Insect Nervous System Research Articles

Directional vibration sensing in the termite Macrotermes natalensis.

Although several behavioural studies demonstrate the ability of insects to localise the source of vibrations, it is still unclear how insects are able to perceive directional information from vibratory signals on solid substrates, because time-of-arrival and amplitude difference between receptory structures are thought to be too small to be processed by insect nervous systems. The termite Macrotermes natalensis communicates using vibrational drumming signals transmitted along subterranean galleries. When soldiers are attacked by predators, they tend to drum with their heads against the substrate and create a pulsed vibration. Workers respond by a fast retreat into the nest. Soldiers in the vicinity start to drum themselves, leading to an amplification and propagation of the signal. Here we show that M. natalensis makes use of a directional vibration sensing in the context of colony defence. In the field, soldiers are recruited towards the source of the signal. In arena experiments on natural nest material, soldiers are able to localise the source of vibration. Using two movable platforms allowing us to vibrate the legs of the left and right sides of the body with a time delay, we show that the difference in time-of-arrival is the directional cue used for orientation. Delays as short as 0.2 ms are sufficient to be detected. Soldiers show a significant positive tropotaxis to the platform stimulated earlier, demonstrating for the first time perception of time-of-arrival delays and vibrotropotaxis on solid substrates in insects.

Na+-K+-ATPase trafficking induced by heat shock pretreatment correlates with increased resistance to anoxia in locusts.

The sensitivity of insect nervous systems to anoxia can be modulated genetically and pharmacologically, but the cellular mechanisms responsible are poorly understood. We examined the effect of a heat shock pretreatment (HS) on the sensitivity of the locust (Locusta migratoria) nervous system to anoxia induced by water immersion. Prior HS made locusts more resistant to anoxia by increasing the time taken to enter a coma and by reducing the time taken to recover the ability to stand. Anoxic comas were accompanied by surges of extracellular potassium ions in the neuropile of the metathoracic ganglion, and HS reduced the time taken for clearance of excess extracellular potassium ions. This could not be attributed to a decrease in the activity of protein kinase G, which was increased by HS. In homogenates of the metathoracic ganglion, HS had only a mild effect on the activity of Na(+)-K(+)-ATPase. However, we demonstrated that HS caused a threefold increase in the immunofluorescent localization of the α-subunit of Na(+)-K(+)-ATPase in metathoracic neuronal plasma membranes relative to background labeling of the nucleus. We conclude that HS induced trafficking of Na(+)-K(+)-ATPase into neuronal plasma membranes and suggest that this was at least partially responsible for the increased resistance to anoxia and the increased rate of recovery of neural function after a disturbance of K(+) homeostasis.

Trace amines inhibit insect odorant receptor function through antagonism of the co-receptor subunit

Many insect behaviors are driven by olfaction, making insect olfactory receptors (ORs) appealing targets for insect control. Insect ORs are odorant-gated ion channels, with each receptor thought to be composed of a representative from a large, variable family of odorant binding subunits and a highly conserved co-receptor subunit (Orco), assembled in an unknown stoichiometry. Synthetic Orco directed agonists and antagonists have recently been identified. Several Orco antagonists have been shown to act via an allosteric mechanism to inhibit OR activation by odorants. The high degree of conservation of Orco across insect species results in Orco antagonists having broad activity at ORs from a variety of insect species and suggests that the binding site for Orco ligands may serve as a modulatory site for compounds endogenous to insects or may be a target of exogenous compounds, such as those produced by plants. To test this idea, we screened a series of biogenic and trace amines, identifying several as Orco antagonists. Of particular interest were tryptamine, a plant-produced amine, and tyramine, an amine endogenous to the insect nervous system. Tryptamine was found to be a potent antagonist of Orco, able to block Orco activation by an Orco agonist and to allosterically inhibit activation of ORs by odorants. Tyramine had effects similar to those of tryptamine, but was less potent. Importantly, both tryptamine and tyramine displayed broad activity, inhibiting odorant activation of ORs of species from three different insect orders (Diptera, Lepidoptera and Coleoptera), as well as odorant activation of six diverse ORs from a single species (the human malaria vector mosquito, Anopheles gambiae). Our results suggest that endogenous and exogenous natural compounds serve as Orco ligands modulating insect olfaction and that Orco can be an important target for the development of novel insect repellants.

Trace amines inhibit insect odorant receptor function through antagonism of the co-receptor subunit.

