Cross-Sectional 4D-Printing: Upscaling Self-Shaping Structures with Differentiated Material Properties Inspired by the Large-Flowered Butterwort (Pinguicula grandiflora).

Extrusion-based 4D-printing, which is an emerging field within additive manufacturing, has enabled the technical transfer of bioinspired self-shaping mechanisms by emulating the functional morphology of motile plant structures (e.g., leaves, petals, capsules). However, restricted by the layer-by-layer extrusion process, much of the resulting works are simplified abstractions of the pinecone scale's bilayer structure. This paper presents a new method of 4D-printing by rotating the printed axis of the bilayers, which enables the design and fabrication of self-shaping monomaterial systems in cross sections. This research introduces a computational workflow for programming, simulating, and 4D-printing differentiated cross sections with multilayered mechanical properties. Taking inspiration from the large-flowered butterwort (Pinguicula grandiflora), which shows the formation of depressions on its trap leaves upon contact with prey, we investigate the depression formation of bioinspired 4D-printed test structures by varying each depth layer. Cross-sectional 4D-printing expands the design space of bioinspired bilayer mechanisms beyond the XY plane, allows more control in tuning their self-shaping properties, and paves the way toward large-scale 4D-printed structures with high-resolution programmability.

Chemical Gradients in Polymer-Modified Paper Sheets-Towards Single-Layer Biomimetic Soft Robots.

Biomimetic actuators are typically constructed as functional bi- or multilayers, where actuating and resistance layers together dictate bending responses upon triggering by environmental stimuli. Inspired by motile plant structures, like the stems of the false rose of Jericho (Selaginella lepidophylla), we introduce polymer-modified paper sheets that can act as soft robotic single-layer actuators capable of hygro-responsive bending reactions. A tailored gradient modification of the paper sheet along its thickness entails increased dry and wet tensile strength and allows at the same time for hygro-responsiveness. For the fabrication of such single-layer paper devices, the adsorption behavior of a cross-linkable polymer to cellulose fiber networks was first evaluated. By using different concentrations and drying procedures fine-tuned polymer gradients throughout the thickness can be achieved. Due to the covalent cross-linking of polymer with fibers, these paper samples possess significantly increased dry and wet tensile strength properties. We furthermore investigated these gradient papers with respect to a mechanical deflection during humidity cycling. The highest humidity sensitivity is achieved using eucalyptus paper with a grammage of 150 g m-2 modified with the polymer dissolved in IPA (~13 wt%) possessing a polymer gradient. Our study presents a straightforward approach for the design of novel hygroscopic, paper-based single-layer actuators, which have a high potential for diverse soft robotic and sensor applications.

Digital image correlation techniques for motion analysis and biomechanical characterization of plants.

Temporally and spatially complex 3D deformation processes appear in plants in a variety of ways and are difficult to quantify in detail by classical cinematographic methods. Furthermore, many biomechanical test methods, e.g. regarding compression or tension, result in quasi-2D deformations of the tested structure, which are very time-consuming to analyze manually regarding strain fields. In materials testing, the contact-free optical 2D- or 3D-digital image correlation method (2D/3D-DIC) is common practice for similar tasks, but is still rather seldom used in the fundamental biological sciences. The present review aims to highlight the possibilities of 2D/3D-DIC for the plant sciences. The equipment, software, and preparative prerequisites are introduced in detail and advantages and disadvantages are discussed. In addition to the analysis of wood and trees, where DIC has been used since the 1990s, this is demonstrated by numerous recent approaches in the contexts of parasite-host attachment, cactus joint biomechanics, fruit peel impact resistance, and slow as well as fast movement phenomena in cones and traps of carnivorous plants. Despite some technical and preparative efforts, DIC is a very powerful tool for full-field 2D/3D displacement and strain analyses of plant structures, which is suitable for numerous in-depth research questions in the fields of plant biomechanics and morphogenesis.

The cracking of Scots pine (Pinus sylvestris) cones.

Pine cones show functionally highly resilient, hygroscopically actuated opening and closing movements, which are repeatable and function even in millions of years old, coalified cones. Although the functional morphology and biomechanics behind the individual seed scale motions are well understood, the initial opening of the cone, which is often accompanied by an audible cracking noise, is not. We therefore investigated the initial opening events of mature fresh cones of Scots pine (Pinus sylvestris) and their subsequent motion patterns. Using high-speed and time lapse videography, 3D digital image correlation techniques, force measurements, thermographic and chemical-rheological resin analyses, we are able to draw a holistic picture of the initial opening process involving the rupture of resin seals and very fast seed scale motion in the millisecond regime. The rapid cone opening was not accompanied by immediate seed release in our experiments and, therefore, cannot be assigned to ballistochory. As the involved passive hydraulic-elastic processes in cracking are very fine-tuned, we hypothesize that they are under tight mechanical-structural control to ensure an ecologically optimized seed release upon environmental conditions suitable for wind dispersal. In this context, we propose an interplay of humidity and temperature to be the external "drivers" for the initial cone opening, in which resin works as a crucial chemical-mechanical latch system.

