Localization of myoinhibitory peptide immunoreactivity in Manduca sexta and Bombyx mori, with indications that the peptide has a role in molting and ecdysis
1 Division of Neurobiology, University of Arizona, Box 210077, Tucson, AZ
85721-0077, USA
2 Insect Biocontrol Laboratory, USDA, ARS, PSI, BARC-West, Beltsville, MD
20705, USA
* Author for correspondence (e-mail: ntd{at}neurobio.arizona.edu)
Accepted 13 January 2003
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Summary |
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Key words: tobacco hornworm, Manduca sexta, Bombyx mori, CCAP, ecdysteroids, prothoracicostatic peptide, ecdysterostatic hormone, neurosecretion, allatostatin, epiproctodeal gland
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Introduction |
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In the later stages of adult development there is a rapid decline in
ecdysteroid titer, and this decline has been shown to be essential for
initiating the programmed cell death that accompanies adult metamorphosis
(Schwartz and Truman, 1983).
During the molt from the fourth to the fifth larval instar, the drop in the
ecdysteroid peak is even more precipitous
(Bollenbacher et al., 1981
;
Langelan et al., 2000
). This
rapid decline in ecdysteroid titer is essential for activating endocrine cells
that initiate ecdysis behavior
(Sláma, 1980
;
Truman et al., 1983
;
Truman and Morton, 1990
;
Hewes and Truman, 1994
;
it
an et al.,
1999
; Kingan and Adams,
2000
). In addition, the decline in ecdysteroid titer is essential
for the initiation of cuticular melanization in larvae
(Curtis et al., 1984
).
The rapidly rising phase of the ecdysteroid titer results from activation
of the prothoracic glands by prothoracicotropic hormone (PTTH; see review by
Bollenbacher and Granger,
1985). Several factors that inhibit rather than stimulate
ecdysteroid production have been identified in insects (reviewed by
Gäde et al., 1997
;
Hua et al., 1999
;
Dedos et al., 2001
), and these
factors may be involved in promoting the rapidly declining phase of the
ecdysteroid peaks. A neuropeptide that acts, in vitro, on the
prothoracic glands to inhibit PTTH-stimulated secretion of ecdysone by the
silkworm Bombyx mori is of special interest to our study
(Hua et al., 1999
;
Dedos et al., 2001
). Hua et al.
(1999
) found this
prothoracicostatic peptide to be identical to a myoinhibitory peptide, Mas-MIP
I (Manduca sexta myoinhibitory peptide I), which was first isolated
and identified from M. sexta by Blackburn et al.
(1995
).
Mas-MIP I belongs to the W2W9amide peptide family that is characterized by
tryptophan residues at the 2 and 9 positions
(Lorenz et al., 2000).
Peptides of this family have been identified in a locust
(Schoofs et al., 1991
), moths
(Blackburn et al., 1995
,
2001
;
Hua et al., 1999
), a cricket
(Lorenz et al., 1995
), a stick
insect (Lorenz et al., 2000
)
and a cockroach (Predel et al.,
2001
). In addition, a gene for these peptides was cloned in B.
mori (Hua et al., 2000
)
and Drosophila melanogaster
(Williamson et al., 2001
).
These various reports suggest that the W2W9amide peptides are of widespread
occurrence in insects. Moreover, bioassays indicate that, in addition to the
prothoracicostatic effect mentioned above, these peptides may inhibit myogenic
contraction of visceral muscles in locusts, moths and cockroaches
(Schoofs et al., 1991
;
Blackburn et al., 1995
;
Predel et al., 2001
) and JH
and ovarian ecdysteroid synthesis in crickets (Lorenz et al.,
1995
,
2000
).
As yet, the endocrinal source of the MIP/prothoracicostatic peptide has not
been demonstrated conclusively. Triseleva and Golubeva
(1998), using an antiserum
raised to Mas-MIP I, found MIP-immunoreactive (MIP-IR) cells in the
subesophageal and abdominal ganglia and on the hindgut of adults of the
tobacco budworm Heliothis virescens, and Hua et al.
(2000
) labeled a pair of
MIP-IR neurosecretory cells in the brain of B. mori. Therefore, using
an antiserum to Mas-MIP I (Triseleva and
Golubeva, 1998
), we have undertaken to identify the MIP-IR
neuroendocrine cells that may be a source of an MIP/prothoracicostatic hormone
in M. sexta. The principal focus of our study has been the
fourth-instar larval stage, because the hormonal control of molting of this
stage has been well studied and because this stage is uncomplicated by
metamorphic changes. We have also examined MIP immunoreactivity in
fourth-instar larvae of B. mori.
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Materials and methods |
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Eggs of Bombyx mori L. were obtained from Carolina Biological Supply Company (Burlington, NC, USA), and the larvae were fed freshly collected mulberry leaves and kept under the same conditions as above.
