Distribution and daily oscillation of GABA in the circadian system of the cockroach Rhyparobia maderae.

Gamma-aminobutyric acid (GABA) is the prevalent inhibitory neurotransmitter in nervous systems promoting sleep in both mammals and insects. In the Madeira cockroach, sleep-wake cycles are controlled by a circadian clock network in the brain's optic lobes, centered in the accessory medulla (AME) with its innervating pigment-dispersing factor (PDF) expressing clock neurons at the anterior-ventral rim of the medulla. GABA is present in cell clusters that innervate different circuits of the cockroach's AME clock, without colocalizing in PDF clock neurons. Physiological, immunohistochemical, and behavioral assays provided evidence for a role of GABA in light entrainment, possibly via the distal tract that connects the AME's glomeruli to the medulla. Furthermore, GABA was implemented in clock outputs to multiple effector systems in optic lobe and midbrain. Here, GABAergic brain circuits were analyzed further, focusing on the circadian system in search for sleep/wake controlling brain circuits. All GABA-immunoreactive neurons of the cockroach brain were also stained with an antiserum against the GABA-synthesizing enzyme glutamic acid decarboxylase. We found strong overlap of the distribution of GABA-immunoreactive networks with PDF clock networks in optic lobes and midbrain. Neurons in five of the six soma groups that innervate the clock exhibited GABA immunoreactivity. The intensity of GABA immunoreactivity in the distal tract showed daily fluctuations with maximum staining intensity in the middle of the day and weakest staining at the end of the day. Quantification via enzyme-linked immunosorbent assay and quantitative liquid chromatography coupled to electrospray ionization tandem mass spectrometry, likewise, showed higher GABA levels in the optic lobe during the inactivity phase of the cockroach during the day and lower levels during its activity phase at dusk. Our data further support the hypothesis that light- and PDF-dependently the circadian clock network of the cockroach controls GABA levels and thereby promotes sleep during the day.

Enhanced Coverage of Insect Neuropeptides in Tissue Sections by an Optimized Mass-Spectrometry-Imaging Protocol.

Mass spectrometry imaging (MSI) of neuropeptides has become a well-established method with the ability to combine spatially resolved information from immunohistochemistry with peptidomics information from mass spectrometric analysis. Several studies have conducted MSI of insect neural tissues; however, these studies did not detect neuropeptide complements in manners comparable to those of conventional peptidomics. The aim of our study was to improve sample preparation so that MSI could provide comprehensive and reproducible neuropeptidomics information. Using the cockroach retrocerebral complex, the presented protocol produces enhanced coverage of neuropeptides at 15 µm spatial resolution, which was confirmed by parallel analysis of tissue extracts using electrospray-ionization MS. Altogether, more than 100 peptide signals from 15 neuropeptide-precursor genes could be traced with high spatial resolution. In addition, MSI spectra confirmed differential prohormone processing and distinct neuropeptide-based compartmentalization of the retrocerebral complex. We believe that our workflow facilitates incorporation of MSI in neuroscience-related topics, including the study of complex neuropeptide interactions within the CNS.

Agatoxin-like peptides in the neuroendocrine system of the honey bee and other insects.

We investigated the peptide inventory of the corpora cardiaca (CC) of the honey bee, Apis mellifera, by direct tissue profiling using MALDI-TOF MS combined with proteomic approaches focusing on cysteine-containing peptides. An agatoxin-like peptide (ALP) was identified as a component of the glandular part of the CC and was associated with the presence of the adipokinetic hormone in mass spectra. Although abundant in the CC, ALP does not belong to the toxins observed in the venom gland of A. mellifera. Homologs of ALP are highly conserved in major groups of arthropods and in line with this we detected ALP in the CC of non-venomous insects such as cockroaches and silverfish. In the American cockroach, Periplaneta americana, ALP was also identified in the CNS and stomatogastric nervous system. This is the first report that establishes the presence of ALPs in the neuroendocrine tissues of insects and further studies are necessary to reveal common functions of these peptides, e.g. as antimicrobial agents, ion channel modulators or classical neuropeptides. BIOLOGICAL SIGNIFICANCE: Among the messenger molecules of the nervous system, neuropeptides represent the structurally most diverse class and basically participate in the regulation of all physiological processes. The set of neuropeptides, their functions and spatial distribution are particularly well-studied in insects. Until now, however, several potential neuropeptide receptors remained orphan, which indicates the existence of so far unknown ligands. In our study, we used proteomic methods such as cysteine modification, enzymatic digestion and peptide derivatization, combined with direct tissue profiling by MALDI-TOF mass spectrometry, for the discovery of novel putative messenger molecules in the neuroendocrine system. The described presence of agatoxin-like peptides in the nervous system of the honey bee and other insects was overseen so far and is thus a remarkable addition to the very well studied neuropeptidome of insects. It is not yet clear, if these toxin-like peptides act as antimicrobial agents, ion channel modulators or classical neuropeptides.

