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Arthropod respiration depends on the tracheal system running from spiracles at the body surface through the body and appendages. Here, three species of stick insects (Carausius morosus, Ramulus artemis, Sipyloidea sipylus) are investigated for the tracheae in the prothorax and foreleg. The origin of the tracheae from the mesothoracic spiracle that enter the foreleg is identified: five tracheae originate from the mesothoracic spiracle, of which two enter the foreleg (supraventral trachea, trachea pedalis anterior). These two tracheae run separately through the leg to the femur-tibia joint where they fuse, but in the proximal tibia split again into two tracheae. The leg tracheae in stick insects are homologous to those in Tettigoniidae (bushcrickets). Stick insects have two chordotonal organs in the proximal tibia (subgenual organ and distal organ) which locate dorsally of the leg trachea. The tracheal system shows no adaptation specific to the propagation of airborne sound, like enlarged spiracles or tracheal volumes. Tracheal vesicles form in the tibia proximally to the mechanosensory organs, but no tracheal sacks or expansions occur at the level of the sensory organs that could mediate the detection of airborne sound or amplify substrate vibrations transmitted in the hemolymph fluid. Rather, the morphological characteristics indicate a respiratory function.

Like several other arthropod species, stick insects use their antennae for tactile exploration of the near-range environment and for spatial localisation of touched objects. More specifically, Carausius morosus continuously moves its antennae during locomotion and reliably responds to antennal contact events with directed movements of a front leg. Here we investigate the afferent projection patterns of antennal hair fields (aHF), proprioceptors known to encode antennal posture and movement, and to be involved in antennal movement control. We show that afferents of all seven aHF of C. morosus have terminal arborisations in the dorsal lobe (DL) of the cerebral (=supraoesophageal) ganglion, and descending collaterals that terminate in a characteristic part of the gnathal (=suboesophageal) ganglion. Despite differences of functional roles among aHF, terminal arborisation patterns show no topological arrangement according to segment specificity or direction of movement. In the DL, antennal motoneuron neurites show arborizations in proximity to aHF afferent terminals. Despite the morphological similarity of single mechanoreceptors of aHF and adjacent tactile hairs on the pedicel and flagellum, we find a clear separation of proprioceptive and exteroceptive mechanosensory neuropils in the cerebral ganglion. Moreover, we also find this functional separation in the gnathal ganglion.

Insects are highly capable walkers, but many questions remain regarding how the insect nervous system controls locomotion. One particular question is how information is communicated between the ‘lower level’ ventral nerve cord (VNC) and the ‘higher level’ head ganglia to facilitate control. In this work, we seek to explore this question by investigating how systems traditionally described as ‘positive feedback’ may initiate and maintain stepping in the VNC with limited information exchanged between lower and higher level centers. We focus on the ‘reflex reversal’ of the stick insect femur-tibia joint between a resistance reflex (RR) and an active reaction in response to joint flexion, as well as the activation of populations of descending dorsal median unpaired (desDUM) neurons from limb strain as our primary reflex loops. We present the development of a neuromechanical model of the stick insect (Carausius morosus) femur-tibia (FTi) and coxa-trochanter joint control networks ‘in-the-loop’ with a physical robotic limb. The control network generates motor commands for the robotic limb, whose motion and forces generate sensory feedback for the network. We based our network architecture on the anatomy of the non-spiking interneuron joint control network that controls the FTi joint, extrapolated network connectivity based on known muscle responses, and previously developed mechanisms to produce ‘sideways stepping’. Previous studies hypothesized that RR is enacted by selective inhibition of sensory afferents from the femoral chordotonal organ, but no study has tested this hypothesis with a model of an intact limb. We found that inhibiting the network’s flexion position and velocity afferents generated a reflex reversal in the robot limb’s FTi joint. We also explored the intact network’s ability to sustain steady locomotion on our test limb. Our results suggested that the reflex reversal and limb strain reinforcement mechanisms are both necessary but individually insufficient to produce and maintain rhythmic stepping in the limb, which can be initiated or halted by brief, transient descending signals. Removing portions of this feedback loop or creating a large enough disruption can halt stepping independent of the higher-level centers. We conclude by discussing why the nervous system might control motor output in this manner, as well as how to apply these findings to generalized nervous system understanding and improved robotic control.

Previous studies have demonstrated that myofibrillar ATPase (mATPase) enzyme activity in muscle fibers determines their contraction properties. We analyzed mATPase activities in muscles of the front, middle and hind legs of the orthopteran stick insect (Carausius morosus) to test the hypothesis that differences in muscle fiber types and distributions reflected differences in their behavioral functions. Our data show that all muscles are composed of at least three fiber types, fast, intermediate and slow, and demonstrate that: (1) in the femoral muscles (extensor and flexor tibiae) of all legs, the number of fast fibers decreases from proximal to distal, with a concomitant increase in the number of slow fibers. (2) The swing phase muscles protractor coxae and levator trochanteris, have smaller percentages of slow fibers compared to the antagonist stance muscles retractor coxae and depressor trochanteris. (3) The percentage of slow fibers in the retractor coxae and depressor trochanteris increases significantly from front to hind legs. These results suggest that fiber-type distribution in leg muscles of insects is not identical across leg muscles but tuned towards the specific function of a given muscle in the locomotor system.

During walking, the leg motor system must continually adjust to changes in mechanical conditions, such as the inclination of the ground. To understand the underlying control, it is important to know how changes in leg muscle activity relate to leg kinematics (movements) and leg dynamics (forces, torques). Here, we studied these parameters in hindlegs of stick insects (Carausius morosus) during level and uphill/downhill (±45deg) walking, using a combination of electromyography, 3D motion capture and ground reaction force measurements. We find that some kinematic parameters including leg joint angles and body height vary across walking conditions. However, kinematics vary little compared with dynamics: horizontal leg forces and torques at the thorax-coxa joint (leg protraction/retraction) and femur-tibia joint (leg flexion/extension) tend to be stronger during uphill walking and are reversed in sign during downhill walking. At the thorax-coxa joint, the different mechanical demands are met by adjustments in the timing and magnitude of antagonistic muscle activity. Adjustments occur primarily in the first half of stance after the touch-down of the leg. When insects transition from level to incline walking, the characteristic adjustments in muscle activity occur with the first step of the leg on the incline, but not in anticipation. Together, these findings indicate that stick insects adjust leg muscle activity on a step-by-step basis so as to maintain a similar kinematic pattern under different mechanical demands. The underlying control might rely primarily on feedback from leg proprioceptors signaling leg position and movement. © 2019. Published by The Company of Biologists Ltd.

Force feedback from Campaniform sensilla (CS) on insect limbs helps to adapt motor outputs to environmental conditions, but we are only beginning to reveal the neural control mechanisms that mediate these influences. We studied CS groups that affect control of the thoraco-coxal joint in the stick insect Carausius morosus by applying horizontal and vertical forces to the leg stump. Motor effects of ablation of CS groups were evaluated by recording extracellularly from protractor (ProCx) and retractor (RetCx) nerves. Extracellular recordings showed that the effects of stimulating the sensilla were consistent with their broad ranges of directional sensitivity: for example, RetCx firing in response to posterior bending could be reduced by ablating several groups of trochanteral CS, whereas ablation of tibial and femoral sensilla had little effect. In contrast, ProCx motor neuron activity upon anteriorly directed stimuli was affected mainly by ablating a single CS group (G2). Dye fills of trochanteral, femoral and tibial CS groups with fluorescent dyes revealed a common projection pattern with little group specificity. These findings support the idea that the influences of CS feedback are determined by the activities of pre-motor interneurons, facilitating fast and task-dependent adaptation to changing environmental conditions.