Staging of fourth-instar larvae of M. sexta
Fourth-instar larvae were used to study the secretory cycle of the
MIP-immunoreactive (MIP-IR) epiproctodeal gland. This instar may be divided
into pre-molting and molting phases, and the initiation of the molting phase
is photo-gated. Molting of Gate I, fourth-instar larvae begins with the
release of PTTH late in the second scotophase after ecdysis, and molting of
Gate II larvae begins with release of PTTH early in the third scotophase
(Truman, 1972). Gate II larvae
were used to study the epiproctodeal glands during the molting cycle because
the onset of the cycle is well synchronized by the photo-gate. Groups of Gate
II larvae were isolated by the method described by Fain and Riddiford
(1975
). Glands from day 0, day
1 and day 2 of Gate II larvae were used to study the pre-molting phase of the
instar.
In order to study the glands at specific stages during the molting phase,
we used the external markers described by Fain and Riddiford
(1975), Curtis et al.
(1984
) and Langelan et al.
(2000
). These markers are
based on: (1) separation of the seventh abdominal spiracular plate from the
old cuticle (spiracular apolysis) and subsequent development of this pharate
spiracular plate of the fifth-instar larva; (2) slippage of the pharate head
capsule; (3) tanning of the pharate crochets of the prolegs; and (4)
appearance of air in the old head capsule (airheads) shortly before ecdysis to
the fifth instar.
Endocrine glands from the following stages of molting were studied: (1)
early onset of spiracular apolysis, which begins several hours after the
release of PTTH and is recognized by a gradual lightening of color in a small,
crescent-shaped area immediately above the spiracle
(Langelan et al., 2000); (2)
approximately 4 h prior to head-capsule slippage, the time at which the
pharate spiracular plate is approximately 250 µm longer than that of the
fourth-instar larva; (3) onset of head-capsule slippage, which occurs
approximately 12 h after the onset of spiracular apolysis and is recognized by
the separation of the stemmata from the old cuticle, as the pharate head
begins a slow retraction into the prothoracic exoskeleton of the fourth-instar
larva; (4) 5 h and (5) 10 h after the onset of head-capsule slippage, stages
which were obtained by timing larvae from the onset of head-capsule slippage;
(6) tanning of the crochets, which occurs approximately 18 h after
head-capsule slippage; and (7) airheads, which appear approximately 28 h after
the onset of head-capsule slippage.
Immunocytochemistry
The insects were anesthetized by chilling on crushed ice, and tissues were
dissected in cold, physiological saline solution
(Pichon et al., 1972). The
tissues were then fixed for 12 h in cold 4% paraformaldehyde and immunostained
as whole mounts or as 200-µm sections cut on a Vibratome (Technical
Products International, St Louis, MO, USA). The fluorescence
immunocytochemistry was performed as described previously
(Davis et al., 1993
). Briefly,
the tissues were processed as follows. After fixation and after incubations in
the primary and secondary antisera, respectively, the tissues were washed
thoroughly in phosphate-buffered saline (pH 7.2) containing 1% Triton X-100
(PBST). Normal donkey serum (10%) in PBST was used as the blocking solution
and as the diluent for the various antisera. The polyclonal antiserum to
Mas-MIP I was raised as described by Triseleva and Golubeva
(1998
) and, using whole mounts
of the CNS of third-instar larvae of M. sexta, we tested the
immunostaining by this antiserum at dilutions ranging from 1:500 to 1:10 000.
Because a dilution of 1:2000 appeared to give optimum results, this dilution
of the antiserum was used routinely. It was applied for approximately 12 h at
room temperature and with gentle rotation. In addition, antisera to a
cockroach-type allatostatin, to crustacean cardioactive peptide (CCAP) and to
molluscan small cardioactive peptide B (SCPB) were used under the
same conditions. Goat anti-rabbit immunoglobulin G (IgG) conjugated to
tetramethyl rhodamine isothiocyanate (TRITC) or to fluorescein isothiocyanate
(FITC), and goat anti-mouse IgG conjugated to FITC (Jackson ImmunoResearch
Laboratories, West Grove, PA, USA) were used as secondary antisera and applied
at a dilution of 1:200 for 12 h at room temperature and with gentle
rotation.
Double-immunostaining to identify the MIP-IR neuroendocrine cells of the
adult brain was accomplished using the rabbit polyclonal antiserum to MIP and
the mouse monoclonal antibody to SCPB, and these antisera were
labeled, respectively, with TRITC-conjugated goat anti-rabbit IgG and
FITC-conjugated goat anti-mouse IgG. In addition, double-staining of
neuroendocrine cells was performed using FITC-conjugated, goat anti-rabbit IgG
to label the anti-MIP, and then, after treatment with normal rabbit serum to
block any unbound goat anti-rabbit IgG binding sites, the tissue was
immunostained with TRITC-conjugated, rabbit anti-allatostatin
(Davis et al., 1997). The
method described by Nichols et al.
