Non-invasive characterization of the elastic protein resilin in insects using Raman spectroscopy.

Resilin is an extremely efficient elastic protein found in the moving parts of insects. Despite many years of resilin research, we are still only just starting to understand its diversity, native structures, and functions. Understanding differences in resilin structure and diversity could lead to the development of bioinspired elastic polymers, with broad applications in materials science. Here, to better understand resilin structure, we offer a novel methodology for identifying resilin-rich regions of the insect cuticle using non-invasive Raman spectroscopy in a model species, the desert locust (Schistocerca gregaria). The Raman spectrum of the resilin-rich semilunar process of the hind leg was compared with that of nearby low-resilin cuticle, and reference spectra and peaks assigned for these two regions. The main peaks of resilin include two bands associated with tyrosine at 955-962 and 1141-1203 cm-1 and a strong peak at 1615 cm-1, attributed to the α-Amide I group associated with dityrosine. We also found the chitin skeletal modes at ~485-567 cm-1 to be significant contributors to spectra variance between the groups. Raman spectra were also compared to results obtained by fluorescence spectroscopy, as a control technique. Principal component analysis of these resulting spectra revealed differences in the light-scattering properties of resilin-rich and resilin-poor cuticular regions, which may relate to differences in native protein structure and relative abundance.

Quantification of bush-cricket acoustic trachea mechanics using Atomic Force Microscopy nanoindentation.

Derived from the respiratory tracheae, bush-crickets' acoustic tracheae (or ear canals) are hollow tubes evolved to transmit sounds from the external environment to the interior ear. Due to the location of the ears in the forelegs, the acoustic trachea serves as a structural element that can withstand large stresses during locomotion. In this study, we report a new Atomic Force Microscopy Force Spectroscopy (AFM-FS) approach to quantify the mechanics of taenidia in the bush-cricket Mecopoda elongata. Mechanical properties were examined over the longitudinal axis of hydrated taenidia, by indenting single fibres using precision hyperbolic tips. Analysis of the force-displacement (F-d) extension curves at low strains using the Hertzian contact model showed an Elastic modulus distribution between 13.9 MPa to 26.5 GPa, with a mean of 5.2 ± 7 GPa and median 1.03 GPa. Although chitin is the primary component of stiffness, variation of elasticity in the nanoscale suggests that resilin significantly affects the mechanical properties of single taenidia fibres (38% of total data). For indentations up to 400 nm, an intricate chitin-resilin response was observed, suggesting structural optimization between compliance and rigidity. Finite-element analysis on composite materials demonstrated that the Elastic modulus is sensitive to the percentage of resilin and chitin content, their location and structural configuration. Based on our results, we propose that the distinct moduli of taenidia fibres indicate sophisticated evolution with elasticity playing a key role in optimization. STATEMENT OF SIGNIFICANCE: In crickets and bush-crickets, the foreleg tracheae have evolved into acoustic canals, which transport sound to the ears located on the tibia of each leg. Tracheae are held open by spiral cuticular micro-fibres called taenidia, which are the primary elements of mechanical reinforcement. We developed an AFM-based method to indent individual taenidia at the nanometre level, to quantify local mechanical properties of the interior acoustic canal of the bush-cricket Mecopoda elongata, a model species in hearing research. Taenidia fibres were immobilized on a hard substrate and the indenter directly approached the epicuticle surface. This is the first characterization of the nano-structure of unfixed tracheal taenidia, and should pave the way for further in vivo mechanical investigations of auditory structures.

Ear pinnae in a neotropical katydid (Orthoptera: Tettigoniidae) function as ultrasound guides for bat detection.

Early predator detection is a key component of the predator-prey arms race and has driven the evolution of multiple animal hearing systems. Katydids (Insecta) have sophisticated ears, each consisting of paired tympana on each foreleg that receive sound both externally, through the air, and internally via a narrowing ear canal running through the leg from an acoustic spiracle on the thorax. These ears are pressure-time difference receivers capable of sensitive and accurate directional hearing across a wide frequency range. Many katydid species have cuticular pinnae which form cavities around the outer tympanal surfaces, but their function is unknown. We investigated pinnal function in the katydid Copiphora gorgonensis by combining experimental biophysics and numerical modelling using 3D ear geometries. We found that the pinnae in C. gorgonensis do not assist in directional hearing for conspecific call frequencies, but instead act as ultrasound detectors. Pinnae induced large sound pressure gains (20-30 dB) that enhanced sound detection at high ultrasonic frequencies (>60 kHz), matching the echolocation range of co-occurring insectivorous gleaning bats. These findings were supported by behavioural and neural audiograms and pinnal cavity resonances from live specimens, and comparisons with the pinnal mechanics of sympatric katydid species, which together suggest that katydid pinnae primarily evolved for the enhanced detection of predatory bats.

Sexual repurposing of juvenile aposematism in locusts.