Adipokinetic hormone (AKH) is an insect neuropeptide that plays crucial roles in a variety of physiological functions such as regulation of heartbeat frequency, blood hemolymph trehalose levels, and protein synthesis. It exerts its functions through binding to its cognate G protein-coupled receptor (GPCR), named adipokinetic hormone receptor (AKHR). The aim of this study is to characterize AKHR of stick insect, Carausius morosus, which becomes an agricultural and forest pest during its outbreaks, and to screen pesticide candidates that would act through inhibition of AKHR. To this aim, the sequence of the receptor and its ligand were obtained from previously published transcriptome data and homology modeling, molecular docking, and molecular dynamics (MD) simulations were combined to find the ligand-binding pocket of AKHR. As a result, crucial residues in ligand binding were identified. These residues were located at the 6th and 7th transmembrane (TM) domains and the 2nd extracellular loop (ECL) of AKHR model. In order to propose pesticide candidates, virtual screening was performed, and candidate ligands were obtained. Considering the binding energies and the stability of the interaction between the ligand and the receptor, four hit compounds were selected. In conclusion, this study revealed a possible ligand-binding pocket of AKHR and proposed some high-affinity small-molecules to block its function, which would further facilitate pesticide design studies against the same receptor of various pests.

Claws are the most widespread attachment devices in animals, but comparatively little is known about the mechanics of claw attachment. A key morphological parameter in determining attachment ability is claw sharpness; however, there is a conflict between sharpness and fracture resistance. Sharper claws can interlock on more surfaces but are more likely to break. Body size interacts with this conflict such that larger animals should have much blunter claws and consequently poorer attachment ability than smaller animals. This expected size-induced reduction in attachment performance has not previously been investigated, and it is unclear how animals deal with this effect, and whether it indeed exists. We explored the scaling of claw sharpness with body size using four insect species (Nauphoeta cinerea, Gromphadorhina portentosa, Atta cephalotes and Carausius morosus) each covering a large size range. The scaling of claw sharpness varied significantly between species, suggesting that they face different pressures regarding claw function. Attachment forces were measured for A. cephalotes and G. portentosa (which had different scaling of claw sharpness) on several rough surfaces using a centrifuge setup. As expected, attachment performance was poorer in larger animals. Firstly, larger animals were more likely to slip, although this effect depended on the scaling of claw sharpness. Secondly, when they gripped, they attached with smaller forces relative to their weight. This size-induced reduction in attachment performance has significant implications for the attachment ability of larger animals on rough surfaces.

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.

Active tactile exploration behaviour is constrained to a large extent by the morphological and biomechanical properties of the animal's somatosensory system. In the model organismCarausius morosus, the main tactile sensory organs are long, thin, seemingly delicate, but very robust antennae. Previous studies have shown that these antennae are compliant under contact, yet stiff enough to maintain a straight shape during active exploration. Overcritical damping of the flagellum, on the other hand, allows for a rapid return to the straight shape after release of contact. Which roles do the morphological and biomechanical adaptations of the flagellum play in determining these special mechanical properties? To investigate this question, we used a combination of biomechanical experiments and numerical modelling. A set of four finite-element (FE) model variants was derived to investigate the effect of the distinct geometrical and material properties of the flagellum on its static (bending) and dynamic (damping) characteristics. The results of our numerical simulations show that the tapered shape of the flagellum had the strongest influence on its static biomechanical behaviour. The annulated structure and thickness gradient affected the deformability of the flagellum to a lesser degree. The inner endocuticle layer of the flagellum was confirmed to be essential for explaining the strongly damped return behaviour of the antenna. By highlighting the significance of two out of the four main structural features of the insect flagellum, our study provides a basis for mechanical design of biomimetic touch sensors tuned to become maximally flexible while quickly resuming a straight shape after contact.

As nocturnal animals, stick insects sample near-range information by active tactile exploration using their antennae. A previous study on the use of visual and tactile cues showed that antennectomy in stick insects strongly affects climbing efficiency, whereas blindfolding does not (Dürr et al., 2003, Int.J.Robot.Res., 22:151-167). However, this study used a setup with very low luminance and visual contrast. Since it is known that stick insects reliably orient themselves towards visual landmarks (Kalmus, 1937, Z.vergl.Physiol., 25:494-508), we wondered whether stick insects can exploit visual cues during climbing, provided that visual contrast and luminance were similar to experimental conditions of visual orientation experiments.To test this, we let animals climb an obstacle (height: 24 mm). On the front of the obstacle we placed one of three visual patterns (a horizontal or vertical black bar on white background, or a checkerboard). Stick insects were first tested intact and then grouped into two cohorts: In cohort 1 animals were tested with both antennae cut, then retested after blindfolding. In cohort 2, animals were tested blindfolded first, then with additional antennectomy. For each cohort and test session, we analysed the minimum distance to the obstacle, head orientation, and foot placement. Our expectation was that sighted animals with cut antennae would react similarly to intact animals, and differently from blindfolded, antennectomized animals. Our results proved both expectations wrong in that blindfolding had little or no effect on the parameters observed. We conclude that stick insects make little or no use of visual cues during climbing.

Goal-directed, unrestrained motor behaviour in a variable and unpredictable environment requires animals to adjust their limb movements appropriately. In mammals, neuromuscular strategies with functionally distinct transition strides between level and slope walking have been found (Gottschall & Nichols, 2011, Phil.Trans. R.Soc.L.B, 366(1570), 1565-1579). So far, this has not been described for insects, where slope-dependent changes have been studied mainly in steady state situations with differing slope, during upside-down walking, or in climbing a stair of given height. With the present study, we evaluate the behavioural adjustments of unrestrained walking stick insects (Carausius morosus) in response to a step change in slope of the walkway (±45°). Larsen et al. (1995, J.Comp.Physiol.A, 176(2), 229-238) suggested that stance phases can be divided into an early support and a later propulsive part, in line with the protractor coxae muscles showing braking activity during early stance (Graham & Wendler, 1981, Physiol.Entomol., 6(2), 161-174). Here, we analyse the hind leg protraction/retraction system of the stick insect, using simultaneous recordings of whole-body kinematics and muscle activity in freely walking animals. Our results suggest an early load-dependent activation of both muscles with delayed retractor activity in case of only little load, supporting the idea of a biphasic power stroke. The situation, however, is quite different for leg kinematics, where overall differences among steady state situations were small, and the most prominent differences occurred only during the transitional steps between level walking and slope walking. Further studies may address the contribution of other muscles (e.g., depressor trochanteris) and legs.

Thanatosis is a common phenomenon in which prey appear to feign death when attacked by predators. It was once widely believed that thanatosis exploited predators' tendencies to avoid dead prey. However, this hypothesis has never been tested, and its feasibility has been questioned to the point that it has been largely abandoned [1,2]. Here, I show that naive birds quickly learned that dead Indian stick insects Carausius morosus were unpalatable, and subsequently rejected live insects that displayed thanatosis, but not those that failed to show thanatosis. Thanatosis had no effect on the behavior of birds that had never experienced dead insects, or those that had experienced dead insects whose resemblance to thanatosic insects had been destroyed. Therefore, thanatosis clearly caused predators to avoid prey that they mistakenly perceived to be dead.

