Insect exoskeletons react to hypergravity.

A typical feature of biological materials is their ability to adapt to mechanical load. However, it is not known whether the cuticle exoskeleton, one of the most common biological structures, also shares this trait. Here, we show direct experimental evidence that prolonged exposure to hypergravity conditions affects the morphology and biomechanics of an insect exoskeleton. Locusts were raised for several weeks in a custom-designed centrifuge at various levels of hypergravity. Biomechanical measurements and X-ray microtomography show that up to 3 g load Young's modulus of the tibiae increased by about 67%. Higher gravitational loads however decreased the survival rate, body mass and endocuticle thickness. These results directly show that cuticle exoskeletons can react to hypergravity. This ability has so far only been known for bone endoskeletons and plants. Our findings thus add important context to the discussion on general ultimate factors in the evolution of adaptive biological materials and skeletal systems.

3D escape: an alternative paradigm for spatial orientation studies in insects.

Arthropods and in particular insects show a great variety of different exoskeletal sensors. For most arthropods, spatial orientation and gravity perception is not fully understood. In particular, the interaction of the different sensors is still a subject of ongoing research. A disadvantage of most of the experimental methods used to date to study the spatial orientation of arthropods in behavioral experiments is that the body or individual body parts are fixed partly in a non-natural manner. Therefore, often only the movement of individual body segments can be used to evaluate the experiments. We here present a novel experimental method to easily study 3D-escape movements in insects and analyze whole-body reaction. The animals are placed in a transparent container, filled with a lightweight substrate and rotating around two axes. To verify our setup, house crickets (Acheta domesticus) with selectively manipulated gravity-perceiving structures were analyzed. The spatial orientation behavior was quantified by measuring the time individuals took to escape toward the surface and the angular deviation toward the gravitational vector. These experiments confirm earlier results and therefore validated our experimental setup. Our new approach thus allows to investigate several comprehensive questions regarding the spatial orientation of insects and other animals.

Semi-automated differentiation of insect exo- and endocuticle in X-ray microtomography.

One of the most versatile and complex biological composite materials, the insect exoskeleton shows a huge range in biomechanical properties. The cuticle exoskeleton can be differentiated into two main histologically different layers with distinct properties: the outer, more sclerotized exocuticle and inner, softer endocuticle. For most biomechanical research questions, it is of great importance to be able to selectively characterize geometrical features of these layers. However, most conventional preparation methods (cross-sections, histological staining, SEM) require complex and destructive sample preparation, which provides only two-dimensional information. Here, we present a novel, simple staining method using X-ray microtomography to distinguish between exo- and endocuticle in a 3D environment without sample destruction. We illustrate the power of our method using locust (Locusta migratoria) hindleg tibia, a well characterized biomechanical sample. Our method allows an easy and direct measurement of exo- and endocuticle and their respective geometric features. Applying our method will help to understand the biomechanical role of exo- and endocuticle within an insect exoskeleton and will allow us to understand its composition and morphological features in more detail.

Insect wing damage: causes, consequences and compensatory mechanisms.

The evolution of wings has played a key role in the success of insect species, allowing them to diversify to fill many niches. Insect wings are complex multifunctional structures, which not only have to withstand aerodynamic forces but also need to resist excessive stresses caused by accidental collisions. This Commentary provides a summary of the literature on damage-reducing morphological adaptations in wings, covering natural causes of wing collisions, their impact on the structural integrity of wings and associated consequences for both insect flight performance and life expectancy. Data from the literature and our own observations suggest that insects have evolved strategies that (i) reduce the likelihood of wing damage and (ii) allow them to cope with damage when it occurs: damage-related fractures are minimized because wings evolved to be damage tolerant and, in the case of wing damage, insects compensate for the reduced aerodynamic efficiency with dedicated changes in flight kinematics.

Epidermal Cell Surface Structure and Chitin-Protein Co-assembly Determine Fiber Architecture in the Locust Cuticle.

The geometrical similarity of helicoidal fiber arrangement in many biological fibrous extracellular matrices, such as bone, plant cell wall, or arthropod cuticle, to that of cholesteric liquid mesophases has led to the hypothesis that they may form passively through a mesophase precursor rather than by direct cellular control. In search of direct evidence to support or refute this hypothesis, here, we studied the process of cuticle formation in the tibia of the migratory locust, Locusta migratoria, where daily growth layers arise by the deposition of fiber arrangements alternating between unidirectional and helicoidal structures. Using focused ion beam/scanning electron microscopy (FIB/SEM) volume imaging and scanning X-ray scattering, we show that the epidermal cells determine an initial fiber orientation, from which the final architecture emerges by the self-organized co-assembly of chitin and proteins. Fiber orientation in the locust cuticle is therefore determined by both active and passive processes.

