Tài liệu Báo cáo khoa học: Tachykinin-related peptide precursors in two cockroach species Molecular cloning and peptide expression in brain neurons and intestine docx

Tachykinin-related peptide precursors in two cockroach
species
Molecular cloning and peptide expression in brain neurons and
intestine
Reinhard Predel1, Susanne Neupert1, Steffen Roth1, Christian Derst1 and Dick R. Nassel2
ă
1 Saxon Academy of Sciences, Research Group Jena, Germany
2 Department of Zoology, Stockholm University, Sweden

Keywords
brain-gut peptides; insect neuropeptide;
neurochemistry; mass spectrometry;
Periplaneta americana; Leucophaea maderae
Correspondence
R. Predel, Saxon Academy of Sciences,
Research Group Jena, Erbertstraße 1,
07743 Jena, Germany
Tel: +49 3641 949191
Fax: +49 3641 949192
E-mail:
(Received 1 March 2005, revised 22 April
2005, accepted 6 May 2005)
doi:10.1111/j.1742-4658.2005.04752.x

Tachykinins and tachykinin-related peptides (TKRPs) play major roles in
signaling in the nervous system and intestine of both invertebrates and vertebrates. Here we have identified cDNAs encoding precursors of multiple
TKRPs from the cockroaches Leucophaea maderae and Periplaneta americana. All nine LemTKRPs that had been chemically isolated in earlier
experiments could be identified on the precursor of L. maderae. Four previously unidentified LemTKRPs were found in addition on the precursor.
The P. americana cDNA displayed an open reading frame very similar to
that of L. maderae with 13 different TKRPs. MALDI-TOF mass spectra
from tissues of both species confirms the presence of all the TKRPs encoded on the precursor plus two additional peptides that are cleavage products of the N-terminally extended TKRPs. A tissue-specific distribution of

TKRPs was observed in earlier experiments at isolation from brain and
midgut of L. maderae. Our data do not suggest a differential gene
expression but a different efficacy in processing of LemTKRP-2 and
Lem ⁄ PeaTKRP-3 in the brain and intestine, respectively. This results in a
gut-specific accumulation of these extended peptides, whereas in the brain
their cleavage products, LemTKRP-1 and LemTKRP-311)19, are most
abundant. Mass spectrometric analysis demonstrated the occurrence of the
different TKRPs in single glomeruli of the tritocerebrum and in cells of the
optical lobe.

Tachykinins constitute a family of multifunctional
neuropeptides whose signaling mechanisms seem to be
partially conserved through evolution [1–5]. Although
the tachykinin peptides display only limited sequence
identities when comparing invertebrates and mammals,
their G-protein-coupled receptors (GPCRs) display
more striking similarities, suggesting ancestral relationships [4,5]. The three principal mammalian tachykinins, substance P, neurokinin A and neurokinin B, are
processed from two precursors, preprotachykinin A
and B and they act with preferential affinities on three

different GPCRs, NK1–NK3 [5]. More recently, additional tachykinins, the hemokinins, were identified on
a third precursor encoding gene, preprotachykinin C,
expressed in hematopoietic cells of mouse, rat and
humans [6,7].
In invertebrates the tachykinins exist in two major
forms: (a) the tachykinin-related peptides (TKRPs; previously termed TRPs) that differ from the mammalian
tachykinins by having a C-terminus FXGXRamide
(X ¼ variable residues), rather than FXGLMamide
and (b) invertebrate tachykinins (Inv-TKs) with an

Abbreviations
ESI-Q-TOF MS, electrospray ionization quadrupole time-of-flight mass spectrometry; GPCR, G-protein-coupled receptors; TKRP, tachykininrelated peptide.

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FXGLMamide C-terminus [2,4]. As there is no evidence
that the insect and molluscan Inv-TKs display biological
activity in the native organism, it is likely that the
TKRPs are the principal endogeneous tachykinins in
invertebrates. The TKRPs are known to exist in multiple forms encoded by a single gene in each species studied so far [2,4]. Thus there are six TKRPs encoded on
the Drosophila melanogaster gene dtk [8], seven in the
honey bee Apis mellifera [9], three in the mosquito
Anopheles gambiae [10], seven in the echiuroid worm
Urechis unicinctus [11,12], and seven identical copies of a
single form in the crayfish and spiny lobster [13].
The largest number of TKRP isoforms in a single
species was isolated biochemically from the cockroach
Leucophaea maderae. In this species nine different
TKRPs (LemTKRP-1–9) were identified [14,15].
Immunocytochemistry revealed the distribution of
LemTKRP-like immunoreactivity in numerous neurons
of the central nervous system and peripheral ganglia,
as well as in the intestine and in some neurons innervating skeletal muscles [16]. With antisera specific to
several LemTKRPs (1,2,7) it could be demonstrated
that these are colocalized in the same neurons of the