Allatostatins (AST) are neuropeptides with variable function ranging from regulation of developmental processes to the feeding behavior in insects. They exert their effects by binding to cognate GPCRs, called Allatostatin receptors (AlstR), which emerge as promising targets for pesticide design. However, AlstRs are rarely studied. This study is the first reported structural study on AlstR-AST interaction. In this work, the first C type AlstR from the stick insect Carausius morosus (CamAlstR-C) was identified and its interaction with type C AST peptide was shown to be physically consistent with the experimental results. The proposed structure of CamAlstR-C revealed a conserved motif within the third extracellular loop, which, together with the N-terminus is essential for ligand binding. In this work, computational studies were combined with molecular and nano-scale approaches in order to introduce an unknown GPCR-ligand system. Consequently, the data obtained provided a reliable target region for future agonist/inverse agonist studies on AlstRs.

Animals and humans need to move deftly and flexibly to adapt to environmental demands. Despite a large body of work on the neural control of walking in invertebrates and vertebrates alike, the mechanisms underlying the motor flexibility that is needed to adjust the motor behavior remain largely unknown. Here, we investigated optomotor-induced turning and the neuronal mechanisms underlying the differences between the leg movements of the two body sides in the stick insect Carausius morosus. We present data to show that the generation of turning kinematics in an insect are the combined result of descending unilateral commands that change the leg motor output via task-specific modifications in the processing of local sensory feedback as well as modification of the activity of local central pattern generating networks in a body-side-specific way. To our knowledge, this is the first study to demonstrate the specificity of such modifications in a defined motor task. DOI: http://dx.doi.org/10.7554/eLife.13799.001, eLife digest Walking along a curve or turning is a complex manoeuvre for the nervous system, as it must coordinate different leg movements on each side of the body. Rhythmic processes such as walking are controlled by networks of neurons called central pattern generators. The resulting movements can be adjusted by feedback from sense organs in response to environmental conditions. For example, sensory feedback that provides information about the load placed on each leg, allows the animal to control the duration of a stance. How the nerve cells, or neurons, involved in these processes work together to produce complex, flexible movements such as turning is largely unknown. Previous work on how the brain negotiates turning movements has been carried out mostly in animals that swim or fly. To understand what happens during walking, Gruhn et al. monitored stick insects that walked in a curve on a slippery surface, and recorded the electrical activity within the animals' nervous system as they turned. By comparing the activity of the nervous system on each side of the body while the insects walked a curve, Gruhn et al. found that the nervous system uses at least three different mechanisms to produce the different movements on the inside and outside. Firstly, the sensory feedback signals that communicate the load on the leg are processed in the legs on the outside of the curve to support forward steps, while they are processed on the inside legs to support forward, sideward, and backward steps. Secondly, the motor activity produced by the central pattern generator is modulated to be stronger for the muscle that moves the leg backward on the outside of the curve. At the same time, this activity is stronger for the muscle that moves the leg forward on the inside of the curve. Thirdly, signals from a front leg influence the movement of the other legs on the same side of the body. This influence is strong on the inside and weak on the outside of the curve. Together or separately, these three mechanisms could provide the animal with the means to perform turns in all their different curvatures. Future work will need to work out exactly which local neurons process the signals sent from the brain to control movement. DOI: http://dx.doi.org/10.7554/eLife.13799.002

Optimization of sensory processing during development can be studied by using photoreceptors of hemimetabolous insects (with incomplete metamorphosis) as a research model. We have addressed this topic in the stick insectCarausius morosus, where retinal growth after hatching is accompanied by a diurnal-to-nocturnal shift in behavior, by recording from photoreceptors of first instar nymphs and adult animals using the patch-clamp method. In the nymphs, ommatidia were smaller and photoreceptors were on average 15-fold less sensitive to light than in adults. The magnitude of A-type K+current did not increase but the delayed rectifier doubled in adults compared with nymphs, the K+current densities being greater in the nymphs. By contrast, the density of light-induced current did not increase, although its magnitude increased 8.6-fold, probably due to the growth of microvilli. Nymph photoreceptors performed poorly, demonstrating a peak information rate (IR) of 2.9 ± 0.7 bits/s versus 34.1 ± 5.0 bits/s in adults in response to white-noise stimulation. Strong correlations were found between photoreceptor capacitance (a proxy for cell size) and IR, and between light sensitivity and IR, with larger and more sensitive photoreceptors performing better. In adults, IR peaked at light intensities matching irradiation from the evening sky. Our results indicate that biophysical properties of photoreceptors at each age stage and visual behavior are interdependent and that developmental improvement in photoreceptor performance may facilitate the switch from the diurnal to the safer nocturnal lifestyle. This also has implications for how photoreceptors achieve optimal performance.

In the present study, the tarsal attachment pads (euplantulae) of two stick insect species (Phasmatodea) were compared. While the euplantulae of Cuniculina impigra (syn. Medauroidea extradentata) are smooth, those of Carausius morosus bear small nubs on their surfaces. In order to characterize the adhesive and frictional properties of both types of euplantulae, adhesion and friction measurements on smooth (Ra=0.054 μm) and rough (Ra=1.399 μm) substrates were carried out. The smooth pads of C. impigra generated stronger adhesion on the smooth substrate than on the rough one. The adhesive forces of the structured pads of C. morosus did not differ between the two substrates. Friction experiments showed anisotropy for both species with higher values for proximal pulls than for distal pushes. In C. impigra, friction was stronger on the smooth than on the rough surface for both directions, whereas in C. morosus friction was stronger on the smooth surface only for pushes. This shows that smooth attachment pads are able to generate relatively stronger adhesion and friction on a flat smooth surface than on a rough one. In contrast, nubby pads have similar adhesion on both substrates, and also show no difference in friction in the pulling direction. This leads to the conclusion that smooth pads are specialized for rather smooth substrates, whereas nubby pads are better adapted to generate stronger forces on a broader range of surfaces.

Anatomical features of leg motoneurons, dorsal unpaired median (DUM) cells, and sensory neurons in stick insect mesothoracic ganglia were examined using fluorescent dye backfills of lateral nerves. Structures were analyzed in whole-mounts of ganglia and transverse sections. Numbers of motoneurons and details of their structure by far exceed previously published data. The general neuroanatomical layout of motoneurons matches the general orthopteran pattern. Cell bodies of excitatory motoneurons form clusters in the lateral cortex, dendrites branch mainly in the dorsal neuropil. We identified nine DUM cells, six of which have axons in nerve nl5. Most sensory fibers terminate in the ventral association center (VAC). Twenty-three small cell bodies located close to the soma of the fast extensor tibiae motoneuron likely belong to strand receptors. Labeled structures are compared with previously published data from stick insects and other orthopterous insects.

