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.

Transcriptomic, peptidomic, and mass spectrometry imaging analysis of the brain in the ant Cataglyphis nodus.

Behavioral flexibility is an important cornerstone for the ecological success of animals. Social Cataglyphis nodus ants with their age-related polyethism characterized by age-related behavioral phenotypes represent a prime example for behavioral flexibility. We propose neuropeptides as powerful candidates for the flexible modulation of age-related behavioral transitions in individual ants. As the neuropeptidome of C. nodus was unknown, we collected a comprehensive peptidomic data set obtained by transcriptome analysis of the ants' central nervous system combined with brain extract analysis by Q-Exactive Orbitrap mass spectrometry (MS) and direct tissue profiling of different regions of the brain by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. In total, we identified 71 peptides with likely bioactive function, encoded on 49 neuropeptide-, neuropeptide-like, and protein hormone prepropeptide genes, including a novel neuropeptide-like gene (fliktin). We next characterized the spatial distribution of a subset of peptides encoded on 16 precursor proteins with high resolution by MALDI MS imaging (MALDI MSI) on 14 µm brain sections. The accuracy of our MSI data were confirmed by matching the immunostaining patterns for tachykinins with MSI ion images from consecutive brain sections. Our data provide a solid framework for future research into spatially resolved qualitative and quantitative peptidomic changes associated with stage-specific behavioral transitions and the functional role of neuropeptides in Cataglyphis ants.

Descending octopaminergic neurons modulate sensory-evoked activity of thoracic motor neurons in stick insects.

Neuromodulatory neurons located in the brain can influence activity in locomotor networks residing in the spinal cord or ventral nerve cords of invertebrates. How inputs to and outputs of neuromodulatory descending neurons affect walking activity is largely unknown. With the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and immunohistochemistry, we show that a population of dorsal unpaired median (DUM) neurons descending from the gnathal ganglion to thoracic ganglia of the stick insect Carausius morosus contains the neuromodulatory amine octopamine. These neurons receive excitatory input coupled to the legs' stance phases during treadmill walking. Inputs did not result from connections with thoracic central pattern-generating networks, but, instead, most are derived from leg load sensors. In excitatory and inhibitory retractor coxae motor neurons, spike activity in the descending DUM (desDUM) neurons increased depolarizing reflexlike responses to stimulation of leg load sensors. In these motor neurons, descending octopaminergic neurons apparently functioned as components of a positive feedback network mainly driven by load-detecting sense organs. Reflexlike responses in excitatory extensor tibiae motor neurons evoked by stimulations of a femur-tibia movement sensor either are increased or decreased or were not affected by the activity of the descending neurons, indicating different functions of desDUM neurons. The increase in motor neuron activity is often accompanied by a reflex reversal, which is characteristic for actively moving animals. Our findings indicate that some descending octopaminergic neurons can facilitate motor activity during walking and support a sensory-motor state necessary for active leg movements.NEW & NOTEWORTHY We investigated the role of descending octopaminergic neurons in the gnathal ganglion of stick insects. The neurons become active during walking, mainly triggered by input from load sensors in the legs rather than pattern-generating networks. This report provides novel evidence that octopamine released by descending neurons on stimulation of leg sense organs contributes to the modulation of leg sensory-evoked activity in a leg motor control system.

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.

Quantification of Biogenic Amines from Individual GFP-Labeled Drosophila Cells by MALDI-TOF Mass Spectrometry.