The neuropeptide SIFamide in the brain of three cockroach species.

The sequence as well as the distribution pattern of SIFamide in the brain of different insects is highly conserved. As a general rule, at least four prominent SIFamide-immunoreactive somata occur in the pars intercerebralis. They arborize throughout the brain and the ventral nerve cord. Whereas SIFamide is implicated in mating and sleep regulation in Drosophila, other functions of this peptide remain largely unknown. To determine whether SIFamide plays a role in the circadian system of cockroaches, we studied SIFamide in Rhyparobia (= Leucophaea) maderae (Blaberidae), Periplaneta americana (Blattidae), and Therea petiveriana (Polyphagidae). Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry revealed identical SIFamide sequences (TYRKPPFNGSIFamide) in the three species. In addition to four large immunoreactive cells in the pars intercerebralis (group 1), smaller SIFamide-immunoreactive somata were detected in the pars intercerebralis (group 2), in the superior median protocerebrum (group 3), and in the lateral protocerebrum (group 4). Additional cells in the optic lobe (group 5) and posterior protocerebrum (group 6) were stained only in P. americana. Almost the entire protocerebrum was filled with a beaded network of SIFamide-immunoreactive processes that especially strongly invaded the upper unit of the central body. Double-label experiments did not confirm colocalizations with γ-aminobutyric acid (GABA) or the circadian coupling peptide pigment-dispersing factor (PDF). In contrast to locusts, colocalization of SIFamide and histamine immunoreactivity occurred not in group 1, but in group 4 cells. Because the accessory medulla displayed SIFamide immunoreactivity and injections of SIFamide delayed locomotor activity rhythms circadian time-dependently, SIFamide plays a role in the circadian system of cockroaches. J. Comp. Neurol. 524:1337-1360, 2016. © 2015 Wiley Periodicals, Inc.

Identification and expression of capa gene in the fire ant, Solenopsis invicta.

Recent genome analyses suggested the absence of a number of neuropeptide genes in ants. One of the apparently missing genes was the capa gene. Capa gene expression in insects is typically associated with the neuroendocrine system of abdominal ganglia; mature CAPA peptides are known to regulate diuresis and visceral muscle contraction. The apparent absence of the capa gene raised questions about possible compensation of these functions. In this study, we re-examined this controversial issue and searched for a potentially unrecognized capa gene in the fire ant, Solenopsis invicta. We employed a combination of data mining and a traditional PCR-based strategy using degenerate primers designed from conserved amino acid sequences of insect capa genes. Our findings demonstrate that ants possess and express a capa gene. As shown by MALDI-TOF mass spectrometry, processed products of the S. invicta capa gene include three CAPA periviscerokinins and low amounts of a pyrokinin which does not have the C-terminal WFGPRLa motif typical of CAPA pyrokinins in other insects. The capa gene was found with two alternative transcripts in the CNS. Within the ventral nerve cord, two capa neurons were immunostained in abdominal neuromeres 2-5, respectively, and projected into ventrally located abdominal perisympathetic organs (PSOs), which are the major hormone release sites of abdominal ganglia. The ventral location of these PSOs is a characteristic feature and was also found in another ant, Atta sexdens.

Neuropeptidergic input pathways to the circadian pacemaker center of the Madeira cockroach analysed with an improved injection technique.

