Biology of the CAPA peptides in insects.

CAPA peptides have been isolated from a broad range of insect species as well as an arachnid, and can be grouped into the periviscerokinin and pyrokinin peptide families. In insects, CAPA peptides are the characteristic and most abundant neuropeptides in the abdominal neurohemal system. In many species, CAPA peptides exert potent myotropic effects on different muscles such as the heart. In others, including blood-sucking insects able to transmit serious diseases, CAPA peptides have strong diuretic or anti-diuretic effects and thus are potentially of medical importance. CAPA peptides undergo cell-type-specific sorting and packaging, and are the first insect neuropeptides shown to be differentially processed. In this review, we discuss the current knowledge on the structure, distribution, receptors and physiological actions of the CAPA peptides.

Unique accumulation of neuropeptides in an insect: FMRFamide-related peptides in the cockroach, Periplaneta americana.

FMRFamides belong to the most extensively studied neuropeptides in invertebrates and exhibit diverse physiological effects on different target organs, such as muscles, intestine and the nervous system. This study on the American cockroach confirms for the first time that extended FMRFamides occur in non-dipteran insects. By means of tandem mass spectrometry, these neuropeptides were structurally elucidated, and sequence information was used for subsequent cloning of the cockroach FMRFamide gene. This precursor gene encodes for 24 putative peptides and shows sufficient similarity with the Drosophila FMRFamide gene. Of the 24 peptides, 23 were detected by mass spectrometric methods; it is the highest number of neuropeptide forms shown to be expressed from a single precursor in any insect. The expression was traced back to single neurons in the thoracic ganglia. The unique accumulation of these FMRFamide-related peptides in thoracic perisympathetic organs provides the definite evidence for a tagma-specific distribution of peptidergic neurohormones in neurohaemal release sites of the insect CNS. Excitatory effects of the cockroach FMRFamides were observed on antenna-heart preparations. In addition, the newly described FMRFamides reduce the spike frequency of dorsal-unpaired median neurons and reduce the intracellular calcium concentration, which may affect the peripheral release of the biogenic amine octopamine.

Peptidergic neurohemal system of an insect: mass spectrometric morphology.

Neuropeptides are by far the most diverse group of messenger molecules in insects. To understand cell signaling and function, it is essential to reveal the complete neuropeptide profile of a single neuron/nerve/neurohemal organ first. In this study, matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry was used to analyze the peptidergic system of an insect, focussing on the neurohemal structures. Major neurohemal organs were investigated, including the retrocerebral complex, perisympathetic organs, and all nerves supplying these organs with neurosecretions. Additionally, peripheral neurohemal release sites such as the dilator muscle of the antennal circulatory organ and lateral heart nerves were studied, as well as parts of the stomatogastric nervous system. The following neuropeptide families were analyzed: kinins, allatostatins, leucomyosuppressin, corazonin, adipokinetic hormones, myoinhibitory peptide, sulfakinins, periviscerokinins, YLSamide, VEAacid, SKNacid, proctolin, the head peptide, and pyrokinins. Beyond a contribution to a map of the distribution of neuropeptides in a neurohemal system, the following conclusions can be drawn from these experiments. (1) Nearly all abundant peaks in the different mass spectra represent peptides that have already been identified. (2) Although only adult males were used in this study, variations in the peptide abundances were observed that are possibly correlated with different physiological/developmental conditions. (3) Peptides have a body-region-specific distribution in the neurohemal system. (4) A clear compartmentalisation of the retrocerebral complex could be observed.

Myoinhibitory neuropeptides in the American cockroach.

A large number of myostimulatory neuropeptides from neurohaemal organs of the American cockroach have been described since 1989. These peptides, isolated from the retrocerebral complex and abdominal perisympathetic organs, are thought to be released as hormones. To study the coordinated action of these neuropeptides in the regulation of visceral muscle activity, it might be necessary to include myoinhibitors as well, however, not a single myoinhibitory neuropeptide of the American cockroach has been described so far. To fill this gap, we describe the isolation of LMS (leucomyosuppressin) and Pea-MIP (myoinhibitory peptide) from neurohaemal organs of the American cockroach. LMS was very effective in inhibiting phasic activity of all visceral muscles tested. It was found in the corpora cardiaca of different species of cockroaches, as well as in related insect groups, including mantids and termites. Pea-MIP which is strongly accumulated in the corpora cardiaca was not detected with a muscle bioassay system but when searching for tryptophane-containing peptides using a diode-array detector. This peptide caused only a moderate inhibition in visceral muscle assays. The distribution of Pea-MIP in neurohaemal organs and cells supplying these organs with Pea-MIP immunoreactive material, is described. Additionally to LMS and Pea-MIP, a member of the allatostatin peptide family, known to exhibit inhibitory properties in other insects, was tested in visceral muscle assays. This allatostatin was highly effective in inhibiting spontaneous activity of the foregut, but not of other tested visceral muscles of the American cockroach.

