Suboesophageal Ganglion Research Articles

The complex neurochemistry of the cockroach antennal heart

The innervation of the antennal heart of the cockroach Periplaneta americana was studied with immunocytochemical techniques on both the light and electron microscopic levels. The antennal heart is innervated by two efferent systems, both using one biogenic amine in combination with neuropeptides. In one, we found co-localization of serotonin with proctolin and allatostatin. These fibers most likely originate from paired neurons located in the suboesophageal ganglion. In the second system, we found octopamine co-localized with the short neuropeptide F. The source of this second system is dorsal unpaired median (DUM) neurons, also located in the suboesophageal ganglion. The possible effects of these neuromediators on different targets are discussed.

NERVOUS SYSTEM OF A PIGEON SLENDER LOUSE COLUMBICOLA COLUMBAE (PHTHIRAPTERA: ISCHNOCERA)

The nervous system of a pigeon slender louse, Columbicola columbae, has been studied in greater detail. C. columbae is a permanent, obligatory, and wingless insect. The central nervous system consists of a series of ganglia that supply nerves to successive body segments. The three main ganglia in the head, protocerebrum, deutocerebrum, and tritocerebrum, are fused to form the brain or supraesophageal ganglion. The nervous system of the pigeon slender louse, C. columbae (Phthiraptera: Ischnocera), consists of a U-shaped compact cerebral ganglion, subesophageal ganglion, and three thoracic ganglia. The ventral nerve cord and subesophageal ganglion are also located in the head. The three thoracic ganglions are placed in pro-, meso-, and meta-thorax. The frontal ganglion is situated anterior to the brain at a considerable distance.. KEYWORDS :Brain, Columbicola columbae, Nervous system, Pigeon slender louse

A new species of Simulium (Asiosimulium) (Diptera: Simuliidae) from northeastern Thailand, with its phylogenetic relationships with related species in the subgenus Asiosimulium.

Simulium (Asiosimulium) khongchiamense sp. nov. is described based on females, males, pupae, and mature larvae collected from Khong Chiam District, Ubon Ratchathani Province, northeastern Thailand. It is characterized in the female by the medium-long sensory vesicle, scutum with 3 dark longitudinal vittae and elongate cercus; in the male by the number of upper-eye (large) facets in 17 or 18 vertical rows and 18 or 19 horizontal rows, hind basitarsus moderately enlarged and ventral plate with the posterior margin moderately concave medially; in the pupa by the head and thoracic integument sparsely covered with tubercles and gill of arborescent type with 32 or 33 filaments; and in the larva by the postgenal cleft deep, reaching the posterior margin of the hypostoma and sheath of the subesophageal ganglion dark pigmented. DNA analysis based on COI gene of all known species of the subgenus Asiosimulium, except for S. shanense and S. suchitrae, indicated that this new species can be clearly differentiated from all other related species (S. phurueaense, S. oblongum, S. saeungae, S. furvum, and S. wanchaii) with interspecific genetic distances ranging between 4.79% and 19.18%. This is the eighth species of the subgenus Asiosimulium. Taxonomic notes are given to distinguish this new species from the 7 known species members in its same subgenus. Additionally, keys to species of all members in the subgenus Asiosimulium are provided.

Caterpillars (Manduca sexta) parasitized by the wasp Cotesia congregata stop chewing despite an intact motor system.

The parasitic wasp Cotesia congregata suppresses feeding in its host, the caterpillar Manduca sexta, during specific periods of wasp development. We examined both feeding behaviour and the neurophysiology of the mandibular closer muscle in parasitized and unparasitized control M. sexta to determine how the wasp may accomplish this. To test whether the wasps activated a pre-existing host mechanism for feeding cessation, we examined the microstructure of feeding behaviour in caterpillars that stopped feeding due to illness-induced anorexia or an impending molt. These microstructures were compared to that shown by parasitized caterpillars. While there were overall differences between parasitized and unparasitized caterpillars, the groups showed similar progression in feeding microstructure as feeding ended, suggesting a common pattern for terminating a meal. Parasitized caterpillars also consumed less leaf area in 100 bites than control caterpillars at around the same time their feeding microstructure changed. The decline in food consumption was accompanied by fewer spikes/burst and shorter burst durations in chewing muscle electromyograms. Similar extracellular results were obtained from the motorneuron of the mandibular closer muscle. However, chewing was dramatically re-activated in non-feeding parasitized caterpillars if the connectives posterior to the subesophageal ganglion were severed. The same result was observed in unparasitized caterpillars given the same treatment. Our results suggest that the reduced feeding in parasitized caterpillars was not due to damage to the central pattern generator (CPG) for chewing, motor nerves or chewing muscles, but was more likely due to a suppression of chewing CPG activity by ascending or descending inputs.