Understanding how animals control locomotion in different behaviors requires understanding both the kinematics of leg movements and the neural activity underlying these movements. Stick insect leg kinematics differ in forward and backward walking. Describing leg muscle activity in these behaviors is a first step toward understanding the neuronal basis for these differences. We report here the phasing of EMG activities and latencies of first spikes relative to precise electrical measurements of middle leg tarsus touchdown and liftoff of three pairs ( protractor/retractor coxae, levator/depressor trochanteris, extensor/flexor tibiae) of stick insect middle leg antagonistic muscles that play central roles in generating leg movements during forward and backward straight walking. Forward walking stance phase muscle (depressor, flexor, and retractor) activities were tightly coupled to touchdown, beginning on average 93 ms prior to and 9 and 35 ms after touchdown, respectively. Forward walking swing phase muscle (levator, extensor, and protractor) activities were less tightly coupled to liftoff, beginning on average 100, 67, and 37 ms before liftoff, respectively. In backward walking the protractor/retractor muscles reversed their phasing compared with forward walking, with the retractor being active during swing and the protractor during stance. Comparison of intact animal and reduced two- and one-middle-leg preparations during forward straight walking showed only small alterations in overall EMG activity but changes in first spike latencies in most muscles. Changing body height, most likely due to changes in leg joint loading, altered the intensity, but not the timing, of depressor muscle activity.

The dorsal heart of the Indian stick insect, Carausius morosus, is responsible for the anterograde flow of hemolymph to the aorta and into the body cavity. The contraction frequency of the insect heart is known to be influenced by several substances of neural source. Here, a semi-exposed heart assay was employed to study the effect of an aminergic substance (octopamine) and three neuropeptides (C. morosus hypertrehalosemic hormone [Carmo-HrTH], crustacean cardioactive peptide [CCAP], and proctolin) on heart contraction. The contraction frequency was measured as beats per minute in adults ligated between the head and the prothorax. All three investigated neuropeptides had a stimulatory effect on heart contraction that lasted approximately 6 min, after which the normal heart beat rate was restored. Proctolin and CCAP stimulated the rate of heart beat also in unligated stick insects, whereas Carmo-HrTH was active only in ligated insects. The latter could suggest that when the stick insect is not ligated, a competing substance may be released from the head of C. morosus; the competing substance is, apparently, not physiologically active but it binds or blocks access to the receptor of Carmo-HrTH-II, thereby rendering the HrTH peptide "not active." In ligated stick insects, 6.7 × 10-8 M Carmo-HrTH-II significantly increased the heart beat rate; higher doses resulted in no further increase, suggesting the saturation of the HrTH receptor. Octopamine inhibited the rate at which the heart contracted in a dose-dependent manner; inhibition was achieved with 10-4 M of octopamine.

Spontaneous and experimentally induced pseudotumor formation in Carausius morosus impairs the midgut tissue homeostasis. Spontaneous pseudotumor formation begins by the break down of a single or a small group of columnar cells (CCs) and is followed by the degeneration of neighboring CCs. There are not only marked similarities but also decisive differences between normal dying CCs in healthy specimens and the degeneration of CCs leading to pseudotumors: in both cases, the apical cell parts with the nucleus are extruded into the midgut lumen, but only during of pseudotumor formation an "amorphous substance" originates from the basal parts of the CCs. Hemocytes are attracted to this substance and form a nodule-like aggregation, which is responsible for the phenotype of pseudotumors. Pseudotumor infestation has also an impact on the midgut nidi, which consist of an intestinal stem cell and several CC progenitor cells. In healthy specimens only one progenitor cell per nidus differentiates at a time, but, several to all progenitor cells differentiate simultaneously in pseudotumor-infested specimens. Extirpation of the ingluvial ganglion in healthy specimens results in an immediate onset of pseudotumor formation and a dramatic acceleration of pseudotumor growth. Importantly, the ultrastructural characteristics of spontaneous and experimentally induced pseudotumors are identical. This supports the idea that the stomatogastric nervous system plays an integral role in the maintenance of midgut tissue homeostasis.

We performed electrophysiological and behavioral experiments in single-leg preparations and intact animals of the stick insect Carausius morosus to understand mechanisms underlying the control of walking speed. At the level of the single leg, we found no significant correlation between stepping velocity and spike frequency of motor neurons (MNs) other than the previously shown modification in flexor (stance) MN activity. However, pauses between stance and swing motoneuron activity at the transition from stance to swing phase and stepping velocity are correlated. Pauses become shorter with increasing speed and completely disappear during fast stepping sequences. By means of extra- and intracellular recordings in single-leg stick insect preparations we found no systematic relationship between the velocity of a stepping front leg and the motoneuronal activity in the ipsi- or contralateral mesothoracic protractor and retractor, as well as flexor and extensor MNs. The observations on the lack of coordination of stepping velocity between legs in single-leg preparations were confirmed in behavioral experiments with intact stick insects tethered above a slippery surface, thereby effectively removing mechanical coupling through the ground. In this situation, there were again no systematic correlations between the stepping velocities of different legs, despite the finding that an increase in stepping velocity in a single front leg is correlated with a general increase in nerve activity in all connectives between the subesophageal and all thoracic ganglia. However, when the tethered animal increased walking speed due to a short tactile stimulus, provoking an escape-like response, stepping velocities of ipsilateral legs were found to be correlated for several steps. These results indicate that there is no permanent coordination of stepping velocities between legs, but that such coordination can be activated under certain circumstances.

Stick insect (Carausius morosus) leg muscles contract and relax slowly. Control of stick insect leg posture and movement could therefore differ from that in animals with faster muscles. Consistent with this possibility, stick insect legs maintained constant posture without leg motor nerve activity when the animals were rotated in air. That unloaded leg posture was an intrinsic property of the legs was confirmed by showing that isolated legs had constant, gravity-independent postures. Muscle ablation experiments, experiments showing that leg muscle passive forces were large compared with gravitational forces, and experiments showing that, at the rest postures, agonist and antagonist muscles generated equal forces indicated that these postures depended in part on leg muscles. Leg muscle recordings showed that stick insect swing motor neurons fired throughout the entirety of swing. To test whether these results were specific to stick insect, we repeated some of these experiments in cockroach (Periplaneta americana) and mouse. Isolated cockroach legs also had gravity-independent rest positions and mouse swing motor neurons also fired throughout the entirety of swing. These data differ from those in human and horse but not cat. These size-dependent variations in whether legs have constant, gravity-independent postures, in whether swing motor neurons fire throughout the entirety of swing, and calculations of how quickly passive muscle force would slow limb movement as limb size varies suggest that these differences may be caused by scaling. Limb size may thus be as great a determinant as phylogenetic position of unloaded limb motor control strategy.

Legged locomotion results from a combination of central pattern generating network (CPG) activity and intralimb and interlimb sensory feedback. Data on the neural basis of interlimb coordination are very limited. We investigated here the influence of stepping in one leg on the activities of neighboring-leg thorax–coxa (TC) joint CPGs in the stick insect (Carausius morosus). We used a new approach combining single-leg stepping with pharmacological activation of segmental CPGs, sensory stimulation, and additional stepping legs. Stepping of a single front leg could activate the ipsilateral mesothoracic TC CPG. Activation of the metathoracic TC CPG required that both ipsilateral front and middle legs were present and that one of these legs was stepping. Unlike the situation in real walking, ipsilateral mesothoracic and metathoracic TC CPGs activated by front-leg stepping fired in phase with the front-leg stepping. Local (intralimb) sensory feedback from load sensors could override this intersegmental influence of front-leg stepping, shifting retractor motoneuron activity relative to the front-leg step cycle and thereby uncoupling them from front-leg stepping. These data suggest that front-leg stepping in isolation would result in in-phase activity of all ipsilateral legs, and functional stepping gaits (in which the three ipsilateral legs do not step in synchrony) emerge because of local load sensory feedback overriding this in-phase influence.

