Sequence and expression of the CAPA/CAP2b gene in the tobacco hawkmoth, Manduca sexta
Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA
* Author for correspondence (e-mail: loi{at}uoneuro.uoregon.edu)
Accepted 12 July 2004
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Summary |
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The spatial and temporal expression pattern of the CAPA gene in the Manduca central nervous system (CNS) was determined in all major post-embryonic stages using in situ hybridization techniques. The CAPA gene is expressed in a total of 27 pairs of neurons in the post-embryonic Manduca CNS. A total of 16 pairs of cells is observed in the brain, two pairs in the sub-esophageal ganglion (SEG), one pair in the third thoracic ganglion (T3), one pair in each unfused abdominal ganglion (A1A6) and two pairs in the fused terminal ganglion. The mRNA from the CAPA gene is present in nearly every ganglion in each post-embryonic stage. The number of cells expressing the CAPA gene varies during post-embryonic life, starting at 54 cells in first-instar larvae and declining to a minimum of 14 cells midway through adult development.
Key words: CAP2b, cardioacceleratory peptide, Manduca sexta, insect peptide, insect neuropeptide, tobacco hawkmoth, insect peptide gene, Drosophila melanogaster, CAPA gene.
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Introduction |
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The second and only other member of the CAP group of peptides to have been
sequenced to date is CAP2b (pELYAFPRVamide; renamed in the present paper as
Mas-CAPA-1). Like CCAP, Mas-CAPA-1 is involved in numerous physiological roles
in a diverse set of insects. Mas-CAPA-1 was first identified in the moth
Manduca sexta as causing an increase in heart rate when applied
in vivo and in vitro (Tublitz and Truman,
1985a,b
,c
,d
;
Tublitz et al., 1991
).
Mas-CAPA-1 has also been shown to stimulate the rate of fluid secretion when
applied to Malpighian tubules from Manduca and the fruit fly
Drosophila melanogaster (Skaer et
al., 2002
; Davies et al.,
1995
,
1997
).
Mas-CAPA-1 appears to be related to several other insect peptides with
CAPA-like structures. The fruit fly, Drosophila melanogaster,
contains a deduced peptide, Drm-CAPA-1 (GANMGLYFPRVamide), with a high degree
of structural similarity, particularly at the C-terminus, to Mas-CAPA-1
(Kean et al., 2002). Although
ineffective on the Drosophila heart (N.J.T., unpublished), this
peptide is a potent activator of fluid secretion by the Drosophila
Malpighian tubules (Kean et al.,
2002
). CAPA-like peptides with actions on the hyperneural muscle
have been isolated from the perisympathetic organs of the cockroach
Leucophaea maderae and the locust Locusta migratoria
(Predel et al., 1995
;
Predel and Gade, 2002
). There
is increasing evidence that Mas-CAPA-1 and Drm-CAPA-1 are part of the CAPA
family of peptides found in a variety of insects. The CAPA peptides as a group
appear to be part of a larger superfamily of structurally related modulatory
neuropeptides that include the periviscerokinins in insects
(Wegener et al., 2002
) and the
small cardioactive peptides in molluscs
(Whim et al., 1993
).
Despite the increased interest in the CAPA peptides, there is little
information about CAPA gene structure and gene expression. The release of the
Drosophila genome sequence provided the first glimpse into the
sequence of a CAPA-encoding gene (Kean et
al., 2002). The aim of the present study was to further address
this issue in the moth Manduca sexta, where much of the early work on
CAPA peptides was performed. Using standard molecular methods, we have
isolated and sequenced a CAPA-encoding gene in Manduca. We also
present in situ hybridization data on the spatial and temporal
expression pattern of this gene in all post-embryonic stages in
Manduca.
