. Fisher and
J. W. Truman, unpublished observations).
Effects of CO2 on the timing of ecdysis in larvae and
pupae
Brief exposure to CO2 after ETH injection proved to be a
non-invasive method of triggering early ecdysis. As seen in
Fig. 7A, a 1 min exposure to
CO2 given 20 min after ETH injection resulted in the onset of
ecdysis within 4 min after the treatment. This response was significantly
faster than that seen in neck-ligated larvae (P<0.001), but not
from that of thorax-ligated animals (P>0.5; see
Fig. 4). This suggested that
CO2 may relieve the inhibition provided by the SOG and the thoracic
ganglia. Application of CO2 to pharate pupae similarly triggered
premature ecdysis (data not shown). The response to CO2 did not
appear to be due to simple anoxia since there was no significant change in the
timing of ecdysis in larvae treated with N2 compared with controls
(P>0.5).
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The response of larvae to CO2 was subsequently tested at various times after ETH injection (Fig. 7B). Treatment always consisted of a 1 min exposure to the gas, and the time of onset of ecdysis was then determined. At 10 min after ETH injection, application of CO2 did not advance the onset of ecdysis compared with controls (P>0.5). In contrast, CO2 triggered the rapid onset of ecdysis when applied 15, 20 or 25 min after ETH injection, at times when cGMP staining was apparent in the CCAP-containing neurons. At each successive treatment time, the lag between CO2 application and the onset of ecdysis was reduced, although this was not significantly different.
Effects of CO2 on the timing of ecdysis in adults
For adult eclosion, the delay between the appearance of cGMP in the cell
27/704 group and the onset of ecdysis behaviours is 2-3 h
(Ewer and Truman, 1997). To
determine whether CO2 affected the timing of adult eclosion,
animals were first entrained with a light and thermal regime, which resulted
in the majority of moths (approximately 90 %) eclosing within a narrow window
of time (09:00-13:00 h; see Fig.
9A). Adult ecdysis was generally completed within less than 1 min,
with abdominal and thoracic behaviours occurring within seconds of each other.
These events are depicted as open and filled black circles, respectively,
overlaid because of the rapid succession of the two behaviours (see
Fig. 9A). Eclosion began with
one or two waves of peristaltic contractions that moved anteriorly along the
abdomen. These rapidly evolved into a two-phase movement involving the
retraction of the entire abdomen followed by its forceful extension
(Fig. 8A). The extension was
coupled to a vigorous flexing of the wing bases. The latter served to rupture
the sutures over the head and thoracic cuticle, and the abdominal extensions
helped to push the animal out of the rigid pupal cuticle. These behaviours
have been described in detail by Mesce and Truman
(1988
). Once the moths had
escaped from the pupal cuticle, they searched for a perch, upon which they
remained to inflate their wings.
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Entrained pharate adults were given a 1 min pulse of CO2 at various times prior to ecdysis. If CO2 was applied at 07:30 h, animals did not respond to the treatment. The moths subsequently emerged within their expected period and their behaviour was indistinguishable from that of controls (compare overlaid open and filled pink and black circles; Fig. 9B). With successively later pulses of CO2, a variety of responses was observed (Fig. 9C-E). A proportion of the animals ignored the CO2 treatment and emerged at their expected times (overlaid pink circles). A large group of pharate adults responded with the initiation of abdominal peristaltic movements within minutes of the CO2 treatment.
In some cases, the initial abdominal peristalsis quickly progressed to the two-phase abdominal movements coordinated with the strong thoracic flexing of the wing bases (early overlaid pink circles). These movements usually brought about the rapid shedding of the entire cuticle. In many of the treated animals, however, the abdominal peristalsis occurred in isolation with little or no thoracic activity (isolated open pink circles). These movements occasionally ruptured the abdominal cuticle. The peristaltic movements were transient and the animals then became quiescent, although every 5 min or so, 2-3 waves of abdominal peristalsis were noted which then rapidly disappeared. The quiescence lasted for 20-90 min, at which time the remaining abdominal cuticle was removed by initiation of the retraction/extension movements, and thoracic shrugging resulted in the shedding of the thoracic and head portions of the cuticle (isolated filled pink circles). The moths that showed a split ecdysis behaviour (separate open and filled pink circles) appeared to finish ecdysis approximately 30-60 min earlier than the controls.
