Brochosome size variation and its influence on leafhopper (Hemiptera: Cicadellidae) wing wettability.

Insect wing surfaces have nano- and microscale features that enable multi-functionality. Leafhoppers (Hemiptera: Cicadellidae) are unique in that they produce and excrete nanoscale particles, called brochocosomes, that are spread onto the integument by the insect. Brochosomes are extra-cuticular, removable, and make the integument both superhydrophobic and anti-reflective, 2 greatly desired properties in engineering and material science fields. Adaptations like this have captured the interest of researchers looking to draw inspiration from the natural world to create novel solutions and optimize current technologies. Here, we compare brochosome size and wettability across 8 species of leafhoppers using scanning electron microscopy and microgoniometry. We demonstrate that brochosome size is variable within species and that size and wettability are both significantly impacted by species. We report the extent of variability in each case and examine the relationship between brochosome size, body length, and hydrophobicity. In discussing potential applications for brochosomes, we comment on an emerging and rapid analysis technique for evaluating small biological particles. Additionally, we discuss a few recent brochosome-inspired designs and comment on important considerations. Our work provides valuable insight on a unique system that can guide the design of functionalized materials with enhanced hydrophobic and self-cleaning properties.

Insect-scale jumping robots enabled by a dynamic buckling cascade.

Millions of years of evolution have allowed animals to develop unusual locomotion capabilities. A striking example is the legless-jumping of click beetles and trap-jaw ants, which jump more than 10 times their body length. Their delicate musculoskeletal system amplifies their muscles' power. It is challenging to engineer insect-scale jumpers that use onboard actuators for both elastic energy storage and power amplification. Typical jumpers require a combination of at least two actuator mechanisms for elastic energy storage and jump triggering, leading to complex designs having many parts. Here, we report the new concept of dynamic buckling cascading, in which a single unidirectional actuation stroke drives an elastic beam through a sequence of energy-storing buckling modes automatically followed by spontaneous impulsive snapping at a critical triggering threshold. Integrating this cascade in a robot enables jumping with unidirectional muscles and power amplification (JUMPA). These JUMPA systems use a single lightweight mechanism for energy storage and release with a mass of 1.6 g and 2 cm length and jump up to 0.9 m, 40 times their body length. They jump repeatedly by reengaging the latch and using coiled artificial muscles to restore elastic energy. The robots reach their performance limits guided by theoretical analysis of snap-through and momentum exchange during ground collision. These jumpers reach the energy densities typical of the best macroscale jumping robots, while also matching the rapid escape times of jumping insects, thus demonstrating the path toward future applications including proximity sensing, inspection, and search and rescue.

Electron Tomography and Machine Learning for Understanding the Highly Ordered Structure of Leafhopper Brochosomes.

Insects known as leafhoppers (Hemiptera: Cicadellidae) produce hierarchically structured nanoparticles known as brochosomes that are exuded and applied to the insect cuticle, thereby providing camouflage and anti-wetting properties to aid insect survival. Although the physical properties of brochosomes are thought to depend on the leafhopper species, the structure-function relationships governing brochosome behavior are not fully understood. Brochosomes have complex hierarchical structures and morphological heterogeneity across species, due to which a multimodal characterization approach is required to effectively elucidate their nanoscale structure and properties. In this work, we study the structural and mechanical properties of brochosomes using a combination of atomic force microscopy (AFM), electron microscopy (EM), electron tomography, and machine learning (ML)-based quantification of large and complex scanning electron microscopy (SEM) image data sets. This suite of techniques allows for the characterization of internal and external brochosome structures, and ML-based image analysis methods of large data sets reveal correlations in the structure across several leafhopper species. Our results show that brochosomes are relatively rigid hollow spheres with characteristic dimensions and morphologies that depend on leafhopper species. Nanomechanical mapping AFM is used to determine a characteristic compression modulus for brochosomes on the order of 1-3 GPa, which is consistent with crystalline proteins. Overall, this work provides an improved understanding of the structural and mechanical properties of leafhopper brochosomes using a new set of ML-based image classification tools that can be broadly applied to nanostructured biological materials.