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Materials and methods |
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Polymerase chain reaction (PCR) screening for a CAPA-containing transcript from a Manduca sexta cDNA library
All oligonucleotides used in the PCRs described in this paper were made by
Gibco (Life Technologies Inc., Grand Island, NY, USA). Taq polymerase and
buffer were purchased from Clontech (Titanium advantaq; BD Biosciences,
Clontech, Palo Alto, CA, USA), and dNTPs were purchased from Promega (Madison,
WI, USA). The following were used for each 15 µl PCR sample: 0.3 µl of
10 mmol1 dNTPs, 1.5 µl of PCR buffer, 0.3 µl of Taq
polymerase, 0.3 µl of each primer (concentration, 10 pmol
µl1) and sufficient sterile water to bring the reaction
volume to 15 µl. PCRs were performed in a Peltier PTC-200Thermocycler (MJ
Research Inc., Incline Village, NV, USA) using program settings described
below. Manduca sexta 8 Zap cDNA and genomic DNA were made from whole
nerve cords of day 3 fifth-instar larvae by Stratagene Corporation (La Jolla,
CA, USA) and re-amplified as necessary.
PCRs
As a first step in obtaining the CAPA gene sequence, two sets of
PCRs were performed with a degenerate oligonucleotide and a universal primer
using re-amplified Manduca cDNA from fifth-instar nerve cords as the
DNA template. The first set of PCR was performed with a degenerate primer
designed using the amino acid sequence of Manduca sexta CAPA-1. The
degenerate primer was constructed to recognize the sense strand of a putative
CAPA-encoding gene and had a sequence of A/GC/TTITAC/TGCITTC/TCC(G/A/T/C)A/CG
(see Fig. 1; bp
203221). The universal primer was constructed to recognized the vector
arm at the 5' end of the insert and had the sequence
GTAATACGACTCACTATAGGGC. The PCR conditions for the initial PCR were 2 min at
94°C, 30 s at 60°C and 1.5 min at 72°C for the initial cycle,
followed by 34 cycles of 1 min at 94°C, 30 s at 60°C and 1.5 min at
72°C. The reaction was terminated by a 10 min period at 72°C and held
at 4°C. The PCR product was separated by gel electrophoresis using a 5%
polyacrylamide gel. DNA was visualized under ultraviolet illumination after a
5 min incubation with 15 µl of ethidium bromide in 100 ml of gel running
buffer and a rinse in distilled water. The products from this initial PCR were
subcloned and sequenced as described below.
|
The second set of PCRs was performed to determine the remaining sequence of the CAPA mRNA. The same PCR conditions and cDNA template were used as described above except for the primers. One primer was constructed using the consensus sequence obtained from the first set of PCRs. This primer recognized the antisense strand at 250271 bp downstream of the CAPA coding region and had a sequence of CGTCTAAATGCTGTTCAGGTCGCAGG. The second primer was a universal primer that recognized the 3' arm of the vector and had the sequence AATTAACCCTCACTAAAGGG. The products from this initial PCR were separated electrophoretically following the procedure described in the previous paragraph. The PCR products of interest were subcloned and sequenced as described below.
Subcloning and transformation
All PCR bands of interest were subcloned and transformed to generate DNA
templates for sequencing. For subcloning only, PCR products from the initial
and second PCR runs were electrophoresed on 1% low-melt agarose gel (FMC
Bioproducts, Rockland, ME, USA) in E buffer (48 g of Trizma base, 7.4 g of
disodium EDTA dissolved in 600 ml of water, adjusted to pH 8 with acetic
acid). The pieces of gel containing the bands of interest were individually
excised, melted and cloned into a PCR2.1-TOPO vector using a TOPO TA cloning
kit (Invitrogen, Carlsbad, CA, USA) following the procedure specified by the
manufacturer.
DNA sequencing
Cloned PCR products were purified using a Genemate miniprep kit (MOBIO
Laboratories, Solana Beach, CA, USA). PCR was performed to confirm that the
insert contained the PCR product of interest. Purified products were sequenced
at the University of Oregon DNA Sequencing Facility using the vectors M13
forward and M13 reverse (Invitrogen) on a Beckman CEQ 8000 capillary sequencer
(Beckman Coulter, Fullerton, CA, USA).