Chemonastic Stalked Glands in the Carnivorous Rainbow Plant Byblis gigantea LINDL. (Byblidaceae, Lamiales).

Carnivorous rainbow plants (Byblis, Byblidaceae, Lamiales) possess sticky flypaper traps for the capture, retention, and digestion of prey (mainly small insects). The trapping system is based on a multitude of millimeter-sized glandular trichomes (also termed stalked glands), which produce adhesive glue drops. For over a century, the trapping system of Byblis was considered passive, meaning that no plant movement is involved. Recently, a remarkable discovery was made: the stalked glands of Byblis are indeed capable of reacting to chemical (protein) stimuli with slow movement responses. This prompted us to investigate this phenomenon further with a series of experiments on the stimulation, kinematics, actuation, and functional morphology of the stalked glands of cultivated Byblis gigantea plants. Measured stalked gland lengths and densities on the trap leaves are similar to the data from the literature. Motion reactions could only be triggered with chemical stimuli, corroborating the prior study on the stalked gland sensitivity. Reaction time (i.e., time from stimulation until the onset of motion) and movement duration are temperature-dependent, which hints towards a tight physiological control of the involved processes. The stalked gland movement, which consist of a sequence of twisting and kinking motions, is rendered possible by the components of the stalk cell wall and is furthermore anatomically and mechanically predetermined by the orientation of cellulose microfibrils in the cell wall. Successive water displacement processes from the stalk cell into the basal cells actuate the movement. The same kinematics could be observed in stalked glands drying in air or submersed in a saturated salt solution. Stimulated and dried stalked glands as well as those from the hypertonic medium were capable of regaining their initial shape by rehydration in water. However, no glue production could be observed afterwards. The long-time overlooked chemonastic movements of stalked glands may help Byblis to retain and digest its prey; however, further research is needed to shed light on the ecological characteristics of the rainbow plant's trapping system.

Shapeshifting in the Venus flytrap (Dionaea muscipula): Morphological and biomechanical adaptations and the potential costs of a failed hunting cycle.

The evolutionary roots of carnivory in the Venus flytrap (Dionaea muscipula) stem from a defense response to plant injury caused by, e.g., herbivores. Dionaea muscipula aka. Darwin's most wonderful plant underwent extensive modification of leaves into snap-traps specialized for prey capture. Even the tiny seedlings of the Venus flytrap already produce fully functional, millimeter-sized traps. The trap size increases as the plant matures, enabling capture of larger prey. The movement of snap-traps is very fast (~100-300 ms) and is actuated by a combination of changes in the hydrostatic pressure of the leaf tissue with the release of prestress (embedded energy), triggering a snap-through of the trap lobes. This instability phenomenon is facilitated by the double curvature of the trap lobes. In contrast, trap reopening is a slower process dependent on trap size and morphology, heavily reliant on turgor and/or cell growth. Once a prey item is caught, the trap reconfigures its shape, seals itself off and forms a digestive cavity allowing the plant to release an enzymatic cocktail to draw nutrition from its captive. Interestingly, a failed attempt to capture prey can come at a heavy cost: the trap can break during reopening, thus losing its functionality. In this mini-review, we provide a detailed account of morphological adaptations and biomechanical processes involved in the trap movement during D. muscipula hunting cycle, and discuss possible reasons for and consequences of trap breakage. We also provide a brief introduction to the biological aspects underlying plant motion and their evolutionary background.

Smooth or with a Snap! Biomechanics of Trap Reopening in the Venus Flytrap (Dionaea muscipula).

Fast snapping in the carnivorous Venus flytrap (Dionaea muscipula) involves trap lobe bending and abrupt curvature inversion (snap-buckling), but how do these traps reopen? Here, the trap reopening mechanics in two different D. muscipula clones, producing normal-sized (N traps, max. ≈3 cm in length) and large traps (L traps, max. ≈4.5 cm in length) are investigated. Time-lapse experiments reveal that both N and L traps can reopen by smooth and continuous outward lobe bending, but only L traps can undergo smooth bending followed by a much faster snap-through of the lobes. Additionally, L traps can reopen asynchronously, with one of the lobes moving before the other. This study challenges the current consensus on trap reopening, which describes it as a slow, smooth process driven by hydraulics and cell growth and/or expansion. Based on the results gained via three-dimensional digital image correlation (3D-DIC), morphological and mechanical investigations, the differences in trap reopening are proposed to stem from a combination of size and slenderness of individual traps. This study elucidates trap reopening processes in the (in)famous Dionaea snap traps - unique shape-shifting structures of great interest for plant biomechanics, functional morphology, and applications in biomimetics, i.e., soft robotics.