Best Practices of Bioinspired Design: Key Themes and Challenges.

Bioinspired design (BID) is an interdisciplinary research field that can lead to innovations to solve technical problems. There have been many attempts to develop a framework to de-silo engineering and biology and implement processes to enable BID. In January of 2022, we organized a symposium at the 2022 Society of Integrative and Comparative Biology Annual Meeting to bring together educators and practitioners of BID. The symposium aimed to: a) consolidate best practices in teaching bioinspiration, b) create and sustain effective multidisciplinary teams, c) summarize best approaches to conduct problem-based or solution-driven fundamental research, and d) bring bioinspired design innovations to market. During the symposium, several themes emerged. Here we highlight three critical themes that need to be addressed for BID to become a truly interdisciplinary strategy that benefits all stakeholders and results in innovation. First, there is a need for a usable methodology that leads to proper abstraction of biological principles for engineering design. Second, the utilization of engineering models to test biological hypotheses is essential for the continued engagement of biologists in BID. And third, the necessity of proven team-science strategies that will lead to successful collaborations between engineers and biologists. Accompanying this introduction is a variety of perspectives and research articles highlighting best practices in bioinspired design research and product development and guides that can highlight the challenges and facilitate interdisciplinary collaborations in the field of bioinspired design.

Particulate-Droplet Coalescence and Self-Transport on Superhydrophobic Surfaces.

Particulate transport from surfaces governs a variety of phenomena including fungal spore dispersal, bioaerosol transmission, and self-cleaning. Here, we report a previously unidentified mechanism governing passive particulate removal from superhydrophobic surfaces, where a particle coalescing with a water droplet (∼10 to ∼100 µm) spontaneously launches. Compared to previously discovered coalescence-induced binary droplet jumping, the reported mechanism represents a more general capillary-inertial dominated transport mode coupled with particle/droplet properties and is typically mediated by rotation in addition to translation. Through wetting and momentum analyses, we show that transport physics depends on particle/droplet density, size, and wettability. The observed mechanism presents a simple and passive pathway to achieve self-cleaning on both artificial as well as biological materials as confirmed here with experiments conducted on butterfly wings, cicada wings, and clover leaves. Our findings provide insights into particle-droplet interaction and spontaneous particulate transport, which may facilitate the development of functional surfaces for medical, optical, thermal, and energy applications.

Addressing Diverse Motivations to Enable Bioinspired Design.

Bioinspired design (BID) is an inherently interdisciplinary practice that connects fundamental biological knowledge with the capabilities of engineering solutions. This paper discusses common social challenges inherent to interdisciplinary research, and specific to collaborating across the disciplines of biology and engineering when practicing BID. We also surface best practices that members of the community have identified to help address these challenges. To accomplish this goal, we address challenges of bioinspiration through a lens of recent findings within the social scientific study of interdisciplinary teams. We propose three challenges faced in BID: (1) complex motivations across collaborating researchers, (2) misperceptions of relationships and benefits between biologists and engineers, and (3) institutionalized barriers that disincentivize interdisciplinary work. We advance specific recommendations for how to address each of these challenges.

Staying Dry and Clean: An Insect's Guide to Hydrophobicity.

Insects demonstrate a wide diversity of microscopic cuticular and extra-cuticular features. These features often produce multifunctional surfaces which are greatly desired in engineering and material science fields. Among these functionalities, hydrophobicity is of particular interest and has gained recent attention as it often results in other properties such as self-cleaning, anti-biofouling, and anti-corrosion. We reviewed the historical and contemporary scientific literature to create an extensive review of known hydrophobic and superhydrophobic structures in insects. We found that numerous insects across at least fourteen taxonomic orders possess a wide variety of cuticular surface chemicals and physical structures that promote hydrophobicity. We discuss a few bioinspired design examples of how insects have already inspired new technologies. Moving forward, the use of a bioinspiration framework will help us gain insight into how and why these systems work in nature. Undoubtedly, our fundamental understanding of the physical and chemical principles that result in functional insect surfaces will continue to facilitate the design and production of novel materials.

Nonlinear elasticity and damping govern ultrafast dynamics in click beetles.