Light entrainment pathways synchronize the circadian clock of almost all species of the animal and plant kingdom to the daily light dark cycle. In the Madeira cockroach Rhyparobia (Leucophaea) maderae, the circadian clock is located in the accessory medulla of the brain's optic lobes. The clock has abundant neuropeptides with unknown functions. Previous studies suggested that myoinhibitory peptides (MIPs), orcokinins (ORCs), and allatotropin (AT) take part in light input pathways to the circadian clock. As the sequences of AT and ORCs of R. maderae have not yet been determined, with matrix-assisted laser desorption/ionization-time of flight mass spectrometry, the respective Rhyparobia peptides were characterized. To search for light-like phase-shifting inputs to the circadian clock, Rhyparobia-MIP-1, Rhyparobia-AT, and Rhyparobia-ORC were injected at different circadian times, combined with locomotor activity assays. An improved, less invasive injection method was developed that allowed for the analysis of peptide effects within <2 weeks after injection. Rhyparobia-MIP-1 and Rhyparobia-AT injections resulted in dose-dependent monophasic phase response curves with maximum delays at the beginning of the subjective night, similar to light-dependent phase delays. In contrast to Manduca sexta-AT, Rhyparobia-AT did not phase advance locomotor activity rhythms. Only injections of Rhyparobia-ORCs resulted in a biphasic light-like phase response curve. Thus, it is hypothesized that Rhyparobia-MIP-1 and -AT are candidates for relaying light-dependent delays and/or non-photic inputs to the clock, whereas Rhyparobia-ORCs might be part of the light-entrainment pathways relaying phase delays and advances to the circadian clock of the Madeira cockroach.

Myoinhibitory peptides in the brain of the cockroach Leucophaea maderae and colocalization with pigment-dispersing factor in circadian pacemaker cells.

Myoinhibitory peptides (MIPs) are a family of insect W(X(6))Wamides with inhibitory effects on visceral muscles and juvenile hormone synthesis. Although MIPs are widely distributed within the nervous system, a detailed analysis of their distribution and function in insect brains is still missing. We analyzed the distribution of MIPs in the brain of the cockroach Leucophaea maderae. We focused on the accessory medulla (AMe), a small neuropil near the medulla that acts as the master circadian clock. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and Nano-LC electrospray ionization (ESI) mass spectrometry revealed five Lem-MIPs in preparations of the AMe and corpora cardiaca. The complete sequences of two of these peptides were identified. Immunocytochemistry revealed wide distribution of MIP-related peptides in the cockroach brain. The superior median protocerebrum, parts of the central complex, and the tritocerebrum showed particularly dense immunostaining. In contrast, only a few local interneurons were stained in the antennal lobe and a few extrinsic neurons in the mushroom body, including a giant neuron innervating the calyces. The noduli of the AMe showed dense immunostaining, and neurons in all AMe cell groups except the anterior neurons were labeled. Pigment-dispersing factor- (PDF) and MIP immunostaining was colocalized in two neurons of the AMe. No colocalization of MIP- and PDF immunostaining was detected in the anterior optic commissure, but two small PDF-immunoreactive commissural fibers near the posterior optic commissure showed colocalized MIP immunostaining. The results suggest that several MIPs participate in different functional circuits of the circadian system and are involved in multiple brain circuits of the Madeira cockroach.

Genomics, transcriptomics, and peptidomics of Daphnia pulex neuropeptides and protein hormones.

We report 43 novel genes in the water flea Daphnia pulex encoding 73 predicted neuropeptide and protein hormones as partly confirmed by RT-PCR. MALDI-TOF mass spectrometry identified 40 neuropeptides by mass matches and 30 neuropeptides by fragmentation sequencing. Single genes encode adipokinetic hormone, allatostatin-A, allatostatin-B, allatotropin, Ala(7)-CCAP, CCHamide, Arg(7)-corazonin, DENamides, CRF-like (DH52) and calcitonin-like (DH31) diuretic hormones, two ecdysis-triggering hormones, two FIRFamides, one insulin, two alternative splice forms of ion transport peptide (ITP), myosuppressin, neuroparsin, two neuropeptide-F splice forms, three periviscerokinins (but no pyrokinins), pigment dispersing hormone, proctolin, Met(4)-proctolin, short neuropeptide-F, three RYamides, SIFamide, two sulfakinins, and three tachykinins. There are two genes for a preprohormone containing orcomyotropin-like peptides and orcokinins, two genes for N-terminally elongated ITPs, two genes (clustered) for eclosion hormones, two genes (clustered) for bursicons alpha, beta, and two genes (clustered) for glycoproteins GPA2, GPB5, three genes for different allatostatins-C (two of them clustered) and three genes for IGF-related peptides. Detailed comparisons of genes or their products with those from insects and decapod crustaceans revealed that the D. pulex peptides are often closer related to their insect than to their decapod crustacean homologues, confirming that branchiopods, to which Daphnia belongs, are the ancestor group of insects.