Many insect behaviors are driven by olfaction, making insect olfactory receptors (ORs) appealing targets for insect control. Insect ORs are odorant-gated ion channels, with each receptor thought to be composed of a representative from a large, variable family of odorant binding subunits and a highly conserved co-receptor subunit (Orco), assembled in an unknown stoichiometry. Synthetic Orco directed agonists and antagonists have recently been identified. Several Orco antagonists have been shown to act via an allosteric mechanism to inhibit OR activation by odorants. The high degree of conservation of Orco across insect species results in Orco antagonists having broad activity at ORs from a variety of insect species and suggests that the binding site for Orco ligands may serve as a modulatory site for compounds endogenous to insects or may be a target of exogenous compounds, such as those produced by plants. To test this idea, we screened a series of biogenic and trace amines, identifying several as Orco antagonists. Of particular interest were tryptamine, a plant-produced amine, and tyramine, an amine endogenous to the insect nervous system. Tryptamine was found to be a potent antagonist of Orco, able to block Orco activation by an Orco agonist and to allosterically inhibit activation of ORs by odorants. Tyramine had effects similar to those of tryptamine, but was less potent. Importantly, both tryptamine and tyramine displayed broad activity, inhibiting odorant activation of ORs of species from three different insect orders (Diptera, Lepidoptera and Coleoptera), as well as odorant activation of six diverse ORs from a single species (the human malaria vector mosquito, Anopheles gambiae). Our results suggest that endogenous and exogenous natural compounds serve as Orco ligands modulating insect olfaction and that Orco can be an important target for the development of novel insect repellants.

Trace amines inhibit insect odorant receptor function through antagonism of the co-receptor subunit

Many insect behaviors are driven by olfaction, making insect olfactory receptors (ORs) appealing targets for insect control.  Insect ORs are odorant-gated ion channels, with each receptor thought to be composed of a representative from a large, variable family of odorant binding subunits and a highly conserved co-receptor subunit (Orco), assembled in an unknown stoichiometry.  Synthetic Orco directed agonists and antagonists have recently been identified.  Several Orco antagonists have been shown to act via an allosteric mechanism to inhibit OR activation by odorants.  The high degree of conservation of Orco across insect species results in Orco antagonists having broad activity at ORs from a variety of insect species and suggests that the binding site for Orco ligands may serve as a modulatory site for compounds endogenous to insects or may be a target of exogenous compounds, such as those produced by plants.  To test this idea, we screened a series of biogenic and trace amines, identifying several as Orco antagonists.  Of particular interest were tryptamine, a plant-produced amine, and tyramine, an amine endogenous to the insect nervous system.  Tryptamine was found to be a potent antagonist of Orco, able to block Orco activation by an Orco agonist and to allosterically inhibit activation of ORs by odorants.  Tyramine had effects similar to those of tryptamine, but was less potent.  Importantly, both tryptamine and tyramine displayed broad activity, inhibiting odorant activation of ORs of species from three different insect orders (Diptera, Lepidoptera and Coleoptera), as well as odorant activation of six diverse ORs from a single species (the human malaria vector mosquito, Anopheles gambiae ).  Our results suggest that endogenous and exogenous natural compounds serve as Orco ligands modulating insect olfaction and that Orco can be an important target for the development of novel insect repellants.

Electrophysiological Recording From <em>Drosophila</em> Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

A Systematic Nomenclature for the Insect Brain

Despite the importance of the insect nervous system for functional and developmental neuroscience, descriptions of insect brains have suffered from a lack of uniform nomenclature. Ambiguous definitions of brain regions and fiber bundles have contributed to the variation of names used to describe the same structure. The lack of clearly determined neuropil boundaries has made it difficult to document precise locations of neuronal projections for connectomics study. To address such issues, a consortium of neurobiologists studying arthropod brains, the Insect Brain Name Working Group, has established the present hierarchical nomenclature system, using the brain of Drosophila melanogaster as the reference framework, while taking the brains of other taxa into careful consideration for maximum consistency and expandability. The following summarizes the consortium's nomenclature system and highlights examples of existing ambiguities and remedies for them. This nomenclature is intended to serve as a standard of reference for the study of the brain of Drosophila and other insects.

Locomotor behaviour of Blattella germanica modified by DEET.

N,N-diethyl-3-methylbenzamide (DEET) is the active principle of most insect repellents used worldwide. However, its toxicity on insects has not been widely studied. The aim of this work is to study the effects of DEET on the locomotor activity of Blattella germanica. DEET has a dose-dependent repellent activity on B. germanica. Locomotor activity was significantly lower when insects were pre-exposed to 700 µg/cm2 of DEET for 20 or 30 minutes, but it did not change when pre-exposure was shorter. Locomotor activity of insects that were pre-exposed to 2.000 µg/cm2 of DEET for 10 minutes was significantly lower than the movement registered in controls. No differences were observed when insects were pre-exposed to lower concentrations of DEET. A 30-minute pre-exposure to 700 µg/cm2 of DEET caused a significant decrease in locomotor activity. Movement was totally recovered 24 h later. The locomotor activity measured during the exposure to different concentrations of DEET remained unchanged. Insects with decreased locomotor activity were repelled to the same extent than control insects by the same concentration of DEET. We demonstrated that the repellency and modification of locomotor activity elicited by DEET are non-associated phenomena. We also suggested that the reduction in locomotor activity indicates toxicity of DEET, probably to insect nervous system.

