One hundred years of phase polymorphism research in locusts.

One hundred years ago in 1921, Sir Boris Uvarov recognized that two locust species are one species but appearing in two different phases, a solitarious and a gregarious phase. As locust swarms are still a big problem affecting millions of people, basic research has tried to understand the causes for the transition between phases. This phenomenon of phase polymorphism, now called polyphenism, is a very complex multifactorial process and this short review will draw attention to this important aspect of insect research.

Role of the Insect Neuroendocrine System in the Response to Cold Stress.

Insects are the largest group of animals. They are capable of surviving in virtually all environments from arid deserts to the freezing permafrost of polar regions. This success is due to their great capacity to tolerate a range of environmental stresses, such as low temperature. Cold/freezing stress affects many physiological processes in insects, causing changes in main metabolic pathways, cellular dehydration, loss of neuromuscular function, and imbalance in water and ion homeostasis. The neuroendocrine system and its related signaling mediators, such as neuropeptides and biogenic amines, play central roles in the regulation of the various physiological and behavioral processes of insects and hence can also potentially impact thermal tolerance. In response to cold stress, various chemical signals are released either via direct intercellular contact or systemically. These are signals which regulate osmoregulation - capability peptides (CAPA), inotocin (ITC)-like peptides, ion transport peptide (ITP), diuretic hormones and calcitonin (CAL), substances related to the general response to various stress factors - tachykinin-related peptides (TRPs) or peptides responsible for the mobilization of body reserves. All these processes are potentially important in cold tolerance mechanisms. This review summarizes the current knowledge on the involvement of the neuroendocrine system in the cold stress response and the possible contributions of various signaling molecules in this process.

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.

Neural pathways in the pallial nerve and arm nerve cord revealed by neurobiotin backfilling in the cephalopod mollusk Octopus vulgaris.

Here, we report the findings after application of neurobiotin tracing to pallial and stellar nerves in the mantle of the cephalopod mollusk Octopus vulgaris and to the axial nerve cord in its arm. Neurobiotin backfilling is a known technique in other molluscs, but it is applied to octopus for the first time to be best of our knowledge. Different neural tracing techniques have been carried out in cephalopods to study the intricate neural connectivity of their nervous system, but mapping the nervous connections in this taxon is still incomplete, mainly due to the absence of a reliable tracing method allowing whole-mount imaging. In our experiments, neurobiotin backfilling allowed: (1) imaging of large/thick samples (larger than 2 mm) through optical clearing; (2) additional application of immunohistochemistry on the backfilled tissues, allowing identification of neural structures by coupling of a specific antibody. This work opens a series of future studies aimed to the identification of the neural diagram and connectome of octopus nervous system.

A population of descending tyraminergic/octopaminergic projection neurons of the insect deutocerebrum.

In this study, we describe a cluster of tyraminergic/octopaminergic neurons in the lateral dorsal deutocerebrum of desert locusts (Schistocerca gregaria) with descending axons to the abdominal ganglia. In the locust, these neurons synthesize octopamine from tyramine stress-dependently. Electrophysiological recordings in locusts reveal that they respond to mechanosensory touch stimuli delivered to various parts of the body including the antennae. A similar cluster of tyraminergic/octopaminergic neurons was also identified in the American cockroach (Periplaneta americana) and the pink winged stick insect (Sipyloidea sipylus). It is suggested that these neurons release octopamine in the ventral nerve cord ganglia and, most likely, convey information on arousal and/or stressful stimuli to neuronal circuits thus contributing to the many actions of octopamine in the central nervous system.

The functional connectivity between the locust leg pattern generators and the subesophageal ganglion higher motor center.

Higher motor centers and central pattern generators (CPGs) interact in the control of coordinated leg movements during locomotion throughout the animal kingdom. The subesophageal ganglion (SEG) is one of the insect head ganglia reported to have a role in the control of walking behavior. Here we explored the functional relations between the SEG and the thoracic leg CPGs in the desert locust. Backfill staining revealed about 300 SEG descending interneurons (DINs) altogether. Recordings from an in-vitro isolated chain of thoracic ganglia, with intact or severed connections to the SEG, during pharmacological activation were used to determine how the SEG affects the centrally generated motor output to the legs. The SEG was demonstrated to both activate leg CPGs and synchronize their bilateral activity. The role of the SEG in insect locomotion is discussed in light of these findings.

Ex vivo recordings reveal desert locust forelimb control is asymmetric.

Lateralized behaviours are widespread among the animals, including insects with their miniature brains, perhaps being a way of maximising neural capacity (reviewed in [1,2]). However, evidence for functional asymmetries in the neural circuitry itself is scarce. Here, using bilateral simultaneous recordings from the ex vivo nervous system of desert locusts, we show that the neural control of their forelimbs is asymmetric. This asymmetry was retained throughout the experimental period and either with or without the suboesophageal ganglion (SOG). These findings provide evidence for hard-wired neural sidedness and contribute to our understanding of the lateralization observed in in-vivo motor behaviours.