Myostimulatory neuropeptides in cockroaches: structures, distribution, pharmacological activities, and mimetic analogs.

In this brief overview we give the historical background on the discovery of myostimulatory neuropeptides in cockroaches. Related peptides were later found in other insect groups as well. We summarize the current knowledge on primary structures, localization, physiological and pharmacological effects of the different cockroach neuropeptides, including kinins, sulfakinins, pyrokinins, tachykinin-related peptides, periviscerokinins, corazonin, and proctolin. In addition, we briefly comment on the development of mimetic pseudopeptide analogs in the context of their possible use in insect pest management.

Efficacy of native FXPRLamides (pyrokinins) and synthetic analogs on visceral muscles of the American cockroach.

Six pyrokinins, members of a widely distributed neuropeptide family in insects (FXPRLamides), have been identified from the American cockroach. Five of these peptides, Pea-PK-1-5, were tested in different myotropic bioassays, including hyperneural muscle, hindgut, foregut and oviduct. Among these muscles, the hyperneural muscle exhibited the highest sensitivity to pyrokinin applications. The efficacy of the different pyrokinins differed dramatically. No muscle specific effectiveness was obtained; the ranking order in all muscle assays was as follows: PK-1>PK-4>PK-3>PK-2>PK-5. Testing of synthetic analogs revealed the importance of the amino acid at the variable -4 position of the C-terminus. PK-5, the only one of the five tested peptides which is stored in abdominal perisympathetic organs, has probably no myotropic function at all. This is further evidence that these neurohemal release sites are not necessary to compensate the open circulatory system of insects but have rather specific functions which are totally unknown as yet.

Neurosecretion: peptidergic systems in insects.

Insect neuropeptides are produced in less than 1% of the cells of the central nervous system. Despite this, they are important messenger molecules which influence nearly all physiological processes, including behaviour. They can act as transmitters, modulators and classical hormones, and often exhibit pleiotropic functions when released into the haemolymph. The large number of neuropeptides that has been identified from some of the model organisms among insects underlines the complexity of the neurosecretory system; studies about the coordinated actions of these substances are in their preliminary stages. Recent advances in insect neuropeptide research will be reviewed here, concentrating on the distribution of multiple peptide forms in the central nervous system and adjacent neurohaemal organs, and the role of neuropeptides in eclosion behaviour.

Identification of novel periviscerokinins from single neurohaemal release sites in insects MS/MS fragmentation complemented by Edman degradation.

Three novel members of the periviscerokinin family could be identified directly from extracts of single abdominal perisympathetic organs of blaberoid cockroaches by means of electrospray ionization-quadrupole time of flight (ESI-QTOF) MS. Sequences of these periviscerokinins were confirmed by Edman degradation. Their primary structures are GSSGLIPFGRT-NH2 (Lem-PVK-1), GSSGLISMPRV-NH2 (Lem-PVK-2), and GSSGMIPFPRV-NH2 (Lem-PVK-3). Hitherto only known from the American cockroach, this neuropeptide family contains a highly conserved N-terminus whereas, at the C-terminus, only the penultimate amino-acid residue (Arg) has been found in all members of this peptide family. The identified periviscerokinins are the only abundant myoactive peptides in abdominal perisympathetic organs of blaberoid cockroches and they appear to be absent in the retrocerebral complex. Screening of extracts of single abdominal perisympathetic organs (70-90 microm in diameter), from five different species of the suborder Blaberoidea, revealed that they all contain the three neuropeptides which are described here for the first time.

Tagma-specific distribution of FXPRLamides in the nervous system of the American cockroach.