brain [17]. Earlier data also indicated that some isoforms are expressed only in the brain (LemTKRP6,7,8,9) or intestine (LemTKRP-3,4), and some in both
tissues (LemTKRP-1,2,5) [14,15]. To be able to solve
the question of cell- or tissue-specific expression of
LemTKRPs it is critical to identify the gene(s) encoding their precursor(s). In the present paper, we have
cloned the genes encoding the TKRPs of L. maderae
and the American cockroach Periplaneta americana.
MALDI-TOF mass spectra from tissues of both species suggest a different efficacy in processing of two
N-terminally extended TKRPs in the brain and intestine, respectively. This could result in the gut-specific
accumulation of such peptides, whereas in the brain
cleavage products of these TKRPs are abundant. Apart
from these, all TKRPs that can be predicted from the
precursors of the two species were found in the brain as
well as in the midgut. In total, 14 different TKRPs can
be generated in L. maderae and 15 in P. americana.
Mass spectrometric analysis demonstrated the occurrence of the different TKRPs in glomeruli of the tritocerebrum and in cells of the optical lobe.

Results
Cloning of cockroach preprotachykinin cDNAs
Using degenerated PCRs and RACE, we obtained
full length preprotachykinin cDNA sequences for
L. maderae and P. americana. The cDNAs of 1512 bp
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R. Predel et al.

(lem, GenBank accession number AY766011) and
1200 bp (pea, GenBank accession number AY766012)
code for open reading frames of 360 amino acids
(L. maderae) and 366 amino acids (P. americana).
Sequence analysis identified a signal peptide (26aa)

and 13 tachykinin-related peptides (TKRPs) in both
sequences. As biochemical data showed alternative
processing of the first TKRP sequence (giving rise to
LemTKRP-1 and LemTKRP-2) more TKRPs may be
processed out of the precursor molecule. The sequences
of the identified TKRPs are highly conserved between
the two cockroach species, 95.1% of the 144 amino
acids encoding for mature TKRPs are identical in
L. maderae and P. americana, only six amino acid substitutions and one deletion were found (Fig. 1). The
signal peptide sequence (69.2% identity) and the spacer
sequences between the TKRPs (64.8% identity) are
much less conserved (Fig. 1). When comparing the
individual TKRP sequences, 10 of 13 peptides are
nona- or decapeptides; only the unprocessed first and
the last encoded peptide are significantly longer
(TKRP-2, 18 amino acids and TKRP-3, 19 amino
acids). In addition, a novel peptide (TKRP-11) consisting of only seven amino acids was found. Nearly all
TKRP sequences are followed by typical amidation ⁄ processing signals (GKK motif) within the precursor, only LemTKRP-4 was followed by the GKR
motif.
Mass spectrometric screening of TKRPs in
brain neurons and tritocerebral glomeruli of
P. americana and L. maderae
Immunocytochemical results have shown a number
of TKRP immmunopositive cells in the brain of L. maderae [16]. Mainly due to good accessibility, immunoreactive glomeruli of the tritocerebrum and neurons in
the vicinity (inner side) of the accessory medulla were
chosen for mass spectrometric analysis. The tritocerebral neuropil receives sensory input from the labrum
via the labral nerves [18]. Cells and glomeruli were individually dissected for both species and prepared for
MALDI-TOF MS.
Resulting mass spectra from tritocerebral glomeruli
suggested that these glomeruli contain the majority of

the TKRPs encoded on the precursors (Fig. 2). The
orthologs LemTKRP-3 ⁄ PeaTKRP-3 could not be
detected in any preparation where an otherwise good
signal-to-noise ratio was obtained. However, a cleavage product of TKRP-3 (LemTKRP-311)19) was
found in both species. From the other extended TKRP
(LemTKRP-2) very weak ion signals could be detected
only in a few preparations from L. maderae (not
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Tachykinin-related peptides in cockroaches