(1995a
,b
)
was used for double-immunostaining for CCAP
(Davis et al., 1993
) and MIP.
The tissue was first treated with a rabbit antiserum to CCAP and labeled with
an excess (1:100) of Rhodamine Red-X-conjugated Fab fragment of anti-rabbit
IgG (H+L) from goat (Jackson ImmunoResearch Laboratories). Then, after
thorough washing, the tissue was treated with rabbit antiserum to MIP and
labeled with FITC-conjugated anti-rabbit IgG from goat.
To test the capacity of the MIP antiserum to recognize epitopes of Mas-MIP
I, the working dilution of the antiserum was subjected to liquid-phase
preadsorption for 24 h at 4°C in 104 mol
l1 synthetic Mas-MIP I; this treatment eliminated all
specific immunostaining. Davis et al.
(1993,
1997
) previously characterized
the immunostaining of M. sexta tissues by antisera to CCAP and
allatostatin and to the antibody to SCPB.
In addition to the immunostaining, the RNA in the gland cells was stained
with Acridine Orange according to the histochemical method of Kiernan
(1990).
Neuronal tracing
The axonal projections to their targets (orthograde) were traced by filling
through stumps of the proctodeal nerve. Biocytin (Sigma, St Louis, MO, USA)
was used for filling towards peripheral targets and was labeled with
TRITC-conjugated streptavidin (Jackson ImmunoResearch Laboratories), following
the methods of Consoulas et al.
(1999). The filling period was
12 days at approximately 5°C.
Laser scanning confocal microscopy
Digitized images of the immunostained and filled preparations were obtained
using a Nikon E800 confocal microscope equipped with green He/Ne and argon
lasers and the appropriate filters. Optical sections (5 µm) of
digitized images were merged and processed using software programs of Simple
PCI (Compix Inc., Cranberry Township, PA, USA), Corel Draw and Corel
Photopaint (Corel Corp., Ottawa, Ontario, Canada). Prints were made using a
Tektronix Phaser SDX. Where needed, the digitized images were modified only to
merge files, enhance contrast and provide color.
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Results |
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Because no MIP-immunoreactive neurons were found in the frontal ganglion or in the recurrent nerve (not shown), it appears that MIP does not function as a myoactive peptide in the stomatogastric nervous system.
Ganglia of the larval ventral nerve cord contained several MIP-IR somata, and, of these, a pair of MIP-IR neurons in the abdominal ganglia were identified as the 704 interneurons (Fig. 1C). These cells are discussed in further detail below. Neurons of the ventral nerve cord cells did not project to the perivisceral organs (PVO; Fig. 1C), and, thus, there was no evidence of release of MIP-IR neuropeptides from neuroendocrine cells into the ventral nerve cord of larvae. Except for the terminal abdominal ganglion (TAG), MIP-IR processes did not extend into nerves of the ganglia of the ventral nerve cord. Therefore, there was no evidence that MIP-IR neurons in these pre-terminal ganglia innervate any peripheral organs, including the prothoracic glands.
The MIP antiserum labeled several large, mid-line and lateral neurons in the TAG (Fig. 1D), and processes of these cells extended into dorsal nerve 8 (curved arrow) and the terminal nerve (straight arrow). These MIP-IR cells will be considered in a separate publication, but it should be noted here that they do not project to neurohemal release sites and, thus, are not neuroendocrine. Instead, they mostly innervate muscles of the hindgut (Fig. 1E) via projections into the terminal nerve and thence into the proctodeal nerve. The larval hindgut and its innervation by the proctodeal nerve are depicted in Fig. 2.
|
The epiproctodeal glands of M. sexta larvae
The antiserum to MIP labeled two pairs of large, multinuclear,
neuroendocrine cells on the proctodeal nerve of all larval and pupal stages.
Although these are peripheral neurosecretory cells, we will show that they
have a very specialized structure and a secretory function that appears to be
independent of the nervous system. We therefore have chosen to name them the
epiproctodeal glands. Each gland is attached to the proctodeal nerve just
anterior to the junction of the rectum and hindgut (Figs
2,
3A) and, at this location, the
gland and proctodeal nerve are anchored tightly to the hindgut by
connective-tissue fibers. The structure of the cells of this neuroendocrine
gland were first described by Reinecke et al.
(1978), who noted that they
are multinucleate (Fig. 3B) and
contain many dense-core vesicles.
|
Many varicose processes extend from the gland cells onto the surface of the proctodeal nerve and its branches (Fig. 3A). These processes do not extend onto the terminal nerve, and no processes were found extending onto the musculature of the hindgut. These superficial, varicose processes are typical of those known to serve for neurohemal release; therefore, the immunoreactivity of the epiproctodeal glands indicates that they release an MIP-like peptide into the hemolymph.