SAUV-A Bio-Inspired Soft-Robotic Autonomous Underwater Vehicle.

Autonomous and remotely operated underwater vehicles allow us to reach places which have previously been inaccessible and perform complex repair, exploration and analysis tasks. As their navigation is not infallible, they may cause severe damage to themselves and their often fragile surroundings, such as flooded caves, coral reefs, or even accompanying divers in case of a collision. In this study, we used a shallow neural network, consisting of interlinking PID controllers, and trained by a genetic algorithm, to control a biologically inspired AUV with a soft and compliant exoskeleton. Such a compliant structure is a versatile and passive solution which reduces the accelerations induced by collisions to 56% of the original mean value acting upon the system, thus, notably reducing the stress on its components and resulting reaction forces on its surroundings. The segmented structure of this spherical exoskeleton protects the encased system without limiting the use of cameras, sensors or manipulators.

What goes up must come down: biomechanical impact analysis of falling locusts.

Many insects are able to precisely control their jumping movements. Once in the air, the properties of the actual landing site, however, are almost impossible to predict. Falling insects thus have to cope with the situation at impact. In particular, for insects jumping to escape predators, a controlled landing movement appears to be a major evolutionary advantage. A quick recovery into an upright and stable body posture minimizes the time to prepare for the next escape jump. In this study, we used high-speed recordings to investigate the falling and in particular the impact behavior of Schistocerca gregaria locusts, a common model organism for studies on the biomechanics of jumping. Detailed impact analyses of free-falling locusts show that most insects typically crashed onto the substrate. Although free-falling locusts tended to spread their legs, they mostly fell onto the head and thorax first. The presence of wings did not significantly reduce impact speed; however, it did affect the orientation of the body at impact and significantly reduced the time to recover. Our results also show that alive warm locusts fell significantly faster than inactive or dead locusts. This indicates a possible tradeoff between active control versus reduced speed. Interestingly, alive insects also tended to perform a characteristic bending movement of the body at impact. This biomechanical adaptation might reduce the rebound and shorten the time to recover. The adhesive pads also play an important role in reducing the time to recover by allowing the insect to anchor itself to the substrate.

Morphology, shape variation and movement of skeletal elements in starfish (Asterias rubens).

Starfish (order: Asteroidea) possess a complex endoskeleton composed of thousands of calcareous ossicles. These ossicles are embedded in a body wall mostly consisting of a complex collagen fiber array. The combination of soft and hard tissue provides a challenge for detailed morphological and histological studies. As a consequence, very little is known about the general biomechanics of echinoderm endoskeletons and the possible role of ossicle shape in enabling or limiting skeletal movements. In this study, we used high-resolution X-ray microscopy to investigate individual ossicle shape in unprecedented detail. Our results show the variation of ossicle shape within ossicles of marginal, reticular and carinal type. Based on these results we propose an additional classification to categorize ossicles not only by shape but also by function into 'connecting' and 'node' ossicles. We also used soft tissue staining with phosphotungstic acid successfully and were able to visualize the ossicle ultrastructure at 2-µm resolution. We also identified two new joint types in the aboral skeleton (groove-on-groove joint) and between adambulacral ossicles (ball-and-socket joint). To demonstrate the possibilities of micro-computed tomographic methods in analyzing the biomechanics of echinoderm skeletons we exemplarily quantified changes in ossicle orientation for a bent ray for ambulacral ossicles. This study provides a first step for future biomechanical studies focusing on the interaction of ossicles and soft tissues during ray movements.

Investigating the efficiency of a bio-inspired insect repellent surface structure.

Most insects with smooth or hairy adhesive pads have very little problems in attaching to smooth substrates. A careful selection of surface roughness, however, can effectively limit the contact area of the adhesive organs with the surface. In comparison to conventional toxin-based insect repelling methods, biologically inspired micro- and nanostructured insect repellent surface structures, thus, offer a great potential to effective and environmentally-friendly control insect pests. We here present a simple experimental approach to qualitatively and quantitatively analyse the efficiency of a micro- and nanorough surface structure. Nauphoeta cinerea and Gastrophysa viridula as model organisms for insects with smooth and hairy adhesive pads, respectively, were placed in an enclosed environment. The escape movements of freely running insects over either the repellent or a control surfaces were counted and analyzed in detail. The tested surface structures were able to significantly reduce the escape of cockroaches with smooth adhesive pads by 44.1%. Interestingly, the data indicates that N. cinerea might use mechanical cues from the antenna to discriminate between surfaces before making contact with the adhesive pads. G. viridula with hairy adhesive pads were not significantly affected by the surface structure. By carefully adjusting 'critical' surface topography parameters relevant for insect adhesion, more efficient and selective repellent surfaces might be achieved. Such nanostructure-based insect repellent surfaces could also help to utilize recruitment behavior of certain insect species and might present a novel approach to effectively control insect pests.