Structural and Molecular Properties of Insect Type II Motor Axon Terminals.

A comparison between the axon terminals of octopaminergic efferent dorsal or ventral unpaired median neurons in either desert locusts (Schistocerca gregaria) or fruit flies (Drosophila melanogaster) across skeletal muscles reveals many similarities. In both species the octopaminergic axon forms beaded fibers where the boutons or varicosities form type II terminals in contrast to the neuromuscular junction (NMJ) or type I terminals. These type II terminals are immunopositive for both tyramine and octopamine and, in contrast to the type I terminals, which possess clear synaptic vesicles, only contain dense core vesicles. These dense core vesicles contain octopamine as shown by immunogold methods. With respect to the cytomatrix and active zone peptides the type II terminals exhibit active zone-like accumulations of the scaffold protein Bruchpilot (BRP) only sparsely in contrast to the many accumulations of BRP identifying active zones of NMJ type I terminals. In the fruit fly larva marked dynamic changes of octopaminergic fibers have been reported after short starvation which not only affects the formation of new branches ("synaptopods") but also affects the type I terminals or NMJs via octopamine-signaling (Koon et al., 2011). Our starvation experiments of Drosophila-larvae revealed a time-dependency of the formation of additional branches. Whereas after 2 h of starvation we find a decrease in "synaptopods", the increase is significant after 6 h of starvation. In addition, we provide evidence that the release of octopamine from dendritic and/or axonal type II terminals uses a similar synaptic machinery to glutamate release from type I terminals of excitatory motor neurons. Indeed, blocking this canonical synaptic release machinery via RNAi induced downregulation of BRP in neurons with type II terminals leads to flight performance deficits similar to those observed for octopamine mutants or flies lacking this class of neurons (Brembs et al., 2007).

Octopaminergic innervation and a neurohaemal release site in the antennal heart of the locust Schistocerca gregaria.

A detailed account is given by the octopaminergic innervation of the antennal heart in Schistocerca gregaria using various immunohistochemical methods. Anterograde axonal filling illustrates the unilateral innervation on the medial ventral surface of the pumping muscle of the antennal heart via the paired corpora cardiaca nerve III. In addition, antibody staining revealed that ascending axons of this nerve terminate at the ampullae of the antennal heart forming synaptoid structures and extensive neurohaemal release sites. Due to the innervation by two dorsal unpaired median neurons, the presence of the biogenic amines octopamine and tyramine could be visualized by immunocytochemistry in an insect antennal heart for the first time. The data suggest that tyramine acts as a precursor and not purely as an independent transmitter. While the octopaminergic fibers innervating the pumping muscle of the antennal heart indicate a cardioregulatory role, we conclude that octopamine released from the neurohaemal area is pumped into the antennae and an involvement in the modulation of the antennal sensory sensitivity is discussed.

Professor Ernst Bresslau, founder of the Zoology Departments at the Universities of Cologne and Sao Paulo: lessons to learn from his life history.

In this article, the life history of the founding father of the departments of Zoology at the Universities of Cologne and Sao Paulo, Prof. Ernst Bresslau, is described on occasion of the establishing of the "Ernst Bresslau Guest Professorship" at the University of Cologne. His main scientific achievements are discussed, in particular his research on the evolutionary origin of the mammary apparatus, in addition to his broad interest in biological topics. Among the many technical advancements that he introduced was the micro slow-motion camera developed together with the Zeiss Company which allowed to film ciliary beats at high speeds.

Assessing segmental versus non-segmental features in the ventral nervous system of onychophorans (velvet worms).

BACKGROUND: Due to their phylogenetic position as one of the closest arthropod relatives, studies of the organisation of the nervous system in onychophorans play a key role for understanding the evolution of body segmentation in arthropods. Previous studies revealed that, in contrast to the arthropods, segmentally repeated ganglia are not present within the onychophoran ventral nerve cords, suggesting that segmentation is either reduced or might be incomplete in the onychophoran ventral nervous system. RESULTS: To assess segmental versus non-segmental features in the ventral nervous system of onychophorans, we screened the nerve cords for various markers, including synapsin, serotonin, gamma-aminobutyric acid, RFamide, dopamine, tyramine and octopamine. In addition, we performed retrograde fills of serially repeated commissures and leg nerves to localise the position of neuronal somata supplying those. Our data revealed a mixture of segmental and non-segmental elements within the onychophoran nervous system. CONCLUSIONS: We suggest that the segmental ganglia of arthropods evolved by a gradual condensation of subsets of neurons either in the arthropod or the arthropod-tardigrade lineage. These findings are in line with the hypothesis of gradual evolution of segmentation in panarthropods and thus contradict a loss of ancestral segmentation within the onychophoran lineage.