FXPRLamide (pyrokinin) distribution in the central nervous system and major neurohaemal organs of the American cockroach and related cockroach species was investigated using immunocytochemistry and MALDI-TOF mass spectrometry. Six isoforms (Pea-PK-1 through -6) were found in different neurohaemal release sites. Pea-PK-1-4 and Pea-PK-6 are all stored in the retrocerebral complex and are all produced in cells located in both the suboesophageal ganglion (SOG) and the tritocerebrum. These pyrokinins were found to be concentrated in and around the corpora allata. No other known peptides were detectable in such high concentrations in this neurohaemal organ. They reach the corpora cardiaca/allata via the nervi corporis cardiaci-1 (NCC-1), NCC-3, and nervi corporis allati-2 (NCA-2). Abdominal perisympathetic organs contained only Pea-PK-5 and low quantities of the sequence-related Pea-PK-6. Neither Pea-PK-5 nor -PK-6 was detected in thoracic perisympathetic organs. It is likely that the expression of pyrokinins in the central nervous system is tagma (body region)-specific. Pea-PK-6 was identified during this study as follows: Ser-Glu-Ser-Glu-Val-Pro-Gly-Met-Trp-Phe-Gly-Pro-Arg-Leu-NH(2).

Allatostatins from the retrocerebral complex and antennal pulsatile organ of the American cockroach: structural elucidation aided by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

The occurrence of allatostatins in retrocerebral complexes and antennal pulsatile organs of the American cockroach, Periplaneta americana, was investigated. Previously, molecular cloning of the P. americana allatostatin gene had predicted 14 peptides of this family [Ding et al., Comparison of the allatostatin neuropeptide precursors in the distantly related cockroaches Periplaneta americana and Diploptera punctata. Eur J Biochem 1997;234:737-746], however, only two forms had been identified by peptide isolation procedures [Weaver et al., Identification of two allatostatins from the CNS of the cockroach Periplaneta americana: novel members of a family of neuropeptide inhibitors of insect juvenile hormone biosynthesis. Comp Biochem Physiol 1994;107(C):119-127]. Using an extract of only 200 corpora cardiaca/corpora allata, we have found that at least 11 allatostatins occur in the retrocerebral complex. These peptides were already separated from other substances of the crude extract in the first HPLC step with heptafluorobutyric acid as organic modifier, and subsequently identified by MALDI-TOF mass spectrometry. Moreover, we have demonstrated the occurrence of nearly all allatostatins, including the cleavage product of Pea-AST-2 (LPVYNFGL-NH2), in antennal pulsatile organs of males and females. Allatostatins are predominant neuropeptides in these organs. Additionally, only two other known peptides could be identified in these organs by mass screening: proctolin and leucomyosuppressin. The function of allatostatins in antennal pulsatile organs remains unclear. We assume a release into the hemolymph via the ampullac, which could act as neurohemal release sites. The method described for the identification of allatostatins is a very fast method for neuropeptide screening in neurohemal tissues.

Post-translational modifications of the insect sulfakinins: sulfation, pyroglutamate-formation and O-methylation of glutamic acid.

We identified and chemically characterized the two major forms of sulfakinins from an extract of 800 corpora cardiaca/corpora allata complexes of the American cockroach, Periplaneta americana. Bioactivity during the purification was monitored by measuring heart beat frequency in a preparation in situ. By Edman degradation analysis and MS, these main forms were identified as having the primary structures Pea-SK [EQFDDY(SO(3)H)GHMRFamide] and Lem-SK-2 [pQSDDY(SO(3)H)GHMRFamide]. The sulfation was confirmed by UV, MS and peptide synthesis. In addition, post-translationally modified sulfakinins of both major forms were isolated and identified. Firstly, nonsulfated forms of these peptides are present in considerable amounts in the corpora cardiaca/allata. Secondly, the N-terminally blocked Pea-SK and the nonblocked Lem-SK-2 occur naturally in neurohaemal release sites. Thirdly, modified Pea-SK with O-methylated glutamic acid occurs which is not an artefact of peptide purification. The major forms of the sulfakinins were shown to be highly active on both the heart and hindgut with threshold concentrations of approximately 5 x 10(-10) M (heart) and 2 x 10(-9) M (hindgut).

Quantification of periviscerokinin-1 in the nervous system of the American cockroach, Periplaneta americana. An insect neuropeptide with unusual distribution.