Many small animals use springs and latches to overcome the mechanical power output limitations of their muscles. Click beetles use springs and latches to bend their bodies at the thoracic hinge and then unbend extremely quickly, resulting in a clicking motion. When unconstrained, this quick clicking motion results in a jump. While the jumping motion has been studied in depth, the physical mechanisms enabling fast unbending have not. Here, we first identify and quantify the phases of the clicking motion: latching, loading, and energy release. We detail the motion kinematics and investigate the governing dynamics (forces) of the energy release. We use high-speed synchrotron X-ray imaging to observe and analyze the motion of the hinge's internal structures of four Elater abruptus specimens. We show evidence that soft cuticle in the hinge contributes to the spring mechanism through rapid recoil. Using spectral analysis and nonlinear system identification, we determine the equation of motion and model the beetle as a nonlinear single-degree-of-freedom oscillator. Quadratic damping and snap-through buckling are identified to be the dominant damping and elastic forces, respectively, driving the angular position during the energy release phase. The methods used in this study provide experimental and analytical guidelines for the analysis of extreme motion, starting from motion observation to identifying the forces causing the movement. The tools demonstrated here can be applied to other organisms to enhance our understanding of the energy storage and release strategies small animals use to achieve extreme accelerations repeatedly.

Neurophysiological and behavioral responses of blacklegged ticks to host odors.

The blacklegged tick, Ixodes scapularis (Ixodida, Ixodidae), is one of the major disease vectors in the United States, and due to multiple human impact factors, such as decreasing forest size for land development and climate change, it has expanded its range and established across the United States. Throughout the life cycle, ticks locate hosts for their blood-meal, and although the ecologies of this tick and their hosts have been studied in depth, the sensory physiology behind host location largely remains unexplored. Here, we report establishing a robust paradigm to isolate and identify odors from the natural milieu for I. scapularis. We performed single sensillum recordings (SSR) from the olfactory sensilla on the tick tarsi, and used the SSR system as a biological detector to isolate natural compounds that elicited biological activity. The SSR setup was further tested in tandem with gas chromatography (GC) wherein the ticks' olfactory sensillum activity served as a biological detector. The GC-SSR recordings from the wall pore sensilla in the Haller's organ, and further identification of the biologically active deer gland constituents by GC-mass spectrometry (GC-MS) revealed methyl substituted phenols as strong chemostimuli, as compared to ethyl or propyl substitutions. The strongest electrophysiological activity was elicited by m- cresol followed by p- cresol. Ethyl- and propylphenols with any of the three substitutions (ortho, meta or para), did not induce any neurophysiological activity. Finally, a behavioral analysis in a dual-choice olfactometer of all these phenols at three different doses revealed no significant behavioral response, except for p- cresol at -3 dilution. Overall, this study contributes to our understanding of I. scapularis tick's neurophysiology and provides a robust platform to isolate and identify natural attractants and repellents.

Dissolvable Template Nanoimprint Lithography: A Facile and Versatile Nanoscale Replication Technique.

Nanoimprinting lithography (NIL) is a next-generation nanofabrication method, capable of replicating nanostructures from original master surfaces. Here, we develop highly scalable, simple, and nondestructive NIL using a dissolvable template. Termed dissolvable template nanoimprinting lithography (DT-NIL), our method utilizes an economic thermoplastic resin to fabricate nanoimprinting templates, which can be easily dissolved in simple organic solvents. We used the DT-NIL method to replicate cicada wings which have surface nanofeatures of ∼100 nm in height. The master, template, and replica surfaces showed a >∼94% similarity based on the measured diameter and height of the nanofeatures. The versatility of DT-NIL was also demonstrated with the replication of re-entrant, multiscale, and hierarchical features on fly wings, as well as hard silicon wafer-based artificial nanostructures. The DT-NIL method can be performed under ambient conditions with inexpensive materials and equipment. Our work opens the door to opportunities for economical and high-throughput nanofabrication processes.

Latching of the click beetle (Coleoptera: Elateridae) thoracic hinge enabled by the morphology and mechanics of conformal structures.