Fig. 1. Amino acid sequences of the P. americana (Pea) and L. maderae (Lem) TKRP precursors. Identified peptides are labeled in gray; designations do not follow (for historical reasons) the position in the precursors, thus numbering of the TKRPs is given below the amino acids.
As seen in the consensus sequence in the middle, only six amino acid substitutions and one deletion were found in the peptide-encoding
regions.

shown). Glomeruli of both species, however, contained
a cleavage product of TKRP-2 (LemTKRP-29)17)
which is identical to LemTKRP-1. None of the numerous known cockroach peptides was observed in mass
spectra of tritocerebral glomeruli from the two species,
but a number of unknown substances were represented
(Fig. 2).
In a second step, glomeruli (50–60 lm) of the tritocerebrum were individually dissected (Fig. 3). In these
experiments, it could be confirmed that each of the
TKRP containing glomeruli indeed contained all the
peptides found in the complete cluster of tritocerebral
glomeruli (Fig. 4).
Mass spectra of TKRP-producing cells in the optic

lobe of both cockroach species revealed the presence
of all TKRPs found in the tritocerebral glomeruli
(Fig. 5). This suggests that the peptide pattern of
TKRPs in the brain is not site-specific. Again, the two
extended TKRPs (TKRP-2 and -3) were not detectable. Mass spectra of these cells (15–20 lm) showed a
much lower signal intensity than those of the tritocerebral glomeruli. The adjoining unknown substances typical of the tritocerebral TKRP-glomeruli were also
found in mass spectra from preparations of optic lobe
cell bodies. To confirm the identity of the mass signals
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of the TKRPs proposed from the precursor, an extract
of the tissue from 50 tritocerebral neuropil areas of
P. americana was prepared for ESI-Q-TOF MS. The
doubly charged ion species of 534.3 ([M + H]+ of
1067.6) was chosen for tandem fragmentation, as it
was suspected to contain two TKRPs with identical
masses (LemTKRP-7 and -12) whose expression could
not be verified with MALDI-MS. Although the full
scan of the solution after rinsing the purification capillary with 20% acetonitrile ⁄ 5% formic acid revealed
only a very weak signal at 534.3 (with the other
TKRPs mostly below the threshold), the CID spectrum clearly confirmed that both peptides were present
in the sample (Fig. 6). The peptide sequences, designations and masses of all TKRPs from both cockroach
species are listed in Table 1.
Screening for TKRPs in the midgut
of P. americana
A midgut-specific expression of TKRPs was found for
L. maderae in earlier experiments [15]. Our analysis of
TKRP-expressing neurons of the brain indicates that
only the long TKRPs (TKRP-2 and -3) are candidates for midgut-specific expression. As a direct mass
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R. Predel et al.

Fig. 2. MALDI-TOF mass spectra typical
of tritocerebral TKRP-containing glomeruli.
TKRPs are numbered. (A) L. maderae,
(B) P. americana.

spectrometric screening of peptides from gut tissues
failed, we analyzed fractions obtained after HPLCseparation of an extract from 20 midguts of P. americana. MALDI-TOF mass spectra revealed the
occurrence of all TKRPs which were detected in
the brain neurons also in the midgut. This includes the
aforementioned cleavage products of TKRP-2 and -3.
In addition, distinct mass signals typical of unprocessed
TKRP-2 and TKRP-3 were observed. All TKRPs
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typical of P. americana (PeaTKRP-3,4,6,9,10,14) were
subsequently chosen for tandem fragmentation which
confirmed the predicted sequences (not shown).

Discussion
Studies using cockroaches greatly contributed to our
knowledge about neuropeptides of invertebrates. It
was with L. maderae that the identification of many
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R. Predel et al.