Functional Analysis of Pheromone Biosynthesis Activating Neuropeptide Receptor Isoforms in Maruca vitrata.

Insect sex pheromones are volatile chemicals that induce mating behavior between conspecific individuals. In moths, sex pheromone biosynthesis is initiated when pheromone biosynthesis-activating neuropeptide (PBAN) synthesized in the suboesophageal ganglion binds to its receptor on the epithelial cell membrane of the pheromone gland. To investigate the function of PBAN receptor (PBANR), we identified two PBANR isoforms, MviPBANR-B and MviPBANR-C, in the pheromone glands of Maruca vitrata. These two genes belong to G protein-coupled receptors (GPCRs) and have differences in the C-terminus but share a 7-transmembrane region and GPCR family 1 signature. These isoforms were expressed in all developmental stages and adult tissues. MviPBANR-C had the highest expression level in pheromone glands among the examined tissues. Through in vitro heterologous expression in HeLa cell lines, only MviPBANR-C-transfected cells responded to MviPBAN (≥5 µM MviPBAN), inducing Ca2+ influx. Sex pheromone production and mating behavior were investigated using gas chromatography and a bioassay after MviPBANR-C suppression by RNA interference, which resulted in the major sex pheromone component, E10E12-16:Ald, being quantitatively reduced compared to the control, thereby decreasing the mating rate. Our findings indicate that MviPBANR-C is involved in the signal transduction of sex pheromone biosynthesis in M. vitrata and that the C-terminal tail plays an important role in its function.

Redundant actions of neuropeptides encoded by the dh-pban gene for larval color pattern formation in the oriental armyworm Mythimnaseparata

The pyrokinin (PK)/pheromone biosynthesis-activating neuropeptide (PBAN) family, which is defined by a conserved C-terminal pentapeptide (FXPRLamide), is involved in many physiological processes in insects. In the oriental armyworm Mythimna separata, the larvae exhibit a variety of color patterns in response to changes in population density, which are caused by melanization and a reddish coloration hormone (MRCH), which is a member of the FXPRLamide neuropeptides. Interestingly, in some lepidopteran insects, MRCH is known as a PBAN, which activates the pheromone gland to produce sex pheromones. PBAN is encoded by a single gene, dh-pban, which encodes additional FXPRLamide neuropeptides, such as the diapause hormone (DH) and subesophageal ganglion neuropeptides (SGNPs). To determine the roles of the dh-pban gene, which produces multiple types of FXPRLamide neuropeptides after post-transcriptional cleavage of the precursor protein, we performed CRISPR/Cas9-mediated targeted mutagenesis in M. separata. We demonstrated that knockout armyworm larvae lost density-dependent cuticular melanization and retained yellow body color, even when reared under crowded conditions. Moreover, our rescue experiments using the synthetic peptides showed that not only PBAN but also β- and γ-SGNPs significantly induce the cuticular melanization in a dose dependent manner. Taken together, our results provide genetic evidence that neuropeptides encoded by the single dh-pban gene act redundantly to control density-dependent color pattern formation in M. separata.

The morphology of the free-living females of Strepsiptera (Insecta).