This study was undertaken to reveal the quantitative distribution of the insect neuropeptide periviscerokinin-1 (Pea-PVK-1) in the central nervous system of Periplaneta americana and to demonstrate that neurons stained in a previous immunohistochemical study contain authentic Pea-PVK-1. For this, we combined ELISA, HPLC, and MALDI-TOF mass spectrometry. The high specificity of the used antiserum enabled the quantification of Pea-PVK-1 in unseparated tissue extracts. No cross-reactivities with other insect neuropeptides were detected in ELISA. Only two immunoreactive fractions, coeluting with synthetic Pea-PVK-1 in its oxidized and nonoxidized form, were found in HPLC-separated extracts of the brain, suboesophageal ganglion, metathoracic ganglion, second abdominal ganglion with or without perisympathetic organ, and terminal ganglion. By using MALDI-TOF mass spectrometry, we were able to confirm the existence of authentic Pea-PVK-1 in these fractions. The abdominal perisympathetic organs contained 6.3 pmol Pea-PVK-1 per animal; another 1.3 pmol were found in the abdominal ganglia. More than 90% of the total 8.2 pmol in the central nervous system was found in the abdominal ganglia and their perisympathetic organs. The corpora cardiaca and corpora allata did not contain immunoreactive material, suggesting that Pea-PVK-1 is not released by the cephalic neurohaemal system. The quantitative distribution of Pea-PVK-1 differs considerably from that of other known insect neuropeptides.

Differential distribution of pyrokinin-isoforms in cerebral and abdominal neurohemal organs of the American cockroach.

Different pyrokinin isoforms were identified from major neurohemal organs of the American cockroach. During their isolation they were recognized by bioassay using a hyperneural muscle preparation that is sensitive to pyrokinins. All structures were elucidated by sequence analysis and mass spectrometry. The primary structures of the novel peptides isolated from the retrocerebral complex are LVPFRPRL-NH2 (designated Pea-PK-3) and DHLPHDVYSPRL-NH2 (designated Pea-PK-4). A pyrokinin, labeled Pea-PK-5, was isolated from abdominal perisympathetic organs. Structural analysis of this peptide yielded the sequence GGGGSGETSGMWFGPRL-NH2. The threshold concentrations of the identified pyrokinins for an eliciting effect on contractions of the hyperneural muscle preparations differed dramatically. This indicates that the different distribution of pyrokinin-isoform observed in neurohemal organs may be associated with different functions. This is the first report of a differential distribution of peptide-isoforms in the neurohemal organs of insects.

Periviscerokinin-like immunoreactivity in the nervous system of the American cockroach.

A highly specific polyclonal antiserum has been raised against periviscerokinin, the first neuropeptide isolated from the perisympathetic organs of insects (Predel et al. 1995). In this study, two different neuronal systems with periviscerokinin-like immunoreactivity were distinguished in the central nervous system of the American cockroach: (1) An intrinsic neuronal network, restricted to the head-thoracic region, was formed by intersegmental projecting neurons of the brain, suboesophageal ganglion and metathoracic ganglion. In addition, groups of local interneurons occurred in the proto- and tritocerebrum. (2) A typical neurohormonal system was stained exclusively in the abdomen; it was represented by abdominal perisympathetic organs which were supplied by three cell clusters located in each unfused abdominal ganglion. As revealed by nickel backfills, most neurons with axons entering the perisympathetic organs contained a periviscerokinin-like peptide. Immunoreactive fibres left the perisympathetic organs peripherally, innervated the hyperneural muscle and ran via the link nerves/segmental nerves to the heart and segmental vessels. All visceral muscles innervated by periviscerokinin-immunoreactive fibres were shown to be sensitive to periviscerokinin, whereas the hindgut gave no specific response to this peptide.

The unique neuropeptide pattern in abdominal perisympathetic organs of insects.

We successfully isolated and identified the abundant neuropeptides of the abdominal perisympathetic organs of the American cockroach, including all myoactive compounds. Peptide sequence analysis and mass spectrometry of abundant substances that were not bioactive in different muscle assays yielded the following sequences: TDPLWQLPGAHLEQYLS-NH2 (Pea-YLS-amide), AFLTLTPGSHVDSYVEA-OH (Pea-VEAacid), and SDLTWTYQSPGDPTNSKN-OH (Pea-SKNacid). The given structures led to the conclusion of an unique neuropeptide pattern in abdominal perisympathetic organs. We confirmed this assumption with immunocytochemical studies, using antisera raised against different myotropic neuropeptides of the abdominal perisympathetic organs. Moreover, mass spectrometric methods, developed for the investigation of single neurohemal organs, confirmed the neuropeptide pattern in these organs.

Isolation of periviscerokinin-2 from the abdominal perisympathetic organs of the American cockroach, Periplaneta americana.

