Anti-icing strategies of plant surfaces: the ice formation on leaves visualized by Cryo-SEM experiments.

This paper presents an experimental study on surface icing on leaves in six plant species having different surface micromorphology and wettability properties. Contrary to previous studies on ice crystallization, which have been mainly performed by using infrared video thermography, we applied a Cryo-SEM approach allowing not only characterization of plant surfaces in their native conditions but also visualization of ice crystal formation on the native plant surfaces at the micro- and nanoscales. The Cryo-SEM was also used as an experimental device to freeze water vapor, thaw ice crystals, and freeze fluid water on the plant surface again. The experiments clearly demonstrate that trichome coverage (especially with several distinct layers) and 3D wax projections can be recognized as anti-icing strategies of plants. Trichomes can prevent and delay ice formation by being nucleation points for the formation of ice from vapor and protect the plant surface from overcooling, when fluid water freezes in contact with the leaf surface. The study shows for the first time two important effects that might reduce plant cell freezing rate: the presence of air pockets between wax projections that protect from direct contact between ice crystals and the plant cuticle and elimination of fluid water after thawing and preventing further re-freezing on the surface. The detailed knowledge obtained here is not only important for plant ecology, evolution, and plant protection but also for looking for potential biomimetic strategies that reduce/avoid icing of cultural plants and artificial technical surfaces.

Traction force measurements on male Strepsiptera (Insecta) revealed higher forces on smooth compared with hairy substrates.

The aim of this study was to find out how strongly the parasitic insect Stylopsovinae, which has tarsi equipped with tenent hairs and lacking claws, attaches to different substrates. We investigated adhesion of male S. ovinae to the abdomen of its hymenopteran host (Andrena vaga), the hairier abdomen of a Bombus sp. and two artificial smooth reference surfaces with different degrees of hydrophilicity. In our experiments, the male S. ovinae developed significantly higher forces on smooth surfaces. However, the forces were significantly lower on all the hymenopteran surfaces used in the experiment. The absence of anisotropy in the force grip in cranial/caudal direction relative to the host might indirectly indicate that S. ovinae generate forces by adhesion rather than mechanical interlocking with the host hairs. The tolerance of the attachment system of S. ovinae to the substrate chemistry might be explained by the primary contribution of van der Waals interactions and not capillary forces to adhesion in S. ovinae.

Egg-laying job makes males hot: body temperature measurements in egg-laying tandems of the dragonfly Sympetrum vulgatum using IR camera.

Tandem oviposition, where males guide females in contact, requires quite a substantial muscle activity from males and, therefore, stronger heat production within the male thorax compared to the female thorax. In the present study, an infrared camera equipped with a macrolens was applied in the field, in order to estimate temperature in different body regions of male and female dragonflies Sympetrum vulgatum laying eggs in tandems. In both sexes, the thorax was considerably warmer than other body parts. The male thorax was on average 3-4 °C warmer than that of the female. These observations support previous data that Sympetrum males have a stronger muscular activity and heat production in tandem during the egg-laying process compared to females. The data provide additional evidence that this kind of contact guarding behavior during oviposition is rather costly for males. The tip of the male abdomen was much warmer than its own abdomen in the middle region. This result might be explained by possible heat transfer from the female head to the male abdomen through the contact between male abdominal appendages and the female head. An alternative explanation might be strong activity of the muscles controlling male anal appendages. Finally, this study also demonstrated a strong potential of IR cameras in field studies of dragonfly behavioral physiology.

Anti-adhesive effects of plant wax coverage on insect attachment.

The long period of reciprocal antagonistic coevolution between some insect and plant species has led to the development of plant surface attributes that reduce insect attachment. These features serve as a defence against herbivores, sap-sucking insects and nectar robbers, contribute to a temporary capture of insect pollinators, and prevent the escape of insects from traps of carnivorous plants. This review summarises the literature on attachment-mediated insect-plant interactions. A short introduction to attachment systems of insects is presented and the effect of three-dimensional epicuticular waxes on insect attachment is illustrated by many examples. Special attention is given to the mechanisms of the anti-attachment properties of plant wax structures (the roughness hypothesis, the contamination hypothesis, the fluid-adsorption hypothesis, and the wax-dissolving hypothesis) and their ecological implications.