The morphology of the adult free-living females of Mengenilla moldrzyki and Eoxenos laboulbenei (Strepsiptera, Mengenillidae) was documented with µCT-based 3D reconstructions and histological serial sections. External and internal features of both species are characterized by far-reaching specialization and structural simplification. The well-developed mandibles are moved by large muscles. Other mouthparts and their corresponding musculature are simplified or absent. The brain is partly shifted into the prothorax. It is followed by a single postcephalic postcerebral ganglionic complex also containing the suboesophageal complex and an unpaired abdominal nerve. Postcephalic sclerites are absent, except for the plate-like pronotum and small pleural sclerites. Wings and associated muscles are missing. The lumina of the large midgut and the anterior hindgut are disconnected. Seven bulb-shaped Malpighian tubules in M. moldrzyki is the highest number yet described for Strepsiptera. The 10-segmented abdomen lacks appendages. An unpaired birth organ opens ventrally on abdominal segment VII. The entire body cavity is filled with numerous freely floating eggs, 1386 in the specimen M. moldrzyki and 721 in E. laboulbenei. Genital ducts, defined gonads, and genital glands are missing. The morphology of female Mengenillidae is discussed with respect to sexual dimorphism and structural features of the postembryonic stages. Phylogenetic implications are outlined. This article is protected by copyright. All rights reserved.

Electroconductive copolymer EDOT-pyrrole for intracellular recording experiments in Helix aspersa neurons

The typical electrodes used on neuronal intracellular recording consist fundamentally of Ag/AgCl. However, the conductive conjugated polymers represent a novel material that barely has been investigated on the neuronal intercellular recoding as an electrode. Herein, we demonstrate the feasibility of copolymer EDOT-pyrrole-coated electrodes for neural intracellular recordings. The electrical characterization of the electrophoretic deposition-coated electrodes was crucial to demonstrate the stability in the electrolytic medium. Recordings of action potentials using Helix aspersa subesophageal ganglion neurons showed that the electrodes made by electrophoretic deposition of EDOT-PY 0.9–0.1/PO4 copolymer are sensitive to bioelectrical signals and stable in the application of current with a signal amplitude almost two times compared with the Ag/AgCl value and high signal-to-noise ratio (SNR = 15 dB) against the Ag/AgCl electrodes (SNR = 11 dB). Such characteristics are superior to the commercial Ag/AgCl electrodes, which means a prominent material for applications in electrophysiology and neurophysiology.

Pirenzepine Binding Sites in the Brain of the Honeybee Apis mellifera: Localization and Involvement in Non-Associative Learning.

Simple SummaryThe cholinergic system has been extensively studied in insects as the target of pesticides that affect the functioning of the nicotinic acetylcholine receptors. In the honeybee, the detrimental effects of pesticides are observed in behavior, including associative learning. The current work describes the expression of muscarinic acetylcholine receptors in the honeybee brain. We show that the muscarinic antagonist pirenzepine induces a slow-down of non-associative learning, the habituation of the proboscis extension reflex. We suggest that pirenzepine binding sites located on the neurons of the subesophagous ganglion are involved in the habituation of the reflex.Muscarinic acetylcholine receptors (mAChRs) play a central role in learning and memory in mammals as in honeybees. The results obtained in the honeybee Apis mellifera are based on the detrimental effects of the mAChR antagonists, atropine and scopolamine, on olfactory associative memory. Binding sites for the mAChR antagonist BODIPY® FL pirenzepine were localized in the brain of the honeybee forager. Pirenzepine binding sites were detected indifferently in several somata and neuropilar areas. The highest binding site densities were present in the central complex and in somata of the dorsomedial border of the antennal lobes. An additional binding pattern was found in somata of the subesophageal ganglion. By contrast, Kenyon cell (KC) somata were not stained. Pirenzepine (PZ) effects on non-associative learning were evaluated. Treated animals required more trials for the habituation of the proboscis extension reflex (PER) than controls, and the duration of the PER increased after PZ brain injection. These results suggest that the network mediating habituation of the PER involves PZ binding sites that are not necessarily present on the circuitry mediating olfactory conditioning of the PER.