Because backfills from the base of the terminal nerve to the proctodeal nerve did not stain the cells of the epiproctodeal gland, we concluded that processes of these cells do not extend into the TAG (Fig. 3C). Moreover, these preparations showed that axons from the TAG do not innervate the epiproctodeal glands (Fig. 3C).
While immunostaining the epiproctodeal glands, we noted that their appearance changed during the course of the fourth larval instar. We therefore wished to determine if this variation might represent stages of synthesis and release of neurosecretory products. In order to obtain comparable staining of glands at various stages of the instar, we used identical processing and MIP-immunostaining schedules as well as identical gain and black-level settings for the confocal images. Ten or more glands were stained at each of the three pre-molting and seven molting stages indicated in the Materials and methods. Each image shown in Fig. 4 and Fig. 5 was selected as representative of the designated stage in the premolting or molting phases of the fourth-instar larva.
|
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On day 0, the immunostaining of the neurohemal system of the epiproctodeal gland was distinct (Fig. 4A) and, by the onset of spiracular apolysis, the staining had become intense (Fig. 4B). At approximately 4 h before head-capsule slippage, the immunostaining of the neurohemal system appeared to show a moderate decline (Fig. 4C), and at this time many of the varicosities appeared to be partly vacuous (Fig. 4I). By the onset of head-capsule slippage, the decline in immunostaining was pronounced (Fig. 4D). Five hours after head-capsule slippage, most of the immunostaining of the neurohemal system had disappeared (Fig. 4E), but the cell bodies had become intensely immunoreactive (arrow). The extent of immunostaining of the neurohemal system appeared to increase somewhat by 10 h after head-capsule slippage (Fig. 4F) and had increased conspicuously by the stage of tanning of the crochets (Fig. 4G). By the airhead stage, the immunostaining of the gland (Fig. 4H) was comparable to that of the gland in the pre-molting phase fourth-instar larvae (Fig. 4A).
The intensity of immunostaining of the epiproctodeal-gland cells also appeared to undergo changes related to the premolting and molting stages of the fourth larval instar. Images of these changes were recorded using a 5-µm, optical section made near the middle of typical gland cells of each stage and using the same settings of the confocal microscope for each image. The intensity of MIP-immunostaining of the cytoplasm was low during the pre-molting stage (Fig. 5A,B), but staining became distinctly stronger by the onset of spiracular apolysis (Fig. 5C). By the onset of head-capsule slippage (Fig. 5D), and continuing for at least 5 h (Fig. 5E), the immunostaining of the cytoplasm was intense. Ten hours after head-capsule slippage, the staining had decreased (Fig. 5F), and this decrease in staining continued at the crochet-tanning (Fig. 5G) and airhead (Fig. 5H) stages to become comparable to the staining of the cells at the pre-molting stage.
MIP-IR neuroendocrine cells of adult M. sexta
Immunostaining of Vibratome sections of the brain of adults demonstrated
two pairs of MIP-IR median neurosecretory cells in the brain
(Fig. 3D; green), and the
processes of these cells were found to project to neurohemal release sites in
the corpora cardiaca (Fig. 6A).
In M. sexta, there are only two groups of median neuroendocrine cells
present as two pairs, the type 1A4 and 1A5 cells
(Homberg et al., 1991). The
1A5 cells are labeled uniquely by the monoclonal antibody to
SCPB (Davis et al.,
1997
) and can be distinguished, thereby, from the 1A4
cells. Vibratome sections were double-labeled using rabbit anti-MIP and mouse
anti-SCPB, as indicated in the Materials and methods. The results
shown in Fig. 3D indicated that
the MIP-IR staining (green) is not co-localized with the SCPB-IR
staining (red) of the 1A5 cells
(Fig. 3D), and the MIP-IR
neuroendocrine cells must, therefore, be the 1A4 cells. The
1A4 cells are also known to be allatostatin-immunoreactive
(Davis et al., 1997
) and, as
further proof of the identity of the MIP-IR neuroendocrine cells, brain
sections were doubly stained for allatostatin and MIP. The results indicated
that allatostatin and MIP immunoreactivities are co-localized in the
1A4 neurosecretory cells (Fig.