Facing the Green Threat: A Water Flea's Defenses against a Carnivorous Plant.

Every ecosystem shows multiple levels of species interactions, which are often difficult to isolate and to classify regarding their specific nature. For most of the observed interactions, it comes down to either competition or consumption. The modes of consumption are various and defined by the nature of the consumed organism, e.g., carnivory, herbivory, as well as the extent of the consumption, e.g., grazing, parasitism. While the majority of consumers are animals, carnivorous plants can also pose a threat to arthropods. Water fleas of the family Daphniidae are keystone species in many lentic ecosystems. As most abundant filter feeders, they link the primary production to higher trophic levels. As a response to the high predatory pressures, water fleas have evolved various inducible defenses against animal predators. Here we show the first example, to our knowledge, in Ceriodaphnia dubia of such inducible defenses of an animal against a coexisting plant predator, i.e., the carnivorous bladderwort (Utricularia x neglecta Lehm, Lentibulariaceae). When the bladderwort is present, C. dubia shows changes in morphology, life history and behavior. While the morphological and behavioral adaptations improve C. dubia's survival rate in the presence of this predator, the life-history parameters likely reflect trade-offs for the defense.

The Structural and Mechanical Basis for Passive-Hydraulic Pine Cone Actuation.

The opening and closing of pine cones is based on the hygroscopic behavior of the individual seed scales around the cone axis, which bend passively in response to changes in environmental humidity. Although prior studies suggest a bilayer architecture consisting of lower actuating (swellable) sclereid and upper restrictive (non- or lesser swellable) sclerenchymatous fiber tissue layers to be the structural basis of this behavior, the exact mechanism of how humidity changes are translated into global movement are still unclear. Here, the mechanical and hydraulic properties of each structural component of the scale are investigated to get a holistic picture of their functional interplay. Measurements of the wetting behavior, water uptake, and mechanical measurements are used to analyze the influence of hydration on the different tissues of the cone scales. Furthermore, their dimensional changes during actuation are measured by comparative micro-computed tomography (µ-CT) investigations of dry and wet scales, which are corroborated and extended by 3D-digital image correlation-based displacement and strain analyses, biomechanical testing of actuation force, and finite element simulations. Altogether, a model allowing a detailed mechanistic understanding of pine cone actuation is developed, which is a prime concept generator for the development of biomimetic hygromorphic systems.

The effects of substrate porosity, mechanical substrate properties and loading conditions on the attachment performance of the Mediterranean medicinal leech (Hirudo verbana).

The ectoparasitic lifestyle of the Mediterranean medicinal leech (Hirudo verbana) requires reliable functioning of its attachment organs (i.e. anterior and posterior suction discs) on multiple habitat- and host-specific surfaces under both normal and shear stresses. In addition to some intrinsic properties of the attachment devices, however, only a few extrinsic factors (e.g. substrate roughness and porosity) have been considered in previous studies on leech suckers. Using centrifugal force experiments, we analysed the attachment performance of H. verbana under different types of loading on artificial substrates differing in porosity and their mechanical properties. Whereas the substrate porosity significantly influenced leech attachment under normal and shear loading, the different mechanical properties did not noticeably affect attachment within the considered parameter limits. Furthermore, suction was confirmed to be the primary attachment mechanism independent of the prevailing loading condition. The question of whether the suction cups of H. verbana are adapted to a specific loading condition could not be answered. In any case, our results again highlight the high functional resilience of leech suckers guaranteeing a successful ectoparasitic lifestyle.

Structural gradients and anisotropic hydraulic conductivity in the enigmatic eel traps of carnivorous corkscrew plants (Genlisea spp.).