Abolition of egg diapause by ablation of suboesophageal ganglion in parental females is compatible with genetic engineering methods

Microinjection of genetic material into non-diapause eggs is required for genetic engineering of silkworms. Besides diapause could be useful for maintaining transgenic lines, a drawback of this technology is that most standard silkworm strains and experimental lines of interest produce diapausing eggs. Several approaches have been developed to abolish diapause but none are very efficient. Here, we investigated the ablation of the suboesophageal ganglion (SG) in female pupae, which is a source of the hormone required to trigger egg diapause, as a mean to abolish diapause. We showed that SG-ablation is a reliable method to produce nondiapause eggs. Additionally, the challenge associated with lower fecundity of females with SG ablation was resolved by injecting pilocarpine into the mated female. We also investigated the suitability of nondiapause eggs laid by SG-ablated females for transgenesis, targeted mutagenesis, and induction of parthenogenetic development. Our results demonstrated SG-ablation to be a useful and simple method for expanding the possibilities associated with genetic engineering in silkworms.

Discovery of a hidden form of neuropeptide F and its presence throughout the CNS–gut axis in the mud crab, Scylla olivacea

The mud crab Scylla olivacea (Scyol) is an economically crucial crustacean species in Thailand, due to its high market demand and nutritional value. The neuropeptide F (NPF) has been implicated in the coordinated regulation of feeding and metabolism in invertebrates. While various isoforms of neuropeptide F (NPF) have been previously explored in the mud crab, some knowledge gaps in relation to the NPF family, and ambiguities in the nomenclature from previous reports, remain. In this study, NPF was firstly localized in the central nervous system and gastrointestinal tract of the mud crab, S. olivacea, using a polyclonal antibody against Macrobrachium rosenbergii, Macro-NPF. The NPF immunoreactivity (ir) was detected dominantly in the X-organ/sinus gland complex of the eyestalk (ES) and the various neuronal clusters (cluster 6, 9/11, and 14/15) and neuropils (anteromedial and posteromedial protocerebral neuropils, olfactory and accessory olfactory neuropils, and medial antennule neuropil, columnar neuropil) of the brain (BR), commissural ganglia, and suboesophageal ganglion of the ventral nerve cord (VNC). Interestingly, this study also presented the NPF immunoreactivity (NPF-ir) in the acinar gland-like cell and spindle-shaped epithelial cells of S. olivacea intestine. The full-length Scyol-NPFII was characterized by molecular cloning and revealed 414 nucleotides with 375 nucleotides of an open reading frame which encoded 124 deduced amino acids. A 124-amino acid precursor protein of Scyol-NPFII included a 26-residue signal peptide and a 69-amino acid mature peptide. The Scyol-NPFII showed the highest percentage of hit similarity to S. paramamosain-NPFII and clustered in the NPFII family, separated from the other forms of NPFs in this species. The spatial gene expression in various tissues revealed that Scyol-NPFII was found dominantly in the ES and BR, VNC, heart, intestine, and muscle. This study provided a novel form of NPF in the female mud crab, S. olivacea, which could open the possibility of its functioning in the brain-to-gut controlling axis. This study could provide essential information for further application in the cultured system of S. olivacea in the near future.

Octopamine mediates sugar relief from a chronic-stress-induced depression-like state in Drosophila

Chronic, uncontrollable stress can result in psychiatric syndromes, including anxiety and major depressive disorder, in humans and mammalian disease models.1,2 Similarly, several days of chronic stress can induce depression-associated behavioral alteration in Drosophila accompanied by changes in biogenic amine levels in the adult brain.3-6 In our chronic stress paradigm, flies are subjected to 3days of repetitive phases of 300Hz vibrations combined with overcrowding and food deprivation. This treatment reduces voluntary behavioral activity, including the motivation to climb wide gaps (risk taking) and to stop for sweets (anhedonia), suggesting a depression-like state (DLS). These behavioral changes correlate with decreased serotonin release to the mushroom body (MB), a major behavioral control center in the central brain of the fly.7,8 Stressed flies are relieved from the DLS by feeding the anti-depressant serotonin precursor 5-HTP or the selective serotonin reuptake inhibitor fluoxetine. Notably, feeding sucrose to stressed flies results in elevated serotonin levels in the brain and ameliorates the DLS.3 Here, we show that this sugar relief is mediated by the neurotransmitter octopamine signaled from ventral unpaired medial neurons located in the subesophageal ganglion. The octopamine signaling of sweet sensation is transmitted to the MB via the dopaminergic PAM neurons. In addition, neuronal-silencing experiments reveal that the serotonergic dorsal paired medial (DPM) neurons innervating the MB are essential for sugar relief. Conversely, thermogenetic or optogenetic activation of DPMs can replace sweet sensation, elucidating that serotonergic signaling from DPMs takes part in positively modulating DLS-related behavioral changes.