Damage, repair and regeneration in insect cuticle: The story so far, and possibilities for the future.

The exoskeleton of an insect can contain countless specializations across an individual, across developmental stages, and across the class Insecta. Hence, the exoskeleton's building material cuticle must perform a vast variety of functions. Cuticle displays a wide range of material properties which are determined by several known factors: the amount and orientation of the chitin fibres, the constituents and degree of cross-linking and hydration of the protein matrix, the relative amounts of exo- and endocuticle, and the shape of the structures themselves. In comparison to other natural materials such as wood and mammal bone, relatively few investigations into the mechanical properties of insect cuticle have been carried out. Of these, very few have focussed on the need for repair and its effectiveness at restoring mechanical stability to the cuticle. Insect body parts are often subject to prolonged repeated cyclic loads when running and flying, as well as more extreme "emergency" behaviours necessary for survival such as jumping, wedging (squeezing through small holes) and righting (when overturned). What effects have these actions on the cuticle itself? How close to the limits of failure does an insect push its body parts? Can an insect recover from minor or major damage to its exoskeleton "bones"? No current research has answered these questions conclusively.

Effect of sample treatment on biomechanical properties of insect cuticle.

Experimental limitations often prevent to perform biomechanical measurements on fresh arthropod cuticle samples. Hence, in many cases short- or long-term storage of samples is required. So far, it is not known whether any of the standard lab-techniques commonly used to fix or store insect cuticle samples in any way affects the biomechanical properties of the respective samples. In this paper we systematically address this question for the first time, with a focus on practical, easily accessible and common lab-methods including storage in water, ethanol, glutaraldehyde, freezing and desiccation. We performed a comprehensive and sensitive non-destructive Dynamic Mechanical Analysis (DMA) on locust hind leg tibiae using a three-point-bending setup. Our results show that from all tested treatments, freezing samples at -20 °C was the best option to maintain the original values for Young's modulus and damping properties of insect cuticle. In addition, our results indicate that the damping properties of locust hind legs might be mechanically optimized in respect to the jumping and kicking direction.

Bridging the gap: wound healing in insects restores mechanical strength by targeted cuticle deposition.

If an insect is injured, can it repair its skeleton in a manner which is mechanically strong and viable? Previous work has described the biological processes that occur during repair of insect cuticle, but until now, there has been no biomechanical assessment of the repaired area. We analysed the biomechanics of the injury repair process in the desert locust (Schistocerca gregaria). We show that after an incision, a healing process occurred which almost doubled the mechanical strength of locust tibial cuticle, restoring it to 66% of the original, intact strength. This repair process occurred by targeted cuticle deposition, stimulated by the presence of the injury. The cut surfaces remained unrepaired, but a patch of endocuticle was deposited, reinforcing the area and thus increasing the effective fracture toughness. The deposition rate of endocuticle inside the tibia increased fourfold compared with uninjured controls, but only on the dorsal side, where the incision was placed. The limb is highly loaded during jumping, so this partial restoration of strength will have a profound effect on the fitness of the insect. A finite-element model provided insights into the mechanics of the repair, predicting that the patch material reaches its ultimate strength before the fracture toughness of the existing cuticle is exceeded.

Physical principles of fluid-mediated insect attachment - Shouldn't insects slip?

Insects use either hairy or smooth adhesive pads to safely adhere to various kinds of surfaces. Although the two types of adhesive pads are morphologically different, they both form contact with the substrate via a thin layer of adhesive fluid. To model adhesion and friction forces generated by insect footpads often a simple "wet adhesion" model is used, in which two flat undeformable substrates are separated by a continuous layer of fluid. This review summarizes the key physical and tribological principles that determine the adhesion and friction in such a model. Interestingly, such a simple wet-adhesion model falls short in explaining several features of insect adhesion. For example, it cannot predict the observed high static friction forces of the insects, which enable them to cling to vertical smooth substrates without sliding. When taking a closer look at the "classic" attachment model, one can see that it is based on several simplifications, such as rigid surfaces or continuous layers of Newtonian fluids. Recent experiments show that these assumptions are not valid in many cases of insect adhesion. Future tribological models for insect adhesion thus need to incorporate deformable adhesive pads, non-Newtonian properties of the adhesive fluid and/or partially "dry" or solid-like contact between the pad and the substrate.