PREMISE: Among the sophisticated trap types in carnivorous plants, the underground eel traps of corkskrew plants (Genlisea spp., Lentibulariaceae) are probably the least understood in terms of their functional principle. Here, we provide a detailed analysis of structural and hydraulic features of G. hispidula traps, contributing to the ongoing debate on whether these traps can actively generate water streams to promote prey capture. METHODS: Anatomical and hydraulic traits of detached traps, including inner trap diameters, chamber line element, hair length, glandular pattern, and hydraulic conductivity, were investigated quantitatively using light and electron microscopy, x-ray microtomography, and hydraulic measurements. RESULTS: Hydraulic resistivity in the neck of the trap, from the trap mouth toward the vesicle (digestive chamber) was 10 times lower than in the opposite direction. The comparison of measured and theoretical flow rates suggests that the retrorse hairs inside trap necks also provide considerable resistance against movement of matter toward the vesicle. Hairs showed a gradient in length along the neck, with the shortest hairs near the vesicle. Co-occurrence of quadrifid and bifid glands was limited to a small part of the neck, with quadrifids near the vesicle and bifids toward the trap mouth. CONCLUSIONS: The combination of structural gradients with hydraulic anisotropy suggests the trap is a highly fine-tuned system based on likely trade-offs between efficient prey movement in the trap interior toward the vesicle, prey retention, and spatial digestion capacities and is not counter to the generation of water streams.

Bio-Inspired Motion Mechanisms: Computational Design and Material Programming of Self-Adjusting 4D-Printed Wearable Systems.

This paper presents a material programming approach for designing 4D-printed self-shaping material systems based on biological role models. Plants have inspired numerous adaptive systems that move without using any operating energy; however, these systems are typically designed and fabricated in the form of simplified bilayers. This work introduces computational design methods for 4D-printing bio-inspired behaviors with compounded mechanisms. To emulate the anisotropic arrangement of motile plant structures, material systems are tailored at the mesoscale using extrusion-based 3D-printing. The methodology is demonstrated by transferring the principle of force generation by a twining plant (Dioscorea bulbifera) to the application of a self-tightening splint. Through the tensioning of its stem helix, D. bulbifera exhibits a squeezing force on its support to provide stability against gravity. The functional strategies of D. bulbifera are abstracted and translated to customized 4D-printed material systems. The squeezing forces of these bio-inspired motion mechanisms are then evaluated. Finally, the function of self-tightening is prototyped in a wrist-forearm splint-a common orthotic device for alignment. The presented approach enables the transfer of novel and expanded biomimetic design strategies to 4D-printed motion mechanisms, further opening the design space to new types of adaptive creations for wearable assistive technologies and beyond.

Self-Actuated Paper and Wood Models: Low-Cost Handcrafted Biomimetic Compliant Systems for Research and Teaching.

The abstraction and implementation of plant movement principles into biomimetic compliant systems are of increasing interest for technical applications, e.g., in architecture, medicine, and soft robotics. Within the respective research and development approaches, advanced methods such as 4D printing or 3D-braiding pultrusion are typically used to generate proof-of-concept demonstrators at the laboratory or demonstrator scale. However, such techniques are generally time-consuming, complicated, and cost-intensive, which often impede the rapid realization of a sufficient number of demonstrators for testing or teaching. Therefore, we have produced comparable simple handcrafted compliant systems based on paper, wood, plastic foil, and/or glue as construction materials. A variety of complex plant movement principles have been transferred into these low-cost physical demonstrators, which are self-actuated by shrinking processes induced by the anisotropic hygroscopic properties of wood or paper. The developed systems have a high potential for fast, precise, and low-cost abstraction and transfer processes in biomimetic approaches and for the "hands-on understanding" of plant movements in applied university and school courses.

Programming sequential motion steps in 4D-printed hygromorphs by architected mesostructure and differential hygro-responsiveness.

Through their anisotropic cellular mesostructure and differential swelling and shrinking properties, hygroscopic plant structures move in response to changes in the environment without consuming metabolic energy. When the movement is choreographed in sequential time steps, either in individual structures or with a coordinated interplay of various structural elements, complex functionalities such as dispersal and protection of seeds are achieved. Inspired by the multi-phase motion in plant structures, this paper presents a method to physically program the timescale and the sequences of shape-change in 4D-printed hygromorphic structures. Using the FDM 3D-printing method, we have developed multi-layered, multi-material functional bilayers that combine highly hygroscopic active layers (printed with hygroscopic bio-composite materials) with hydrophobic restrictive and blocking layers (printed with PLA and TPC materials). The timescale of motion is programmed through the design of the mesostructured layers and 3D-printing process parameters, including thickness (number of printed active layers), porosity (filling ratio of the active layer), and water permeability (filling ratio of the blocking layer). Through a series of experiments, it is shown that the timescale of motion can be extended by increasing the thickness of the active layer, decreasing the porosity of the active layer, or increasing the filling ratio of the hydrophobic restrictive and blocking layers. Similarly, a lower thickness of the active layer and lower filling ratio of all layers result in a faster motion. As a proof of concept, we demonstrate several prototypes that exhibit sequential motion, including an aperture with overlapping elements where each completes its movement sequentially to avoid collision, and a self-locking mechanism where defined areas of the structure are choreographed to achieve a multi-step self-shaping and locking function. The presented method extends the programmability and the functional capabilities of hygromorphic 4D-printing, allowing for novel applications across fields such as robotics, smart actuators, and adaptive architecture.

