Bioreplicated visual features of nanofabricated buprestid beetle decoys evoke stereotypical male mating flights

Recent advances in nanoscale bioreplication processes present the potential for novel basic and applied research into organismal behavioral processes. Insect behavior potentially could be affected by physical features existing at the nanoscale level. We used nano-bioreplicated visual decoys of female emerald ash borer beetles (Agrilus planipennis) to evoke stereotypical mate-finding behavior, whereby males fly to and alight on the decoys as they would on real females. Using an industrially scalable nanomolding process, we replicated and evaluated the importance of two features of the outer cuticular surface of the beetle’s wings: structural interference coloration of the elytra by multilayering of the epicuticle and fine-scale surface features consisting of spicules and spines that scatter light into intense strands. Two types of decoys that lacked one or both of these elements were fabricated, one type nano-bioreplicated and the other 3D-printed with no bioreplicated surface nanostructural elements. Both types were colored with green paint. The light-scattering properties of the nano-bioreplicated surfaces were verified by shining a white laser on the decoys in a dark room and projecting the scattering pattern onto a white surface. Regardless of the coloration mechanism, the nano-bioreplicated decoys evoked the complete attraction and landing sequence of Agrilus males. In contrast, males made brief flying approaches toward the decoys without nanostructured features, but diverted away before alighting on them. The nano-bioreplicated decoys were also electroconductive, a feature used on traps such that beetles alighting onto them were stunned, killed, and collected.Biomimicry of insect visual communication signals has received much recent attention, with growing interest in nanofabrication processes that result in artificially produced structural colors (1) such as those emanating from the ridges on butterfly wing scales (2). The fidelity of the nanoreplication of visual signals with communication value to such organisms has been underexplored, however. Visually induced behavior in arthropods often integrates color and edge-motion detection, with interactions often involving a variety of biotic and abiotic entities, making it difficult to reproduce experimentally (3).Bioreplication of visual signaling structures might be manipulated so as to provide insight into the mechanisms of such signaling processes; however, all currently known examples of bioreplicated nanostructures that have been created to affect behavior involve unicellular movements across particular textured environments (4–7), rather than directed to evoke responses of specialized sensory organs of more complex multicellular organisms. Bioreplicated structures emitting behaviorally effective visual cues also may be useful for such practical purposes as the monitoring and detection of pest species, but the communication efficacy of the bioreplica needs to be validated under field conditions using naturally occurring (i.e., wild) populations.In contrast, biomimicry of chemical signals, such as insect pheromones, has been a burgeoning field for more than half a century. Synthetically reproduced pheromones have been successfully applied under field conditions to manipulate insect behavior for invasive species pest detection, population monitoring of endemic species, and disruption of mating. Thousands of studies have described the essential components of nanoscale levels (nanograms) of semiochemical signals that trigger behavioral responses, such as upwind flight for mating (8), alarm responses (9), and trail following (10). Furthermore, neurophysiological techniques have elucidated how these signals are transduced by peripheral sensory organs (11) and integrated into odor sensations in the higher processing centers of the insect brain (12). In the realm of applied science, these insights have led to trapping protocols for pest population detection, attract-and-kill protocols, and mating disruption (13). Visually attractive features of trapping technologies generally have not been approached with such rigor, however, and are usually optimized by simple manipulations of trap colors without efforts to understand the underlying mechanisms of visual attraction.In an effort to initiate such an approach to manipulation of visual signaling systems, we used an industrially scalable nano-bioreplication technique (14) to produce high-fidelity replicas of the structural features of the cuticle of the hard wing covers (elytra) of an invasive buprestid beetle pest, the emerald ash borer (Agrilus planipennis). This species is a tree-killing pest of Asian origin whose visual signal is emitted by the elytra of a female at rest on an ash leaf in direct sunlight, which triggers attraction of flying males that are patrolling the canopy. Male responses unfold as rapid flights toward the females from heights of up to 2 m, usually terminating with the males alighting directly on the females and attempting to copulate (15). This “paratrooper” descent behavior by flying A. planipennis males in the field can be repeatedly evoked by affixing dead A. planipennis females to ash leaves (15, 16). Furthermore, various other potentially invasive European and North American tree-feeding Agrilus species have been observed performing similar stereotypical inflight descents onto dead beetle decoys affixed to the leaves of preferred host trees (17, 18). Such approaches are often seen to congeneric, heterospecific targets. One such species, the two-spotted oak borer, Agrilus biguttatus, that is similar in size and habits to A. planipennis is known to kill oak trees within its native range in Europe (19), particularly after drought (20) or defoliation events (21).The base colors of many metallic-colored beetles, including buprestid beetles (Fig. 1A), are known to be structurally produced by the repeated alternation of cuticle layers (Fig. 1D) with different refractive indices (22, 23). This periodically multilayered assemblage functions as a quarter-wave Bragg stack reflector in a particular spectral regime (2) and is thus highly effective for creating a color of narrow specificity in sunlight, unlike many naturally occurring pigments. The reflected light is also affected by regular fine-scale topographic features of the surface, including thousands of sharp spicules each emitting green to yellow colors, which are further scattered by numerous spines (Fig. 1 B and C). Many of the physical attributes of the A. planipennis cuticle that produce its attractive visual signal have been replicated by a process that involves the stamping of a polymer quarter-wave Bragg stack reflector with a set of dies cast from the actual elytra of a female A. planipennis (Fig. 2) (14).Open in a separate windowFig. 1.Structural color and surface topography of A. planipennis wings. (A) Optical microscopy showing a dorsal view of the beetle elytron. (B) Higher-magnification optical microscopy showing spines and cilia. (C) Scanning electron micrograph showing a higher-resolution image of the surface topography. (D) Transmission electron micrograph of a cross-section of an elytron, showing four alternating layers of differing refractive indices. (C and D are reprinted with permission from ref. 14.)Open in a separate windowFig. 2.Nano-bioreplicated decoy characteristics. (A) Optical microscopy of the nickel die. (B) Scanning electron micrograph of a nickel die used for bioreplication, showing a similar structure as the A. planipennis surface (Fig. 1), but without the cilia. (C) Optical microscopy of the dorsal view of a nano-bioreplicated A. planipennis decoy that reproduces the surface structure of the beetle and is colored by metallic paint. (A and B are reprinted with permission from ref. 14.)Here we report on direct field observations of A. planipennis and A. biguttatus male behavior toward natural beetle decoys versus three types of synthetic decoys with varying degrees of verisimilitude with respect to the fidelity of bioreplication. These synthetic decoys included: (i) a bioreplicated decoy created by a nanomolding process and colored with a polymer functioning as a Bragg reflector; (ii) another bioreplicated decoy created by a nanomolding process and colored with a metallic green paint; and (iii) a 3D-printed decoy consisting of a smooth polymer surface without a nanomolded bioreplicated surface structure, also colored with green metallic paint. We investigated whether the nanomolding process could create light-scattering patterns similar to those of real decoys by observing light emissions resulting from the application of a white laser to the surfaces of real and synthetic decoys in a dark room. We hypothesized that a sufficient degree of verisimilitude with respect to color and fine-scale topological features of the elytra could be achieved through the bioreplication process to elicit inflight mating approaches and landings similar to those evoked by real beetles. We also incorporated the bioreplicated decoys into a trapping system in which the electroconductive properties of the decoy are used to electrocute male beetles when they approach and alight on the decoys.