The Greasy Pole Syndrome in Alliaria petiolata (Brassicaceae): The Pubescence and Wax Coverage on Stems Reduce Invasion by Lasius niger Ants.

To reduce negative effects of floral visitation by ants, which do not serve as reliable cross-pollinators, some plants have developed a non-floral, stem-based defense mechanism called greasy pole syndrome. In the present study, we examined the effects of two surface features (trichomes and three-dimensional epicuticular wax coverage) on stems of Alliaria petiolata plants on visiting frequencies, travelled distances, and running velocities of Lasius niger ants. The experiments were performed with stem samples prepared from different (apical and basal) stem portions showing different surface morphologies (smooth control, covered by wax and trichomes + wax, respectively). The control, mechanically wiped stem samples lacking any surface features were significantly more often visited by ants, where they travelled significantly longer distances and moved with significantly higher velocities, compared to the intact stems. The apical and basal stem portions showed no significant differences in the measured parameters. Based on data obtained, we conclude about the main contribution of the wax to the greasy pole function of the A. petiolata stem via reduction of ant adhesion to the wax-bearing stem surface, whereas trichomes presumably serve as the first barrier for ants approaching usually from the ground level and protect the fragile wax coverage from an excessive deterioration.

Insect attachment on waxy plant surfaces: the effect of pad contamination by different waxes.

This study focuses on experimental testing of the contamination hypothesis and examines how the contamination of insect adhesive pads with three-dimensional epicuticular waxes of different plant species contributes to the reduction of insect attachment. We measured traction forces of tethered Chrysolina fastuosa male beetles having hairy adhesive pads on nine wax-bearing plant surfaces differing in both shape and dimensions of the wax structures and examined insect adhesive organs after they have contacted waxy substrates. For comparison, we performed the experiments with the same beetle individuals on a clean glass sample just before (gl1) and immediately after (gl2) the test on a plant surface. The tested insects showed a strong reduction of the maximum traction force on all waxy plant surfaces compared to the reference experiment on glass (gl1). After beetles have walked on waxy plant substrates, their adhesive pads were contaminated with wax material, however, to different extents depending on the plant species. The insects demonstrated significantly lower values of both the maximum traction force and the first peak of the traction force and needed significantly longer time to reach the maximum force value in the gl2 test than in the gl1 test. These effects were especially pronounced in cases of the plant surfaces covered with wax projections having higher aspect ratios. The data obtained clearly indicated the impact of waxy plant surfaces on the insect ability to subsequently attach to the clean smooth surface. This effect is caused by the contamination of adhesive pads and experimentally supports the contamination hypothesis.

Comparative study on mechanical properties and biomineralization of hooks in the diaspores of three epizoochorous plant species.

Most of the plants using epizoochory show adaptations to this diaspore dispersal strategy by having the diaspores covered by barbs, hooks, spines or viscid outgrowths, which allow diaspores to easily attach to an animal surface. Many previous studies have been mainly focused on the dispersal distances and efficiency, or effectiveness of diverse attachment structures depending on their size, anatomy, and morphology. However, the knowledge about the mechanical properties of these structures remains rather poor. In this study, we use a combination of scanning electron microscopy, energy dispersive X-ray element analysis and nanoindentation, to examine the microstructure, biomineralization and mechanical properties of single hooks in Arctium minus, Cynoglossum officinale and Galium aparine. Both the biomineralization and mechanical properties of the hooks strongly differ in examined plant species; mechanical properties depend on the biomineralization pattern, such as the accumulation of silicon and calcium. Elastic modulus and hardness decrease in the series C. officinaleG. aparineA. minus. Anisotropic mechanical properties are found between the radial and longitudinal directions in each single hook. By characterizing the mechanical properties and biomineralization of plant hooks, this paper contributes to the understanding of attachment biomechanics related to seed dispersal. STATEMENT OF SIGNIFICANCE: The dispersal of seeds is essential for plant survival. Many of the plants that use the outside surface of animals to transport the seeds show adaptations to this dispersal strategy by having the seeds covered with hooks. Although these hooks have various sizes, morphologies and anatomical structures, all of them provide mechanical interlocking to animal surfaces. To reduce the risk of interlocking failure, the hooks are usually reinforced by mineralization. However, the relationship between mineralization, mechanical properties and specialized function of plant hooks has been largely overlooked. Here we perform a characterization study on the hooks of three plant species. Our results deepen the current understanding of the mineralization-material-function relationship in specialized hooks of plant seeds.