Fig. 3. Immunofluorescence staining in the deuto- (DC) and tritocerebrum (TC) of P. americana by means of an antiserum against
Lom-TKRP (whole mount preparation). Immunoreactivity is mainly
detectable in glomeruli of the antennal lobe (AL) and the tritocerebral
neuropil (arrow) which receives input from the labral nerves. Scale
bar: 500 lm. The inset shows an isolated tritocerebral glomerulus,
which was subsequently analyzed by MALDI-TOF MS (Fig. 4), immediately before transfer to the sample plate.

novel neuropeptide families started in the 1980s [19].
These findings provided the basis for the subsequent
detection of neuropeptides in other insect species,
among them the American cockroach, locusts and also
D. melanogaster [20,21,22]. In this study, we have
cloned tachykinin-related peptide encoding cDNAs
from P. americana and L. maderae. Today, with the
present identification of the TKRPs from P. americana, no other insect species is known to express a larger number of identified neuropeptides (more than 80
peptides [20,23]) in the CNS. Both cockroach TKRP
precursors contain 13 copies of related TKRPs and no
other predicted peptides with amidation signals. The
L. maderae precursor contains two copies of LemTKRP-9 and single copies of the other 11 peptides,
whereas in P. americana there are 13 different TKRPs.
We also showed that two additional peptides (LemTKRP-1 and LemTKRP-311)19) can be cleaved from
these progenitor sequences in both species, producing
15 different TKRPs in P. americana and 14 in L. madFEBS Journal 272 (2005) 3365–3375 ª 2005 FEBS

Tachykinin-related peptides in cockroaches

erae. This number of TKRPs is large compared to that

in the precursors in the worm U. unicinctus, crayfish,
other insects and mammalians and seems to be in line
with data from other known cockroach neuropeptide
precursors. For instance the allatostatin-A type precursors of different cockroaches display a higher number
of allatostatin forms than those in D. melanogaster
and A. gambiae [10,24], and the recently identified precursor for FMRFamide-related peptides of P. americana contains the largest number of neuropeptides
known to be encoded on a single precursor in any
insect [20].
Although P. americana and L. maderae belong to
different suborders of the order Blattodea, their TKRP
precursors display striking similarities in their peptide
encoding regions (Fig. 1). Most TKRPs are identical,
and others display minor amino acid substitutions.
Two N-terminally extended peptides (TKRP-2 and -3)
are present in both species and the other peptides are
nona- or decapeptides, except LemTKRP-11 which is a
heptapeptide. It is also noteworthy that all the LemTKRPs and their P. americana orthologs, except LemTKRP-3 and 11, have a proline in the second position
(XPX-) of their N-terminus. This proline provides
some resistance to nonspecialized amino peptidases,
but also renders the peptides sensitive to proline-specific dipeptidyl peptidase (DPP IV) attack [25,26].
A comparison with the open reading frames of other
invertebrate TKRP encoding genes reveals that the
largest similarities are in the actual peptide progenitor
sequences, whereas signal peptides and spacing
sequences between peptides are much more divergent.
Between the two cockroach genes the similarities are
also much less distinct in the nonpeptide coding
regions (64.8% similarities in spacer regions, 69.8%
similarities in signal peptide). Compared to other
cloned TKRP precursors there are some major differences in nonpeptide coding sequences. For instance in

the worm U. unicinctus and the mosquito A. gambiae,
the TKRP sequences are separated only by the dibasic
amino acids and amidation signals [10,12]; whereas in
the cockroaches, honeybee [9], crayfish, spiny lobster
[13] and fruit fly [8], there are sequences of varying
length between the TKRPs. Only in the D. melanogaster TKRP precursor, Dtk, other putative amidated
peptides (unrelated to TKRPs) could be predicted. It
is, however, not likely that these additional putative
peptides are cleaved from the Drosophila precursor
[27].
The most N-terminally located peptide LemTKRP-2
does not have dibasic cleavage sites N-terminally. Thus
this peptide has to be cleaved directly from the signal
peptide (probably by a signal peptidase). A similar
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R. Predel et al.

Fig. 4. MALDI-TOF mass spectrum typical
of a single tritocerebral TKRP-containing
glomerulus of L. maderae; TKRPs are numbered. Both ion species and relative
abundances are very similar to spectra from
preparations of complete TKRP-containing
neurophils (Fig. 2A).