Cell-cell communication plays a crucial role in orchestrating and modulating neural circuits. To understand such interactions, it is vital to determine and quantify the involved messenger molecules such as neuropeptides and biogenic amines on the level of single cells. In this study, we used single-cell mass spectrometry (SCMS) to qualify and quantify octopamine (OA) and tyramine (TA) from isolated single cells from intact brains of the fruit fly Drosophila melanogaster. Our workflow involved targeted GFP-guided single-cell microdissection, on-plate chemical derivatization with 4-hydroxy-3-methoxycinnamaldehyde (CA) or 2,5-dimethyl-1 H-pyrrole-3,4-dicarbaldehyde (DPD) for increasing ion stability and ion signal intensity, and isotopically marked internal standards for quantification by MALDI-TOF MS. We were able to determine a limit of detection for OA of 1 fmol/µL, for TA of 2.5 fmol/µL and a lower limit of quantification (LLOQ) of 10 fmol/µL for both substances. SCMS of GFP-labeled somata from ventral midline neurons of the labial neuromere (VMlb) of the gnathal ganglion revealed an OA titer of 17.38 fmol/µL and a TA titer (∼2.5 fmol/µL) lower than the LLOQ, independent of sex. However, using a genetically altered driver line devoid of OA, TßhnM18/Tdc2 > GFP, we confirmed TA in these cells. Furthermore, cold-anesthetization of flies caused a significant increase in OA content in VMlb somata. We compared OA titers of somata from two different OA/TA cell clusters to demonstrate the usefulness of targeted SCMS in advancing our understanding of OA/TA signaling in behavior and physiology. An influence on the detection of neuropeptides by our derivatized SCMS method could be excluded.

Analysis of Single Neurons by Perforated Patch Clamp Recordings and MALDI-TOF Mass Spectrometry.

Single-cell mass spectrometry has become an established technique to study specific molecular properties such as the neuropeptide complement of identified neurons. Here, we describe a strategy to characterize, by MALDI-TOF mass spectrometry, neurochemical composition of neurons that were identified by their electrophysiological and neuroanatomical characteristics. The workflow for the first time combined perforated patch clamp recordings with dye loading by electroporation for electrophysiological and neuroanatomical characterization as well as chemical profiling of somata by MALDI-TOF mass spectrometry with subsequent immunohistochemistry. To develop our protocol, we used identified central olfactory neurons from the American cockroach Periplaneta americana. First, the combined approach was optimized using a relative homogeneous, well-characterized neuron population of uniglomerular projection neurons, which show acetylcholine esterase immunoreactivity. The general applicability of this approach was verified on local interneurons, which are a diverse neuron population expressing highly differentiated neuropeptidomes. Thus, this study shows that the newly established protocol is suitable to comprehensively analyze electrophysiological, neuroanatomical, and molecular properties of single neurons. We consider this approach an important step to foster single-cell analysis in a wide variety of neuron types.

Profiling neurotransmitters in a crustacean neural circuit for locomotion.

Locomotor systems are widely used to study rhythmically active neural networks. These networks have to be coordinated in order to produce meaningful behavior. The crayfish swimmeret system is well suited to investigate such coordination of distributed neural oscillators because the neurons and their connectivity for generating and especially for coordinating the motor output are identified. The system maintains a fixed phase lag between the segmental oscillators, independent of cycle period. To further the understanding of the system's plasticity for keeping the phase lag fixed, we profiled the neurotransmitters used by the Coordinating Neurons, which are necessary and sufficient for coordination of the segmental oscillators. We used a combination of electrophysiological, immunohistochemical, and mass spectrometric methods. This arrangement of methods ensured that we could screen for several specific neurotransmitters, since a single method is often not suitable for all neurotransmitters of interest. In a first step, to preselect neurotransmitter candidates, we investigated the effect of substances known to be present in some swimmeret system neurons on the motor output and coordination. Subsequently, we demonstrated electrophysiologically that the identified synapse between the Coordinating Neurons and their target is mainly chemical, but neither glutamate antagonist nor γ-aminobutyric acid antagonist application affected this synapse. With immunohistochemical experiments, we provide strong evidence that the Coordinating Neurons are not serotonergic. Single-cell MALDI-TOF mass spectrometry with subsequent principal component analysis identified acetylcholine as the putative neurotransmitter for both types of Coordinating Neurons.

Transcriptomic and Neuropeptidomic Analysis of the Stick Insect, Carausius morosus.