We investigated insects Carausius morosus walking whilst hanging upside down along a narrow 3 mm horizontal beam. At the end of the beam, the animal takes a 180 degrees turn. This is a difficult situation because substrate area is small and moves relative to the body during the turn. We investigated how leg movements are organised during this turn. A non-contact of either front leg appears to indicate the end of the beam. However, a turn can only begin if the hind legs stand in an appropriate position relative to each other; the outer hind leg must not be placed posterior to the inner hind leg. When starting the turn, both front legs are lifted and usually held in a relatively stable position and then the inner middle leg performs a swing-and-search movement: The leg begins a swing, which is continued by a searching movement to the side and to the rear, and eventually grasps the beam. At the same time the body is turned usually being supported by the outer middle leg and both hind legs. Then front legs followed by the outer middle leg reach the beam. A scheme describing the turns based on a few simple behavioural elements is proposed.

Animals have evolved adhesive structures on their legs to cling to the substrate during locomotion. Here we characterise the ultrastructure and mechanical properties of adhesive pads in Carausius morosus (Phasmatodea) using atomic force microscopy (AFM) as well as transmission and scanning electron microscopy (TEM, SEM). The smooth adhesive arolium has a soft cuticle consisting of principal rods, which branch into finer fibres near the surface. Indentation experiments showed that the pad material consists of distinct layers with different mechanical properties. The 100-300 nm thick outermost layer consisting of the cuticulin envelope and the epicuticle is extremely soft and resilient (mean effective Young's modulus 12 kPa), while the subjacent procuticle is a much stiffer material (mean effective Young's modulus 625 kPa). AFM contact mode imaging revealed that the cuticle is mechanically anisotropic, which can be explained by its fibrillar inner structure. We propose that the described layered structure of smooth adhesive pads, consisting of materials decreasing in stiffness towards the outer surface, represents a superior design to conform and adhere to substrates with roughnesses at different length scales. This design principle could be easily implemented in technical adhesives, and thus has a potential to inspire biomimetic applications.

A key element of walking is the coordinated interplay of multiple limbs to achieve a stable locomotor pattern that is adapted to the environment. We investigated intersegmental coordination of walking in the stick insect, Carausius morosus by examining the influence a single stepping leg has on the motoneural activity of the other hemiganglia, and whether this influence changes with the walking direction. We used a reduced single leg walking preparation with only one intact front, middle, or hind leg. The intact leg performed stepping movements on a treadmill, thus providing intersegmental signals about its stepping to the other hemiganglia. The activity of coxal motoneurons was simultaneously recorded extracellularly in all other segments. Stepping sequences of any given single leg in either walking direction were accompanied by an increase in coxal motoneuron (MN) activity of all other segments, which was mostly modulated and slightly in phase with stance of the walking leg. In addition, forward stepping of the front leg and, to a lesser extent, backward stepping of the hind leg elicited alternating activity in mesothoracic coxal MNs. Forward and backward stepping of the middle leg did not elicit alternating activity in coxal MNs in any other hemiganglia, indicating that the influence of middle leg stepping is qualitatively different from that of forward front and backward hind leg stepping. Our results indicate that in an insect walking system individual segments differ with respect to their intersegmental influences and thus cannot be treated as similar within the chain of segmental walking pattern generators. Consequences for the current concepts on intersegmental coordination are discussed.

Leg movements of stick insects (Carausius morosus) making turns towards visual targets are examined in detail, and a dynamic model of this behaviour is proposed. Initial results suggest that front legs shape most of the body trajectory, while the middle and hind legs just follow external forces (Rosano H, Webb B, in The control of turning in real and simulated stick insects, vol. 4095, pp 145-156, 2006). However, some limitations of this explanation and dissimilarities in the turning behaviour of the insect and the model were found. A second set of behavioural experiments was made by blocking front tarsi to further investigate the active role of the other legs for the control of turning. The results indicate that it is necessary to have different roles for each pair of legs to replicate insect behaviour. We demonstrate that the rear legs actively rotate the body while the middle legs move sideways tangentially to the hind inner leg. Furthermore, we show that on average the middle inner and hind outer leg contribute to turning while the middle outer leg and hind inner leg oppose body rotation. These behavioural results are incorporated into a 3D dynamic robot simulation. We show that the simulation can now replicate more precisely the turns made by the stick insect.

This article describes basic parameters characterizing walking of the stick insect Aretaon asperrimus to allow a comparative approach with other insects studied. As in many other animals, geometrical parameters such as step amplitude and leg extreme positions do not vary with walking velocity. However, the relation between swing duration and stance duration is quite constant, in contrast to most insects studied. Therefore, velocity profiles during swing vary with walking velocity whereas time course of leg trajectories and leg angle trajectories are independent of walking velocity. Nevertheless, A. asperrimus does not show a classical tripod gait, but performs a metachronal, or tetrapod, gait, showing phase values differing from 0.5 between ipsilateral neighbouring legs. As in Carausius morosus, the detailed shape of the swing trajectory may depend on the form of the substrate. Effects describing coordinating influences between legs have been found that prevent the start of a swing as long as the posterior leg performs a swing. Further, the treading on tarsus reflex can be observed in Aretaon. No hint to the existence of a targeting influence has been found. Control of rearward walking is easiest interpreted by maintaining the basic rules but an anterior-posterior reversal of the information flow.

Like many flightless, obligatory walking insects, the stick insect Carausius morosus makes intensive use of active antennal movements for tactile near range explora- tion and orientation. The antennal joints of C. morosus have a peculiar oblique and non-orthogonal joint axis arrange- ment. Moreover, this arrangement is known to differ from that in crickets (Ensifera), locusts (Caelifera) and cock- roaches (Blattodea), all of which have an orthogonal joint axis arrangement. Our hypothesis was that the situation found in C. morosus represents an important evolutionary trait of the order of stick and leaf insects (Phasmatodea). If this was true, it should be common to other species of the Phasmatodea. The objective of this comparative study was to resolve this question. We have measured the joint axis orientation of the head-scape and scape-pedicel joints along with other parameters that affect the tactile efficiency of the antenna. The obtained result was a complete kinematic description of the antenna. This was used to determine the size and location of kinematic out-of-reach zones, which are indicators of tactile acuity. We show that the oblique and non-orthogonal arrangement is common to eight species from six sub-families indicating that it is a synapomorphic character of the Euphasmatodea. This character can improve tactile acuity compared to the situation in crickets, locusts and cockroaches. Finally, because molecular data of a recent study indicate that the Phasmatodea may have evolved as flightless, obligatory walkers, we argue that the antennal joint axis arrangement of the Euphasmatodea reflects an evolutionary adaptation to tactile near range exploration during terrestrial locomotion.