Once the entire cuticle had been shed, all the moths perched and inflated
their wings in a manner similar to controls (pink and black crosses). For
example, the time from complete shedding of the cuticle until the completion
of wing expansion and rotation of wings to the `moth' position
(Truman and Endo, 1974) was
94±2 min (N=31) for controls and 93±1 min
(N=24) for animals treated with CO2 at 9:30 h. For the
subgroup of CO2-treated animals that showed premature peristalsis
behaviour, the time was 97±4 min (N=12) between the final
completion of ecdysis and the termination of wing spreading. These values were
not significantly different (P>0.5).
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Discussion |
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Double-labeling experiments (Fig. 2B,
C) show that these lateral neurons are not the diuretic
hormone/leucokinin-containing L3,4 cells
(Chen et al., 1994), as was
suggested recently (Zitnan and Adams,
2000
). This is not surprising, because the paired lateral
leucokinin-containing cells are not detected in the first or second abdominal
ganglia (Chen et al., 1994
),
and it is in these ganglia that cGMP-IR is strongest in these ventrolateral
neurons. This and the fact that these neurons project ipsilaterally through
the ventral nerves (Fig. 2A,
arrowhead) suggest that they are lateral neurons L2 and
L5 instead, based on the nomenclature of Davis et al.
(1993
). In Manduca
sexta larvae, cells in this lateral cluster contain cardioacceleratory
peptides (other than CCAP) (Tublitz and
Sylwester, 1990
), but during metamorphosis they change their
peptide content to bursicon (Taghert and
Truman, 1982
). While such cardioacceleratory peptides and bursicon
have been attributed to this cluster
(Tublitz and Sylwester, 1990
;
Taghert and Truman, 1982
),
immunological confirmation of the peptide content of the L2 and
L5 cells is still required. The time of appearance of cGMP in these
cells, at the time of ecdysis, suggests that they may play a role in the
postecdysial phase of the behavioural sequence.
A role for FMRFamide-related peptides during ecdysis has been suggested on
the basis of immunohistochemical patterns of expression
(Miao et al., 1998). However,
on the basis of cGMP-IR, cells that contain FMRFamide-related peptides appear
not to be direct targets of EH. These peptides are not found in the cell
27/704 group nor did we find them in the L2,5 cells
(Fig. 2B). A set of thoracic
neurons show changes in expression levels of FMRFamide-related peptides before
and after ecdysis (Miao et al.,
1998
), but these are not among the cells that show cGMP-IR during
ecdysis (data not shown). Hence, activation of FMRFamide-positive neurons may
be caused by ETH or CCAP, but most probably not directly by EH.
The role of descending inhibition in the timing of ecdysis onset
A model for the interactions between the hormones regulating the ecdysis
sequence in insects suggests that peptides from peripheral glands and from the
central nervous system interact to trigger the pre-ecdysis and ecdysis
behaviours (e.g. Gammie and Truman,
1999). Declining ecdysteroid titres initiate the release of
various peptides from the epitracheal glands. These peptides activate
pre-ecdysis behaviours. One of these peptides, ETH, also promotes the release
of EH from the brain (Ewer et al.,
1997
; Gammie and Truman,
1999
). EH stimulates the CCAP-containing neurons, cells 27 and
704, in the ventral nerve cord via an increase in cGMP levels
(Ewer et al., 1994
;
Gammie and Truman, 1997a
). It
may also stimulate the L2 and L5 neurons via
cGMP. As discussed above, these cells may contain cardioacceleratory peptides
or bursicon (Tublitz and Sylwester,
1990
), but their role in the ecdysis sequence is unclear. CCAP
from cells 27 and 704 then releases the ecdysis motor program
(Gammie and Truman, 1999
). The
onset of the ecdysis phase, however, involves an interplay between excitatory
and inhibitory influences (Baker et al.,
1999
; Ewer and Truman,
1997
; Gammie and Truman,
1997b
; Zitnan and Adams,
2000
). It is clear that the brain is required for the onset of the
ecdysis behaviour itself (Novicki and
Weeks, 1996
), but this requirement ends with the release of EH
(Ewer et al., 1997
;
Novicki and Weeks, 1996
).