PCR screening for a CAPA gene from a Manduca sexta genomic DNA
PCR was performed on genomic Manduca DNA to determine the presence
of introns within the coding region of the CAPA gene. The genomic
library was made by Stratagene Corporation from DNA extracted from
fifth-instar Manduca larvae. One primer recognized a region upstream
of the putative start codon. The second primer was designed to recognize a
region downstream of the putative stop codon. These two primers ensured that
the resultant PCR product spanned the entire coding region. All procedures
from PCR to subcloning and sequencing were as described above.
In situ hybridization
The in situ hybridization protocol used here was identical to that
published elsewhere (Loi et al.,
2001) except that the in situ probes recognized portions
of the Mas-CAPA-1 mRNA. The probes were constructed using the DIG (genius 4)
RNA labeling kit purchased from Roche Molecular Biochemicals (Indianapolis,
IN, USA). Two probes were used; one (
366 bp) recognized the beginning of
the mRNA to
306 bp into the coding region, and the other (
212 bp)
recognized the portion of the CAPA gene from
390 bp after the
start codon to
158 bp after the stop codon. Because the results from the
two probes were identical and because the cells expressing the transcripts
corresponded to cells labeled with a Mas-CAPA-1 antibody (N.J.T., unpublished
results), negative controls using a sense probe were not performed.
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Results |
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Spatial expression of the CAPA gene in the Manduca CNS
Two probes were used to investigate the expression of the CAPA
gene. One recognized 366 bp of the 5' end of the mRNA. The other was
made against a 216 bp region in the 3' non-coding region. Both probes
labeled the same cells, with differences only in the intensity and background.
Because the results from the two probes were identical in preliminary studies,
the temporal and spatial expression data presented here were obtained with the
shorter probe. The shorter probe labeled a maximum of 54 cells in the early
larvae stage and only 14 cells in the pharate adult stage
(Fig. 2). The spatial
expression of the Manduca CAPA gene is described in detail in the
following sections.
|
Brain
The CAPA in situ probe labeled 1516 pairs of cells in the
brain of 1st- and 2nd-instar caterpillars. Of these, 78 pairs of cells
are located dorsally, five pairs are situated ventrally and four pairs are
found near the middle (Figs 2,
3,
4AC). The number
of cells expressing the CAPA gene declines as the animal matures
(Figs 3,
4). By early 5th instar, there
are five pairs of CAPA-expressing cells, and this number increases to
12 pairs of cells by wandering day 1 (W1). The number of CAPA cells again
declines after W1 and, by pharate adult, only one pair is left
(Fig. 4D). Due to the
reorganization of the brain during metamorphosis, it was impossible to
determine if the pair expressing the CAPA gene in the pharate adult
brain bears any relationship to any of the in situ positive cells in
the larval brain.
|
|
Sub-esophageal ganglion (SEG)
There are two pairs of CAPA-expressing cells in the SEG. Both
pairs of cells are located along the midline; one pair is located anteriorly
and is substantially smaller than the other pair, which is located posteriorly
(Fig. 5). The anterior pair
stops expressing the CAPA transcript message after molting into the
5th instar. The posterior pair continues to express the CAPA gene
into the pharate adult (Fig.
4D) and adult stages (adult day 1; not shown).
|
Thoracic ganglia
Expression of the CAPA gene transcript was not observed in the 1st
and 2nd thoracic ganglia of any post-embryonic stage in Manduca. It
is expressed in a pair of lateral cells in the 3rd thoracic ganglion
(Fig. 6) in 1st-, 2nd- and
3rd-instar larvae. These cells stop expressing the CAPA transcript after the
HCP stage in the 4th-instar larva and do not express it in any subsequent
stage.
|
Abdominal ganglia
The 1st abdominal ganglion (A1) has a pair of posterior midline cells that
label with the CAPA probe only in 1st-instar larvae.