We recorded fast extensor tibiae motor neuron activity during single-legged treadmill walking in the stick insect, Carausius morosus. We used this activity to stimulate the extensor muscle motor nerve, observed the resulting extensor muscle contractions under isotonic conditions, and quantified these contractions with a variety of measures. Extensor contractions induced in this manner were highly variable, with contraction measures having SDs of 12 to 51%, and ranges of 82 to 275%, when expressed as percentages of the means, an unexpectedly wide range for a locomotory pattern. Searches for correlations among the contraction measures showed that, in general, this high variability is not reduced by contraction measure covariation. Comparing responses (to identical input) across animals showed that extensor muscles from different animals generally significantly differed from one another. However, correlation analyses on these data suggested that these differences do not indicate that multiple extensor muscle subtypes exist. Extensor muscles instead appear to belong to a single class, albeit one with high animal to animal variability. These data thus provide another well-quantified example (along with Aplysia feeding) of a repetitive but highly variable motor pattern (in contrast to the high rhythmicity and stereotypy present in most other well-quantified repetitive motor patterns). We suggest this high variability could be an adaptive combination of locomotion, active sensing, and crypsis arising from the relatively low demand for locomotion in Carausius behavior, the highly fragmented environment the animal inhabits, and its need to avoid predatory attention.

SUMMARYThe control of locomotion requires the ability to adapt movement sequences to the behavioural context of the animal. In hexapod walking, adaptive behavioural transitions require orchestration of at least 18 leg joints and twice as many muscle groups. Although kinematics of locomotion has been studied in several arthropod species and in a range of different behaviours,almost nothing is known about the transition from one behavioural state to another. Implicitly, most studies on context-dependency assume that all parameters that undergo a change during a behavioural transition do so at the same rate. The present study tests this assumption by analysing the sequence of kinematic events during turning of the stick insect Carausius morosus, and by measuring how the time courses of the changing parameters differ between legs. Turning was triggered reliably at a known instant in time by means of the optomotor response to large-field visual motion. Thus, knowing the start point of the transition, the kinematic parameters that initiate turning could be ranked according to their time constants.Kinematics of stick insect walking vary considerably among trials and within trials. As a consequence, the behavioural states of straight walking and curve walking are described by the distributions of 13 kinematic parameters per leg and of orientation angles of head and antennae. The transitions between the behavioural states are then characterised by the fraction of the variance within states by which these distributions differ,and by the rate of change of the corresponding time courses. The antennal optomotor response leads that of the locomotor system. Visually elicited turning is shown to be initiated by stance direction changes of both front legs. The transition from straight to curve walking in stick insects follows different time courses for different legs, with time constants of kinematic parameters ranging from 1.7 s to more than 3 s. Therefore, turning is a behavioural transition that involves a characteristic orchestration of events rather than synchronous parallel actions with a single time constant.

This paper concentrates on the system that controls the femur-tibia joint in the legs of the stick insect, Carausius morosus. Earlier investigations have shown that this joint is subject to a mixture of proportional and differential control whereby the differential part plays a prominent role. Experiments presented here suggest another interpretation: single legs of a stick insect were systematically perturbed using devices of different compliance and compensatory forces and movements monitored. When the compliance is high (soft spring), forces are generated that return the leg close to its original position. When the compliance is low (stiff spring), larger forces are generated but sustained changes in position occur that are proportional to the force that is applied. Selective ablation of leg sense organs showed that the leg did not maintain its position after elimination of afferents of the femoral chordotonal organ. Ablation of leg campaniform sensilla had no effect. These data support the idea that different control strategies are used, depending upon substrate compliance. In particular, what we and other authors have called a differential controller, is now considered as an integral controller that ldquointelligently gives uprdquo when the correlation between motor output and movement of the leg is low.

Legged locomotion through natural environments is very complex and variable. For example, leg kinematics may differ strongly among species, but even within the same species it is adaptive and context-dependent. Inter-species differences in locomotion are often difficult to interpret, because both morphological and ecological differences among species may be strong and, as a consequence, confound each other's effects. In order to understand better how body morphology affects legged locomotion, we compare unrestrained whole-body kinematics of three stick insect species with different body proportions, but similar feeding ecology: Carausius morosus, Aretaon asperrimus and Medauroidea extradentata (= Cuniculina impigra). In order to co-vary locomotory context, we introduced a gradually increasing demand for climbing by varying the height of stairs on the set-up. The species were similar in many aspects, for example in using distinct classes of steps, with minor differences concerning the spread of corrective short steps. Major differences were related to (1) antenna length, (2) segment lengths of thorax and head, and (3) the ratio of leg length over body length: (1) Whereas all species continuously moved their antennae, only Medauroidea executed high swing movements with its front legs to search for obstacles in the near-range environment. (2) Whereas all species adjusted their body inclination, the range in which body segments moved differed considerably, with longer thorax segments tending to be moved more. (3) Finally, leg posture, time courses of leg joint angles and intra-leg coordination differed most strongly in long-legged Medauroidea.

Two species of stick insects are currently known to be present in the Macaronesian archipelagos: Clonopsis gallica (Charpentier) (Phasmatodea: Bacillidae) on the Canary Islands and in the Azores and Carausius morosus (Sinéty) (Phasmatidae) in the Azores. Here, we provide the first reliable records of the presence and distribution of C. gallica and C. morosus on Madeira Island. Egg and adult stages are briefly described along with some notes on the life history of these species in captivity. Data on islandwide distribution are based on specimens donated by the public in response to an article published in a daily newspaper. This method of data collection raised great popular interest in stick insects. The role of newspapers as a means of communicating awareness in biodiversity issues is discussed.

Successful ecdysis in insects depends on proper timing and sequential activation of an elaborate series of motor programs driven by a relatively conserved network of neuropeptides. The behaviors must be activated at the appropriate times to ensure successful loosening and shedding of the old cuticle, and can be influenced by environmental cues in the form of immediate sensory feedback and by circadian rhythms. We assessed the behaviors, components of the neural network and the circadian basis of ecdysis in the stick insect, Carausius morosus. C. morosus showed many of the characteristic pre-ecdysis and ecdysis behaviors previously described in crickets and locusts. Ecdysis was described in three phases, namely the (i) preparatory or pre-ecdysis phase, (ii) the ecdysial phase, and (iii) the post-ecdysis or exuvial phase. The frequencies of push-ups and sways during the preparatory phase were quantified as well as durations of all the phases. The regulation of ecdysis appeared to act via elevation of cGMP, as described in many other insects, although eclosion hormone-like immunoreactivity was not noted using a lepidopteran antiserum. Finally, C. morosus showed a circadian rhythm to the onset of ecdysis, with ecdysis occurring just prior to or at lights on. Ecdysis could be induced precociously with mechanical stimulation.