One of the most thoroughly studied insect species, with respect to locomotion behavior, is the stick insect Carausius morosus. Although detailed information exists on premotor networks controlling walking, surprisingly little is known about neuropeptides, which are certainly involved in motor activity generation and modulation. So far, only few neuropeptides were identified from C. morosus or related stick insects. We performed a transcriptome analysis of the central nervous system to assemble and identify 65 neuropeptide and protein hormone precursors of C. morosus, including five novel putative neuropeptide precursors without clear homology to known neuropeptide precursors of other insects ( Carausius neuropeptide-like precursor 1, HanSolin, PK-like1, PK-like2, RFLamide). Using Q Exactive Orbitrap and MALDI-TOF mass spectrometry, 277 peptides including 153 likely bioactive mature neuropeptides were confirmed. Peptidomics yielded a complete coverage for many of the neuropeptide propeptides and confirmed a surprisingly high number of heterozygous sequences. Few neuropeptide precursors commonly occurring in insects, including those of insect kinins and sulfakinins, could neither be found in the transcriptome data nor did peptidomics support their presence. The results of our study represent one of the most comprehensive peptidomic analyses on insects and provide the necessary input for subsequent experiments revealing neuropeptide function in greater detail.

Single Cell Peptidomics: Approach for Peptide Identification by N-Terminal Peptide Derivatization.

In recent years, single cell microanalysis techniques have moved into the center stage to study fundamental intracellular interactions and cell-cell communication events, and have led to a better understanding of physiological processes and behavioral patterns. The availability of more sensitive, robust, and precise mass spectrometers improved the detection and characterization of putative neuroactive substances from individual cells. For sequence characterization, particularly when working with samples as small as a single cell, the most crucial step to obtain usable data is sample preparation. For some studies, genetic or molecular data are not available to confirm an amino acid sequence of a putative neuropeptide, and it is necessary to sequence the peptide from the mass spectrometry analysis alone (i.e., de novo sequencing). In this chapter, a protocol is described for de novo sequencing of neuropeptides from individual single cells by N-terminal derivatization using 4-sulfophenyl isothiocyanate and subsequent mass spectrometric analysis.

Neuropeptide Mapping of Dimmed Cells of Adult Drosophila Brain.

Neuropeptides are structurally highly diverse messenger molecules that act as regulators of many physiological processes such as development, metabolism, reproduction or behavior in general. Differentiation of neuropeptidergic cells often corresponds with the presence of the transcription factor DIMMED. In the central nervous system of the fruit fly Drosophila melanogaster, DIMMED commonly occurs in neuroendocrine neurons that release peptides as neurohormones but also in interneurons with complex branching patterns. Fly strains with green fluorescence protein (GFP)-expressing dimmed cells make it possible to systematically analyze the processed neuropeptides in these cells. In this study, we mapped individual GFP-expressing neurons of adult D. melanogaster from the dimmed (c929)>GFP line. Using single cell mass spectrometry, we analyzed 10 types of dimmed neurons from the brain/gnathal ganglion. These cells included neuroendocrine cells with projection into the retrocerebral complex but also a number of large interneurons. Resulting mass spectra not only provided comprehensive data regarding mature products from 13 neuropeptide precursors but also evidence for the cellular co-localization of neuropeptides from different neuropeptide genes. The results can be implemented in a neuroanatomical map of the D. melanogaster brain. Graphical Abstract ᅟ.

Neuropeptidomics of the Bed Bug Cimex lectularius.

The bed bug Cimex lectularius is a globally distributed human ectoparasite with fascinating biology. It has recently acquired resistance against a broad range of insecticides, causing a worldwide increase in bed bug infestations. The recent annotation of the bed bug genome revealed a full complement of neuropeptide and neuropeptide receptor genes in this species. With regard to the biology of C. lectularius, neuropeptide signaling is especially interesting because it regulates feeding, diuresis, digestion, as well as reproduction and also provides potential new targets for chemical control. To identify which neuropeptides are translated from the genome-predicted genes, we performed a comprehensive peptidomic analysis of the central nervous system of the bed bug. We identified in total 144 different peptides from 29 precursors, of which at least 67 likely present bioactive mature neuropeptides. C. lectularius corazonin and myosuppressin are unique and deviate considerably from the canonical insect consensus sequences. Several identified neuropeptides likely act as hormones, as evidenced by the occurrence of respective mass signals and immunoreactivity in neurohemal structures. Our data provide the most comprehensive peptidome of a Heteropteran species so far and in comparison suggest that a hematophageous life style does not require qualitative adaptations of the insect peptidome.

Different processing of CAPA and pyrokinin precursors in the giant mealworm beetle Zophobas atratus (Tenebrionidae) and the boll weevil Anthonomus grandis grandis (Curculionidae).