The 2nd abdominal ganglion (A2) has a similar pair of posterior midline cells that express the CAPA gene from the 1st instar onwards. CAPA gene expression in these cells is very variable; only a small percentage (530%) of cells express the gene within any given stage of development (Fig. 7A,B). In addition to the high variability of CAPA gene expression in these cells, the CAPA gene is often transiently expressed. For example, the CAPA transcript is undetectable during the first 3 days of the 5th instar (D13), yet reappears at W1. The CAPA transcript in these cells disappears again in early pupae, only to reappear in the pharate adult stage.
|
The 3rd, 4th, 5th and 6th abdominal ganglia (A36) each contain a pair of posterior midline cells that are labeled by the CAPA probe. Transcript expression is very robust in these cells in all ganglia and remains detectable from the 1st instar until adult day 1 (Fig. 7C).
Terminal ganglion
The CAPA gene is expressed in a pair of anterior lateral cells in
both terminal ganglion 7 and 8 (T78). The message is detectable only
until D0 4th instar (Fig.
8A,B). At metamorphosis, the 6th abdominal ganglion fuses with the
terminal ganglion, and a pair of cells expressing the CAPA transcript are
present in the A6 neuromere of pharate adult terminal ganglia
(Fig. 8C). These cells are
likely to be the same CAPA-positive cells found in the A6 in larvae
(Figs 2,
3).
|
Temporal expression of the CAPA gene in the Manduca CNS
Expression of the CAPA gene varies greatly across developmental
stages in Manduca (Fig.
3). The maximum number of cells expressing the CAPA gene
(54 cells) is found in 1st-instar larvae. As larvae mature and go through
various molts, the number of cells expressing the CAPA gene declines.
There is a significant increase in cells positive for the CAPA gene
on W1 of the last larval instar (5th instar). Following pupation, the number
of neurons expressing the CAPA gene product drops precipitously,
reaching a minimum of 14 cells in day 1 adults.
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Discussion |
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One obvious question arising from these data is the relationship between
the two new peptides (Mas-CAPA-2 and Mas-PK-1) described in the present study
and the remaining three unsequenced CAPs (CAP1a, CAP1b and CAP2c). Early work
on the CAPs in Manduca localized CAP bioactivity to the segmentally
iterated perivisceral organs (PVOs; also known as the perisympathetic organs)
in the Manduca ventral nerve cord (Tublitz and Truman,
1985a,b
,c
,d
).
Wegener et al. (2002
) used
MALDI-TOF mass spectral analysis to demonstrate the presence in larval
Manduca PVOs of four proteinaceous molecules: CCAP,
Mas-CAPA-1/Mas-CAPA-1 and two other unknown molecules. These two unknown
molecules have monoisotopic molecular masses (1346.4 and 1388.5 Da) nearly
identical to those of the two novel peptides encoded by the Manduca
CAPA gene (Mas-CAPA-2, predicted monoisotopic mass=1388.7 Da; Mas-PK-1,
predicted monoisotopic mass=1346.6 Da). These data provide direct empirical
evidence supporting the hypothesis that the Manduca CAPA propeptide
gene product is processed into three peptides, all of which are expressed in
and presumably released from the PVOs. However, further experimentation is
obviously required to determine the exact relationship, if any, between the
two novel peptides on the CAPA gene and the CAPs.
Rationale for naming DGVLNLYPFPRVa Mas-CAPA-2
The structure of one of the deduced peptides encoded by the CAPA
gene, DGVLNLYPFPRVa, is highly similar at the C-terminus to that of Mas-CAPA-1
(pELYAFPRVa). The Drosophila capability gene encodes two peptides,
Drm-CAPA-1 and Drm-CAPA-2, with C-terminal sequences similar to Mas-CAPA-1 and
Mas-CAPA-2 (DGVLNLYPFPRVa; Fig.