Which Sugar to Take and How Much to Take? Two Distinct Decisions Mediated by Separate Sensory Channels.

In Drosophila melanogaster, gustatory receptor neurons (GRNs) for sugar taste coexpress various combinations of gustatory receptor (Gr) genes and are found in multiple sites in the body. To determine whether diverse sugar GRNs expressing different combinations of Grs have distinct behavioral roles, we examined the effects on feeding behavior of genetic manipulations which promote or suppress functions of GRNs that express either or both of the sugar receptor genesGr5a (Gr5a+ GRNs) and Gr61a (Gr61a+ GRNs). Cell-population-specific overexpression of the wild-type form of Gr5a (Gr5a+) in the Gr5a mutant background revealed that Gr61a+ GRNs localized on the legs and internal mouthpart critically contribute to food choice but not to meal size decisions, while Gr5a+ GRNs, which are broadly expressed in many sugar-responsive cells across the body with an enrichment in the labella, are involved in both food choice and meal size decisions. The legs harbor two classes of Gr61a expressing GRNs, one with Gr5a expression (Gr5a+/Gr61a+ GRNs) and the other without Gr5aexpression (Gr5a−/Gr61a+ GRNs). We found that blocking the Gr5a+ class in the entire body reduced the preference for trehalose and blocking the Gr5a- class reduced the preference for fructose. These two subsets of GRNsare also different in their central projections: axons of tarsal Gr5a+/Gr61a+ GRNs terminate exclusively in the ventral nerve cord, while some axons of tarsal Gr5a−/Gr61a+ GRNs ascend through the cervical connectives to terminate in the subesophageal ganglion. We propose that tarsal Gr5a+/Gr61a+ GRNs and Gr5a−/Gr61a+ GRNs represent functionally distinct sensory pathways that function differently in food preference and meal-size decisions.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo.

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

NEUROSECRETION IN OLIGOCHAETES - A LESS STUDIED SUBJECT IN INVERTEBRATE RESEARCH

The concept of neurosecretion was first developed by Ernst Scharrer (1905-1965) and Berta Scharrer (1906-1995) between 1928 and 1937. The neurosecretory cells are neuronal in structure and glandular in function. Typically, the neurosecretory cells are “tear drop” in appearance with a long axon. The neurosecretory products (neurosecretion/neurohormone) are proteinaeous in nature which is consistent with the ultrastructure of the cells
 In earthworm species, irrespective of their ecological categories, the neurosecretory cells in their distribution, structure and functions in the central nervous system are more or less similar in different species of earthworms. The central nervous system in tropical earthworms reveals mainly two types of neurosecretory cells: A type (outer cortical tier of deeply stained AF positive cells with distinct axonal processes) and B type (light stained cells lying in between A type cells and central fibrous neuropile). In addition to A and B cells, an “islet” of neurosecretory cells called S cells or Hubl cells are present at the base of circum esophageal connectives of subesophageal ganglia. Both A and B cells discharge their secretion (neurohormone) at the margin of highly vascularised fibrous neuropile called “zone of accumulation” which may be considered as “elementary neuro-haemal organ”. Neurosecretion in Oligochaetes has great role in food intake, osmoregulation, thermal acclimation, regeneration, copulation and cocoon production.