3E, yellow).
|
In adults, as in larvae, no evidence was found of neurohemal release of MIP from the abdominal ganglia (Fig. 6BD). About mid-way in the development of the pharate adult, two pairs of neurons, additional to the 704 interneurons of larvae, were labeled for MIP-IR, but their projections indicated that they are interneurons rather than neuroendocrine cells (Fig. 6B). By near the time of adult eclosion, the immunoreactivity of all of these cells had decreased markedly (Fig. 6C) such that, in the fully mature adult, there was almost no MIP immunoreactivity in the abdominal ganglia (Fig. 6D). In the TAG of the pharate adult near mid-way in development, the interneurons, as well as the visceromotor neurons that innervate the hindgut, were weakly labeled (Fig. 6E), and in the fully mature adult, there was almost no MIP immunoreactivity in the TAG (Fig. 6F).
The epiproctodeal glands persisted in the pupa and developing, pharate adult (Fig. 6G). In addition to the nerves of the hindgut, a very extensive meshwork of MIP-IR neurohemal processes developed on each side of the anterior rectal chamber (Fig. 6G,H), and, as in the larval stage, these processes originated from the epiproctodeal gland cells. By mid-way through metamorphosis, MIP-immunostaining of the epiproctodeal gland cells was barely discernible (Fig. 6G), and by the adult stage, the varicosities of the neurohemal system appeared to be partly depleted (Fig. 6H).
MIP-IR neuroendocrine cells of B. mori larvae
Because the prothoracicostatic effect of MIP has been demonstrated only in
B. mori (Hua et al.,
1999), it was of interest to identify the MIP-IR neuroendocrine
cells in this species. A single median neurosecretory cell was immunostained
in each hemisphere of the larval brain
(Fig. 7A; arrowhead), and this
cell projects ipsilaterally (arrow) to neurohemal release sites in the corpora
cardiaca (Fig. 7B). As in
M. sexta, no neuroendocrine cells were found in the ganglia of the
ventral nerve cord, and no evidence was found indicating innervation of
prothoracic glands by MIP-IR axons. MIP-immunostaining demonstrated the
epiproctodeal glands and their neurohemal system
(Fig. 7C), and their appearance
in the day 2, fourth-instar larva was much the same as that of M.
sexta.
|
MIP immunoreactivity of the 704 interneurons in larvae of M.
sexta and B. mori
In the abdominal ganglia of larvae of M. sexta, the antiserum to
MIP labeled lateral cells located in a position very similar to that of a pair
previously shown to be CCAP-immunoreactive and identified as interneurons 704
and neurosecretory cells 27 (Davis et al.,
1993). Double labeling with anti-MIP and anti-CCAP demonstrated
that immunoreactivities to these antisera are co-localized in the 704
interneurons (Fig. 8A,B) but
not in the CCAP-IR cells 27 (Fig.
8A).
|
Gammie and Truman (1997)
found that at the time of ecdysis, the level of CCAP immunoreactivity
decreases abruptly in the 704 neurons, and so we wished to know if a similar
change occurred in the MIP immunoreactivity of the 704 neurons. Thirty
abdominal ganglia of third-instar larvae at the stage of crochet tanning and
thirty at the time of ecdysis were immunostained in tandem, and their confocal
images were prepared using the same settings for gain and darkness. Ganglia
stained several hours before ecdysis consistently showed strong
MIP-immunostaining of processes in the neuropil and the somata of interneurons
704 (Fig. 8C), but at ecdysis
the ganglia consistently showed a pronounced decline in immunostaining
(Fig. 8D). The MIP
immunoreactivity of the 704 interneurons persisted in the pharate adult
(Fig. 6B,C) but was no longer
present in the fully mature adult (Fig.
6D).
Unlike the abdominal ganglia of M. sexta, two pairs of lateral neurons were immunostained in B. mori (Fig. 8E), and it was not immediately apparent if either of these were comparable with the 704 neurons. Therefore, double-immunostaining was performed with the antisera to MIP and CCAP. Immunostaining of abdominal ganglia of B. mori for CCAP labeled pairs of lateral cells comparable with cells 27 and 704 in M. sexta (Fig. 8F). Double-staining indicated that CCAP-IR is co-localized with MIP-IR in the anterior pair of MIP-IR cells, and, therefore, these cells are the 704 interneurons of B. mori (Fig. 8G; yellow). The possible release of an MIP-like peptide from these neurons at ecdysis in B. mori was not studied.
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Discussion |
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In the fourth-instar larva of M. sexta, the epiproctodeal glands
undergo pronounced changes in secretory activity, and these changes are
synchronized with the molting cycle. Immunostaining indicated that during the
latter part of this cycle, an MIP-like peptide begins to accumulate in the
neurohemal system of the glands (Fig.
4F,G) and that this accumulation continues into the pre-molting
period (Fig. 4A,H). In
fourth-instar larvae, immunostaining of the neurohemal system is most intense
just before spiracular apolysis (Fig.
4B), apparently indicating a maximum accumulation of the MIP-like
peptide at this time; Langelan et al.
(2000) have shown that this
time is near the onset of release of ecdysone from the prothoracic glands. At
approximately 4 h before head-capsule slippage, the intensity of
MIP-immunostaining started to decline (Fig.