Complexity and diversity of motion amplification and control strategies in motile carnivorous plant traps.

Similar to animals, plants have evolved mechanisms for elastic energy storage and release to power and control rapid motion, yet both groups have been largely studied in isolation. This is exacerbated by the lack of consistent terminology and conceptual frameworks describing elastically powered motion in both groups. Iconic examples of fast movements can be found in carnivorous plants, which have become important models to study biomechanics, developmental processes, evolution and ecology. Trapping structures and processes vary considerably between different carnivorous plant groups. Using snap traps, suction traps and springboard-pitfall traps as examples, we illustrate how traps mix and match various mechanisms to power, trigger and actuate motions that contribute to prey capture, retention and digestion. We highlight a fundamental trade-off between energetic investment and movement control and discuss it in a functional-ecological context.

Snapshot prey spectrum analysis of the phylogenetically early-diverging carnivorous Utricularia multifida from U. section Polypompholyx (Lentibulariaceae).

Utricularia multifida is carnivorous bladderwort from Western Australia and belongs to a phylogenetically early-diverging lineage of the genus. We present a prey spectrum analysis resulting from a snapshot sampling of 17 traps-the first of this species to our knowledge. The most abundant prey groups were Ostracoda, Copepoda, and Cladocera. The genus cf. Cypretta (Cyprididae, Ostracoda) was the predominant prey. However, a high variety of other prey organisms with different taxonomic backgrounds was also detected. Our results indicate that U. multifida may potentially be specialized in capturing substrate-bound prey. Future approaches should sample plants from different localities to allow for robust comparative analyses.

Form, Structure, and Function: How Plants vs. Animals Solve Physical Problems.

Plants and animals have evolved solutions for a wide range of mechanical problems, such as adhesion and dispersal. Several of these solutions have been sources for bio-inspiration, like the Lotus Effect for self-cleaning surfaces or Velcro for adhesion. This symposium brought together plant and animal biomechanics researchers who tackle similar problems in different systems under the unifying theme of structure-function relations with relevance to bio-inspiration. For both communities it holds true that the structural systems, which have evolved in the respective organisms to address the mechanical challenges mentioned above, are often highly complex. This requires interdisciplinary research involving "classical" experimental biology approaches in combination with advanced imaging methods and computational modeling. The transfer of such systems into biomimetic technical materials and structures comes with even more challenges, like scalability issues and applicability. Having brought all these topics under one umbrella, this symposium presented the forefront of biophysical basic and application-oriented international research with the goal of facilitation knowledge transfer across systems and disciplines.

Functional-morphological analyses of the delicate snap-traps of the aquatic carnivorous waterwheel plant (Aldrovanda vesiculosa) with 2D and 3D imaging techniques.

BACKGROUND AND AIMS: The endangered aquatic carnivorous waterwheel plant (Aldrovanda vesiculosa) catches prey with 3-5-mm-long underwater snap-traps. Trapping lasts 10-20 ms, which is 10-fold faster than in its famous sister, the terrestrial Venus flytrap (Dionaea muscipula). After successful capture, the trap narrows further and forms a 'stomach' for the digestion of prey, the so-called 'sickle-shaped cavity'. To date, knowledge is very scarce regarding the deformation process during narrowing and consequent functional morphology of the trap. METHODS: We performed comparative analyses of virtual 3D histology using computed tomography (CT) and conventional 2D histology. For 3D histology we established a contrasting agent-based preparation protocol tailored for delicate underwater plant tissues. KEY RESULTS: Our analyses reveal new structural insights into the adaptive architecture of the complex A. vesiculosa snap-trap. In particular, we discuss in detail the arrangement of sensitive trigger hairs inside the trap and present actual 3D representations of traps with prey. In addition, we provide trap volume calculations at different narrowing stages. Furthermore, the motile zone close to the trap midrib, which is thought to promote not only the fast trap closure by hydraulics but also the subsequent trap narrowing and trap reopening, is described and discussed for the first time in its entirety. CONCLUSIONS: Our research contributes to the understanding of a complex, fast and reversible underwater plant movement and supplements preparation protocols for CT analyses of other non-lignified and sensitive plant structures.