A microCT-based atlas of the central nervous system and midgut in sea spiders (Pycnogonida) sheds first light on evolutionary trends at the family level

BackgroundPycnogonida (sea spiders) is the sister group of all other extant chelicerates (spiders, scorpions and relatives) and thus represents an important taxon to inform early chelicerate evolution. Notably, phylogenetic analyses have challenged traditional hypotheses on the relationships of the major pycnogonid lineages (families), indicating external morphological traits previously used to deduce inter-familial affinities to be highly homoplastic. This erodes some of the support for phylogenetic information content in external morphology and calls for the study of additional data classes to test and underpin in-group relationships advocated in molecular analyses. In this regard, pycnogonid internal anatomy remains largely unexplored and taxon coverage in the studies available is limited.ResultsBased on micro-computed X-ray tomography and 3D reconstruction, we created a comprehensive atlas of in-situ representations of the central nervous system and midgut layout in all pycnogonid families. Beyond that, immunolabeling for tubulin and synapsin was used to reveal selected details of ganglionic architecture. The ventral nerve cord consistently features an array of separate ganglia, but some lineages exhibit extended composite ganglia, due to neuromere fusion. Further, inter-ganglionic distances and ganglion positions relative to segment borders vary, with an anterior shift in several families. Intersegmental nerves target longitudinal muscles and are lacking if the latter are reduced. Across families, the midgut displays linear leg diverticula. In Pycnogonidae, however, complex multi-branching diverticula occur, which may be evolutionarily correlated with a reduction of the heart.ConclusionsSeveral gross neuroanatomical features are linked to external morphology, including intersegmental nerve reduction in concert with trunk segment fusion, or antero-posterior ganglion shifts in partial correlation to trunk elongation/compaction. Mapping on a recent phylogenomic phylogeny shows disjunct distributions of these traits. Other characters show no such dependency and help to underpin closer affinities in sub-branches of the pycnogonid tree, as exemplified by the tripartite subesophageal ganglion of Pycnogonidae and Rhynchothoracidae. Building on this gross anatomical atlas, future studies should now aim to leverage the full potential of neuroanatomy for phylogenetic interrogation by deciphering pycnogonid nervous system architecture in more detail, given that pioneering work on neuron subsets revealed complex character sets with unequivocal homologies across some families.

A conditional GABAergic synaptic vesicle marker for Drosophila

BackgroundThroughout the animal kingdom, GABA is the principal inhibitory neurotransmitter of the nervous system. It is essential for maintaining the homeostatic balance between excitation and inhibition required for the brain to operate normally. Identification of GABAergic neurons and their GABA release sites are thus essential for understanding how the brain regulates the excitability of neurons and the activity of neural circuits responsible for numerous aspects of brain function including information processing, locomotion, learning, memory, and synaptic plasticity, among others. New methodSince the structure and features of GABA synapses are critical to understanding their function within specific neural circuits of interest, here we developed and characterized a conditional marker of GABAergic synaptic vesicles for Drosophila, 9XV5-vGAT. Results9XV5-vGAT is validated for conditionality of expression, specificity for localization to synaptic vesicles, specificity for expression in GABAergic neurons, and functionality. Its utility for GABAergic neurotransmitter phenotyping and identification of GABA release sites was verified for ellipsoid body neurons of the central complex. In combination with previously reported conditional SV markers for acetylcholine and glutamate, 9XV5-vGAT was used to demonstrate fast neurotransmitter phenotyping of subesophageal ganglion neurons. Comparison with existing methodsThis method is an alternative to single cell transcriptomics for neurotransmitter phenotyping and can be applied to any neurons of interest represented by a binary transcription system driver. ConclusionA conditional GABAergic synaptic vesicle marker has been developed and validated for GABA neurotransmitter phenotyping and subcellular localization of GABAergic synaptic vesicles.