Nanoscale mesh acts as anti-adhesive surface against particulate contamination in eyes of whiteflies.

In many insects the surface of the eye is nanostructured by arrays of protuberances termed ommatidial gratings which provide the cuticle with anti-reflective, anti-wetting and self-cleaning properties. The hypothesised anti-contamination role of the gratings against dust and pollen results from theoretical predictions on grating geometry and experiments on synthetic replicas of ommatidia surfaces but has not yet been proven in an animal. Whiteflies are biological test beds for anti-contamination surfaces because they deliberately distribute wax particles extruded from abdominal plates over their entire bodies. The numerous particles protect the animal against water evaporation and radiation, but may severely impair vision. Using scanning electron microscopy (SEM) and CryoSEM, we here show that the cornea of whiteflies exhibits ~ 220 nm wide mesh-like structures forming hexagonal gratings with thin ~ 40 nm connecting walls. Quantitative measurements of wax particles on the eye show that the nanostructures reduce particle contamination by more than ~ 96% compared to other areas of the cuticle. Altogether, our study is the first description of a predicted optimized grating geometry for anti-contamination in an arthropod. The findings serve as evidence of the high effectiveness of nanostructured surfaces for reducing contact area and thus adhesion forces between biological surfaces and contaminating particles.

Wax Protrusions on Anti-Adhesive Plant Surfaces and Their Interactions with Insect Adhesive Pads: A Mechanical Interpretation.

Insect attachment devices enhance adhesion to complex-geometry substrates by increasing the real contact area. In nature, insects mainly interact with plant surfaces that are often covered by 3D wax structures. Here, we describe, discuss, and give a mechanical interpretation of plant waxes and the possible fracture mechanisms of these wax structures during their interactions with the adhesive pads of insects. It is argued that these plant surface microstructures significantly influence insect adhesion through reducing the contact area and contaminating the insect pads.

Petals Reduce Attachment of Insect Pollinators: A Case Study of the Plant Dahlia pinnata and the Fly Eristalis tenax.

In order to understand whether the petal surface in "cafeteria"-type flowers, which offer their nectar and pollen to insect pollinators in an open way, is adapted to a stronger attachment of insect pollinators, we selected the plant Dahlia pinnata and the hovering fly Eristalis tenax, both being generalist species according to their pollinator's spectrum and diet, respectively. We combined cryo scanning electron microscopy examination of leaves, petals, and flower stems with force measurements of fly attachment to surfaces of these plant organs. Our results clearly distinguished two groups among tested surfaces: (1) the smooth leaf and reference smooth glass ensured a rather high attachment force of the fly; (2) the flower stem and petal significantly reduced it. The attachment force reduction on flower stems and petals is caused by different structural effects. In the first case, it is a combination of ridged topography and three-dimensional wax projections, whereas the papillate petal surface is supplemented by cuticular folds. In our opinion, these "cafeteria"-type flowers have the petals, where the colour intensity is enhanced due to papillate epidermal cells covered by cuticular folds at the micro- and nanoscale, and exactly these latter structures mainly contribute to adhesion reduction in generalist insect pollinators.

The tight attachment achieved by the male discoidal setae is possibly a counter-adaptation to the grease layer on female integument surfaces in green dock beetles.

Green dock beetles Gastrophysa viridula exhibit sexual dimorphism in tarsal attachment setae: females have only pointed, lanceolate and spatula-like setae, while males additionally possess discoidal ones. The sexual dimorphism is probably attributed to the necessity of male discoidal setae to adhere to the smooth back of the female during copulation. We aimed to understand its possible mechanism of attachment with G. viridula. Pull-off forces of both females and males were measured on (i) alive females, (ii) dead and dried females, and (iii) resin replicas of fresh females. The attachment ability tended to increase on dead and replicated female surfaces in both sexes, which indicates that the epicuticular grease layer on the integument of alive intact beetles decreases the attachment. This tendency was prominent in females. The present study clearly showed that in G. viridula discoidal setae enable the males to adhere stronger to female surfaces. The divergent performance found between the sexes differing in their setal composition is probably caused by the stiffness difference between the setae types and by the specific shape of the setal tips. A peculiar reproductive biology in G. viridula is probably attributed to this remarkable divergence of labour in their attachment pads between the sexes.