Elaterid beetles have evolved to 'click' their bodies in a unique maneuver. When this maneuver is initiated from a stationary position on a solid substrate, it results in a jump not carried out by the traditional means of jointed appendages (i.e. legs). Elaterid beetles belong to a group of organisms that amplify muscle power through morphology to produce extremely fast movements. Elaterids achieve power amplifications through a hinge situated in the thoracic region. The actuating components of the hinge are a peg and mesosternal lip, two conformal parts that latch to keep the body in a brace position until their release, the 'click', that is the fast launch maneuver. Although prior studies have identified this mechanism, they were focused on the ballistics of the launched body or limited to a single species. In this work, we identify specific morphological details of the hinges of four click beetle species - Alaus oculatus, Parallelostethus attenuatus, Lacon discoideus and Melanotus spp. - which vary in overall length from 11.3 to 38.8 mm. Measurements from environmental scanning electron microscopy (ESEM) and computerized tomography (CT) were combined to provide comparative structural information on both exterior and interior features of the peg and mesosternal lip. Specifically, ESEM and CT reveal the morphology of the peg, which is modeled as an Euler-Bernoulli beam. In the model, the externally applied force is estimated using a micromechanical experiment. The equivalent stiffness, defined as the ratio between the applied force and the peg tip deflection, is estimated for all four species. The estimated peg tip deformation indicates that, under the applied forces, the peg is able to maintain the braced position of the hinge. This work comprehensively describes the critical function of the hinge anatomy through an integration of specific anatomical architecture and engineering mechanics for the first time.

Ultrascalable Multifunctional Nanoengineered Copper and Aluminum for Antiadhesion and Bactericidal Applications.

Biofouling disrupts the surface functionality and integrity of engineered substrates. A variety of natural materials such as plant leaves and insect wings have evolved sophisticated physical mechanisms capable of preventing biofouling. Over the past decade, several reports have pinpointed nanoscale surface topography as an important regulator of surface adhesion and growth of bacteria. Although artificial nanoengineered features have been used to create bactericidal materials that kill adhered bacteria, functional surfaces capable of synergistically providing antiadhesion and bactericidal properties remain to be developed. Furthermore, fundamental questions pertaining to the need for intrinsic hydrophobicity to achieve bactericidal performance and the role of structure length scale (nano vs micro) are still being explored. Here, we demonstrate highly scalable, cost-effective, and efficient nanoengineered multifunctional surfaces that possess both antiadhesion and bactericidal properties on industrially relevant copper (Cu) and aluminum (Al) substrates. We characterize antiadhesion and bactericidal performance using a combination of scanning electron microscopy (SEM), atomic force microscopy (AFM), live/dead bacterial staining and imaging, as well as solution-phase and Petrifilm measurements of bacterial viability. Our results showed that nanostructures created on both Cu and Al were capable of physical deformation of adhered Escherichia coli bacteria. Bacterial viability measurements on both Cu and Al indicated a complex interaction between the antiadhesion and bactericidal nature of these materials and their surface topography, chemistry, and structure. Increased superhydrophobicity greatly decreased bacterial adhesion while not significantly influencing surface bactericidal performance. Furthermore, we observed that more densely packed nanoscale structures improved antiadhesion properties when compared to larger features, even over extended time scales of up to 24 h. Our data suggests that the superhydrophobic Al substrate possesses superior antiadhesion and bactericidal effects, even over long time courses. The techniques and insights presented here will inform future work on antiadhesion and bactericidal multifunctional surfaces and enable their rational design.

A foreleg transcriptome for Ixodes scapularis ticks: Candidates for chemoreceptors and binding proteins that might be expressed in the sensory Haller's organ.