Rigidity and Flexibility: The Central Basis of Inter-Leg Coordination in the Locust.

Many motor behaviors, and specifically locomotion, are the product of an intricate interplay between neuronal oscillators known as central pattern generators (CPGs), descending central commands, and sensory feedback loops. The relative contribution of each of these components to the final behavior determines the trade-off between fixed movements and those that are carefully adapted to the environment. Here we sought to decipher the endogenous, default, motor output of the CPG network controlling the locust legs, in the absence of any sensory or descending influences. We induced rhythmic activity in the leg CPGs in isolated nervous system preparations, using different application procedures of the muscarinic agonist pilocarpine. We found that the three thoracic ganglia, each controlling a pair of legs, have different inherent bilateral coupling. Furthermore, we found that the pharmacological activation of one ganglion is sufficient to induce activity in the other, untreated, ganglia. Each ganglion was thus capable to impart its own bilateral inherent pattern onto the other ganglia via a tight synchrony among the ipsilateral CPGs. By cutting a connective and severing the lateral-longitudinal connections, we were able to uncouple the oscillators' activity. While the bilateral connections demonstrated a high modularity, the ipsilateral CPGs maintained a strict synchronized activity. These findings suggest that the central infrastructure behind locust walking features both rigid elements, which presumably support the generation of stereotypic orchestrated leg movements, and flexible elements, which might provide the central basis for adaptations to the environment and to higher motor commands.

Drosophila FoxP mutants are deficient in operant self-learning.

Intact function of the Forkhead Box P2 (FOXP2) gene is necessary for normal development of speech and language. This important role has recently been extended, first to other forms of vocal learning in animals and then also to other forms of motor learning. The homology in structure and in function among the FoxP gene members raises the possibility that the ancestral FoxP gene may have evolved as a crucial component of the neural circuitry mediating motor learning. Here we report that genetic manipulations of the single Drosophila orthologue, dFoxP, disrupt operant self-learning, a form of motor learning sharing several conceptually analogous features with language acquisition. Structural alterations of the dFoxP locus uncovered the role of dFoxP in operant self-learning and habit formation, as well as the dispensability of dFoxP for operant world-learning, in which no motor learning occurs. These manipulations also led to subtle alterations in the brain anatomy, including a reduced volume of the optic glomeruli. RNAi-mediated interference with dFoxP expression levels copied the behavioral phenotype of the mutant flies, even in the absence of mRNA degradation. Our results provide evidence that motor learning and language acquisition share a common ancestral trait still present in extant invertebrates, manifest in operant self-learning. This 'deep' homology probably traces back to before the split between vertebrate and invertebrate animals.

Selective neuronal staining in tardigrades and onychophorans provides insights into the evolution of segmental ganglia in panarthropods.

BACKGROUND: Although molecular analyses have contributed to a better resolution of the animal tree of life, the phylogenetic position of tardigrades (water bears) is still controversial, as they have been united alternatively with nematodes, arthropods, onychophorans (velvet worms), or onychophorans plus arthropods. Depending on the hypothesis favoured, segmental ganglia in tardigrades and arthropods might either have evolved independently, or they might well be homologous, suggesting that they were either lost in onychophorans or are a synapomorphy of tardigrades and arthropods. To evaluate these alternatives, we analysed the organisation of the nervous system in three tardigrade species using antisera directed against tyrosinated and acetylated tubulin, the amine transmitter serotonin, and the invertebrate neuropeptides FMRFamide, allatostatin and perisulfakinin. In addition, we performed retrograde staining of nerves in the onychophoran Euperipatoides rowelli in order to compare the serial locations of motor neurons within the nervous system relative to the appendages they serve in arthropods, tardigrades and onychophorans. RESULTS: Contrary to a previous report from a Macrobiotus species, our immunocytochemical and electron microscopic data revealed contralateral fibres and bundles of neurites in each trunk ganglion of three tardigrade species, including Macrobiotus cf. harmsworthi, Paramacrobiotus richtersi and Hypsibius dujardini. Moreover, we identified additional, extra-ganglionic commissures in the interpedal regions bridging the paired longitudinal connectives. Within the ganglia we found serially repeated sets of serotonin- and RFamid-like immunoreactive neurons. Furthermore, our data show that the trunk ganglia of tardigrades, which include the somata of motor neurons, are shifted anteriorly with respect to each corresponding leg pair, whereas no such shift is evident in the arrangement of motor neurons in the onychophoran nerve cords. CONCLUSIONS: Taken together, these data reveal three major correspondences between the segmental ganglia of tardigrades and arthropods, including (i) contralateral projections and commissures in each ganglion, (ii) segmentally repeated sets of immunoreactive neurons, and (iii) an anteriorly shifted (parasegmental) position of ganglia. These correspondences support the homology of segmental ganglia in tardigrades and arthropods, suggesting that these structures were either lost in Onychophora or, alternatively, evolved in the tardigrade/arthropod lineage.