9; Kean et al.,
2002), respectively. The structural homologies and similar
intragenic locations of Drm-CAPA-1 and Manduca Mas-CAPA-1 persuaded
us to rename Manduca Mas-CAPA-1 as Mas-CAPA-1. Using the same
reasoning, and in order to remain consistent with the peptide naming system in
the Drosophila capability gene
(Kean et al., 2002
), we are
assigning the name of Mas-CAPA-2 to DGVLNLYPFPRVa.
|
Classification of Mas-CAPA-1 (pELYAFPRVa) and Mas-CAPA-2 (DGVLNLYPFPRVa)
Peptide classification has traditionally been based on structural and
functional similarities. Due to the absence of detailed functional analyses
for many peptides, peptide classification schemes are generally based on
common elements in their primary sequences, usually at the C-terminus.
C-terminal sequence similarities have been used to identify many peptide
families, including the FMRFamides
(Nichols et al., 1999), the
small cardioactive peptides (SCPs; Perry
et al., 1999
) and the pyrokinins
(Clynen et al., 2003
). A
greater challenge is presented when a novel peptide has a C-terminus with
similarities to more than one peptide family. This is the case for all three
deduced peptides encoded by the Manduca CAPA gene described in this
paper. Each has a primary sequence with some similarity to the pyrokinins, the
periviscerokinins (PVKs) and the SCPs
(Table 1). Mas-CAPA-1 and
Mas-CAPA-2 will be considered first; Mas-PK-1 will be discussed in the
subsequent section.
|
As discussed above, Mas-CAPA-1 and Mas-CAPA-2 share highly similar
C-terminal sequences, each ending in LYAXPRVa, where X is either A or P. Both
are presumably amidated at the C-terminus based on the presence of a glycine
at their carboxyl terminals. These two Manduca peptides share a high
degree of structural homology with two D. melanogaster peptides,
Drm-CAPA-1 and Drm-CAPA-2, both encoded on the Drosophila capability
gene (Table 1;
Kean et al., 2002). Although
the functional significance of Mas-CAPA-2 has yet to be elucidated, it is
known that Mas-CAPA-1 triggers an increase in heart rate in Manduca
(Tublitz et al., 1991
).
Mas-CAPA-1 also stimulates an increase in fluid secretion when applied to
isolated Drosophila Malpighian tubules, as do Drm-CAPA-1 and
Drm-CAPA-2 (Davies et al.,
1995
,
1997
;
Kean et al., 2002
). These
data, taken together, clearly suggest that Mas-CAPA-1, Mas-CAPA-2, Drm-CAPA-1
and Drm-CAPA-2 should be grouped together as a family of CAPA related peptides
(Table 1). The identifying
structural motif for this family is the C-terminal amino acid sequence of
LYAFPRVa.
The question remains, however, whether the CAPA family of peptides should
be a separate peptide family or grouped within an existing peptide family.
Table 1 lists the known peptide
families with structural similarities to the CAPA peptides. The pyrokinins,
the PVKs and the ecdysis-triggering hormones all have residues in common with
the four CAPA-like peptides in Manduca and Drosophila. Of
these, the CAPA peptides have the closest structural homology with the PVKs, a
group of peptides originally isolated from the cockroach Leucophaea
maderae (Predel et al.,
1995; Wegener et al.,
2001
) and subsequently identified in several other insect species
(Predel and Gade, 2002
). Early
work on the PVKs raised the possibility that they required specific N-terminal
and C-terminal motifs, but, with the recent identification of additional PVKs,
this notion has been modified in favor of a GLXXXPRVa PVK signature motif at
the C-terminus (Wegener et al.,
2002
). Interestingly, the primary sequences of many PVKs deviate
substantially from this motif (Predel and
Gade, 2002
; Table
1). Wegener et al.
(2002
) used structural data
and the similar location of PVK-immunopositive neurons in several insect
species including Manduca to argue for the inclusion of Mas-CAPA-1 as
a PVK family member. Although their evidence is consistent with their proposed
classification scheme, the paucity of additional physiological and
pharmacological data prevents us from agreeing, at least at present, with the
proposal to include the CAPA peptides as members of the PVK peptide family. It
is entirely possible that future results will support this interpretation.