Little is known about the molecular basis for the olfactory capabilities of the sensory Haller's organ on the forelegs of ticks. We first expanded the known repertoire of Ionotropic Receptors (IRs), a variant lineage of the ionotropic glutamate receptors, encoded by the black-legged Ixodes scapularis genome from 15 to 125. We then undertook a transcriptome study of fore- and hind-legs of this tick in an effort to identify candidate chemoreceptors differentially expressed in forelegs as likely to be involved in Haller's organ functions. We primarily identified members of the IR family, specifically Ir25a and Ir93a, as highly and differentially expressed in forelegs. Several other IRs, as well as a few members of the gustatory receptor family, were expressed at low levels in forelegs and might contribute to the sensory function of Haller's organ. In addition, we identified eight small families of secreted proteins, with sets of conserved cysteines, which might function as binding proteins. The genes encoding these Microplusin-Like proteins and two previously described Odorant Binding Protein-Like proteins share a common exon-intron structure, suggesting that they all evolved from a common ancestor and represent an independent origin of binding proteins with potential roles comparable to the ChemoSensory Proteins and Odorant Binding Proteins of insects. We also found two Niemann-Pick Type C2 proteins with foreleg-biased expression, however we were unable to detect foreleg-biased expression of a G-Protein-Coupled pathway previously proposed to mediate olfaction in the tick Haller's organ.

Spatially resolved chemical analysis of cicada wings using laser-ablation electrospray ionization (LAESI) imaging mass spectrometry (IMS).

Laser-ablation electrospray ionization (LAESI) imaging mass spectrometry (IMS) is an emerging bioanalytical tool for direct imaging and analysis of biological tissues. Performing ionization in an ambient environment, this technique requires little sample preparation and no additional matrix, and can be performed on natural, uneven surfaces. When combined with optical microscopy, the investigation of biological samples by LAESI allows for spatially resolved compositional analysis. We demonstrate here the applicability of LAESI-IMS for the chemical analysis of thin, desiccated biological samples, specifically Neotibicen pruinosus cicada wings. Positive-ion LAESI-IMS accurate ion-map data was acquired from several wing cells and superimposed onto optical images allowing for compositional comparisons across areas of the wing. Various putative chemical identifications were made indicating the presence of hydrocarbons, lipids/esters, amines/amides, and sulfonated/phosphorylated compounds. With the spatial resolution capability, surprising chemical distribution patterns were observed across the cicada wing, which may assist in correlating trends in surface properties with chemical distribution. Observed ions were either (1) equally dispersed across the wing, (2) more concentrated closer to the body of the insect (proximal end), or (3) more concentrated toward the tip of the wing (distal end). These findings demonstrate LAESI-IMS as a tool for the acquisition of spatially resolved chemical information from fragile, dried insect wings. This LAESI-IMS technique has important implications for the study of functional biomaterials, where understanding the correlation between chemical composition, physical structure, and biological function is critical. Graphical abstract Positive-ion laser-ablation electrospray ionization mass spectrometry coupled with optical imaging provides a powerful tool for the spatially resolved chemical analysis of cicada wings.

Morphometric Analysis of Chemoreception Organ in Male and Female Ticks (Acari: Ixodidae).

The Haller's organ plays a crucial role in a tick's ability to detect hosts. Even though this sensory organ is vital to tick survival, the morphology of this organ is not well understood. The objective of this study was to characterize variation in the morphological components of the Haller's organ of three medically important tick species using quantitative methods. The Haller's organs of Ixodes scapularis Say (Ixodida: Ixodidae) (black-legged tick), Amblyomma americanum (L.) (Ixodida: Ixodidae) (lone star tick), and Dermacentor variabilis (Say) (Ixodida: Ixodidae) (American dog tick) were morphologically analyzed using environmental scanning electron microscopy and geometric morphometrics, and the results were statistically interpreted using canonical variate analysis. Our data reveal significant, quantitative differences in the morphology of the Haller's organ among all three tick species and that in D. variabilis the sensory structure is sexually dimorphic. Studies like this can serve as a quantitative basis for further studies on sensor physiology, behavior, and tick species life history, potentially leading to novel methods for the prevention of tick-borne disease.

Exploring the Role of Habitat on the Wettability of Cicada Wings.