Developmental and activity-dependent plasticity of filiform hair receptors in the locust.

A group of wind sensitive filiform hair receptors on the locust thorax and head makes contact onto a pair of identified interneuron, A4I1. The hair receptors' central nervous projections exhibit pronounced structural dynamics during nymphal development, for example, by gradually eliminating their ipsilateral dendritic field while maintaining the contralateral one. These changes are dependent not only on hormones controlling development but on neuronal activity as well. The hair-to-interneuron system has remarkably high gain (close to 1) and makes contact to flight steering muscles. During stationary flight in front of a wind tunnel, interneuron A4I1 is active in the wing beat rhythm, and in addition it responds strongly to stimulation of sensory hairs in its receptive field. A role of the hair-to-interneuron in flight steering is thus suggested. This system appears suitable for further study of developmental and activity-dependent plasticity in a sensorimotor context with known connectivity patterns.

Dynamic neural control of insect muscle metabolism related to motor behavior.

Skeletal muscle innervation differs between vertebrates and insects. Insect muscle fibers exhibit graded electrical potentials and are innervated by excitatory, inhibitory, and also neuromodulatory motoneurons. The latter form a unique class of unpaired neurons with bilaterally symmetrical axons that release octopamine to alter the efficacy of synaptic transmission and regulate muscle energy metabolism by activating glycolysis. Octopaminergic neurons that innervate muscles with a high energy demand, for example, flight muscles that move the wings of a locust up and down, are active during rest but are inhibited during flight and its preparatory phase, a jump. Therefore, it is argued that these neurons are involved in providing locusts with the necessary fuel at takeoff, but then may aid the switch to lipid oxidation during flight. In general, the octopaminergic system may switch the whole organism from a tonic to a dynamic state.

Neuromodulatory unpaired median neurons in the New Zealand tree weta, Hemideina femorata.

Wetas are ancient Gondwanan orthopterans (Anostostomatidae) with many species endemic to New Zealand. Like all Orthoptera they possess efferent neuromodulatory dorsal unpaired median (DUM) neurons, with bilaterally symmetrical axons, that are important components of motor networks. These neurons produce overshooting action potentials and are easily stimulated by a variety of external mechanosensory stimuli delivered to the body and appendages. In particular, stimulation of the antennae, mouth parts, tarsi and femora of the legs, abdomen, cerci and ovipositor is very effective in activating DUM neurons in the metathoracic ganglion of wetas. In addition, looming visual stimuli or light on-, light off-stimuli excite many metathoracic DUM neurons. These DUM sensory reflex pathways remain viable after the prothoracic to subesophageal connective is cut, whereas in locusts such reflex pathways are interrupted by the ablation. This suggests that, in wetas, sensory reflex pathways for DUM activation are organized in a less centralized fashion than in locusts, and may therefore reflect a plesiomorphic evolutionary state in the weta. In addition, many weta DUM neurons exhibit slow rhythmic bursting which also persists following the connective ablation.

A revision of brain composition in Onychophora (velvet worms) suggests that the tritocerebrum evolved in arthropods.

BACKGROUND: The composition of the arthropod head is one of the most contentious issues in animal evolution. In particular, controversy surrounds the homology and innervation of segmental cephalic appendages by the brain. Onychophora (velvet worms) play a crucial role in understanding the evolution of the arthropod brain, because they are close relatives of arthropods and have apparently changed little since the Early Cambrian. However, the segmental origins of their brain neuropils and the number of cephalic appendages innervated by the brain--key issues in clarifying brain composition in the last common ancestor of Onychophora and Arthropoda--remain unclear. RESULTS: Using immunolabelling and neuronal tracing techniques in the developing and adult onychophoran brain, we found that the major brain neuropils arise from only the anterior-most body segment, and that two pairs of segmental appendages are innervated by the brain. The region of the central nervous system corresponding to the arthropod tritocerebrum is not differentiated as part of the onychophoran brain but instead belongs to the ventral nerve cords. CONCLUSIONS: Our results contradict the assumptions of a tripartite (three-segmented) brain in Onychophora and instead confirm the hypothesis of bipartite (two-segmented) brain composition. They suggest that the last common ancestor of Onychophora and Arthropoda possessed a brain consisting of protocerebrum and deutocerebrum whereas the tritocerebrum evolved in arthropods.