IDENTIFICATION, SEQUENCE AND EXPRESSION OF A CRUSTACEAN CARDIOACTIVE PEPTIDE (CCAP) GENE IN THE MOTH MANDUCA SEXTA
P. K. LOI1,*,
S. A. EMMAL1,
Y. PARK2 and
N. J. TUBLITZ1
1
Institute of Neuroscience, University of Oregon, Eugene, OR 97403,
USA
2
Department of Entomology, University of California, Riverside, CA 92521,
USA
*
e-mail:
tublitz{at}uoneuro.uoregon.edu
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Fig. 1. Complete sequence of the Manduca sexta crustacean cardioactive
peptide (CCAP) gene. Nucleotide and predicted peptide sequences are listed for
the coding region, which starts at nucleotide 1. Blue, CCAP-encoding region;
green, dibasic post-translational cleavage sites; red, glycine used for
C-terminal amidation; yellow shading, consensus signal peptide sequence;
asterisk, stop codon. Intron position and length are indicated above the
nucleotide sequence. Intron sequences are shown separately at the bottom, with
underlined GT and AG for the intron donor and acceptor consensus sequences,
respectively.
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Fig. 2. Expression of the crustacean cardioactive peptide (CCAP) transcript in the
brain of Manduca sexta. (A) Ventral view of the brain in a day 1
first-instar larva showing a cluster of three anterior pairs of cells
(arrows), one anterio-medial pair (concave arrow) and a large ventral pair in
the mid protocerebrum (arrowheads). The sixth pair of cells was located on the
dorsal side (convex arrow; out of focus). (B) Dorsal view of a brain in a day
2 fourth-instar larva. The `sixth dorsal pair' of cells is indicated by convex
arrows, and the convex arrowheads point to the two pairs of cells that started
expressing CCAP after reaching the fourth instar. (C) Posterior view of an
adult brain showing two pairs of cells (arrows). Scale bars, 50 µm.
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Fig. 3. Expression of the crustacean cardioactive peptide (CCAP) transcript in the
subesophageal ganglion (SEG) of Manduca sexta. (A) Ventral view of an
air-filled third-instar SEG showing a pair of posteriolaterally located cells
(concave arrows) and a pair of smaller midline cells (arrows). (B) Ventral
view of an air-filled fourth-instar larval SEG showing the previously
discussed four cells (described in A) and an anteriolateral pair (convex
arrows; left cell out of focus). (C) Ventral view of an SEG from a day 0
fourth-instar larva showing the posteriolaterally located cells (concave
arrows), the anteriolateral cells (convex arrows) and a single anterior cell
to the left of the midline (arrowhead). (D) Posterior view of an adult SEG
showing only one pair of CCAP-expressing cells (arrows). Inset: diagram of the
adult brain showing the area from where D was obtained (box; X indicates
positions of cells). Scale bars, 50 µm.
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Fig. 4. Expression of the crustacean cardioactive peptide (CCAP) transcript in the
thoracic ganglia (T1-T3) of Manduca sexta. (A) Ventral view of T1
from a day 0 fourth-instar larva showing a pair of lateral cells (arrows)
labeled by the in situ hybridization probe for CCAP. (B) Ventral view
of a wandering day 3 fifth-instar T1 showing a pair of labeled lateral cells
(concave arrows) and a second pair of more anterior cells (convex arrows). (C)
Ventral view of T1 from a day 10 pupa (P10) showing two pairs of lateral cells
(concave arrows). (D) The anterior portion (T2) of the fused thoracic ganglion
of a P10 pupa. The concave arrows point to two pairs of lateral cells. (E) The
posterior portion of the fused thoracic ganglion in a P10 pupa with two pairs
of lateral cells (concave arrows) in the T3 neuromere. The dotted line marks
the approximate boundary between T3 and A1 neuromeres. (F) The fused thoracic
ganglion of a day 0 adult. The lateral cells in T2 and T3 are indicated by
concave arrows. Scale bars, 50 µm.
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Fig. 5. Variable nature of crustacean cardioactive peptide (CCAP) transcript
expression in a bilateral pair of lateral neurosecretory cells, NS-L(T), in
the thoracic ganglion of Manduca sexta. (A) The lateral cells in T3
of a third-instar larva. (B) T3 of another third-instar larva with no labeled
cells. (C) The lateral cells in T1 of a day 3 fifth-instar larva with two
cells labeled. (D) T1 of another day 3 fifth-instar larva with no labeled
cells. Scale bars, 50 µm.
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Fig. 6. Expression of the crustacean cardioactive peptide (CCAP) transcript in the
abdominal ganglia (A1-A6) of Manduca sexta. (A) Cells labeled with
the in situ probe in the first abdominal ganglion (A1) from a day 2
second-instar larva. (B) Labeled cells in A2 of a day 1 fifth-instar larva.
(C) Labeled cells in A2 of a day 3 fifth-instar larva. (D) Labeled cells in A4
of a day 0 pupa (P0). (E) Labeled cells in the posterior portion (T3, A1 and
A2) of the fused thoracic ganglion from a day 10 pupa (P10). (F) Labeled cells
in A3 of a P10 pupa. (G-I) Three different planes of focus of A3 in a day 0
adult, from ventral (G) to dorsal (I). (A-I) Lateral cells, concave arrows;
anterior cells, convex arrows; midline cells, arrows; ventral unpaired
neurons, arrowheads. Scale bars, 50 µm.
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Fig. 7. Expression of the crustacean cardioactive peptide (CCAP) transcript in the
terminal ganglion of Manduca sexta. (A) Day 2 second-instar larva.
(B) Air-filled fourth-instar larva. (C) Day 3 fifth-instar larva. (D)
Wandering day 3 fifth-instar larva. (E) Day 10 pupa. (F) Day 0 adult. (A-D)
Terminal ganglia of larvae are formed by the fusion of the seventh abdominal
ganglion (denoted as A7) and abdominal ganglia 8-10 (denoted as A8). Lateral
cells, concave arrows; anterior cells, convex arrows; A8 cells started to
express the CCAP transcript after the head capsule stage of a fourth-instar
larva, concave arrowheads; two pairs of cells that express the CCAP transcript
after wandering, convex arrowheads. (E,F) Terminal ganglia of late pupae
(pupal day 14) and adults (adult day 0) are formed by the fusion of the sixth
abdominal ganglion (denoted A6) with A7 and A8-A10. CCAP-labeled cells in A6,
convex arrowheads; in A7 cells, concave arrows; in A8-A10 cells, arrowheads.
Scale bars, 50 µm.
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Fig. 9. Temporal expression of the crustacean cardioactive peptide (CCAP)
transcript in the Manduca sexta central nervous system. Each central
nervous system region is color-coded. A, adult; L, larva; P, pupa; Ph. Adult,
pharate adult; SEG, subesophageal ganglion; T, thoracic ganglion.
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Fig. 10. Alignment and comparison of deduced amino acid sequences of crustacean
cardioactive peptide (CCAP)-encoding mRNA from Drosophila
melanogaster (DmCCAP) and Manduca sexta (MsCCAP). The
CCAP-coding region: blue; C-terminal glycine, red; putative post-translational
cleavage sites, green. Sequence alignment was obtained using blosum62 with gap
penalty 10 and extension penalty 0.1. Periods (.) are gaps generated for the
alignment. Identical and similar amino acids are in black and gray shaded
boxes, respectively
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© The Company of Biologists Ltd 2001