Fatigue of insect cuticle.

Many parts of the insect exoskeleton experience repeated cyclic loading. Although the cuticle of insects and other arthropods is the second most common natural composite material in the world, so far nothing is known about its fatigue properties, despite the fact that fatigue undoubtedly limits the durability of body parts in vivo. For the first time, we here present experimental fatigue data of insect cuticle. Using force-controlled cyclic loading, we determined the number of cycles to failure for hind legs (tibiae) and hind wings of the locust Schistocerca gregaria, as a function of the applied cyclic stress. Our results show that, although both are made from cuticle, these two body parts behave very differently. Wing samples showed a large fatigue range, failing after 100,000 cycles when we applied 46% of the stress needed for instantaneous failure [the ultimate tensile strength (UTS)]. Legs, in contrast, were able to sustain a stress of 76% of the UTS for the same number of cycles to failure. This can be explained by the difference in the composition and structure of the material, two factors that, amongst others, also affect the well-known behaviour of engineering composites. Final failure of the tibiae occurred via one of two different failure modes--propagation in tension or buckling in compression--indicating that the tibia is 'optimized' by evolution to resist both failure modes equally. These results are further discussed in relation to the evolution and normal use of these two body parts.

Shape optimization in exoskeletons and endoskeletons: a biomechanics analysis.

This paper addresses the question of strength and mechanical failure in exoskeletons and endoskeletons. We developed a new, more sophisticated model to predict failure in bones and other limb segments, modelled as hollow tubes of radius r and thickness t. Five failure modes were considered: transverse fracture; buckling (of three different kinds) and longitudinal splitting. We also considered interactions between failure modes. We tested the hypothesis that evolutionary adaptation tends towards an optimum value of r/t, this being the value which gives the highest strength (i.e. load-carrying capacity) for a given weight. We analysed two examples of arthropod exoskeletons: the crab merus and the locust tibia, using data from the literature and estimating the stresses during typical activities. In both cases, the optimum r/t value for bending was found to be different from that for axial compression. We found that the crab merus experiences similar levels of bending and compression in vivo and that its r/t value represents an ideal compromise to resist these two types of loading. The locust tibia, however, is loaded almost exclusively in bending and was found to be optimized for this loading mode. Vertebrate long bones were found to be far from optimal, having much lower r/t values than predicted, and in this respect our conclusions differ from those of previous workers. We conclude that our theoretical model, though it has some limitations, is useful for investigating evolutionary development of skeletal form in exoskeletons and endoskeletons.

Veins improve fracture toughness of insect wings.

During the lifetime of a flying insect, its wings are subjected to mechanical forces and deformations for millions of cycles. Defects in the micrometre thin membranes or veins may reduce the insect's flight performance. How do insects prevent crack related material failure in their wings and what role does the characteristic vein pattern play? Fracture toughness is a parameter, which characterises a material's resistance to crack propagation. Our results show that, compared to other body parts, the hind wing membrane of the migratory locust S. gregaria itself is not exceptionally tough (1.04±0.25 MPa√m). However, the cross veins increase the wing's toughness by 50% by acting as barriers to crack propagation. Using fracture mechanics, we show that the morphological spacing of most wing veins matches the critical crack length of the material (1132 µm). This finding directly demonstrates how the biomechanical properties and the morphology of locust wings are functionally correlated in locusts, providing a mechanically 'optimal' solution with high toughness and low weight. The vein pattern found in insect wings thus might inspire the design of more durable and lightweight artificial 'venous' wings for micro-air-vehicles. Using the vein spacing as indicator, our approach might also provide a basis to estimate the wing properties of endangered or extinct insect species.

Fracture toughness of locust cuticle.

Insect cuticle is one of the most common biological materials, yet very little is known about its mechanical properties. Many parts of the insect exoskeleton, such as the jumping legs of locusts, have to withstand high and repeated loading without failure. This paper presents the first measurements of fracture toughness for insect cuticle using a standard engineering approach. Our results show that the fracture toughness of cuticle in locust hind legs is 4.12 MPa m(1/2) and decreases with desiccation of the cuticle. Stiffness and strength of the tibia cuticle were measured using buckling and cantilever bending and increased with desiccation. A combination of the cuticle's high toughness with a relatively low stiffness of 3.05 GPa results in a work of fracture of 5.56 kJ m(-2), which is amongst the highest of any biological material, giving the insect leg an exceptional ability to tolerate defects such as cracks and damage. Interestingly, insect cuticle achieves these unique properties without using reinforcement by a mineral phase, which is often found in other biological composite materials. These findings thus might inspire the development of new biomimetic composite materials.