4C). By the onset of head-capsule slippage, the decline in
immunostaining was very pronounced and was almost complete 5 h after
head-capsule slippage (Fig.
4D,E). It seems reasonable to conclude that this rapid loss of
MIP-immunostaining in the neurohemal system is the result of massive release
of the MIP-like peptide into the hemolymph. This period of rapid secretion
(from approximately 4 h before to several hours after head-capsule slippage)
corresponds exactly to the time in the molting cycle at which there is a rapid
decline in the ecdysteroid titer of the fourth-instar larva
(Langelan et al., 2000
). Hua
et al. (1999
) have shown that
MIP has a prothoracicostatic effect in B. mori, and we have
demonstrated that the epiproctodeal glands are the principal source of
secretion of an MIP-like peptide in B. mori and M. sexta.
Therefore, we believe that this period of massive secretion by the proctodeal
gland is largely responsible for the rapid decline of the ecdysteroid peak in
fourth-instar larvae.
Immunostaining also appeared to demonstrate a cycle of MIP synthesis by the epiproctodeal gland cells. During the premolt period, there is a low level of immunostaining in the cytoplasm (Fig. 5A,B), apparently indicating that, as the neurohemal system reaches an optimum level of accumulation of the MIP-like peptide, there is a low level of synthesis (Fig. 4B). By the onset of spiracular apolysis i.e. early in the molting cycle the intensity of immunostaining of the gland cells increased (Fig. 5C), apparently indicating an increase in the rate of synthesis of the MIP-like peptide. The immunostaining of the cells was intense during the first few hours of head-capsule slippage (Fig. 5D,E), indicating a high rate of synthesis at this time. Then the staining indicated a gradual decline in the rate of synthesis of the MIP-like peptide (Fig. 5FH) until it reached a level similar to that of the premolting phase.
The MIP-immunostaining suggested that the initiation of synthesis and release of the MIP-like peptide occurred at about the same time. The release was completed, however, in a relatively short period, while it appeared that considerable time was required for the gland cells to synthesize and transport the peptide so as to replenish the depleted neurohemal system. Our evidence from backfills indicated that the epiproctodeal glands are not innervated (Fig. 3C). Therefore, it seems very likely that the control of the glands is hormonal. Ecdysteroids are promising candidates as the source of this control, and the sudden rise in the ecdysteroid titer may initiate both synthesis and release of the MIP-like peptide by the proctodeal gland. If this hypothesis proves to be correct, then the mechanism responsible for the decline in the ecdysteroid titer in the molting cycle of the fourth-instar larva may be viewed as a delayed negative-feedback loop; ecdysteroids initiate the release of a prothoracicostatic hormone, and this hormone then inhibits release of ecdysteroids.
In the adult of M. sexta, the 1A4 median neuroendocrine
cells were shown to be MIP-immunoreactive, and these cells are also labeled by
antisera to allatostatin and diuretic hormone
(Davis et al., 1997).
Therefore, an MIP-like peptide may be co-released with allatostatin and
diuretic hormone in the adult, but its function in the adult remains to be
studied. The appearance of the epiproctodeal glands in the adult suggests that
they are not functional, but it has been shown in adults of M. sexta
that MIP inhibits the myogenic rhythm of contraction of the hindgut
(Blackburn et al., 1995
).
Therefore, the target of MIP released from the 1A4 median
neuroendocrine cells of the brain might be the hindgut.
In larvae of M. sexta, the antiserum to MIP labeled a small group
of interneurons in the brain and ventral ganglia and visceromotor neurons in
the terminal ganglion but did not label any neuroendocrine cells in the CNS.
We have shown that the lateral pairs of 704 interneurons in the abdominal
ganglia of larvae of M. sexta, and comparable neurons in B.
mori, are MIP-immunoreactive, and this immunoreactivity is colocalized
with that of CCAP. Ewer et al.
(1998) have demonstrated cell
death of the 704 interneurons in fully mature M. sexta adults, and,
as expected, we have found that in adults, these cells are not labeled by
MIP-immunostaining.
In M. sexta, the level of MIP-IR in the 704 interneurons decreases
at the time of larval ecdysis, and Gammie and Truman
(1997) reported a similar
decrease in CCAP-IR. Moreover, these authors showed that application of CCAP
to de-sheathed abdominal ganglia results in generation of rhythmic bursts of
impulses associated with the ecdysis motor program, apparently indicating that
release of CCAP from the 704 interneurons activates this motor program. Our
results strongly suggest that MIP and CCAP are co-released at the time of
ecdysis and that MIP may also be involved in the initiation of the ecdysis
motor program.
