Sequencing and biological effects of an adipokinetic/hypertrehalosemic peptide in the stick insect, Baculum extradentatum

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.     

The regulation of cardiac activity by nitric oxide (NO) in the Vietnamese stick insect, Baculum extradentatum

This study examines the role of the unconventional gaseous signaling molecule nitric oxide (NO) on the regulation of heart rate in the Vietnamese stick insect, Baculum extradentatum. Using nicotinamide dinucleotide hydrogen phosphate (NADPH)-diaphorase histochemistry, as well as immunohistochemistry and Western blotting with an antibody against NO synthetase (NOS), we identified the presence of NOS in hemocytes present throughout the lumen of the dorsal vessel. We propose that NO is delivered to heart muscle tissue via hemocytes circulating within the hemolymph. In the present study, stimulation of NO levels by the application of the NO donor MAHMA-NONOate and l-arginine led to a dose-dependent decrease in heart rate. Treatment of tissues with the NOS inhibitor, L-NAME, in equimolar concentrations with l-arginine, led to a recovery of heart rate, without modifying heart rate on its own. Finally guanosine 3',5'-cyclic monophosphate (cGMP) analog, 8-bromo-cGMP, elicited similar inhibitory effects on stick insect heart rate as did the guanylate cyclase activator, YC-1, and the phosphodiesterase inhibitor, dipyridamole, indicating that cGMP is most likely the second messenger in the stick insect NO signaling pathway. Contrary to the cardioexcitatory effect of NO on other insect hearts, we have found that NO inhibits stick insect heart rate independently from any nervous system input, in a similar inhibitory fashion as that of vertebrate hearts.     

The presence and distribution of crustacean cardioactive peptide in the central and peripheral nervous system of the stick insect, Baculum extradentatum

Crustacean cardioactive peptide (CCAP)-like immunoreactivity was localized and quantified in the central and peripheral nervous system of the Vietnamese stick insect, Baculum extradentatum, using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). The brain, frontal ganglion, suboesophageal ganglion and ventral nerve cord displayed neurons and processes with CCAP-like immunoreactivity. The brain, in comparison to the other parts of the central nervous system, contained the greatest amount of CCAP (167 +/- 18 fmol), and showed CCAP-like staining in neurons, neuropil regions and the central complex. There were also CCAP-like varicosities and processes associated with the corpus cardiacum. The alimentary canal of B. extradentatum contained CCAP with the largest amount localized in the midgut (1110 +/- 274 fmol CCAP equivalents). The midgut contained numerous endocrine-like cells which stained positively for CCAP, whereas the foregut and hindgut revealed an extensive network of CCAP-like immunoreactive axons and varicosities. Based on physiological assays, the hindgut of the stick insect was found to be sensitive to CCAP, showing dose-dependent increases in contractions with threshold at 10(-10) M CCAP and maximal response at 5 x 10(-7) M CCAP. There were negligible quantities of CCAP in the oviducts and no CCAP-like immunoreactivity was associated with the oviducts. CCAP had no effect on spontaneous contractions of the oviducts. The presence of CCAP in the central nervous system, the stomatogastric nervous system, the corpus cardiacum and the alimentary canal, suggest broad ranging roles for CCAP in B. extradentatum.     

Effect of magnetic fields on antioxidative defense and fitness-related traits of Baculum extradentatum (insecta, phasmatodea)

This study aimed to determine the effect of magnetic fields on the antioxidative defense and fitness-related traits of Baculum extradentatum. Following exposure to magnetic fields, antioxidative defense (superoxide dismutase (SOD), catalase (CAT) activities, and total glutathione (GSH) content) and fitness-related traits (egg mortality, development dynamics, and mass of nymphs) were monitored in nymphs. The experimental groups were: control (kept out of influence of the magnets), a group exposed to a constant magnetic field (CMF) of 50 mT, and a group exposed to an alternating magnetic field (AMF) of 50 Hz, 6 mT. We found increased SOD and CAT activities in animals exposed to constant and AMFs, whereas GSH activity was not influenced by experimental magnetic fields. No differences were found in egg mortality between control and experimental groups. Significant differences in the time of development between the control and the CMF group were observed, as well as between the CMF and the AMF group. No differences were found in the mass of the nymphs between the three experimental groups. In conclusion, CMF and AMF have the possibility to modulate the antioxidative defense and some of the fitness-related traits in B. extradentatum.     