Newly laid eggs of stick insects comprise a unique fluid ooplasm that is gradually partitioned into a number of yolk granules by invasion of secondary vitellophages. This study aimed at establishing how yolk granules become acidified in the course of embryonic development. Data show that acidified yolk granules are rather scarce and randomly distributed in vitellophages of early embryos, while they tend to increase gradually in number as development proceeds to completion. Yolk granule acidification is progressively more inhibited in the presence of increasing concentrations of chloroquine, monensin and bafilomycin. A pro-protease was identified cytochemically and by immunoblotting in yolk extracts of progressively more advanced embryos. A specific monoclonal antibody raised against this pro-protease helped to demonstrate that it is gradually processed to yield a lower molecular weight polypeptide as development proceeds to completion. This latter polypeptide was identified as a protease using electrophoresis in polyacrylamide gels containing yolk extracts. Simultaneous administration of a fluorescent substrate for cysteine protease and an acidotropic probe produced superimposable labelling patterns, suggesting that only acidified yolk granules possess a proteolytic activity. On the other hand, yolk granules probed simultaneously for acidification and latent pro-protease yielded labelling patterns partially superimposed. Pro-protease labelling is gradually lost as yolk granules are progressively more acidified during development. Distinct labelling patterns were also obtained in vitellophages processed for the simultaneous detection of pro-protease and protease, suggesting that the two activities are expressed by different yolk granule populations, and that one is gradually converted into the other as time goes by.

Three related nucleotide sequences, encoding mature proteins of 108-113 amino acids, have been obtained from antennal cDNA of the Phasmid Eurycantha calcarata. Among these, one is also expressed in the tarsi as demonstrated by N-terminal sequence and mass spectrometric analyses of protein samples isolated from both organs. PCR experiments performed with specific primers, showed that this species is also expressed in the mouth organs and in the cuticle, while the other two are antennal specific. All three isoforms are similar to Drosophila OS-D and other proteins reported in several insect orders, but one of them is significantly different from the other two. The best conserved elements are the N-terminal region and the four cysteine residues. Accurate ESMS measurements indicated that all cysteines are involved in two disulphide bonds and ruled out the occurrence of additional post-translational modifications. Polyclonal antibodies, raised against the purified protein, did not react with proteins of the same class expressed in another Phasmid species, Carausius morosus, and in the orthopteran Schistocerca gregaria, nor did antibodies against these proteins recognise those of E. calcarata.

Synthesis and secretion of vitellogenin (Vg) polypeptides were studied in egg-laying females of the stick insect Carausius morosus following in vivo exposure to [35S]-methionine and acetyl-N-[3H]-glucosamine. The specificity of radioisotope incorporation was assessed by in vitro inhibition with tunicamycin and carbohydrate extraction with endo-glycosidase H. Vg polypeptides change in molecular weight during synthesis in the fat body and are not further modified upon transfer to the haemolymph or to the oocyte, suggesting that they are already fully glycosylated prior to secretion. Radioactivity in the fat body was initially distributed over cisternae of the rough endoplasmic reticulum and gradually transferred to the Golgi apparatus. Within an hour of exposure, electron-dense granules budding from the trans-Golgi network became preferentially labeled. Radioactivity in the ovarian follicle was restricted to the yolk granules of the cortical ooplasm and to the amorphous material lying within the intercellular channels of the follicular epithelium. This amorphous material was also shown to react positively when tested with a monoclonal antibody raised specifically against a Vg polypeptide.

During active leg movements of an insect leg, the activity of the motoneuron pools of each individual leg joint is generated by the interaction between signals from central rhythm generating sources, peripheral signals as well as coordinating signals from other leg joints and legs. The nature of the synaptic drive from the central rhythm generators onto the motoneuron pools of the individual leg joints during rhythmic motor activity of the stick insect (Carausius morosus) middle leg has been investigated. In the isolated mesothoracic ganglion central rhythm generators were activated pharmacologically by topical application of the muscarinic agonist pilocarpine. Motoneurons supplying the femur-tibia (FT) joint were investigated in detail. Recordings from neuropil processes of these motoneurons revealed that patterning of their rhythmic activity is based on cyclic hyperpolarizing synaptic inputs. These inputs are in clear antiphase for extensor and flexor motoneurons. DCC (discontinuous current clamp) and dSEVC (discontinuous single electrode voltage clamp) recordings showed reversal potentials of the inhibitory inputs between -80 to -85 mV (FETi, N=7; Flex MN, N=3). After intracellular injection of TEA rhythmic inhibition in FETi was decreased by about 84% (N=4). Both findings indicate that the cyclic inhibition is mediated by potassium ions. Thus, it appears that central rhythm generators pattern motor activity in antagonistic tibial motoneuron pools by cyclic alternating inhibition.

Adhesive organs like arolia of insects allow these animals to climb on different substrates by creating high adhesion forces. According to the Dahlquist criterion, adhesive organs must be very soft exhibiting an effective Young's modulus of below 100 kPa to adhere well to substrates. Such a low effective Young's modulus allows the adhesive organs to make almost direct contact to the substrate and results in van der Waals forces beside capillary forces. In previous studies the effective Young's moduli of adhesive organs were determined using indentation tests yielding their structure to be very soft indeed. However, adhesive organs show a layered structure, thus the measured values comprise the effective Young's moduli of several layers of the adhesive organs. In this study, a new approach is illustrated to measure the Young's modulus of the outermost layer of the arolium, i.e. of the epicuticle, of the stick insect Carausius morosus. As a result of the epicuticle being supported by upright fibres tensile tests allow the determination of the Young's modulus of the epicuticle hardly affected by subjacent layers. In our tensile tests arolia of stick insects adhering on a latex membrane were stretched by stretching the membrane while the elongation of the contact area between an arolium and the membrane was recorded. For analysis mathematical models of the mechanical system were developed. When fed with the observed elongations, these models yield estimates for the Young's modulus of the epicuticle of about a hundred megapascal. Thus in arolia a very thin layer (~225 nm) of a rather stiff material, which is less susceptible to abrasion, makes contact to the substrates while the inner fibrous structure of arolia is responsible for their softness.

Soluble proteins of low molecular weight have been purified from chemosensory organs of five species of Phasmids. On the basis of their N-terminal amino acid sequences, two classes can be identified. Polypeptides of 14 and 15 kDa, expressed in the antennae and legs of Eurycantha calcarata and Extatosoma tiaratum, as well as in the antennae of Carausius morosus, bear a close similarity (around 45% identity) with a soluble protein associated with the sensilla coeloconica of Drosophila melanogaster. Two proteins of 19 and 18 kDa, isolated from the antennae and the maxillary palpi, respectively, of Acrophylla wuelfingi, are 59 and 75% identical, in their N-terminal region, to a 19 kDa antennal protein of Carausius morosus. Similarity between members of the two classes is not significant, being limited to two to three identical amino acids in the most favorable cases. Finally, a 17 kDa protein, specifically expressed in the antennae of Sipyloidea sipylus, did not show any homology with other proteins. The expression in sensory organs and the characteristics of these proteins may suggest a function in chemosensory transduction.

SummaryIn its natural habitat, Carausius morosus climbs on the branches of bushes and trees. Previous work suggested that stick insects perform targeting movements with their hind legs to find support more easily. It has been assumed that the animals use position information from the anterior legs to control the touchdown position of the ipsilateral posterior legs. Here we address the questions if not only the hind but also the middle leg performs targeting, and if targeting is still present in a walking animal when influences of mechanical coupling through the ground are removed. If this were the case, it would emphasize the role of underlying neuronal mechanisms. We studied whether targeting occurred in both legs, when the rostral neighboring leg, i.e. either middle- or front leg, was placed at defined positions relative to the body, and analyzed targeting precision for dependency on the targeted position. Under these conditions, the touchdown positions of the hind legs show correlation to the position of the middle leg parallel and perpendicular to the body axis while only weak correlation exists between the middle and front legs, and only in parallel to the body axis. In continuously walking tethered animals targeting accuracy of hind and middle legs parallel to the body axis was barely different. However, targeting became significantly more accurate perpendicular to the body axis. Our results suggest that a neural mechanism exists for controlling the touchdown position of the posterior leg but that the strength of this mechanism is segment-specific and dependent on the behavioral context in which it is used.