Fig. 5. MALDI-TOF mass spectrum typical
of TKRP-containing cells in the optic lobe of

P. americana. TKRPs are numbered. Ion
intensities from these preparations are
much lower than those found in spectra
from tritocerebral glomeruli. The relative
abundance of the different TKRPs, however,
is comparable with the situation in
tritocerebral glomeruli.

unusual location adjacent to the signal peptide was for
instance seen for the neuropeptide proctolin in D. melanogaster [28]. Another feature of the LemTKRP-2
sequence (17-mer) is that it contains a dibasic cleavage
site at which the peptide can be cleaved to obtain the
9mer LemTKRP-1. This cleavage does occur, as we
could demonstrate both LemTKRP-1 and LemTKRP2 in the P. americana midgut in this investigation and
both peptides were chemically isolated from the brain
and intestine of L. maderae [14,15]. The other N-ter3370

minally extended peptides LemTKRP-3 and PeaTKRP-3 (19mers) also contain a dibasic cleavage
signal (Lys-Lys), and in the brain only the truncated
version was observed in both species, whereas the
19-mer was identified in the L. maderae [15] and
P. americana midgut.
Hence, with the extended TKRP-2 and -3 as exceptions, all of the predicted cockroach TKRPs could
be reproducibly detected by MALDI-TOF MS in
tritocerebral glomeruli and cells near the accessory
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Fig. 6. CID (collision-induced dissociation) spectrum (600–1000
atomic mass units) of TKRPs at [M + 2H]+ of 534.3
([M + H]+:1067.6; Fig. 3B) from a methanolic extract of 50 tritocerebral glomeruli using a nanospray source. Before this experiment, the
sample was purified on a capillary filled with Luna C18 material and
the spectrum was taken after rinsing the capillary with 20% (v ⁄ v)
acetonitrile ⁄ 5% (v ⁄ v) formic acid. Only the y-type fragment ions in
the higher mass range are given. The fragments clearly confirmed
the presence of TKRP-7 (white labeling on dark background) and 12
(dark labeling on white background) in the sample that could otherwise not be separated due to mass identity.

medulla of the optic lobe. The unequivocal detection
of TKRPs in single neurons and even glomeruli of the
brain demonstrates the power of modern MS technology when combined with proper identification and
sample preparation. Mass spectra also provide the evi-

dence that interneurons may contain amounts of neuropeptides comparable with those of neuroendocrine
cells which produce peptide hormones such as FMRF
amide-related peptides-producing cells in the thoracic
ganglia [20].
Earlier studies on L. maderae dealt with the problem
of brain-gut peptides and suggested a differential distribution of some TKRPs (see introduction). As shown
in this study, no brain-specific TKRP-gene products
exist. However, the N-terminally extended TKRPs
could not (Lem ⁄ PeaTKRP-3), or only with very low
signal intensity (LemTKRP-2), be identified in the
brain by mass spectrometry. Thus, the previously
documented absence in the brain of LemTKRP-3
[14,17] could be confirmed in L. maderae and was also

true for P. americana. The trace amounts of LemTKRP-2 in the brain of L. maderae, however, indicate
that the differential distribution of the extended
TKRPs and their truncated forms may be attributed
mainly to a differential efficacy in further cleavage of
TKRP-2 and -3. Thus, the unprocessed TKRP-2 and
-3 are probably present in the brain, but in a much
lower concentration than in the midgut and require
some biochemical enrichment to be detectable. This is
the likely explanation why LemTKRP-2 could be
chemically isolated from both brain and intestine in
L. maderae [14,15].
The finding that N-terminally extended TKRPs are
predominantly expressed only in the gut and not in
the brain are in line with findings of enrichment of

Table 1. Peptide sequences, designations and masses of all TKRPs from L. maderae and P. americana. To fit with the P. americana orthologs, LemTKRP-9 with two identical copies on the precursor occurs twice. The truncated versions of TKRP-2 and -3 are included in the table;
for historical reasons the cleavage product of TKRP-2 (TKRP-29)17) retains its own designation, namely LemTKRP-1. Non-identical amino
acids are indicated in bold.
Leucophaea maderae

Periplaneta americana

Peptides

Peptide sequence

[M + H]+, m ⁄ z

Peptides

Peptide sequence

[M + H]+, m ⁄ z

LemTKRP-1
LemTKRP-2
LemTKRP-3
LemTKRP-311)19
LemTKRP-4
LemTKRP-5
LemTKRP-6
LemTKRP-7
LemTKRP-8
LemTKRP-9
LemTKRP-10
LemTKRP-11
LemTKRP-12
LemTKRP-13
LemTKRP-9

APSGFLGVRa
APEESPKRAPSGFLGVRa
NGERAPGSKKAPSGFLGTRa
APSGFLGTRa
APSGFMGMRa
APAMGFQGVRa
APAAGFFGMRa
VPASGFFGMRa
GPSMGFHGMRa
APSMGFQGMRa