Sensory control of leg movement in the stick insect Carausius morosus

Hind legs with crossed receptor-apodemes of the femoral chordotonal organ when making a step during walking often do not release the ground after reaching the extreme posterior position. After putting a clamp on the trochanter (stimulation of the campaniform sensilla) the leg is no longer protracted during walking. However, during searching-movements the same leg is moved very far forwards. The anatomical situation of the campaniform sensilla on the trochanter and the sensory innervation of the trochanter is described. After removal of the hair-rows and continuously stimulating the hair-plate at the thorax-coxa-joint the extreme anterior and posterior positions of the leg in walking are displaced in the posterior direction. Front and middle legs operated in this way sometimes do not release the ground at the end of retraction. In searching-movements the same leg is moved in a normal way. If only one side of a decerebrated animal goes over a step, then on the other side a compensatory effect is observed. The main source of this compensatory information appears to be the BF1-hair-plates. If the animal has to drag a weight the extreme anterior and posterior positions of the middle and hind legs are displaced in the anterior direction. Crossing the receptor-apodeme of the femoral chordotonal organ, when it causes the leg to remain in the protraction phase, displaces the extreme posterior position of the ipsilateral leg in front of the operated one in the posterior direction. Influences of different sources on the extreme posterior position can superimpose. A model is presented which combines both a central programme and peripheral sensory influence. The word programme used here means that it does not only determine the motor output but also determines the reactions to particular afferences. The fact that the reaction to a stimulus depends on the internal state of the CNS is also represented by the model.Supported by Deutsche Forschungsgemeinschaft     

Intersegmental transfer of sensory signals in the stick insect leg muscle control system

Intersegmental coordination during locomotion in legged animals arises from mechanical couplings and the exchange of neuronal information between legs. Here, the information flow from a single leg sense organ of the stick insect Cuniculina impigra onto motoneurons and interneurons of other legs was investigated. The femoral chordotonal organ (fCO) of the right middle leg, which measures posture and movement of the femur-tibia joint, was stimulated, and the responses of the tibial motoneuron pools of the other legs were recorded. In resting animals, fCO signals did not affect motoneuronal activity in neighboring legs. When the locomotor system was activated and antagonistic motoneurons were bursting in alternation, fCO stimuli facilitated transitions from flexor to extensor activity and vice versa in the contralateral leg. Following pharmacological treatment with picrotoxin, a blocker of GABA-ergic inhibition, the tibial motoneurons of all legs showed specific responses to signals from the middle leg fCO. For the contralateral middle leg we show that fCO signals encoding velocity and position of the tibia were processed by those identified local premotor nonspiking interneurons known to contribute to posture and movement control during standing and voluntary leg movements. Interneurons received both excitatory and inhibitory inputs, so that the response of some interneurons supported the motoneuronal output, while others opposed it. Our results demonstrate that sensory information from the fCO specifically affects the motoneuronal activity of other legs and that the layer of premotor nonspiking interneurons is a site of interaction between local proprioceptive sensory signals and proprioceptive signals from other legs.     

The dissection of a stick insect

The application of the carbon-14 ‘light and dark bottle’ technique for school or college use is described. Details are given for a simplified method suitable for a three-hour laboratory session. The method requires either a Geiger or liquid scintillation counter. Examples of class results are provided along with suggestions for further applications in an ecological context.     

A dynamic model of thoracic differentiation for the control of turning in the stick insect

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 work was supported by CONACYT México and the European Commission under project FP6-2003-IST2-004690 SPARK.     

Coactivation of leg reflexes in the stick insect

Each leg of a standing stick insect acts as a height controller. The leg contains several joints. Most of these joints are known to be controlled by feedback loops which are the basis of resistance reflexes (review Bässler 1983). This leads to the question of whether the resistance reflex of the whole leg can be understood as a simple, vectorial sum of the individual reflexes provided by the different joints, or whether additional properties emerge by simultaneous stimulation of several joints. Force measurements were performed while passively moving the middle leg tarsus of a fixed stick insect (Carausius morosus) stepwise to different positions. From the dynamic and static forces the torques developed by each joint were calculated. They were compared with the torques developed when only a single joint was moved by the same amount. The comparison shows that for a large range of positions there are no differences between both situations. Differences occur in two cases. First, the muscle system controlling the coxa-trochanter joint seems to be more strongly excited when the entire leg is moved than when only the one joint is moved. This change increases the linearity of the whole system for small deviations from the zero position. Second, the torque developed by the extensor tibiae system for negative steps (corresponding to increased body height), and the levator of coxa and trochanter for positive steps, decreases rather than increases when the whole leg is moved to extreme positions. This contributes to a decrease in the slope of the force-height characteristic and thus to a more non-linear behaviour of the whole system for the extreme positions. It is well known that the amplification factors of resistance reflexes in the leg show a large variation (Bässler 1972a; Kittmann 1991). Our results indicate that any change of the amplification factor influences the reflexes in all leg joints in the same way.     