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. J. Comp. Neurol. 522:592–608, 2014.

Cytokine Spätzle binds to the Drosophila immunoreceptor Toll with a neurotrophin-like specificity and couples receptor activation

Drosophila Toll functions in embryonic development and innate immunity and is activated by an endogenous ligand, Spätzle (Spz). The related Toll-like receptors in vertebrates also function in immunity but are activated directly by pathogen-associated molecules such as bacterial endotoxin. Here, we present the crystal structure at 2.35-Å resolution of dimeric Spz bound to a Toll ectodomain encompassing the first 13 leucine-rich repeats. The cystine knot of Spz binds the concave face of the Toll leucine-rich repeat solenoid in an area delineated by N-linked glycans and induces a conformational change. Mutagenesis studies confirm that the interface observed in the crystal structure is relevant for signaling. The asymmetric binding mode of Spz to Toll is similar to that of nerve growth factor (NGF) in complex with the p75 neurotrophin receptor but is distinct from that of microbial ligands bound to the Toll-like receptors. Overall, this study indicates an allosteric signaling mechanism for Toll in which ligand binding to the N terminus induces a conformational change that couples to homodimerization of juxtamembrane structures in the Toll ectodomain C terminus.

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

Nicotinic Acetylcholine Receptor Gene Family of the Pea Aphid, Acyrthosiphon pisum

The nicotinic acetylcholine receptors (nAchRs) are cholinergic receptors that form ligand-gated ion channels by five subunits in insect and vertebrate nervous systems. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Here, we identified and cloned 11 candidate nAChR subunit genes in Acyrthosiphon pisum using genome-based bioinformatics combined modern molecular techniques. Most A. pisum nAChRs including α1, α2, α3, α4, α6, α8, and β1 show highly sequence identities with the counterparts of other insects examined. Expression profiles analysis showed that all subunit genes were expressed in adult head. At least two subunits have alternative splicing that obviously increase A. pisum nicotinic receptor diversity. This study will be invaluable for exploring the molecular mechanisms of neonicotinoid-like insecticides in sucking pests, and for ultimately establishing the screening platform of novel insecticides.

Amyloid Precursor Proteins Interact with the Heterotrimeric G Protein Go in the Control of Neuronal Migration

Amyloid precursor protein (APP) belongs to a family of evolutionarily conserved transmembrane glycoproteins that has been proposed to regulate multiple aspects of cell motility in the nervous system. Although APP is best known as the source of β-amyloid fragments (Aβ) that accumulate in Alzheimer's disease, perturbations affecting normal APP signaling events may also contribute to disease progression. Previous in vitro studies showed that interactions between APP and the heterotrimeric G protein Goα-regulated Goα activity and Go-dependent apoptotic responses, independent of Aβ. However, evidence for authentic APP-Go interactions within the healthy nervous system has been lacking. To address this issue, we have used a combination of in vitro and in vivo strategies to show that endogenously expressed APP family proteins colocalize with Goα in both insect and mammalian nervous systems, including human brain. Using biochemical, pharmacological, and Bimolecular Fluorescence Complementation assays, we have shown that insect APP (APPL) directly interacts with Goα in cell culture and at synaptic terminals within the insect brain, and that this interaction is regulated by Goα activity. We have also adapted a well characterized assay of neuronal migration in the hawkmoth Manduca to show that perturbations affecting APPL and Goα signaling induce the same unique pattern of ectopic, inappropriate growth and migration, analogous to defective migration patterns seen in mice lacking all APP family proteins. These results support the model that APP and its orthologs regulate conserved aspects of neuronal migration and outgrowth in the nervous system by functioning as unconventional Goα-coupled receptors.

The insecticide imidacloprid causes mortality of the freshwater amphipod Gammarus pulex by interfering with feeding behavior.