GPNMGFMGMRa
MGFMGMRa
GPSVGFFAMRa
APSAGFMGMRa
APSMGFQGMRa

902.52
1796.98
1929.04
904.50
952.45
1032.54
1023.52
1067.55
1075.49
1080.51
1096.48
828.36
1067.55
1023.49
1080.51

LemTKRP-1
LemTKRP-2
PeaTKRP-3
LemTKRP-311)19
PeaTKRP-4
LemTKRP-5
PeaTKRP-6
LemTKRP-7

LemTKRP-8
PeaTKRP-9
PeaTKRP-10
LemTKRP-11
LemTKRP-12
LemTKRP-13
PeaTKRP-14

APSGFLGVRa
APEESPKRAPSGFLGVRa
NGERAPASKKAPSGFLGTRa
APSGFLGTRa
APGSGFMGMRa
APAMGFQGVRa
APASGFFGMRa
VPASGFFGMRa
GPSMGFHGMRa
APSLGFQGMRa
APNMGFMGMRa
MGFMGMRa
GPSVGFFAMRa
APSAGFMGMRa
APSAGFHGMRa

902.52
1796.98
1943.06
904.50
1009.47
1032.54

1039.51
1067.54
1075.49
1062.55
1110.50
828.36
1067.55
1023.49
1029.50

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N-terminally extended tachykinins, neuropeptide c and
neuropeptide K, in the mammalian intestine [29–31].
Another extended TKRP was isolated from the locust
intestine [32]. In mammals, the tissue-specific expression of tachykinins is caused by alternative splicing of
the PPT-A gene and the existence of two further PPT
genes, but probably also by differential post-translational processing [5].
Previous work has indicated that the different LemTKRPs and the D. melanogaster and Locusta migratoria TKRPs display very small differences in their
biological activity in different bioassays [2,4,8,22,33].
As the TKRPs are encoded on a single precursor and
expressed in the same tissue (or even cells), it is possible that several of the TKRPforms are physiologically redundant. This, however, needs to be carefully
investigated. One reason for this is that although only
one putative TKRP receptor is known so far from
L. maderae [34], an additional GPCR of the tachykinin

type has been identified in some other insects [35–38].
In summary, we have found that cockroach genes
for TKRP precursors are the ones encoding the largest
number of copies of different TKRPs of all invertebrates studied so far. Most of the 14–15 peptides predicted on each of the precursors could be identified in
the brain and intestine of the two cockroach species by
mass spectrometry. A possible case of tissue-specific
accumulation of the N-terminally extended peptides
encoded on the genes could be confirmed by mass
spectrometry.

Experimental procedures

R. Predel et al.

part of the preprotachykinin precursors from L. maderae
and P. americana cDNA: forward primer: 5¢-gcnccng
cnatgggnttycarggngt-3¢ encoding for APAMGFQGV (part
of TKRP5); reverse primer: 5¢-ggngcyttyttnswnccnggngcnck
ytcnccrtt-3¢ encoding for NGERAPASKKA (part of
TKRP3).
The partial preprotachykinin cDNA sequences obtained
were used to design primers for 3¢ and 5¢ nested rapid
amplification of cDNA ends (RACE): lemRACE-F1:
5¢-TCGCTGTTGCAGTACCTGGACTCC-3¢; lemRACEB1: 5¢-GTCTACCAAGTCTCGAAGAAAGTCCTGCTG3¢; peaRACE-F1: 5¢-GATGGAGGGCGCGGAGGAT-3¢;
peaRACE-B1: 5¢-CTTGCCCCTCATGCCATGGAAC-3¢.
For both RACE reactions we used Advantage Taq 2
Mixture (Clontech) and a P. americana RACE library constructed previously [39]. A L. maderae RACE library was
prepared from several tissues (brain, ganglia, malpighian
tubules, muscles and intestine) using Trizol Reagent (Invitrogen, Karlsruhe, Germany) for RNA preparation, Oligotex (Qiagen, Hilden, Germany) for polyA + RNA
preparation and the Marathon cDNA Amplification Kit

(Clontech, Heidelberg, Germany) for reverse transcription,
second strand synthesis and adapter ligation. RACE products were cloned into pGEM-T vector (Promega GmbH,
Mannheim, Germany) for sequencing.