Carbohydrate metabolism in the stick insect, Carausius morosus

Age-related changes in some parameters related to carbohydrate metabolism in the stick insect, Carausius morosus, were investigated during the 6th instar and up to day 37 of adult life. Total haemolymph carbohydrate concentration and the fat body glycogen content are low and may be related to the low activity of this insect. Trehalose constitutes about 75–80% of the total blood carbohydrate pool. During the moult, total blood carbohydrate, fat body glycogen and haemolymph volume, decrease while glycogen phosphorylase activity of the fat body is slightly activated. The effects are brought about mainly by reduced feeding activity, but may also be influenced by the shedding and replacement of the cuticle. During starvation, blood homeostasis is maintained at the expense of fat body glycogen via an activation of phosphorylase. During reproduction, although no dramatic changes in fat body glycogen levels occur, blood carbohydrates are maintained and fat body phosphorylase is slightly activated. The possibility is discussed that during moulting and reproduction, blood sugar homeostasis is maintained by a hormonal mechanism controlling glycogen phosphorylase. No circadian rhythm in any parameter investigated is observed.     

Regulation of the body-substrate-distance in the stick insect: Step responses and modelling the control system

The feed back mechanism subserving the regulation of the body-substrate-distance in the stick insect Carausius morosus has been investigated by means of step-like stimuli. Based on the results obtained in open-loop experiments a model is developed which describes the results obtained under closed loop conditions. When the experimental animal is pushed or pulled in dorso-ventral direction an initial fast and a subsequent very slow change of the body height z over the substrate are observed. The late slow response is a nearly linear function of time and can last for more than one hour if the animal is pulled with moderate forces. It withstands less effectively if pushed. During the slow phase of the response sudden changes of z and of the slope of the z(t)-curves occur, presumably due to corresponding changes of the amplification within the feed back loop.     

Development of the compound eyes of the water stick insect, Ranatra linearis

ABSTRACT. Relationships between estimation of predator-prey distance prior to a capture attempt and some features of the compound eye are investigated at all stages of post-embryonic development. Interommatidial angles increase gradually from the anterior and the dorsal regions to the posterior and ventral regions. Facet diameters vary only slightly over the eye surface but increase with age. New ommatidia appear around the borders of eye after each moult. The older ommatidia are pushed away from the border. From one instar to another ommatidia change their direction of view from between 10 to 30 relative to the body axes. This change in direction far exceeds the calculated changes in direction that would be optimal if ommatidia were to continue viewing the same relative directions in space. This suggests a high degree of plasticity of the underlying neuronal networks.     

A model of leg coordination in the stick insect,Carausim morosus

The kinematic model presented in a separate report is used here to investigate several questions concerning the nature of the coordinating mechanisms. First, one or more mechanisms are inactivated in order to compare the relative efficiencies of the different coordinating mechanisms in maintaining proper coordination. Second, the most efficient mechanism, the position-dependent influence, is varied in order to illustrate the consequences for coordination. Third, the strength of the contralateral coupling is varied in order to make predictions about how contralateral legs establish alternation when started from symmetric positions. The consequences of adding reciprocal contralateral inhibition during swing is tested in the same context.     

A model of leg coordination in the stick insect,Carausius morosus

A model of interleg coordination presented in a separate report is evaluated here by perturbing the step pattern in three ways. First, when the initial leg configuration is varied, the simulated leg movements assume a stable coordination from natural starting configurations in a natural way (Fig. 1a). They also rapidly re-establish the normal coordination when started from unnatural configurations (Fig. 1b-d). An explicit hierarchy of natural frequencies for the legs of the three thoracic segments is not required. Second, when the coordination is perturbed by assigning one or more legs a retraction velocity different from the rest, gliding coordination or various integer step ratios can be produced (Figs. 2–4). Third, when the swing of one leg is obstructed, characteristic changes in the stepping of other legs occur (Fig. 5). Overall differences between the step patterns of the model and those of the stick insect are related to the form of the coordinating mechanisms. Errors made by the model, such as overlapping swings by adjacent legs or discrepancies in step timing and step end-points, point out the limitations of a model restricted to kinematic parameters.     

A model of leg coordination in the stick insect,Carausius morosus

Mechanisms dependent upon leg position coordinate the alternate stepping of adjacent ipsilateral and contralateral legs in the stick insect. In this insect, swing duration and step amplitude are independent of walking speed. A simple geometrical model of the leg controller is used here to test different mechanisms for compatibility with these two invariant features. Leg position is the state variable of a relaxation oscillator and position thresholds determine the transitions between swing and stance. The coordination mechanisms alter these thresholds. The position-dependent mechanisms considered differ either in the form or the speed-dependence of the function relating the shift in the posterior threshold of the receiving leg to the position of the sending leg. The results identify parameter combinations leading to alternate stepping with symmetric or asymmetric phase distributions, to shifts in the posterior extreme position as a function of speed, to double stepping or to in-phase stepping. An optimal position-dependent excitatory mechanism is described. Finally the consequences of adding either inhibitory influences or time-dependent excitatory influences are analyzed.