The analysis of inter-leg coordination in insect walking is generally a study of six-legged locomotion. For decades, the stick insect Carausius morosus has been instrumental for unravelling the rules and mechanisms that control leg coordination in hexapeds. We analysed inter-leg coordination in C. morosus that freely walked on straight paths on plane surfaces with different slopes. Consecutive 1.7 s sections were assigned inter-leg coordination patterns (which we call gaits) based on footfall patterns. Regular gaits, i.e. wave, tetrapod or tripod gaits, occurred in different proportions depending on surface slopes. Tetrapod gaits were observed most frequently, wave gaits only occurred on 90 deg inclining slopes and tripod gaits occurred most often on 15 deg declining slopes, i.e. in 40% of the sections. Depending on the slope, 36–66% of the sections were assigned irregular gaits. Irregular gaits were mostly due to multiple stepping by the front legs, which is perhaps probing behaviour, not phase coupled to the middle legs’ cycles. In irregular gaits, middle leg and hindleg coordination was regular, related to quadrupedal walk and wave gaits. Apparently, front legs uncouple from and couple to the walking system without compromising middle leg and hindleg coordination. In front leg amputees, the remaining legs were strictly coordinated. In hindleg and middle leg amputees, the front legs continued multiple stepping. The coordination of middle leg amputees was maladapted, with front legs and hindlegs performing multiple steps or ipsilateral legs being in simultaneous swing. Thus, afferent information from middle legs might be necessary for a regular hindleg stepping pattern.

Insects carry a pair of actively movable feelers that supply the animal with a range of multimodal information. The antennae of the stick insectCarausius morosusare straight and of nearly the same length as the legs, making them ideal probes for near-range exploration. Indeed, stick insects, like many other insects, use antennal contact information for the adaptive control of locomotion, for example, in climbing. Moreover, the active exploratory movement pattern of the antennae is context-dependent. The first objective of the present study is to reveal the significance of antennal contact information for the efficient initiation of climbing. This is done by means of kinematic analysis of freely walking animals as they undergo a tactually elicited transition from walking to climbing. The main findings are that fast, tactually elicited re-targeting movements may occur during an ongoing swing movement, and that the height of the last antennal contact prior to leg contact largely predicts the height of the first leg contact. The second objective is to understand the context-dependent adaptation of the antennal movement pattern in response to tactile contact. We show that the cycle frequency of both antennal joints increases after obstacle contact. Furthermore, inter-joint coupling switches distinctly upon tactile contact, revealing a simple mechanism for context-dependent adaptation.

SUMMARYThe activity of the middle part of the claw retractor muscle was examined in two species of stick insects (Carausius morosus and Cuniculina impigra). We performed electromyographic recordings while the animals were standing on a smooth or a rough surface of a platform in horizontal, vertical or inverted positions, as well as during rotations of the platform. We recorded tonic and phasic motor units. The tonic units were active all the time without significant differences in spike frequency, regardless of the position of the animals (although there was a tendency for higher discharge frequencies to occur during platform rotations). The phasic units were active almost exclusively during platform movement. In contrast to the tonic units, we detected significant differences in the activities of the phasic units; namely, higher spike frequencies during rotations compared with the stationary phases, especially for rotations into ‘more awkward’ positions. A comparison of the two species revealed no difference in muscle activity, despite differences in the animals' tarsal attachment structures. The same was true when comparing the muscle activity of the two species on both the smooth and the rough surfaces.

The corpora cardiaca of the Vietnamese stick insect, Baculum extradentatum, contain a member of the adipokinetic hormone/red pigment-concentrating hormone/hypertrehalosemic hormone (AKH/RPCH/HrTH) family of peptides whose sequence is identical to that originally described for the Indian stick insect, Carausius morosus. This decapeptide, Carmo-HrTH-II (pELTFTPNWGTa), has both hypertrehalosemic and cardioacceleratory activity in B. extradentatum, and hyperlipaemic activity in locusts. Reversed-phase high performance liquid chromatography (RP-HPLC) of corpora cardiaca extract followed by MALDI-TOF MS/MS also revealed a novel modification of a second peptide in B. extradentatum: the tryptophan residue at position 8 is post-translationally modified to kynurenine.

Detection of force increases and decreases is important in motor control. Experiments were performed to characterize the structure and responses of tibial campaniform sensilla, receptors that encode forces through cuticular strains, in the middle leg of the stick insect (Carausius morosus). The sensilla consist of distinct subgroups. Group 6A sensilla are located 0.3 mm distal to the femoro-tibial joint and have oval shaped cuticular caps. Group 6B receptors are 1 mm distal to the joint and have round caps. All sensilla show directional, phasico-tonic responses to forces applied to the tibia in the plane of joint movement. Group 6B sensilla respond to force increases in the direction of joint extension while Group 6A receptors discharge when those forces decrease. Forces applied in the direction of joint flexion produce the reverse pattern of sensory discharge. All receptors accurately encode the rate of change of force increments and decrements. Contractions of tibial muscles also produce selective, directional sensory discharges. The subgroups differ in their reflex effects: Group 6B receptors excite and Group 6A sensilla inhibit tibial extensor and trochanteral depressor motoneurons. The tibial campaniform sensilla can, therefore, encode force increases or decreases and aid in adapting motor outputs to changes in load.

Imidacloprid, sulfoxaflor and two experimental sulfoximine insecticides caused generally depressive symptoms in stick insects, characterized by stillness and weakness, while also variably inducing postural changes such as persistent ovipositor opening, leg flexion or extension and abdomen bending that could indicate excitation of certain neural circuits. We examined the same compounds on nicotinic acetylcholine receptors in stick insect neurons, which have previously been shown to desensitize in the presence of ACh. Brief U-tube application of 10(-4) M solutions of insecticides for 1 s evoked currents that were much smaller than ACh-evoked currents, and depressed subsequent ACh-evoked currents for several minutes, indicating that the compounds are low-efficacy partial agonists that potently desensitize the receptors. Much lower concentrations of insecticides applied in the bath for longer periods did not activate currents, but inhibited ACh-evoked currents via desensitization of the receptors. Previously described fast- and slowly-desensitizing nACh currents, I(ACh1) and I(ACh2) respectively, were each found to consist of two components with differing sensitivities to the insecticides. Imidacloprid applied in the bath desensitized high-sensitivity components, I(ACh1H) and I(ACh2H) with IC(50)s of 0.18 and 0.13 pM, respectively. It desensitized the low-sensitivity slowly desensitizing component, I(ACh2L), with an IC(50) of 2.6 nM, while a component of the fast-desensitizing current, I(ACh1L), was least sensitive, with an IC(50) of 81 nM I(ACh1L) appeared to be insensitive to the three sulfoximines tested, whereas all three sulfoximines potently desensitized I(ACh1H) and both slowly desensitizing components, with IC(50)s between 2 and 7 nM. We conclude that selective desensitization of certain nAChR subtypes can account for the insecticidal actions of imidacloprid and sulfoximines in stick insects.