Sample preparation for mass spectrometry
After dissection of the brain, the ganglionic sheath was partially removed, glomeruli or somata of neurosecretory cells
separated and transferred with the help of a glass capillary
to a stainless steel sample plate for MALDI-TOF MS or
into a chilled solution of 5 lL methanol ⁄ water ⁄ trifluoroacetic acid (90 : 9 : 1, v ⁄ v ⁄ v) for electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS).

Insects
Cockroaches, L. maderae and P. americana, were raised
under a 12 h light, 12 h dark photoperiod at a constant
temperature of 28 °C. They were fed food pellets for rats
and had free access to water. Adult cockroaches of both
sexes were used for experiments.

Cloning of the L. maderae and P. americana TKRP
precursor cDNA
A combined degenerated PCR and RACE strategy was
used to clone full length sequences of cockroach preprotachykinin cDNAs as described recently for preproFMRFamide
cDNA [20]. Based on biochemically identified L. maderae
TKRPs [14,15] several degenerated PCR primers were
designed. PCR reactions were performed with Advantage
Taq 2 Mixture (Clontech, Palo Alto, CA, USA) at low stringency. Among the primer combinations tested one pair of
primers amplified approximately 790 bp fragments encoding

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MALDI-TOF MS

Neurons ⁄ glomeruli were dried on the sample plate and subsequently rinsed with water to reduce salt contamination.
Matrix solution (a-cyano-4-hydroxycinnamic acid dissolved
in methanol–water) was pumped onto the dried preparations over a period of approximately 5 s using a nanoliter
injector (World Precision Instruments, Berlin, Germany).
Each preparation was allowed to dry and then covered with
pure water for a few seconds; the water was then removed
by cellulose paper. At least five preparations each were
prepared for mass spectrometric analysis.

ESI-Q-TOF MS
Following the dissection procedure, 50 lL 0.1% (v ⁄ v) trifluoroacetic acid were added to the 5 lL methanol ⁄ water ⁄
trifluoroacetic acid. The extract was sonicated, centrifuged

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R. Predel et al.

and the methanol evaporated from the supernatant. The
resulting aqueous supernatant was then loaded onto an activated and equilibrated home-made micro column (purification capillary for electrospray mass spectrometry).

MALDI-TOF MS
MALDI-TOF mass spectra were acquired in positive ion
mode on a Voyager Pro DE biospectrometry workstation
(Applied Biosystems, Framingham, MA, USA) equipped
with a pulsed nitrogen laser emitting at 337 nm. Samples
were analyzed in reflectron mode using a delayed extraction
time of 150 ns, 75% grid voltage, 0.002–0.02% guide wire
voltage, and an accelerating voltage of 20 kV. Laser
strength was adjusted to provide the optimal signal-to-noise

ratio. An external mass spectrum calibration was first performed using synthetic cockroach peptides (Pea-pyrokinin2 ⁄ 5; SPPFAPRLa ⁄ GGGGSGETSGMWFGPRLa).

ESI-Q-TOF MS
Nanoelectrospray mass spectra were acquired in the positive-ion mode using the API Qstar Pulsar (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany)
fitted with a Protana (Odense, Denmark) nanoelectrospray
source. Typically 1050–1150 V was applied as an ion spray
voltage. Samples were purified using a homemade spin column. Approximately 1 mg of Luna C18 material (10 lm;
Phenomenex, Aschaffenburg, Germany) was loaded into a
2-cm capillary column with a needle tip. Liquids are passed
through the column by securing the capillary column to a
purification needle holder (Proxeon Biosystems A ⁄ S,
Odense, Denmark) and centrifugation. After the column
was activated with 50% acetonitrile ⁄ 0.1% TFA and equilibrated in 0.1% TFA, the samples were loaded and rinsed
with 5% formic acid. Peptides were eluted from the column
with solutions of 10, 20, and 30% acetonitrile (5% formic
acid) and collected into a metal-coated nanoelectrospray
capillary. The purified samples were then loaded onto the
source and analyzed. After determining the m ⁄ z of the peptides in MS mode, a collision energy (typically 15–40 V)
was applied. The m ⁄ z of interest was isolated and fragmented with the instrument in ‘enhance all’ mode. MS ⁄ MS data
were acquired over 5 min and manually analyzed.