Inhibition, however, does not require the brain, but rather the lower ganglia
(Fig. 4). The sites of
inhibition include the SOG and one or more of the thoracic ganglia. This is
the case for both larvae and pupae (Fig.
4) and is probably also the case for adults
(Ewer and Truman, 1997
;
Mesce and Truman, 1988
). It
will be interesting to determine whether this is a common feature of ecdysis
in insects in general since inhibitory factors from the head that affect
ecdysis behaviours have also been noted in Diptera
(Baker et al., 1999
) and
Orthoptera (Carlson, 1977
).
There are two options for the regulation of inhibition during ecdysis: (i)
EH may co-activate the CCAP cells and the descending inhibitory cells or (ii)
suppression may be tonic, as is seen for behaviours such as walking
(Roeder, 1967), and thereby be
independent of control by EH. While this issue is not easily resolved, we
favour the first option. In Drosophila melanogaster, mutants lacking
EH-positive neurons (EH cell knockouts) show no signs of this descending
inhibition, while the controls show the same pattern of descending inhibition
seen in Manduca sexta (Baker et
al., 1999
). Inclusion of the descending inhibitory neurons among
the EH target cells is also consistent with the anatomy of some of these
neurons and with the dynamics of cGMP seen in these cells. A prominent feature
of the cell 27/704 system is the paired 704 cells in the labial neuromere of
the SOG and in each of the thoracic ganglia. These neurons have axon
collaterals that descend into the abdomen and make apparent contact with the
abdominal 27/704 neurons. Interestingly, these neurons show an increase in
cGMP levels coincident with that of the abdominal 27/704 cells, but their cGMP
levels decline more rapidly in the descending processes compared with the
abdominal neurons (Ewer et al.,
1994
). The rise in intracellular cGMP levels causes an increase in
the excitability of EH target cells
(Gammie and Truman, 1997a
).
Thus, the rapid rise in cGMP levels in the abdominal 27/704 cells, and in
their putative inhibitors, would cause an activation of the network, but CCAP
release from the abdominal neurons would be held in check by the activity of
the descending inhibitors. The early decline in cGMP levels in the inhibitors
would then result in the waning of the suppression, with the eventual `escape'
of the abdominal neurons, leading to the release of CCAP and, hence, ecdysis
(Fig. 10). This hypothesis is
consistent with the results of experiments blocking cGMP breakdown using a
specific cGMP phosphodiesterase inhibitor, Zaprinast
(Fig. 6). Addition of Zaprinast
caused a prolonged latency to the onset of ecdysis, presumably by prolonging
the time that the descending neurons continued to be active because of their
elevated cGMP levels.
|
The increase in cell excitability elicited by cGMP in descending inhibitory neurons, as well as in the abdominal CCAP neurons, is a simplistic model that assumes that increases in firing rates of each neuron alone provide the inhibition noted in Manduca sexta ecdysis. However, it does generate predictions that can be tested experimentally in future studies.
One or more of the thoracic ganglia form a second source of inhibition
modulating the onset of ecdysis. The thoracic ganglia also appear to be
necessary for maintaining the ecdysis motor program once it has commenced
(Fig. 5). They have already
been shown to play a large role in modulating eclosion in adults
(Mesce and Truman, 1988) and
they are the source of neuronal inputs, which modulate the conserved larval
abdominal motor program to produce the adult motor pattern used for shedding
the rigid pupal cuticle and for digging through the soil. Whether they are
also the source of inhibition modulating the timing of ecdysis in adults is
not yet known.
CO2 and release of the ecdysis behaviour
CO2 provides a powerful tool for studying ecdysis
(Fig. 7). Treatment of animals
with a short pulse of CO2 shortly before the onset of ecdysis
results in its premature onset. This response is conserved in larvae
(Fig. 7), pupae (data not
shown) and in adults (Fig. 9).
The response does not appear to be a result of anoxia since treatment with
nitrogen gas (Fig. 7A) or cold
(data not shown) has no effect on larvae.