Adaptive plasticity requires an integrated suite of functional responses to environmental variation, which can include social communication across life stages. Desert locusts (Schistocerca gregaria) exhibit an extreme example of phenotypic plasticity called phase polyphenism, in which a suite of behavioral and morphological traits differ according to local population density. Male and female juveniles developing at low population densities exhibit green- or sand-colored background-matching camouflage, while at high densities they show contrasting yellow and black aposematic patterning that deters predators. The predominant background colors of these phenotypes (green/sand/yellow) all depend on expression of the carotenoid-binding "Yellow Protein" (YP). Gregarious (high-density) adults of both sexes are initially pinkish, before a YP-mediated yellowing reoccurs upon sexual maturation. Yellow color is especially prominent in gregarious males, but the reason for this difference has been unknown since phase polyphenism was first described in 1921. Here, we use RNA interference to show that gregarious male yellowing acts as an intrasexual warning signal, which forms a multimodal signal with the antiaphrodisiac pheromone phenylacetonitrile (PAN) to prevent mistaken sexual harassment from other males during scramble mating in a swarm. Socially mediated reexpression of YP thus adaptively repurposes a juvenile signal that deters predators into an adult signal that deters undesirable mates. These findings reveal a previously underappreciated sexual dimension to locust phase polyphenism, and promote locusts as a model for investigating the relative contributions of natural versus sexual selection in the evolution of phenotypic plasticity.

Oxytocin/vasopressin-like neuropeptide signaling in insects.

The origin of the oxytocin (OT)/vasopressin (VP) signaling system is thought to date back more than 600million years. OT/VP-like peptides have been identified in numerous invertebrate phyla including molluscs, annelids, nematodes and insects. However, to date we only have a limited understanding of the biological role(s) of this GPCR-mediated signaling system in insects. This chapter presents the current knowledge of OT/VP-like neuropeptide signaling in insects by providing a brief overview of insect OT/VP-like neuropeptides, their genetic and structural commonalities, and their experimentally tested and proposed functions. Despite their widespread occurrence across insect orders these peptides (and their endogenous receptors) appear to be absent in common insect model species, such as flies and bees. We therefore explain the known functionalities of this signaling system in three different insect model systems: beetles, locusts, and ants. Additionally, we review the phylogenetic distribution of the OT/VP signaling system in arthropods as obtained from extensive genome/transcriptome mining. Finally, we discuss the unique challenges in the development of selective OT/VP ligands for human receptors and share our perspective on the possible application of insect- and other non-mammalian-derived OT/VP-like peptide ligands in pharmacology.

Increased muscular volume and cuticular specialisations enhance jump velocity in solitarious compared with gregarious desert locusts, Schistocerca gregaria.

The desert locust, Schistocerca gregaria, shows a strong phenotypic plasticity. It can develop, depending upon population density, into either a solitarious or gregarious phase that differs in many aspects of behaviour, physiology and morphology. Prominent amongst these differences is that solitarious locusts have proportionately longer hind femora than gregarious locusts. The hind femora contain the muscles and energy-storing cuticular structures that propel powerful jumps using a catapult-like mechanism. We show that solitarious locusts jump on average 23% faster and 27% further than gregarious locusts, and attribute this improved performance to three sources: first, a 17.5% increase in the relative volume of their hind femur, and hence muscle volume; second, a 24.3% decrease in the stiffness of the energy-storing semi-lunar processes of the distal femur; and third, a 4.5% decrease in the stiffness of the tendon of the extensor tibiae muscle. These differences mean that solitarious locusts can generate more power and store more energy in preparation for a jump than can gregarious locusts. This improved performance comes at a cost: solitarious locusts expend nearly twice the energy of gregarious locusts during a single jump and the muscular co-contraction that energises the cuticular springs takes twice as long. There is thus a trade-off between achieving maximum jump velocity in the solitarious phase against the ability to engage jumping rapidly and repeatedly in the gregarious phase.

Pollen feeding proteomics: Salivary proteins of the passion flower butterfly, Heliconius melpomene.

While most adult Lepidoptera use flower nectar as their primary food source, butterflies in the genus Heliconius have evolved the novel ability to acquire amino acids from consuming pollen. Heliconius butterflies collect pollen on their proboscis, moisten the pollen with saliva, and use a combination of mechanical disruption and chemical degradation to release free amino acids that are subsequently re-ingested in the saliva. Little is known about the molecular mechanisms of this complex pollen feeding adaptation. Here we report an initial shotgun proteomic analysis of saliva from Heliconius melpomene. Results from liquid-chromatography tandem mass-spectrometry confidently identified 31 salivary proteins, most of which contained predicted signal peptides, consistent with extracellular secretion. Further bioinformatic annotation of these salivary proteins indicated the presence of four distinct functional classes: proteolysis (10 proteins), carbohydrate hydrolysis (5), immunity (6), and "housekeeping" (4). Additionally, six proteins could not be functionally annotated beyond containing a predicted signal sequence. The presence of several salivary proteases is consistent with previous demonstrations that Heliconius saliva has proteolytic capacity. It is likely that these proteins play a key role in generating free amino acids during pollen digestion. The identification of proteins functioning in carbohydrate hydrolysis is consistent with Heliconius butterflies consuming nectar, like other lepidopterans, as well as pollen. Immune-related proteins in saliva are also expected, given that ingestion of pathogens is a likely route to infection. The few "housekeeping" proteins are likely not true salivary proteins and reflect a modest level of contamination that occurred during saliva collection. Among the unannotated proteins were two sets of paralogs, each seemingly the result of a relatively recent tandem duplication. These results offer a first glimpse into the molecular foundation of Heliconius pollen feeding and provide a substantial advance towards comprehensively understanding this striking evolutionary novelty.