Hierachical epicuticular wax coverage on leaves of Deschampsia antarctica as a possible adaptation to severe environmental conditions.

Using cryo scanning electron microscopy, the surface micromorphology of vegetative (leaf blade and ligule) and generative (pedicel and outer glume) organs in Deschampsia antarctica, one of the only two flowering plants native to Antarctica, was examined. Whereas the pedicel and outer glume were wax-free, both leaf sides had a prominent epicuticular wax coverage consisting of two superimposed layers: polygonal rodlets formed by fused irregular platelets (the lower wax layer) and membraneous platelets (the upper wax layer). Although the adaxial (inner) and abaxial (outer) leaf surfaces showed a similar microstructure of the wax coverage, they differed in the thickness ratio between lower and upper wax layer. The ligule bore a very loose wax coverage composed of separate scale-like projections or clusters of them. We suppose that the two-layered wax densely covering both leaf surfaces might contribute to the plant adaptation to severe environmental conditions in Antarctica due to an increase of its resistance against cold temperatures, icing, harmful UV radiation, and dehydration. The presence of the epicuticular wax on the abaxial leaf side and the ligule as well as the hierarchical structure of the wax coverage on both leaf surfaces is described in D. antarctica for the first time.

Combined Effect of Different Flower Stem Features on the Visiting Frequency of the Generalist Ant Lasius niger: An Experimental Study.

In order to understand the effects of the morphology and surface texture of flower stems in Smyrnium rotundifolium on the visiting frequency of generalist ants, we conducted experiments with Lasius niger ants running on dry wooden sticks mimicking different types of stems: (1) intact (grooved) sticks; (2) sticks painted with slaked (hydrated) lime (calcium carbonate coverage) imitating plant epicuticular wax coverage; (3) intact sticks with smooth polyester plate-shaped cuffs imitating upper leaves; and (4) intact sticks bearing cuffs painted with slaked lime. Ants were attracted by the sweet sugar syrup droplets placed on a stick tip, and the number of ants visiting the drops was counted. Our data showed significant differences in the visiting frequencies between the different types of stem-mimicking samples. The number of recorded ants progressively decreased in the following order of samples: intact sticks-painted sticks-sticks with intact cuffs-sticks with painted cuffs. These results clearly demonstrated that micro/nanoscopic surface coverages and macroscopic physical barriers, especially if combined, have a negative impact on the attractiveness of stems to ants. This study provides further evidence for the hypothesis that having a diversity of plant stems in the field, generalist ants prefer substrates where their locomotion is less hindered by obstacles and/or surface slipperiness.

Attachment ability of females and males of the ladybird beetle Cryptolaemus montrouzieri to different artificial surfaces.

To test the effects of surface chemistry and surface roughness on the attachment ability of female and male Cryptolaemus montrouzieri beetles that are equipped with hairy adhesive pads, traction force experiments were performed on three artificial substrates: (1) hydrophobic smooth glass, (2) hydrophobic smooth epoxy resin, and (3) hydrophobic microrough epoxy resin. Also the micromorphology of the dorsal body side and adhesive pads in males and females was examined using a scanning electron microscopy. The traction force ranged from 0.13 to 3.60 mN in females and from 0.28 to 3.20 mN in males. The force values obtained on different test substrates showed highly significant differences and decreased in the following order of substrates: glass - smooth epoxy resin - microrough epoxy resin. In both females and males, the effect of surfaces was similar. The obtained results clearly showed that both surface parameters, chemistry and roughness, affected the attachment ability of beetles. Similar microstructure of adhesive pads in both sexes resulted in similar attachment performance of males and females on all test substrates.

Insect-inspired architecture to build sustainable cities.

Materials, structures, surfaces and buildings of insects are of a great scientific interest, but such basic knowledge about the functional principles of these structures is also highly relevant for technical applications, especially in architecture. Some of the greatest challenges for today's architecture are multifunctionality, energy saving and sustainability - problems that insects have partially solved during their evolution. Entomologists have collected a huge amount of information about the structure and function of such living constructions and surfaces. This information can be utilized in order to mimic them for applications in architecture. The main technology areas, in which insect-inspired ideas can be applied, are the following: (1) new materials, (2) constructions, (3) surfaces, (4) adhesives and bonding technology, (5) optics and photonics. A few selected examples are discussed in this short review, but having more than one million described insect species as a source for inspiration, one might expect many more ideas from entomology for insect-inspired biomimetics in architecture. The incorporation of additional knowledge from insect biology into architecture will improve performance of future buildings. However, biologists still do not have a complete understanding of structure-function relationship of insect materials and construction. Hence, many technological areas will benefit from additional basic entomology research. Also the screening for new inspirations from insects is likely to remain an important research field in the near future.