CO2 does not affect the timing of ecdysis if it is applied prior to EH release, when cGMP is not yet detectable in the CCAP neurons, suggesting that CO2 does not act directly on (i) the ecdysis motoneurons, (ii) the muscles involved in ecdysis or (iii) the as yet inactive CCAP neurons. CO2 treatment becomes effective only after EH release. In the context of the EH target cells, CO2 may act directly to further excite EH-activated CCAP cells or it may act to block the descending inhibition onto these cells. We cannot yet distinguish experimentally between these two possibilities. However, once the CO2 has acted, the system cannot revert to its `waiting' mode.
Unlike simple anoxia, CO2 can act as an anesthetic in insects by
blocking synapses (Clark and Eaton,
1983; Perron et al.,
1972
; Sillans and Biston,
1979
). Since insects go through a transient hyperexcitable phase
before they become anesthetized, it may be that inhibitory circuits are more
sensitive to CO2 than are excitatory ones. CO2 has been
shown to inhibit cercal grooming in cockroaches
(Eaton and Farley, 1969
), but
also specifically reduces the efficacy of
-aminobutyric acid (GABA)
activity in inhibitory neurons of honeybees
(Kashin, 1973
). We favour the
hypothesis that the mechanism for CO2 action on the central nervous
system of larval and pupal Manduca sexta during ecdysis is probably
through releasing the inhibition provided by the SOG and thoracic ganglia.
The eclosion behaviour of the adult is strikingly different from that of
larvae and pupae because adults need to shed a rigid sheath of pupal cuticle
and to dig out from their underground pupation cell. This is accomplished
through two-phase, retraction/extension movements of the abdomen in which the
extension phase is coupled with `shrugging' movements at the wing base
(Kammer and Kinnamon, 1977;
Mesce and Truman, 1988
).
Although the adult behaviour is markedly different from the larval and pupal
behaviours, it is, nevertheless, based on the larval ecdysis central pattern
generator (Mesce and Truman,
1988
). This was shown by nerve cord transection or by using a cold
block to uncouple the abdomen from descending units from the thorax. In the
absence of the thoracic input, the abdominal extension phase of the cycle
immediately disappeared and the retraction phase transformed into the
characteristic larval peristaltic wave. In the animals that showed only
abdominal movements after the CO2 treatment
(Fig. 8), the movements were
similar to those seen when the descending thoracic units are blocked. That is,
they were primarily peristaltic in character. These movements did not,
however, persist with any regularity in CO2-treated animals.
In pharate adults that have released EH, decapitation results in the rapid
onset of a normal adult ecdysis behaviour that includes both the thoracic and
abdominal components (Ewer and Truman,
1997). CO2 treatment also triggers premature ecdysis
and, as in larvae and pupae, it appears to do so only in those that have
already released EH (Fig.
9B-D). The CO2 effects differ from decapitation,
however, in that often only the abdominal component is displayed immediately
after treatment. After a lag, CO2-treated animals eventually
activate thoracic components that allow them to shed their entire pupal
cuticle, and this also appears to be early relative to controls
(Fig. 9C-E). These differences
suggest that the larval- and adult-specific components of ecdysis are under
inhibitory control, but that each is under a distinct inhibitory pathway. The
abdominal component of adult ecdysis shows the same type of CO2
sensitivity as seen in larvae and pupae. Thus, as in the earlier stages,
CO2 appears to relieve descending inhibition that controls the
activation of the persisting larval ecdysis pattern generator. A separate set
of descending units may regulate the adult-specific portions of the behaviour
that include the thoracic flexions and coupled abdominal extensions, but these
seem not to be readily activated by CO2 exposure. The thoracic
portion of the behaviour eventually appears with time, and the entire adult
behaviour is typically expressed somewhat earlier than in controls. Thus, the
adult form of the behaviour appears to include not only the larval ecdysis
pattern generator but also some of the neurons that control the timing of its
expression. At metamorphosis, the animal appears to add additional motor
components and also additional regulatory circuitry to produce the final adult
behaviour.
Although CO2 treatment has a marked effect on the timing of ecdysis, it does not appear to affect other aspects of the ecdysis sequence, such as the release of EH itself. Also, the timing of the post-eclosion event, wing expansion, remains undisturbed relative to the time of ecdysis. CO2 treatment may, therefore, turn out to be a non-invasive and far more useful tool than decapitation for studying the inhibitory influences on ecdysis. Moreover, multiple components of eclosion may be uncoupled in adults by this means.
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