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References |
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Blackburn, M. B., Jaffe, H., Kochansky, J. and Raina, A. K. (2001). Identification of four additional myoinhibitory peptides (MIPs) from the ventral nerve cord of Manduca sexta. Arch. Insect Biochem. Physiol. 48, 121-128.
Blackburn, M. B., Wagner, R. M., Kochansky, J. P., Harrison, D. J., Thomas-Laemont, P. and Raina, A. (1995). Identification of two myoinhibitory peptides, with sequence similarities to the galanins, isolated from the ventral nerve cord of Manduca sexta. Regul. Pept. 57,213 -219.[CrossRef][Medline]
Bollenbacher, W. E. and Granger, N. A. (1985). Endocrinology of the prothoracicotropic hormone. In Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol.7 (ed. G. A. Kurkut and L. I. Gilbert), pp.105 -151. New York: Pergamon Press.
Bollenbacher, W. E., Smith, S. L., Goodman, W. and Gilbert, L. I. (1981). Ecdysteroid titer during larvalpupaladult development of the tobacco hornworm, Manduca sexta. Gen. Comp. Endocrinol. 44,302 -306.[Medline]
Consoulas, C., Johnson, R. M., Pflüger, H. J. and Levine, R. B. (1999). Peripheral distribution of presynaptic sites of abdominal motor and modulatory neurons in Manduca sexta larvae. J. Comp. Neurol. 410,4 -19.[CrossRef][Medline]
Curtis, A. T., Hori, M., Green, J. M., Wolfgang, W. J., Hiruma, K. and Riddiford, L. M. (1984). Ecdysteroid regulation of the onset of cuticular melanization in allectomized and black mutant Manduca sexta larvae. J. Insect Physiol. 30,597 -606.
Davis, N. T., Homberg, U., Dircksen, H., Levine, R. B. and Hildebrand, J. G. (1993). Crustacean cardioactive peptide-immunoreactive neurons in the hawkmoth Manduca sexta and changes in their immunoreactivity during postembryonic development. J. Comp. Neurol. 338,612 -627.[Medline]
Davis, N. T., Veenstra, J. A., Feyereisen, R. and Hildebrand, J. G. (1997). Allatostatinlike-immunoreactive neurons in the tobacco hornworm, Manduca sexta, and isolation and identification of a new neuropeptide related to cockroach allatostatins. J. Comp. Neurol. 385,265 -284.[CrossRef][Medline]
Dedos, S. G., Nagata, S., Ito, J. and Takamiya, M. (2001). Action kinetics of a prothoracicostatic peptide from Bombyx mori and its possible signaling pathway. Gen. Comp. Endocrinol. 122,98 -108.[CrossRef][Medline]
Ewer, J., Wang, C.-M., Klukas, K. A., Mesce, K. A., Truman, J. W. and Fahrbach, S. E. (1998). Programmed cell death of identified peptidergic neurons involved in ecdysis behavior in the moth, Manduca sexta. J. Neurobiol. 37,265 -280.[CrossRef][Medline]
Fain, M. J. and Riddiford, L. M. (1975). Juvenile hormone titers in the hemolymph during late larval development of the tobacco hornworm, Manduca sexta. Biol. Bull. 149,506 -521.[Medline]
Gäde, G., Hoffmann, K. H. and Spring, J. H.
(1997). Hormonal regulation in insects: facts, gaps, and future
directions. Physiol. Rev.
77,963
-1032.
Gammie, S. C. and Truman, J. W. (1997).
Neuropeptide hierarchies and the activation of sequential motor behaviors in
the hawkmoth, Manduca sexta. J. Neurosci.
17,4389
-4397.
Hewes, R. S. and Truman, J. W. (1994). Steroid regulation of excitability of identified neurosecretory cells. J. Neurosci. 14,1812 -1819.[Abstract]
Homberg, U., Davis, N. T. and Hildebrand, J. G. (1991). Peptide-immunohistochemistry of neurosecretory cells in the brain and retrocerebral complex of the sphinx moth, Manduca sexta.J. Comp. Neurol. 303,35 -52.[Medline]
Hua, Y.-J., Tanaka, Y. and Kataoka, H. (2000). Molecular cloning of a prothoracicostatic peptide (PTSP) in the larval brain of the silkworm, Bombyx mori. XXI Internat. Cong. Entomol. 2,884 (Abstr.).
Hua, Y.-J., Tanaka, Y., Nakamura, K., Sakakibara, M., Nagata, S. and Kataoka, H. (1999). Identification of a prothoracicostatic peptide in the larval brain of the silkworm, Bombyx mori. J. Biol. Chem. 247,31169 -31173.[CrossRef]
Kiernan, J A. (1990). Histological and Histochemical Methods: Theory and Practice. 2nd edition. Oxford: Pergamon Press.
Kingan, T. G. and Adams, M. E. (2000).