Evolutionary pressure has pushed many extant species to develop micro/nanostructures that can significantly affect wettability and enable functionalities such as droplet jumping, self-cleaning, antifogging, antimicrobial, and antireflectivity. In particular, significant effort is underway to understand the insect wing surface structure to establish rational design tools for the development of novel engineered materials. Most studies, however, have focused on superhydrophobic wings obtained from a single insect species, in particular, the Psaltoda claripennis cicada. Here, we investigate the relationship between the spatially dependent wing wettability, topology, and droplet jumping behavior of multiple cicada species and their habitat, lifecycle, and interspecies relatedness. We focus on cicada wings of four different species: Neotibicen pruinosus, N. tibicen, Megatibicen dorsatus, and Magicicada septendecim and take a comparative approach. Using spatially resolved microgoniometry, scanning electron microscopy, atomic force microscopy, and high speed optical microscopy, we show that within cicada species, the wettability of wings is spatially homogeneous across wing cells. All four species were shown to have truncated conical pillars with widely varying length scales ranging from 50 to 400 nm in height. Comparison of the wettability revealed three cicada species with wings that are superhydrophobic (>150°) with low contact angle hysteresis (<5°), resulting in stable droplet jumping behavior. The fourth, more distantly related species (Ma. septendecim) showed only moderate hydrophobic behavior, eliminating some of the beneficial surface functional aspects for this cicada. Correlation between cicada habitat and wing wettability yielded little connection as wetter, swampy environments do not necessarily equate to higher measured wing hydrophobicity. The results, however, do point to species relatedness and reproductive strategy as a closer proxy for predicting wettability and surface structure and resultant enhanced wing surface functionality. This work not only elucidates the differences between inter- and intraspecies cicada wing topology, wettability, and water shedding behavior but also enables the development of rational design tools for the manufacture of artificial surfaces for energy and water applications.

Influence of oviposition experience on multiparasitism by Pimpla disparis Vierick and Itoplectis conquisitor Say (Hymenoptera: Ichneumonidae).

We evaluated the potential for competition between the exotic ichneumonid parasitoid Pimpla disparis Vierick and the native ichneumonid Itoplectis conquisitor Say, in the form of multiparasitism and destructive host feeding, by examining how previous oviposition experience influenced host selection. Both species commonly attack the host species, bagworm, Thyridopteryx ephemeraeformis (Haworth) (Lepidoptera: Psychidae), in central Illinois. We used in our study adult female parasitoids that were naïve, had previously oviposited into hosts that contained heterospecifics, or had oviposited into hosts that initially were unparasitized. Naïve parasitoids of both species were disinclined to oviposit into hosts that already were parasitized by heterospecific larvae, suggesting that female parasitoids could detect the larvae. However, parasitoids with prior oviposition experience were less selective and oviposited into hosts that already were parasitized and unparasitized hosts. Female P. disparis and I. conquisitor probed parasitized hosts more frequently than unparasitized hosts. Adult female parasitoids of both species rarely directly fed on hosts, but those that did preferred to feed on hosts that already were parasitized. For both parasitoid species, the first larva to colonize a multiparasitized host was the most likely to survive to adulthood.

Quantification and development of teratocytes in novel-association host-parasitoid combinations.

We studied the development of teratocytes derived from two congeneric gregarious endoparasitic species, Cotesia chilonis and C. flavipes, parasitizing two congeneric novel hosts, Diatraea saccharalis and D. grandiosella. The host-parasitoid combinations studied allowed us to investigate relationships between host suitability and teratocyte development. D. saccharalis was a suitable host for both parasitoids, whereas D. grandiosella was suitable for C. chilonis development but often encapsulated C. flavipes progeny. Encapsulation of C. flavipes by D. grandiosella commenced around the time of parasitoid egg hatch, when teratocytes were released into the host's hemolymph. The gregarious parasitoids studied here released about 200 teratocytes per egg. Both absolute and normalized (teratocytes/parasitoid) numbers decreased over time. D. saccharalis supported more C. flavipes-derived teratocytes than D. grandiosella, possibly because of the unsuitability of the latter host. On intermediate assay days the number of C. flavipes-derived teratocytes was greater than for C. chilonis. However, C. chilonis-derived teratocytes grew larger than C. flavipes. Teratocytes in all host-parasitoid combinations doubled in size during parasitoid development. Teratocytes generally grew larger in D. grandiosella, which was a less suitable host.