Localization of myoinhibitory peptide immunoreactivity in Manduca sexta and Bombyx mori, with indications that the peptide has a role in molting and ecdysis
Norman T. Davis1,*,
Michael B. Blackburn2,
Elena G. Golubeva2 and
John G. Hildebrand1
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
View larger version (140K):
[in a new window]
|
Fig. 1. Confocal images of myoinhibitory peptide (MIP)-immunostained whole mounts
of larval organs of Manduca sexta. (A) Brain of a thirdinstar larva,
showing numerous small, MIP-immunoreactive neurons; the size and location of
these cells indicate that they are not neurosecretory cells. (B) Corpus
allatum (CA) and corpus cardiacum (CC) of an early fifth-instar larva, showing
a lack of immunoreactive neurohemal processes. (C) Third abdominal ganglion of
a third-instar larva, showing a pair of interneurons (704) and a lack of
immunoreactive neurohemal processes in the perivisceral organ (PVO). (D)
Terminal abdominal ganglion of a third-instar larva showing lateral cells that
project into the eighth dorsal nerve (curved arrow) and a terminal cluster of
mid-line neurons that project into the terminal nerve (straight arrow). (E)
MIP-immunoreactive endings on muscles of the posterior region of the hindgut.
Scale bars, 100 µm.
|
|
View larger version (23K):
[in a new window]
|
Fig. 2. A depiction of larval hindgut innervation by the proctodeal nerve and the
location of the epiproctodeal gland.
|
|
View larger version (125K):
[in a new window]
|
Fig. 3. Confocal images of the larval epiproctodeal gland (A,B), axons in the
proctodeal nerve (C) and median neuroendocrine cells in the adult brain of
Manduca sexta (D,E). (A) Myoinhibitory peptide (MIP)-immunostaining
of a whole mount of the epiproctodeal gland (EpG) and its neurohemal processes
on the proctodeal nerve (PN) of a third-instar larva. In addition,
MIP-immunoreactive (MIP-IR) axons that extend from neurons in the terminal
ganglion and terminal nerve (TN) can be seen in the proctodeal nerve (arrow).
(B) Acridine Orange staining of a cell in a whole mount of an epiproctodeal
gland of a fourth-instar larva at a late stage of spiracular apolysis. The
stain demonstrates a high concentration of RNA in the cytoplasm at this stage;
the unstained spherical structures in the cell have been shown in previous
studies to be nuclei. (C) Whole mount of a Biocytin-stained, proctodeal nerve
filled from the base of the terminal nerve of a fourth-instar larva. The
location of one of the gland cells (EpG) can be distinguished by its weak
background staining. Arrows indicate the direction of filling with Biocytin.
Note that branches of these axons do not terminate on the epiproctodeal gland,
and, therefore, the gland apparently is not innervated. Moreover, the staining
failed to demonstrate any processes extending from the gland to the terminal
abdominal ganglion. (D) Double-immunostaining of MIP-IR (red) in the
1A4 cells and of small cardioactive peptide B (SCPB)-IR
(green) in the 1A5 median neuroendocrine cells. Vibratome section
(200 µm), frontal plane of the adult brain. (E) Double-immunostaining for
MIP (red) and allatostatin (green), showing that immunoreactivity to these two
peptides is co-localized (yellow) in the 1A4 cells. Vibratome
section (200 µm), frontal plane of the adult brain. Scale bars, 400 µm
(A) and 100 µm (B–E).
|
|
View larger version (76K):
[in a new window]
|
Fig. 4. Confocal images of whole mounts of epiproctodeal glands immunostained at
various times in the molting cycle of fourth-instar larvae, showing a cycle of
depletion and subsequent recovery of myoinhibitory peptide (MIP)-like
immunoreactivity in the neurohemal system. (A) Moderate staining of the
neurohemal system on day 0. (B) Intense immunostaining by the onset of
spiracular apolysis. (C) The immunostaining is almost unchanged at
approximately 4 h before head-capsule slippage. (D) A pronounced decline in
immunostaining is seen by the onset of headcapsule slippage. (E) Most of the
immunostaining of the neurohemal system is lost by 5 h after headcapsule
slippage but the cell bodies are now well stained (arrow). (F) There is a
moderate increase in staining of the neurohemal system by approximately 10 h
after head-capsule slippage. (G) The immunostaining continues to increase at
the crochet-tanning stage. (H) At the airhead stage, immunostaining of the
neurohemal system is comparable with that of the gland of the fourthinstar
larva at day 0. (I) An enlargement of a portion of the neurohemal system from
C, showing partly vacuous varicosities (arrows). Scale bars, 250 µm
(A–H) and 10 µm (I).