However, for the present, the most conservative approach, based on existing
data, is to place the CAPA peptides in their own peptide family. Hence, we
propose that the four CAPA related peptides (Mas-CAPA-1, Mas-CAPA-2,
Drm-CAPA-1 and Drm-CAPA-2) form the basis for a separate CAPA peptide family
in insects with a shared structural motif of LYAFPRVa. It should be noted
that, based on structural homologies, the CAPA peptide family members are most
closely related to the SCPs from molluscs
(Table 1; Perry et al., 1999
). However,
like the PVKs, the relationship between the CAPAs and the SCPs must await
further investigations.
Classification of TEGPGMWFGPRLa (Mas-PK-1)
The C-terminus of TEGPGMWFGPRLa [Mas-Pyrokinin-1 (Mas-PK-1)] matches that
of the FXPRLamides, a peptide superfamily with diverse functions in insects.
Widely distributed across numerous insect species, the FXPRLamides include
melanization peptides (Matsumoto et al.,
1992), pheromone biosynthesis activating neuropeptides (PBANs;
Iglesias et al., 2002
) and egg
diapause induction peptides (Nachman et
al., 1993
). FXPRLamides also include peptides that affect visceral
muscle function. These myotropic peptides, known primarily from studies on
hemimetabolous insects, have been coined the pyrokinins
(Zdarek et al., 2004
). The
pyrokinins have been localized to different neurohemal release sites in the
insect CNS, including the retrocerebral complex and the PVOs (Predel et al.,
1997
,
1999
;
Predel and Gade, 2002
). One of
the peptides encoded by the Drosophila capability gene, Drm-CAPA-3
(TGPSASSGLWFGPRLa), contains the FXPRLa C-terminus motif and has been
tentatively classified as a pyrokinin (Fig.
9; Table 1;
Kean et al., 2002
). The
primary sequence of the third deduced peptide on the Manduca CAPA
gene, TEGPGMWFGPRLa (Mas-PK-1), closely resembles that of Drm-CAPA-3
(Fig. 9;
Table 1). The close homology
between the Manduca CAPA and Drosophila capability genes,
including the homologies between the two Manduca CAPA peptides and
Drm-CAPA-1 and Drm-CAPA-2, suggests a similar homology between Mas-PK-1 and
Drm-CAPA-3. Although the physiological significance of Mas-PK-1 is unclear,
there is sufficient structural information to tentatively classify Mas-PK-1 as
a member of the pyrokinin peptide family. For these reasons, we have named
this peptide Mas-PK-1, using the `1' designation to avoid future confusion
with any as yet unidentified Manduca pyrokinins.
The Manduca CAPA and the D. melanogaster capability genes are homologous
The Manduca CAPA gene shares many characteristics with the
Drosophila capability (CAPA) gene
(Kean et al., 2002;
Fig. 9). Both have similar
length coding regions (146 vs 149 residues), and 64% of the coding region (95
residues) shows alignment between the two genes based on sequence alignment
analysis using DIALIGN 2 (Morgenstern,
1999
). Each gene codes for three similar peptides, all of which
are probably amidated at the C-terminus based on the presence of a C-terminal
glycine at the end of each peptide sequence. Each deduced Manduca
peptide aligns closely with and shares a significant degree of sequence
homology with its counterpart on the Drosophila capability gene
(Fig. 9). Mas-CAPA-1 and the
two Drosophila CAPA gene peptides ending in PRVa (Drm-CAPA-1 and
Drm-CAPA-2) share functional properties, as each increases the rate of fluid
secretion when applied to Drosophila Malpighian tubules (Davies et
al., 1995
,
1997
;
Kean et al., 2002
). Given
these functional and molecular similarities, it is likely that the
Drosophila capability (CAPA) gene is the homologue of the
Manduca gene described in this paper. We also propose that Mas-CAPA-1
and Mas-CAPA-2 are likely to be the homologous peptides in Manduca to
the Drosophila peptides Drm-CAPA-1 and Drm-CAPA-2, respectively,
based on similarities in their primary sequences and respective intragenic
locations.
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Acknowledgments |
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