HPLC
Midguts of adult P. americana were dissected in insect saline
and shortly rinsed with distilled water before being transferred to 200 lL methanol–water–trifluoroacetic acid
(90 : 9 : 1, v ⁄ v ⁄ v, on ice). Following sonication and centrifugation, the collected supernatant was evaporated to dryness
and resuspended in 500 lL 0.1% (v ⁄ v) trifluoroacetic acid.
This solution was applied to an activated and equilibrated

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Tachykinin-related peptides in cockroaches

SEP-PAK C-18 cartridge (Waters, Milford, MA, USA), peptides were eluted with 40% (v ⁄ v) acetonitrile containing
0.1% (v ⁄ v) trifluoroacetic acid. Peptide separation was performed on a Shimadzu HPLC system (Shimadzu, Duisburg,
Germany) equipped with a diode-array detector and using a
˚
Luna RP-C18 column (150 · 4.6 mm, 100 A, 5 lm, Phenomenex, Torrance, CA, USA) with a linear AB gradient of
10–80% B over 35 min (flow rate: 1 mLỈmin)1). Solvent A
was 0.11% (v ⁄ v) trifluoroacetic acid in water, solvent B 60%
(v ⁄ v) acetonitrile containing 0.1% (v ⁄ v) trifluoroacetic acid.
Fractions were collected manually and subsequently analyzed on a MALDI-TOF mass spectrometer.

Immunocytochemistry
Dissected cockroach brains were fixed overnight at 4 °C
with 4% (v ⁄ v) formaldehyde in phosphate-buffered saline
(NaCl ⁄ Pi), pH 7.2. Subsequently, preparations were washed
in NaCl ⁄ Pi)4% (v ⁄ v) Triton X-100 and NaCl ⁄ Pi)1% (v ⁄ v)
Triton X-100 for 24 h, respectively. The preparations were
then incubated for 5 days at 4 °C in anti-LomTKRP serum
(1 : 1000, diluted with NaCl ⁄ Pi 1% (v ⁄ v) Triton X-100 containing 0.25% (w ⁄ v) bovine serum albumin and normal
goat serum). Following overnight washing in 0.1 molỈL)1
Tris ⁄ HCl, 3% (w ⁄ v) NaCl, 1% (v ⁄ v) Triton X-100
(pH 7.6), the fluorochrome-labeled secondary Cy3 antibodies were used directly as a mixture in NaCl ⁄ Pi–bovine
serum albumin (2.5 mgỈmL)1) at a concentration of
1 : 3000 for 4 days. Finally, the preparations were washed
again overnight in 0.1 molỈL)1 Tris-HCl, 3% (w ⁄ v) NaCl,
1% (v ⁄ v) Triton X-100 (pH 7.6) and transferred in glycerin.
For visualization, tissues were dehydrated in ethanol,
cleared in methyl salicylate and mounted in entellan (Euromex Microscopes, Arnhem, the Netherlands). Immunostainings were examined with a confocal laser scanning
microscope (Zeiss LSM 510 Meta system; Jena, Germany),

equipped with a HeliumNeon1 laser (wavelength 543 nm).
Serial optical sections were assembled into combined
images. Images were exported and processed with Adobe
photoshop 7.0 software.

Peptide terminology and acronyms
The old designation of tachykinin-related peptide (TRP)
was changed here for TKRP as suggested in [4]. This was
done to avoid confusion with, for example, the abbreviation for the transient receptor potential cationic channel
(TRP) and tryptophan (Trp). Thus the old acronyms such
as LemTRP-1, were changed to LemTKRP-1. The designations of the P. americana TKRPs were made such that
peptides identical to L. maderae peptides are called LemTKRPs, others are PeaTKRPs. The numbering of peptides
follows that for already identified peptides [14,15], and not
their sequence order on the precursors. Thus novel peptides

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Tachykinin-related peptides in cockroaches

are given numbers TKRP-10–14. Unique PeaTKRPs are
given numbers that are the same as their LemTKRP orthologs on the precursor. Note that the nonamidated pepide
designated LemTRP-10 in an earlier paper [14] is not likely
to be a true TKRP and is disregarded here (thus the LemTKRP-10 identified here is a totally new peptide).

Acknowledgements
We acknowledge the financial assistance of the Deutsche Forschungsgemeinschaft (Predel 595 ⁄ 6–1,2).

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