Mantises exchange angular momentum between three rotating body parts to jump precisely to targets.

Flightless animals have evolved diverse mechanisms to control their movements in air, whether falling with gravity or propelling against it. Many insects jump as a primary mode of locomotion and must therefore precisely control the large torques generated during takeoff. For example, to minimize spin (angular momentum of the body) at takeoff, plant-sucking bugs apply large equal and opposite torques from two propulsive legs [1]. Interacting gear wheels have evolved in some to give precise synchronization of these legs [2, 3]. Once airborne, as a result of either jumping or falling, further adjustments may be needed to control trajectory and orient the body for landing. Tails are used by geckos to control pitch [4, 5] and by Anolis lizards to alter direction [6, 7]. When falling, cats rotate their body [8], while aphids [9] and ants [10, 11] manipulate wind resistance against their legs and thorax. Falling is always downward, but targeted jumping must achieve many possible desired trajectories. We show that when making targeted jumps, juvenile wingless mantises first rotated their abdomen about the thorax to adjust the center of mass and thus regulate spin at takeoff. Once airborne, they then smoothly and sequentially transferred angular momentum in four stages between the jointed abdomen, the two raptorial front legs, and the two propulsive hind legs to produce a controlled jump with a precise landing. Experimentally impairing abdominal movements reduced the overall rotation so that the mantis either failed to grasp the target or crashed into it head first.

Rapid behavioural gregarization in the desert locust, Schistocerca gregaria entails synchronous changes in both activity and attraction to conspecifics.

Desert Locusts can change reversibly between solitarious and gregarious phases, which differ considerably in behaviour, morphology and physiology. The two phases show many behavioural differences including both overall levels of activity and the degree to which they are attracted or repulsed by conspecifics. Solitarious locusts perform infrequent bouts of locomotion characterised by a slow walking pace, groom infrequently and actively avoid other locusts. Gregarious locusts are highly active with a rapid walking pace, groom frequently and are attracted to conspecifics forming cohesive migratory bands as nymphs and/or flying swarms as adults. The sole factor driving the onset of gregarization is the presence of conspecifics. In several previous studies concerned with the mechanism underlying this transformation we have used an aggregate measure of behavioural phase state, Pgreg, derived from logistic regression analysis, which combines and weights several behavioural variables to characterise solitarious and gregarious behaviour. Using this approach we have analysed the time course of behavioural change, the stimuli that induce gregarization and the key role of serotonin in mediating the transformation. Following a recent critique that suggested that using Pgreg may confound changes in general activity with genuine gregarization we have performed a meta-analysis examining the time course of change in the individual behaviours that we use to generate Pgreg. We show that the forced crowding of solitarious locusts, tactile stimulation of the hind femora, and the short-term application of serotonin each induce concerted changes in not only locomotion-related variables but also grooming frequency and attraction to other locusts towards those characteristic of long-term gregarious locusts. This extensive meta-analysis supports and extends our previous conclusions that solitarious locusts undergo a rapid behavioural gregarization upon receiving appropriate stimulation for a few hours that is mediated by serotonin, at the end of which their behaviour is largely indistinguishable from locusts that have been in the gregarious phase their entire lives.

Behavioural phase change in the Australian plague locust, Chortoicetes terminifera, is triggered by tactile stimulation of the antennae.

Density-dependent phase polyphenism is a defining characteristic of the paraphyletic group of acridid grasshoppers known as locusts. The cues and mechanisms associated with crowding that induce behavioural gregarization are best understood in the desert locust, Schistocerca gregaria, and involve a combination of sensory inputs from the head (visual and olfactory) and mechanostimulation of the hind legs, acting via a transient increase in serotonin in the thoracic ganglia. Since behavioural gregarization has apparently arisen independently multiple times within the Acrididae, the important question arises as to whether the same mechanisms have been recruited each time. Here we explored the roles of visual, olfactory and tactile stimulation in the induction of behavioural gregarization in the Australian plague locust, Chortoicetes terminifera. We show that the primary gregarizing input is tactile stimulation of the antennae, with no evidence for an effect of visual and olfactory stimulation or tactile stimulation of the hind legs. Our results show that convergent behavioural responses to crowding have evolved employing different sites of sensory input in the Australian plague locust and the desert locust.