Capa and pyrokinin (pk) genes in hexapods share a common evolutionary origin. Using transcriptomics and peptidomics, we analyzed products of these genes in two beetles, the giant mealworm beetle (Zophobas atratus; Tenebrionidae) and the boll weevil (Anthonomus grandis grandis; Curculionidae). Our data revealed that even within Coleoptera, which represents a very well-defined group of insects, highly different evolutionary developments occurred in the neuropeptidergic system. These differences, however, primarily affect the general structure of the precursors and differential processing of mature peptides and, to a lesser degree, the sequences of the active core motifs. With the differential processing of the CAPA-precursor in Z. atratus we found a perfect example of completely different products cleaved from a single neuropeptide precursor in different cells. The CAPA precursor in abdominal ganglia of this species yields primarily periviscerokinins (PVKs) whereas processing of the same precursor in neurosecretory cells of the subesophageal ganglion results in CAPA-tryptoPK and a novel CAPA-PK. Particularly important was the detection of that CAPA-PK which has never been observed in the CNS of insects before. The three different types of CAPA peptides (CAPA-tryptoPK, CAPA-PK, PVK) each represent potential ligands which activate different receptors. In contrast to the processing of the CAPA precursor from Z. atratus, no indications of a differential processing of the CAPA precursor were found in A. g. grandis. These data suggest that rapid evolutionary changes regarding the processing of CAPA precursors were still going on when the different beetle lineages diverged. The sequence of the single known PVK of A. g. grandis occupies a special position within the known PVKs of insects and might serve asa basis to develop lineage-specific peptidomimetics capable of disrupting physiological processes regulated by PVKs.

Identification of mature peptides from pban and capa genes of the moths Heliothis peltigera and Spodoptera littoralis.

By transcriptome analysis, we identified PBAN and CAPA precursors in the moths Spodoptera littoralis and Heliothis peltigera which are among the most damaging pests of agriculture in tropical and subtropical Africa as well as in Mediterranean countries. A combination of mass spectrometry and immunocytochemistry was used to identify mature peptides processed from these precursors and to reveal their spatial distribution in the CNS. We found that the sites of expression of pban genes, the structure of PBAN precursors and the processed neuropeptides are very similar in noctuid moths. The sequence of the diapause hormone (DH; tryptopyrokinin following the signal peptide), however, contains two N-terminal amino acids more than expected from comparison with already published sequences of related species. Capa genes of S. littoralis and H. peltigera encode, in addition to periviscerokinins, a tryptopyrokinin showing sequence similarity with DH, which is the tryptopyrokinin of the pban gene. CAPA peptides, which were not known from any noctuid moth so far, are produced in cells of abdominal ganglia. The shape of the release sites of these hormones in H. peltigera represents an exceptionally derived trait state and does not resemble the well-structured abdominal perisympathetic organs which are known from many other insects. Instead, axons of CAPA cells extensively ramify within the ventral diaphragm. The novel information regarding the sequences of all mature peptides derived from pban and capa genes of H. peltigera and S. littoralis now enables a detailed analysis of the bioactivity and species-specificity of the native peptides, especially those from the hitherto unknown capa genes, and to explore their interactions with PBAN/DH receptors.

Identification and distribution of products from novel tryptopyrokinin genes in the locust, Locusta migratoria.

A recent analysis of the genome of Locusta migratoria indicated the presence of four novel insect neuropeptide genes encoding for multiple tryptopyrokinin peptides (tryptoPKs); hitherto only known from pyrokinin or capa genes. In our study, mature products of tryptoPK genes 1 and 2 were identified by mass spectrometry; precursor sequences assigned to the tryptoPK genes 3 and 4 are likely partial sequences of a single precursor. The expression of tryptoPK genes 1 and 2 is restricted to two cells in the subesophageal ganglion, exhibiting not only a unique neuropeptidome but also a very distinctive axonal projection. Comparative neuroendocrinology revealed that homologous cells in other insects also produce tryptoPKs but use other genes to generate this pattern. Since capa and pyrokinin genes are discussed as ancestors of the tryptoPK genes, we completed the hitherto only partially known precursor sequences of these genes by means of transcriptome analyses. The distribution of mature products of CAPA and pyrokinin precursors in the CNS is compared with that of tryptoPKs. In addition, a novel pyrokinin-like precursor is described.