Pigment-dispersing hormone in Daphnia interneurons, one type homologous to insect clock neurons displaying circadian rhythmicity.

We report identification of a beta-type pigment-dispersing hormone (PDH) identical in two water flea species, Daphnia magna and Daphnia pulex. It has been identified by cloning of precursors, chromatographic isolation from tissue extracts followed by immunoassays and de novo-mass spectrometric sequencing. The peptide is restricted to a complex system of distinct interneurons in the brain and visual ganglia, but does not occur in neurosecretory cells projecting to neurohemal organs as in decapod crustaceans. Thirteen neuron types individually identified and reconstructed by immunohistochemistry were almost identical in terms of positions and projection patterns in both species. Several neurons invade and form plexuses in visual ganglia and major brain neuropils including the central body. Five neuron types show contralateral pathways and form plexuses in the lateral, dorsal, or postlateral brain neuropils. Others are local interneurons, and a tritocerebral neuron connects the protocerebrum with the neuropil of the locomotory second antenna. Two visual ganglia neuron types lateral to the medulla closely resemble insect medulla lateral circadian clock neurons containing pigment-dispersing factor based upon positional and projectional criteria. Experiments under 12:12 h light/dark cycles and constant light or darkness conditions showed significant circadian changes in numbers and activities of one type of medulla lateral PDH neuron with an acrophase in the evening. This simple PDH system shows striking homologies to PDH systems in decapod crustaceans and well-known clock neurons in several insects, which suggests evolutionary conservation of an ancient peptidergic interneuronal system that is part of biological clocks.

Identification of kinin-related peptides in the disease vector, Rhodnius prolixus.

We have used an in silico approach to identify a gene from the blood-gorging vector, Rhodnius prolixus, that is predicted to produce an insect kinin prepropeptide. The prepropeptide is 398 amino acids in length and can potentially produce a large number of kinin-related peptides following post-translational processing. A comparison with other insect kinin precursor sequences demonstrates greatest conservation at the C-terminal region of the kinin peptides. Multiple peptides predicted from the kinin gene are phenotypically expressed in R. prolixus, as revealed by MALDI-TOF MS MS, including 12 kinins and one kinin precursor peptide (KPP). Six of these peptides are characterized by the typical insect kinin C-terminal motif FX(1)X(2)WGamide and five of these are also found as truncated forms. Five peptides were identified with an atypical, though similar, FX(1)X(2)WAamide C-terminus. There is also peptide with a C-terminal DDNGamide motif and a number of non-amidated peptides.

Direct peptide profiling of brain tissue by MALDI-TOF mass spectrometry.

Direct MALDI-TOF mass spectrometric peptide profiling is increasingly used to analyze the peptide complement in the nervous system of a variety of invertebrate animals from leech to Aplysia and many arthropod species, especially insects and crustaceans. Here, we describe a protocol for direct peptide profiling of defined areas of the central nervous system of insects. With this method, one can routinely and reliably obtain neuropeptide signatures of selected brain areas from various insects.

Direct MALDI-TOF mass spectrometric peptide profiling of neuroendocrine tissue of Drosophila.

Direct MALDI-TOF mass spectrometric peptide profiling is increasingly used to analyze the peptide complement in the nervous system of a variety of invertebrate animals, from leech to Aplysia and many arthropod species, especially insects and crustaceans. Proper sample preparation is often the most crucial step to obtain the necessary data. Here, we describe protocols for the use of MALDI-TOF mass spectrometry to directly analyze the peptidome of neuroendocrine tissues of insects, particularly Drosophila melanogaster, by MALDI-TOF MS.

Peptidomic analysis of single identified neurons.