Porous substrate affects a subsequent attachment ability of the beetle Harmonia axyridis (Coleoptera, Coccinellidae).

According to literature data, porous substrates can cause a reduction of insect attachment ability. We carried out traction experiments with adult ladybird beetles Harmonia axyridis on the smooth solid glass sample and rough porous Al2O3 membrane to prove the primary effect of absorption of the insect pad secretion by porous media, rather than surface roughness, on the attachment force on the porous sample. With each insect individual, a set of five experiments was conducted: (1) on glass; (2) on the porous membrane; (3-5) on glass immediately after the test on the porous surface, then after 30 min and 1 h of recovery time. On the porous substrate, the forces, being similar in females and males, were greatly reduced compared to those measured on glass. A significant difference between the force values obtained in the first (before the test on the porous sample) and second (immediately after the experiment on the porous sample) tests on glass was observed. After 30 min recovery time, beetles completely regained their attachment ability. Females produced significantly lower forces than males in all experiments on glass: the differences are probably caused by the sexual dimorphism in the microstructure of their adhesive pads. The obtained results are of fundamental importance for further application in biomimetics of novel insect-repelling surfaces and in plant protection by using porous materials.

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.

Radial arrangement of apical adhesive sites promotes contact self-alignment of fruits in Commicarpus plants (Nyctaginaceae).

Fruits of the plants from the genus Commicarpus (Nyctaginaceae) use their adhesive properties for dispersal. They can readily stick to various surfaces including skin, fur, and feathers of potential dispersal vectors using the secretion provided by the set of glands arranged radially at the distal end of the cut-cone-shaped fruit. Field observations show that this particular geometry promotes self-alignment of the fruit to various surfaces after initial contact just by one gland is established. Such self-alignment in turn leads to an increase of the number of contacting points and to the enhancement of adhesive contact area. Here, we study this particular geometry from a theoretical point of view, by probing adhesion ability of geometries having from 2 to 7 radially distributed attachment points. The results show that the radial arrangement provides rapid alignment to the surface. The robust adhesion can be reached already at 5 adhesive points and their further increase does not substantially improve the performance. This study is important not only for our understanding of the functional morphology of biological adhesive systems, but also for the development of technical self-aligning adhesive devices.

Oil adsorption ability of three-dimensional epicuticular wax coverages in plants.

Primary aerial surfaces of terrestrial plants are very often covered with three-dimensional epicuticular waxes. Such wax coverages play an important role in insect-plant interactions. Wax blooms have been experimentally shown in numerous previous studies to be impeding locomotion and reducing attachment of insects. Among the mechanisms responsible for these effects, a possible adsorption of insect adhesive fluid by highly porous wax coverage has been proposed (adsorption hypothesis). Recently, a great decrease in insect attachment force on artificial adsorbing materials was revealed in a few studies. However, adsorption ability of plant wax blooms was still not tested. Using a cryo scanning electron microscopy approach and high-speed video recordings of fluid drops behavior, followed by numerical analysis of experimental data, we show here that the three-dimensional epicuticular wax coverage in the waxy zone of Nepenthes alata pitcher adsorbs oil: we detected changes in the base, height, and volume of the oil drops. The wax layer thickness, differing in samples with untreated two-layered wax coverage and treated one-layered wax, did not significantly affect the drop behavior. These results provide strong evidence that three-dimensional plant wax coverages due to their adsorption capability are in general anti-adhesive for insects, which rely on wet adhesion.

Surface roughness rather than surface chemistry essentially affects insect adhesion.

The attachment ability of ladybird beetles Coccinella septempunctata was systematically investigated on eight types of surface, each with different chemical and topographical properties. The results of traction force tests clearly demonstrated that chemical surface properties, such as static/dynamic de-wettability of water and oil caused by specific chemical compositions, had no significant effect on the attachment of the beetles. Surface roughness was found to be the dominant factor, strongly affecting the attachment ability of the beetles.