Using the isolated hyperneural muscle as bioassay, a novel myotropin was isolated from the abdominal perisympathetic organs of Periplaneta americana. This is the second neuropeptide identified from insect perisympathetic organs. Peptide sequence analysis and mass spectrometry yielded the following structure: Gly-Ser-Ser-Ser-Gly-Leu-Ile-Ser-Met-Pro-Arg-Val-NH2. This peptide, named periviscerokinin-2, was confirmed to be amidated by chemical synthesis, bioassay, and comparison of retention times between native and synthetic peptides. A highly specific antiserum was used to determine sites of synthesis in the abdominal ganglia. Besides periviscerokinin-1, periviscerokinin-2 is the only putative myotropic neurohormone from the abdominal perisympathetic organs that is effective in the nanomolar range. This confirms the hypothesis that the neurohormonal system of the ventral nerve cord is remarkably different from that of the brain.

Isolation and structural elucidation of eight kinins from the retrocerebral complex of the American cockroach, Periplaneta americana.

By monitoring the contractile activity of the hindgut of the American cockroach in vitro eight myotropic neuropeptides were isolated from the retrocerebral complex of the American cockroach. Peptide sequence analysis and mass spectrometry yielded the following structures: Arg- Pro-Ser-Phe-Asn-Ser-Trp-Gly-NH2 (Pea-K-1), Asp-Ala-Ser-Phe-Ser-Ser-Trp-Gly-NH2 (Pea-K-2), Asp-Pro-Ser-Phe-Asn-Ser-Trp-Gly-NH2 (Pea-K-3), Gly-Ala-Gln-Phe-Ser-Ser-Trp-Gly-NH2 (Pea-K-4), Ser-Pro-Ala-Phe-Asn-Ser-Trp-Gly-NH2 (Pea-K-5), Asp-Pro-Ala-Phe-Ser-Ser-Trp-Gly-NH2 (Lem-K-7), Gly-Ala-Asp-Phe-Tyr-Ser-Trp-Gly-NH2 (Lem-K-8) and Ala-Phe-Ser-Ser-Trp-Gly-NH2 (Lom-K). The C-terminal sequence Phe-X-Ser-Trp-Gly-NH2 characterized the peptides as members of the insect kinin family. All structures were confirmed by comparison of retention times between synthetic and natural peptides. The threshold concentration for stimulatory effects of the synthetic peptides on the isolated hindgut was about 10(-9) M and there was no significant difference measured between the different kinin forms. These neuropeptides are the first members of the insect kinin-family isolated from the American cockroach. Their occurrence in the retrocerebral complex suggests a physiological role as neurohormone.

Isolation and structural elucidation of two pyrokinins from the retrocerebral complex of the American cockroach.

By monitoring the contractile activity of the hyperneural muscle of the American cockroach in vitro two peptides were isolated from the retrocerebral complex of the American cockroach. Three purification steps using reversed-phase high performance liquid chromatography on C-18 columns containing trifluoroacetic acid or heptafluorobutyric acid as organic modifiers were sufficient to achieve homogeneous peptide preparations. The structures of both peptides were elucidated by a combination of Edman degradation and mass spectrometry which yielded the following structures: His-Thr-Ala-Gly Phe-Ile-Pro-Arg-Leu-NH2 (Pea-PK-1) and Ser-Pro-Pro-Phe-Ala-Pro-Arg-Leu-NH2 (Pea-PK-2). The C-terminal sequence Phe-X-Pro-Arg-Leu-NH2 characterized the peptides as members of the insect pyrokinin family. The synthetic peptides were shown to have the same retention times as the natural peptides. The occurrence of both peptides in the retrocerebral complex suggests a physiological role as neurohormones. The effects of the synthetic pyrokinis were clearly distinguishable in their actions on the hyperneural muscle. Regarding the threshold concentrations, Pea-PK-2 was only 0.3% as active as Pea-PK-1.

Periviscerokinin (Pea-PVK): a novel myotropic neuropeptide from the perisympathetic organs of the American cockroach.

A myotropic neuropeptide was isolated from extracts of 1000 abdominal perisympathetic organs of males of the cockroach, Periplaneta americana. This peptide, termed periviscerokinin, has excitatory actions on the hyperneural muscle of Periplaneta americana. After peptide sequence analysis and mass spectrometry, the structure of this peptide was confirmed by chemical synthesis and bioassay to be Gly-Ala-Ser-Gly-Leu-Ile-Pro-Val-Met-Arg-Asn-NH2. This sequence is different from the other known myotropic peptides in insects. The threshold concentration for stimulatory effects of the synthetic peptide on the isolated hyperneural muscle was about 10(-9) M, suggesting a physiological role as a neurohormone.