Today, commercially available mass spectrometers increasingly meet all the demands of the proteomics community including high throughput, high sensitivity, and significant fragmentation capability for sequence determinations. Therefore, proper sample preparation is often the most crucial step to obtain the necessary data, particularly when working with biological samples. Depending on the size, sample preparation techniques differ and have to be optimized empirically. This is particularly apparent at the single cell level. In this chapter, we describe protocols for the use of MALDI-TOF mass spectrometry to directly analyse the peptidome of single insect neurons.

Conservation of the function counts: homologous neurons express sequence-related neuropeptides that originate from different genes.

By means of single-cell matrix assisted laser desorption/ionization time-of-flight mass spectrometry, we analysed neuropeptide expression in all FXPRLamide/pheromone biosynthesis activating neuropeptide synthesizing neurons of the adult tobacco hawk moth, Manduca sexta. Mass spectra clearly suggest a completely identical processing of the pheromone biosynthesis activating neuropeptide-precursor in the mandibular, maxillary and labial neuromeres of the subesophageal ganglion. Only in the pban-neurons of the labial neuromere, products of two neuropeptide genes, namely the pban-gene and the capa-gene, were detected. Both of these genes expressed, amongst others, sequence-related neuropeptides (extended WFGPRLamides). We speculate that the expression of the two neuropeptide genes is a plesiomorph character typical of moths. A detailed examination of the neuroanatomy and the peptidome of the (two) pban-neurons in the labial neuromere of moths with homologous neurons of different insects indicates a strong conservation of the function of this neuroendocrine system. In other insects, however, the labial neurons either express products of the fxprl-gene or products of the capa-gene. The processing of the respective genes is reduced to extended WFGPRLamides in each case and yields a unique peptidome in the labial cells. Thus, sequence-related messenger molecules are always produced in these cells and it seems that the respective neurons recruited different neuropeptide genes for this motif.

The neuropeptidomics of Ixodes scapularis synganglion.

Ticks (Ixodoidea) likely transmit the greatest variety of human and animal pathogens of any arthropod vector. Despite their medical significance little data is available about the messenger molecules in the central nervous system that coordinate all physiological processes in these animals, including behaviour. In our study, we performed the first comprehensive neuropeptidomic analysis of a tick species by using MALDI-TOF mass spectrometry. Specifically we analyzed the neuropeptides in the synganglion of Ixodes scapularis. The forthcoming sequence of the genome of this species will represent the first genomic analysis of a member of the large subphylum Chelicerata. For our approach we used information from predicted neuropeptide precursor sequences found in EST databases [Christie, AE. Neuropeptide discovery in Ixodoidea: an in silico investigation using publicly accessible expressed sequence tags. Gen Comp Endocrinol 2008;157:174-185] as well as data obtained by complete de novo sequencing. The direct tissue profiling yielded 20 neuropeptides from 12 neuropeptide precursors. The sequences of these neuropeptides are not as unique as predicted; a comparison with the peptidome of other invertebrates shows a close relationship with insect neuropeptides. This work will provide a resource for studying tick neurobiology and will hopefully also help to identify novel targets for tick and tick-borne disease control.

Neuropeptides in Heteroptera: identification of allatotropin-related peptide and tachykinin-related peptides using MALDI-TOF mass spectrometry.

Recently, the peptidomic analysis of neuropeptides from the retrocerebral complex and abdominal perisympathetic organs of polyphagous stinkbugs (Pentatomidae) revealed the group-specific sequences of pyrokinins, CAPA peptides (CAPA-periviscerokinins/PVKs and CAPA-pyrokinin), myosuppressin, corazonin, adipokinetic hormone, and short neuropeptide F. In this study, we used mass spectrometric profiling of nervous tissue from the species-rich taxon Hemiptera to identify products of two previously unobserved neuropeptide genes from these species, namely allatotropin-related peptide (ATRP) and tachykinin-related peptides (TKRPs). Since neither TKRPs nor allatotropin are accumulated in neurohemal organs, immunocytochemical data were analyzed to find potential accumulation sites within the central nervous system. By mass spectrometry, TKRPs were found to be accumulated in the antennal lobes, and ATRP was identified in the most posterior region of the abdominal ventral nerve cord and fourth abdominal nerves. In addition to neuropeptides from stink bugs, TKRPs and ATRP were also identified from the distantly related bugs Oncopeltus fasciatus (Lygaeidae) and Pyrrhocoris apterus (Pyrrhocoridae). In total, six TKRPs and one ATRP from each species could be elucidated by tandem mass spectrometry. The ATRP of all species is sequence-identical with Locusta migratoria accessory gland myotropin-1 (Lom-AG-MT-1), a member of the highly conserved insect allatotropin family.