Ecdysteroids regulate secretory competence in inka cells. J. Exp.
Biol. 203,3011
-3018.
Langelan, R. E., Fisher, J. E., Hiruma, K., Palli, S. R. and Riddiford, L. M. (2000). Patterns of MHR3 expression in the epidermis during a larval molt of the tobacco hornworm Manduca sexta.Dev. Biol. 227,481 -494.[CrossRef][Medline]
Lorenz, M. W., Kellner, R. and Hoffman, K. H.
(1995). A family of neuropeptides that inhibit juvenile hormone
biosynthesis in the cricket, Gryllus bimaculatus. J. Biol.
Chem. 270,21103
-21108.
Lorenz, M. W., Kellner, R., Hoffman, K. H. and Gäde, G. (2000). Identification of multiple peptides homologous to cockroach and cricket allatostatins in the stick insect Carausius morosus.Insect Biochem. Mol. Biol. 30,711 -718.[CrossRef][Medline]
Nichols, R., McCormick, J. and Caserta, L. (1995b). Cellular expression of the Drosophila neuropeptide DPKQDFMRFamide: evidence for differential processing of the FMRFamide polypeptide precursor. J. Mol. Neurosci. 6, 1-10.[CrossRef][Medline]
Nichols, R., McCormick, J., Lim, I. and Starkman, J. (1995a). Spatial and temporal analysis of the Drosophila FMRFamide neuropeptide gene product SDNFMRFamide: evidence for a restricted cellular expression pattern. Neuropeptides 29,205 -213.[Medline]
Nijhout, H. F. (1994). Insect Hormones. Princeton: Princeton University Press.
Pichon, Y., Sattelle, D. B. and Lane, N. J. (1972). Conduction processes in the nerve cord of the moth Manduca sexta in relation to its ultrastructure and haemolymph ionic composition. J. Exp. Biol. 56,717 -734.[Medline]
Predel, R., Rapus, J. and Eckert, M. (2001). Myoinhibitory neuropeptides in the American cockroach. Peptides 22,199 -208.[CrossRef][Medline]
Reinecke, J. P., Gerst, J., O'Gara, B. and Adams, T. S. (1978). Innervation of hindgut muscle of larval Manduca sexta (L.) (Lepidoptera: Sphingidae) by a peripheral neurosecretory neuron. Int. J. Insect Morphol. Embryol. 7, 435-453.
Schoofs, L., Holman, G. M., Hayes, T. K., Nachman, R. J. and De Loof, A. (1991). Isolation, identification and synthesis of locustamyoinhibiting peptide (Lom-MIP), a novel biologically active peptide from Locusta migratoria. Regul. Pept. 36,111 -119.[CrossRef][Medline]
Schwartz, L. M. and Truman, J. W. (1983). Hormonal control of rates of metamorphic development in the tobacco hornworm, Manduca sexta. Dev. Biol. 99,103 -114.[Medline]
Sláma, K. (1980). Homeostatic function of ecdysteroids in ecdysis and oviposition. Acta Entomol. Bohem. 77,145 -168.
Triseleva, T. A. and Golubeva, E. G. (1998). Immunohistochemical localization of myoinhibitory Mas-MIP-I-like peptides in Heliothis virescens (F.) (Lepidoptera, Noctuidae). Biology Bull. 25,588 -591. [Translated from Russian in Triseleva, T. A. and Golubeva, E. G. (1998). Izvestiya Akademii Nauk, Seriya Biologicheskaya, 6, 712-716.]
Truman, J. W. (1972). Physiology of insect rhythms. I. Circadian organization of the endocrine events underlying the moulting cycle of larval tobacco hornworms. J. Exp. Biol. 57,805 -820.
Truman, J. W. and Morton, D. B. (1990). The eclosion hormone system: an example of coordination of endocrine activity during the molting cycle of insects. Prog. Clin. Biol. Res. 342,300 -308.[Medline]
Truman, J. W., Roundtree, D. B., Reiss, S. E. and Schwartz, L. M. (1983). Ecdysteroids regulate the release and action of eclosion hormone in the tobacco hornworm, Manduca sexta (L.). J. Insect Physiol. 29,895 -900.
Williamson, M., Lenz, C., Winther, M. E., Nässel, D. R. and Grimmelikhuijzen, C. J. P. (2001). Molecular cloning, genomic organization, and expression of a B-type (cricket-type) allatostatin preprohormone from Drosophila melanogaster. Biochem. Biophys. Res. Commun. 281,544 -550.[CrossRef][Medline]
it
an, D., Ross, L. S.,
it
anova,
I., Hermesman, J. L., Gill, S. and Adams, M. E. (1999).
Steroid induction of a peptide hormone gene leads to orchestration of a
defined behavioral sequence. Neuron
23,523
-535.[Medline]