|
|
View larger version (102K):
[in a new window]
|
Fig. 5. Confocal images showing 5 µm optical sections through epiproctodeal
gland cells. The images were made at various times in the molting cycle of
fourth-instar larvae, and the changes in immunostaining indicate a period of
active synthesis of the myoinhibitory peptide (MIP)-like peptide early in the
molting cycle. At day 1 (A) and day 2 (B), the gland cells contain very little
MIP-immunoreactive (MIP-IR) material. (C) At the onset of spiracular apolysis,
distinct MIP-like immunoreactivity appears in the cytoplasm. (D) At the onset
and (E) 5 h after head-capsule slippage, the MIP-like immunoreactivity is
intense. (F) At 10 h after head-capsule slippage and (G) at the
crochet-tanning stage, the MIP-like immunoreactivity of the cytoplasm has
diminished. (H) At the airhead stage, the MIP-IR is weak and resembles that of
the gland cells at the start of the fourth instar (A). Scale bar, 50
µm.
|
|
View larger version (148K):
[in a new window]
|
Fig. 6. Confocal images of myoinhibitory peptide-immunoreactivity (MIP-IR) in
Manduca sexta adults. (A) Vibratome cross-section (200 µm) through
the corpora cardiaca (CC) and aorta, showing immunoreactive endings near the
surface of the CC. (B) Whole mount of the fifth abdominal ganglion of a
pharate adult at stage-13 of development, showing strong labeling of the 704
interneurons (arrowhead), plus an anterior and posterior pair of interneurons
(arrows), The latter are not labeled in the larval stages. (C) Whole mount of
the fifth abdominal ganglion of a pharate adult shortly before eclosion,
showing a pronounced reduction of MIP-immunostaining. (D) Whole mount of the
fifth abdominal ganglion of a day-3 adult, showing an almost complete lack of
staining. (E) Whole mount of the terminal abdominal ganglion of a stage-13
pharate adult, showing a reduction in MIP-immunostaining of the interneurons
(arrowheads) and visceromotor neurons (arrows) (compare with
Fig. 1D). (F) Whole mount of a
terminal abdominal ganglion of a day-3 adult, showing an almost complete lack
of MIPimmunostaining. (G) Epiproctodeal gland of a pharate adult at stage-8 of
development, showing intense immunostaining of the neurohemal system on the
wall of the rectum and proctodeal nerve (PN) and weak staining of the gland
cells (arrowhead). (H) Whole mount of the epiproctodeal gland of a day-1
adult, showing diminished staining of the neurohemal system on the proctodeal
nerves and on the wall of the rectum. At this stage, the immunostaining of the
gland cells is too weak to be seen. Scale bars, 100 µm (A,H) and 200 µm
(B–G).
|
|
View larger version (57K):
[in a new window]
|
Fig. 7. Confocal images of whole mounts of myoinhibitory peptide (MIP)-like
immunoreactivity in Bombyx mori. (A) Left hemisphere of the brain of
a third-instar larva, showing an immunoreactive median neurosecretory cell
(arrowhead) and its ipsilateral projection (arrow). (B) Retrocerebral complex
of a third-instar larva, showing MIP-immunoreactive (MIP-IR) processes in the
corpus cardiacum (CC) but not in the corpus allatum (CA). (C) Epiproctodeal
gland of a fourth-instar larva showing an extensive MIP-IR neurohemal system.
Scale bars, 50 µm.
|
|
View larger version (94K):
[in a new window]
|
Fig. 8. Confocal images of whole mounts of the fifth abdominal ganglion of larvae
of M. sexta and B. mori immunostained for crustacean
cardioactive peptide (CCAP; red) and myoinhibitory peptide (MIP; green). (A)
In M. sexta, immunostaining for CCAP demonstrated interneurons 704
and neuroendocrine cells 27, as well as processes in the neuropil and
perivisceral organ (PVO). (B) In the same ganglion shown in A, double-staining
for CCAP (red) and MIP (green) demonstrated that immunoreactivities to these
peptides are colocalized in interneurons 704 (yellow) but not in
neuroendocrine cells 27 (red). Note also that there is co-localization in much
of the neuropil but not in the PVO. (C) In the abdominal ganglia of M.
sexta larvae a few hours before ecdysis, the MIPimmunostaining of
interneurons 704 and of processes in the neuropil is strong. (D) In larvae at
ecdysis, the MIP-immunostaining of cells and processes is very weak,
suggesting that during ecdysis the MIP-like peptide has been released. (E) In
B. mori, MIP-immunostaining labeled two pairs of lateral cells. (F)
CCAP-immunostaining of the same ganglion labeled anterior and posterior
lateral somata comparable to cells 704 and 27, respectively. (G) The
double-staining of this ganglion demonstrated that CCAP and MIP
immunoreactivity is co-localized in interneurons 704 (yellow) but not in cells
27 (red) nor in the posterior lateral MIP-IR cells (green). Scale bars, 100
µm.
|
|
© The Company of Biologists Ltd 2003