Examination of the role of FMRFamide-related peptides in the circadian clock of the cockroach Leucophaea maderae.

The accessory medulla, the circadian clock of the cockroach Leucophaea maderae, is abundant in neuropeptides. Among these neuropeptides are the FMRFamide-related peptides (FaRPs), which generally share the C-terminal RFamide. As a first step toward understanding the functional role of FaRPs in the circadian clock of the cockroach, immunocytochemistry with antisera against various FaRPs, MALDI-TOF mass spectrometry, and injections of two FaRPs combined with running-wheel assays were performed. Prominent FMRFamide-like immunoreactivity was found in maximally four soma clusters associated with the accessory medulla and in most neuropils of the protocerebrum. By MALDI-TOF mass spectrometry, various extended FMRFamides of the cockroach L. maderae were partially identified in thoracic perisympathetic organs, structures known to accumulate extended FMRFamides in insects. By mass match, several of these peptides were also detected in the accessory medulla. Injections of FMRFamide and Pea-FMRFa-7 (DRSDNFIRF-NH(2)) into the vicinity of the accessory medulla caused time-dependent phase-shifts of locomotor activity rhythms at circadian times 8, 18, and 4. Thus, our data suggest a role for the different FaRPs in the control of circadian locomotor activity rhythms in L. maderae.

Single-cell peptidomics of drosophila melanogaster neurons identified by Gal4-driven fluorescence.

Neuropeptides are widespread signal molecules that display a great chemical and functional diversity. Predictions of neuropeptide cleavage from precursor proteins are not always correct, and thus, biochemical identification is essential. Single-cell analysis is valuable to identify peptides processed from a single precursor, but also to determine coexpression of further neuropeptides from other precursors. We have developed an approach to isolate single identified neurons from the fruit fly Drosophila melanogaster for mass spectrometric analysis. By using Gal4 promoter lines to drive green fluorescent protein under UAS control, we identified specific peptidergic neurons. These neurons were isolated singly under a fluorescence microscope and subjected to MALDI-TOF mass spectrometry. Two Gal4 lines were used here to identify pigment-dispersing factor (PDF) and hugin-expressing neurons. We found that the large PDF expressing clock neurons only give rise to a single peptide, PDF. The three different classes of hugin expressing neurons all display the same mass signal, identical to pyrokinin-2. The other peptide predicted from the hugin precursor, hugin gamma, was not detected in any of the cells. Single-cell peptidomics is a powerful tool in Drosophila neuroscience since Gal4 drivers can be produced for all known neuropeptide genes and thus provide detailed information about neuropeptide complements in neurons of interest.

Localization of leucomyosuppressin in the brain and circadian clock of the cockroach Leucophaea maderae.

The myosuppressins (X1DVX2HX3FLRFamide), which reduce the frequency of insect muscle contractions, constitute a subgroup of the FMRFamide-related peptides. In the cockroach Leucophaea maderae, we have examined whether leucomyosuppressin (pQDVDHVFLRFamide) is present in the accessory medulla, viz., the circadian clock, which governs circadian locomotor activity rhythms. Antisera that specifically recognize leucomyosuppressin stain one to three neurons near the accessory medulla. MALDI-TOF mass spectrometry has confirmed the presence of leucomyosuppressin in the isolated accessory medulla. Injections of 1.15 pmol leucomyosuppressin into the vicinity of the accessory medulla at various circadian times have revealed no statistically significant effects on the phase of circadian locomotor activity rhythms. This is consistent with the morphology of the myosuppressin-immunoreactive neurons, which restrict their arborizations to the circadian clock and other optic lobe neuropils. Thus, leucomyosuppressin might play a role in the circadian system other than in the control of locomotor activity rhythms.

Tachykinin-related peptide precursors in two cockroach species.

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-3(11-19), 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.