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.

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.

Identification and distribution of SIFamide in the nervous system of the desert locust Schistocerca gregaria.

SIFamides are a family of highly conserved arthropod neuropeptides. To date, nine orthocopies from different arthropods, most of them insects, have been identified, all consisting of 11-12 amino acid residues. The striking conservation in sequence is mirrored by highly similar morphologies of SIFamide-immunoreactive neurons: immunolabeling in various insect species revealed four immunopositive neurons with somata in the pars intercerebralis and arborizations extending throughout the brain and ventral nervous system. In contrast, the functional role of these neurons and their neuropeptide SIFamide is largely obscure. To provide an additional basis for functional analysis, we identified, by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, a SIFamide peptide in the desert locust Schistocerca gregaria and studied its distribution throughout the nervous system. Identification was supported by analysis of transcriptomic data obtained from another grasshopper, Stenobothrus lineatus. Scg-SIFamide, unlike all SIFamides identified so far, is a pentadecapeptide with an extended and highly modified N-terminus (AAATFRRPPFNGSIFamide). As in other insects, pairs of descending neurons with somata in the pars intercerebralis and ramifications in most areas of the nervous system are SIFamide-immunoreactive. In addition, a small number of local interneurons in the brain and ventral ganglia were immunostained. Double-label experiments showed that the SIFamide-immunoreactive descending neurons are identical to previously characterized primary commissure pioneer (PNP) neurons of the locust brain that pioneer the first commissure in the brain. The data suggest that the descending SIFamide-immunoreactive neurons play a developmental role in organizing the insect central nervous system. J. Comp. Neurol. 523:108-125, 2015. © 2014 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.

Neonatal insulin action impairs hypothalamic neurocircuit formation in response to maternal high-fat feeding.

Maternal metabolic homeostasis exerts long-term effects on the offspring's health outcomes. Here, we demonstrate that maternal high-fat diet (HFD) feeding during lactation predisposes the offspring for obesity and impaired glucose homeostasis in mice, which is associated with an impairment of the hypothalamic melanocortin circuitry. Whereas the number and neuropeptide expression of anorexigenic proopiomelanocortin (POMC) and orexigenic agouti-related peptide (AgRP) neurons, electrophysiological properties of POMC neurons, and posttranslational processing of POMC remain unaffected in response to maternal HFD feeding during lactation, the formation of POMC and AgRP projections to hypothalamic target sites is severely impaired. Abrogating insulin action in POMC neurons of the offspring prevents altered POMC projections to the preautonomic paraventricular nucleus of the hypothalamus (PVH), pancreatic parasympathetic innervation, and impaired glucose-stimulated insulin secretion in response to maternal overnutrition. These experiments reveal a critical timing, when altered maternal metabolism disrupts metabolic homeostasis in the offspring via impairing neuronal projections, and show that abnormal insulin signaling contributes to this effect.

Neuropeptides in the antennal lobe of the yellow fever mosquito, Aedes aegypti.

For many insects, including mosquitoes, olfaction is the dominant modality regulating their behavioral repertoire. Many neurochemicals modulate olfactory information in the central nervous system, including the primary olfactory center of insects, the antennal lobe. The most diverse and versatile neurochemicals in the insect nervous system are found in the neuropeptides. In the present study, we analyzed neuropeptides in the antennal lobe of the yellow fever mosquito, Aedes aegypti, a major vector of arboviral diseases. Direct tissue profiling of the antennal lobe by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry indicated the presence of 28 mature products from 10 different neuropeptide genes. In addition, immunocytochemical techniques were used to describe the cellular location of the products of up to seven of these genes within the antennal lobe. Allatostatin A, allatotropin, SIFamide, FMRFamide-related peptides, short neuropeptide F, myoinhibitory peptide, and tachykinin-related peptides were found to be expressed in local interneurons and extrinsic neurons of the antennal lobe. Building on these results, we discuss the possible role of neuropeptide signaling in the antennal lobe of Ae. aegypti.