In vivo dynamics of the internal fibrous structure in smooth adhesive pads of insects.

Many insects with smooth adhesive pads can rapidly enlarge their contact area by centripetal pulls on the legs, allowing them to cope with sudden mechanical perturbations such as gusts of wind or raindrops. The short time scale of this reaction excludes any neuromuscular control; it is thus more likely to be caused by mechanical properties of the pad's specialized cuticle. This soft cuticle contains numerous branched fibrils oriented almost perpendicularly to the surface. Assuming a fixed volume of the water-filled cuticle, we hypothesized that pulls could decrease the fibril angle, thereby helping the contact area to expand laterally and longitudinally. Three-dimensional fluorescence microscopy on the cuticle of smooth stick insect pads confirmed that pulls significantly reduced the fibril angle. However, the fibril angle variation appeared insufficient to explain the observed increase in contact area. Direct strain measurements in the contact zone demonstrated that pulls not only expand the cuticle laterally, but also add new contact area at the pad's outer edge.

Biomechanics of the stick insect antenna: damping properties and structural correlates of the cuticle.

The antenna of the Indian stick insect Carausius morosus is a highly specialized near-range sensory probe used to actively sample tactile cues about location, distance or shape of external objects in real time. The length of the antenna's flagellum is 100 times the diameter at the base, making it a very delicate and slender structure. Like the rest of the insect body, it is covered by a protective exoskeletal cuticle, making it stiff enough to allow controlled, active, exploratory movements and hard enough to resist damage and wear. At the same time, it is highly flexible in response to contact forces, and returns rapidly to its straight posture without oscillations upon release of contact force. Which mechanical adaptations allow stick insects to unfold the remarkable combination of maintaining a sufficiently invariant shape between contacts and being sufficiently compliant during contact? What role does the cuticle play? Our results show that, based on morphological differences, the flagellum can be divided into three zones, consisting of a tapered cone of stiff exocuticle lined by an inner wedge of compliant endocuticle. This inner wedge is thick at the antenna's base and thin at its distal half. The decay time constant after deflection, a measure that indicates strength of damping, is much longer at the base (τ>25 ms) than in the distal half (τ<18 ms) of the flagellum. Upon experimental desiccation, reducing mass and compliance of the endocuticle, the flagellum becomes under-damped. Analysing the frequency components indicates that the flagellum can be abstracted with the model of a double pendulum with springs and dampers in both joints. We conclude that in the stick-insect antenna the cuticle properties described are structural correlates of damping, allowing for a straight posture in the instant of a new contact event, combined with a maximum of flexibility.

Arachnids secrete a fluid over their adhesive pads.

BACKGROUND: Many arachnids possess adhesive pads on their feet that help them climb smooth surfaces and capture prey. Spider and gecko adhesives have converged on a branched, hairy structure, which theoretically allows them to adhere solely by dry (solid-solid) intermolecular interactions. Indeed, the consensus in the literature is that spiders and their smooth-padded relatives, the solifugids, adhere without the aid of a secretion. METHODOLOGY AND PRINCIPAL FINDINGS: We investigated the adhesive contact zone of living spiders, solifugids and mites using interference reflection microscopy, which allows the detection of thin liquid films. Like insects, all the arachnids we studied left behind hydrophobic fluid footprints on glass (mean refractive index: 1.48-1.50; contact angle: 3.7-11.2°). Fluid was not always secreted continuously, suggesting that pads can function in both wet and dry modes. We measured the attachment forces of single adhesive setae from tarantulas (Grammostola rosea) by attaching them to a bending beam with a known spring constant and filming the resulting deflection. Individual spider setae showed a lower static friction at rest (26%±2.8 SE of the peak friction) than single gecko setae (Thecadactylus rapicauda; 96%±1.7 SE). This may be explained by the fact that spider setae continued to release fluid after isolation from the animal, lubricating the contact zone. SIGNIFICANCE: This finding implies that tarsal secretions occur within all major groups of terrestrial arthropods with adhesive pads. The presence of liquid in an adhesive contact zone has important consequences for attachment performance, improving adhesion to rough surfaces and introducing rate-dependent effects. Our results leave geckos and anoles as the only known representatives of truly dry adhesive pads in nature. Engineers seeking biological inspiration for synthetic adhesives should consider whether model species with fluid secretions are appropriate to their design goals.