If an organism does not feed, it dies of starvation. Even though some insecticides which are used to control pests in agriculture can interfere with feeding behavior of insects and other invertebrates, the link from chemical exposure via affected feeding activity to impaired life history traits, such as survival, has not received much attention in ecotoxicology. One of these insecticides is the neonicotinoid imidacloprid, a neurotoxic substance acting specifically on the insect nervous system. We show that imidacloprid has the potential to indirectly cause lethality in aquatic invertebrate populations at low, sublethal concentrations by impairing movements and thus feeding. We investigated feeding activity, lipid content, immobility, and survival of the aquatic arthropod Gammarus pulex under exposure to imidacloprid. We performed experiments with 14 and 21 days duration, both including two treatments with two high, one day pulses of imidacloprid and one treatment with a low, constant concentration. Feeding of G. pulex as well as lipid content were significantly reduced under exposure to the low, constant imidacloprid concentration (15 µg/L). Organisms were not able to move and feed – and this caused high mortality after 14 days of constant exposure. In contrast, feeding and lipid content were not affected by repeated imidacloprid pulses. In these treatments, animals were mostly immobilized during the chemical pulses but did recover relatively fast after transfer to clean water. We also performed a starvation experiment without exposure to imidacloprid which showed that starvation alone does not explain the mortality in the constant imidacloprid exposure. Using a multiple stressor toxicokinetic-toxicodynamic modeling approach, we showed that both starvation and other toxic effects of imidacloprid play a role for determining mortality in constant exposure to the insecticide.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection(1-3). Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination(4-6), herbivory of food crops(7), and transmission of disease(8,9). Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being. To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)(10-12). These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)(13,14). This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use. Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques(14-16). Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system. Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Neuropathological effect of dimethyl disulfide on neurons of the desert locust Schistocerca gregaria

Botanical insecticides have introduced a new concept in insecticide research. In response to insect attacks, some plants can release volatile compounds that alter insect metabolism and nervous system activity. In the present study, changes in the electrical activity of chemoreceptors and alteration of the fine structure of metathoracic ganglia of desert locust were examined after acute exposure to dimethyl disulfide (DMDS), a sulfur compound released from Allium porrum. Animals were exposed to 1/4 LC50 of DMDS (0.375μl/L air) and electrophysiological and electron-microscopical studies were carried out. Application of DMDS showed an increase in the activity of deterrent cells present in tarsal chemosensilla of locust. On the other hand, evident degenerative changes in the neurons, neuroglia, neuropile and synaptic vesicles were observed in the metathoracic ganglia of DMDS-treated animals. These findings revealed that pest control using DMDS might be feasible and future work is highly recommended in this respect.

The Dilemmas of the Gourmet Fly: The Molecular and Neuronal Mechanisms of Feeding and Nutrient Decision Making in Drosophila

To survive and successfully reproduce animals need to maintain a balanced intake of nutrients and energy. The nervous system of insects has evolved multiple mechanisms to regulate feeding behavior. When animals are faced with the choice to feed, several decisions must be made: whether or not to eat, how much to eat, what to eat, and when to eat. Using Drosophila melanogaster substantial progress has been achieved in understanding the neuronal and molecular mechanisms controlling feeding decisions. These feeding decisions are implemented in the nervous system on multiple levels, from alterations in the sensitivity of peripheral sensory organs to the modulation of memory systems. This review discusses methodologies developed in order to study insect feeding, the effects of neuropeptides and neuromodulators on feeding behavior, behavioral evidence supporting the existence of internal energy sensors, neuronal and molecular mechanisms controlling protein intake, and finally the regulation of feeding by circadian rhythms and sleep. From the discussed data a conceptual framework starts to emerge which aims to explain the molecular and neuronal processes maintaining the stability of the internal milieu.

Erratum to: Evidence for inhibition of cholinesterases in insect and mammalian nervous systems by the insect repellent deet

Erratum to: Evidence for inhibition of cholinesterases in insect and mammalian nervous systems by the insect repellent deet

Evolutionary Adaptation of the Amino Acid and Codon Usage of the Mosquito Sodium Channel following Insecticide Selection in the Field Mosquitoes

Target site insensitivity resulting from point mutations within the voltage-gated sodium channel of the insect nervous system is known to be of primary importance in the development of resistance to pyrethroid insecticides. This study shifts current research paradigms by conducting, for the first time, a global analysis of all the naturally occurring mutations, both nonsynonymous and synonymous mutations, as well as mutation combinations in the entire mosquito sodium channel of Culex quinquefasciatus and analyzing their evolutionary and heritable feature and roles in insecticide resistance. Through a systematic analysis of comparing nucleotide polymorphisms in the entire sodium channel cDNAs of individuals between susceptible and resistant mosquito strains, between field parental mosquitoes and their permethrin selected offspring, and among different mosquito groups categorized by their levels of tolerance to specific permethrin concentrations within and among the mosquito strains of the field parental strains and their permethrin selected offspring, 3 nonsynonymous (A109S, L982F, and W1573R) and 6 synonymous (L852, G891, A1241, D1245, P1249, and G1733) mutations were identified. The co-existence of all 9 mutations, both nonsynonymous and synonymous, and their homozygousity were found to be important factors for high levels of resistance. Our study, for the first time, provide a strong case demonstrating the co-existence of both nonsynonymous and synonymous mutations in the sodium channel of resistant mosquitoes in response to insecticide resistance and the inheritance of these mutations in the offspring of field mosquito strains following insecticide selection.