Abolition of Egg Diapause by Ablation of Suboesophageal Ganglion in Parental Females is Compatible with Genetic Engineering Methods

Abolition of Egg Diapause by Ablation of Suboesophageal Ganglion in Parental Females is Compatible with Genetic Engineering Methods

Cell-specific expression and individual function of prohormone convertase PC1/3 in Tribolium larval growth highlights major evolutionary changes between beetle and fly neuroendocrine systems

BackgroundThe insect neuroendocrine system acts in the regulation of physiology, development and growth. Molecular evolution of this system hence has the potential to allow for major biological differences between insect groups. Two prohormone convertases, PC1/3 and PC2, are found in animals and both function in the processing of neuropeptide precursors in the vertebrate neurosecretory pathway. Whereas PC2-function is conserved between the fly Drosophila and vertebrates, ancestral PC1/3 was lost in the fly lineage and has not been functionally studied in any protostome.ResultsIn order to understand its original functions and the changes accompanying the gene loss in the fly, we investigated PC1/3 and PC2 expression and function in the beetle Tribolium castaneum. We found that PC2 is broadly expressed in the nervous system, whereas surprisingly, PC1/3 expression is restricted to specific cell groups in the posterior brain and suboesophageal ganglion. Both proteases have parallel but non-redundant functions in adult beetles’ viability and fertility. Female infertility following RNAi is caused by a failure to deposit sufficient yolk to the developing oocytes. Larval RNAi against PC2 produced moulting defects where the larvae were not able to shed their old cuticle. This ecdysis phenotype was also observed in a small subset of PC1/3 knockdown larvae and was strongest in a double knockdown. Unexpectedly, most PC1/3-RNAi larvae showed strongly reduced growth, but went through larval moults despite minimal to zero weight gain.ConclusionsThe cell type-specific expression of PC1/3 and its essential requirement for larval growth highlight the important role of this gene within the insect neuroendocrine system. Genomic conservation in most insect groups suggests that it has a comparable individual function in other insects as well, which has been replaced by alternative mechanisms in flies.

Clock-controlled arylalkylamine N-acetyltransferase (aaNAT) regulates circadian rhythms of locomotor activity in the American cockroach, Periplaneta americana, via melatonin/MT2-like receptor.

Melatonin (MEL) orchestrates daily and seasonal rhythms (eg, locomotion, sleep/wake cycles, and migration among other rhythms) in diverse organisms. We investigated the effects of pharmacological doses (0.03-1mM) of exogenous MEL intake in the cockroach, Periplaneta americana, on locomotor activity. As per os MEL concentration increased, cockroach locomotor rhythm in light-dark (LD) cycles became more synchronized. The ratio of night activity to 24-h activity increased and the acrophase (peak) slightly advanced. MEL application also influenced total activity bouts in the free-running rhythm. Since MEL slightly influenced τ in the free-running rhythms, it is not a central element of the circadian pacemaker but must influence mutual coupling of multi-oscillatory system components. Arylalkylamine N-acetyltransferase (aaNAT) regulates enzymatic production of MEL. aaNAT activities vary in circadian rhythms, and the immunoreactive aaNAT (aaNAT-ir) is colocalized with the key clock proteins cycle (CYC)-ir and pigment-dispersing factor (PDF)-ir These are elements of the central pacemaker and its output pathway as well as other circadian landmarks such as the anterior and posterior optic commissures (AOC and POC, respectively). It also partially shares immunohistochemical reactivity with PER-ir and DBT-ir neurons. We analyzed the role of Pamericana aaNAT1 (PaaaNAT1) (AB106562.1) by injecting dsRNAaaNAT1 . qPCR showed a decrease in accumulations of mRNAs encoding PaaaNAT1. The injections led to arrhythmicity in LD cycles and the arrhythmicity persisted in constant dark (DD). Continuous administration of MEL resynchronized the rhythm after arrhythmicity was induced by dsRNAaaNAT1 injection, suggesting that PaaaNAT is the key regulator of the circadian system in the cockroach via MEL production. PaaaNAT1 contains putative E-box regions which may explain its tight circadian control. The receptor that mediates MEL function is most likely similar to the mammalian MT2, because injecting the competitive MT2 antagonist luzindole blocked MEL function, and MEL injection after luzindole treatment restored MT function. Human MT2-ir was localized in the circadian neurons in the cockroach brain and subesophageal ganglion. We infer that MEL and its synthesizing enzyme, aaNAT, constitute at least one circadian output pathway of locomotor activity either as a distinct route or in association with PDF system.