Complex hemolymph circulation patterns in grasshopper wings.

An insect's living systems-circulation, respiration, and a branching nervous system-extend from the body into the wing. Wing hemolymph circulation is critical for hydrating tissues and supplying nutrients to living systems such as sensory organs across the wing. Despite the critical role of hemolymph circulation in maintaining healthy wing function, wings are often considered "lifeless" cuticle, and flows remain largely unquantified. High-speed fluorescent microscopy and particle tracking of hemolymph in the wings and body of the grasshopper Schistocerca americana revealed dynamic flow in every vein of the fore- and hindwings. The global system forms a circuit, but local flow behavior is complex, exhibiting three distinct types: pulsatile, aperiodic, and "leaky" flow. Thoracic wing hearts pull hemolymph from the wing at slower frequencies than the dorsal vessel; however, the velocity of returning hemolymph (in the hindwing) is faster than in that of the dorsal vessel. To characterize the wing's internal flow mechanics, we mapped dimensionless flow parameters across the wings, revealing viscous flow regimes. Wings sustain ecologically important insect behaviors such as pollination and migration. Analysis of the wing circulatory system provides a template for future studies investigating the critical hemodynamics necessary to sustaining wing health and insect flight.

High level of novelty under the hood of convergent evolution.

Little is known about the extent to which species use homologous regulatory architectures to achieve phenotypic convergence. By characterizing chromatin accessibility and gene expression in developing wing tissues, we compared the regulatory architecture of convergence between a pair of mimetic butterfly species. Although a handful of color pattern genes are known to be involved in their convergence, our data suggest that different mutational paths underlie the integration of these genes into wing pattern development. This is supported by a large fraction of accessible chromatin being exclusive to each species, including the de novo lineage-specific evolution of a modular optix enhancer. These findings may be explained by a high level of developmental drift and evolutionary contingency that occurs during the independent evolution of mimicry.

A novel gene REPTOR2 activates the autophagic degradation of wing disc in pea aphid.

Wing dimorphism in insects is an evolutionarily adaptive trait to maximize insect fitness under various environments, by which the population could be balanced between dispersing and reproduction. Most studies concern the regulatory mechanisms underlying the stimulation of wing morph in aphids, but relatively little research addresses the molecular basis of wing loss. Here, we found that, while developing normally in winged-destined pea aphids, the wing disc in wingless-destined aphids degenerated 30-hr postbirth and that this degeneration was due to autophagy rather than apoptosis. Activation of autophagy in first instar nymphs reduced the proportion of winged aphids, and suppression of autophagy increased the proportion. REPTOR2, associated with TOR signaling pathway, was identified by RNA-seq as a differentially expressed gene between the two morphs with higher expression in the thorax of wingless-destined aphids. Further genetic analysis indicated that REPTOR2 could be a novel gene derived from a gene duplication event that occurred exclusively in pea aphids on autosome A1 but translocated to the sex chromosome. Knockdown of REPTOR2 reduced autophagy in the wing disc and increased the proportion of winged aphids. In agreement with REPTOR's canonical negative regulatory role of TOR on autophagy, winged-destined aphids had higher TOR expression in the wing disc. Suppression of TOR activated autophagy of the wing disc and decreased the proportion of winged aphids, and vice versa. Co-suppression of TOR and REPTOR2 showed that dsREPTOR2 could mask the positive effect of dsTOR on autophagy, suggesting that REPTOR2 acted as a key regulator downstream of TOR in the signaling pathway. These results revealed that the TOR signaling pathway suppressed autophagic degradation of the wing disc in pea aphids by negatively regulating the expression of REPTOR2.

Salivary carbonic anhydrase II in winged aphid morph facilitates plant infection by viruses.

Aphids are the most common insect vector transmitting hundreds of plant viruses. Aphid wing dimorphism (winged vs. wingless) not only showcases the phenotypic plasticity but also impacts virus transmission; however, the superiority of winged aphids in virus transmission over the wingless morph is not well understood. Here, we show that plant viruses were efficiently transmitted and highly infectious when associated with the winged morph of Myzus persicae and that a salivary protein contributed to this difference. The carbonic anhydrase II (CA-II) gene was identified by RNA-seq of salivary glands to have higher expression in the winged morph. Aphids secreted CA-II into the apoplastic region of plant cells, leading to elevated accumulation of H+. Apoplastic acidification further increased the activities of polygalacturonases, the cell wall homogalacturonan (HG)-modifying enzymes, promoting degradation of demethylesterified HGs. In response to apoplastic acidification, plants accelerated vesicle trafficking to enhance pectin transport and strengthen the cell wall, which also facilitated virus translocation from the endomembrane system to the apoplast. Secretion of a higher quantity of salivary CA-II by winged aphids promoted intercellular vesicle transport in the plant. The higher vesicle trafficking induced by winged aphids enhanced dispersal of virus particles from infected cells to neighboring cells, thus resulting in higher virus infection in plants relative to the wingless morph. These findings imply that the difference in the expression of salivary CA-II between winged and wingless morphs is correlated with the vector role of aphids during the posttransmission infection process, which influences the outcome of plant endurance of virus infection.

Nonuniform structural properties of wings confer sensing advantages.

Sensory feedback is essential to both animals and robotic systems for achieving coordinated, precise movements. Mechanosensory feedback, which provides information about body deformation, depends not only on the properties of sensors but also on the structure in which they are embedded. In insects, wing structure plays a particularly important role in flapping flight: in addition to generating aerodynamic forces, wings provide mechanosensory feedback necessary for guiding flight while undergoing dramatic deformations during each wingbeat. However, the role that wing structure plays in determining mechanosensory information is relatively unexplored. Insect wings exhibit characteristic stiffness gradients and are subject to both aerodynamic and structural damping. Here we examine how both of these properties impact sensory performance, using finite element analysis combined with sensor placement optimization approaches. We show that wings with nonuniform stiffness exhibit several advantages over uniform stiffness wings, resulting in higher accuracy of rotation detection and lower sensitivity to the placement of sensors on the wing. Moreover, we show that higher damping generally improves the accuracy with which body rotations can be detected. These results contribute to our understanding of the evolution of the nonuniform stiffness patterns in insect wings, as well as suggest design principles for robotic systems.

Detection and measurement of butterfly eyespot and spot patterns using convolutional neural networks.

Butterflies are increasingly becoming model insects where basic questions surrounding the diversity of their color patterns are being investigated. Some of these color patterns consist of simple spots and eyespots. To accelerate the pace of research surrounding these discrete and circular pattern elements we trained distinct convolutional neural networks (CNNs) for detection and measurement of butterfly spots and eyespots on digital images of butterfly wings. We compared the automatically detected and segmented spot/eyespot areas with those manually annotated. These methods were able to identify and distinguish marginal eyespots from spots, as well as distinguish these patterns from less symmetrical patches of color. In addition, the measurements of an eyespot's central area and surrounding rings were comparable with the manual measurements. These CNNs offer improvements of eyespot/spot detection and measurements relative to previous methods because it is not necessary to mathematically define the feature of interest. All that is needed is to point out the images that have those features to train the CNN.

Flow development and leading edge vorticity in bristled insect wings.

Small flying insects such as the tiny thrip Gynaikothrips ficorum have wings with bristles attached to a solid shaft instead of solid membranes. Air passing through the bristle fringe, however, makes bristled insect wings less effective for aerodynamic force production. In this study, we quantified the ability of bristled wings to generate a leading edge vortex (LEV) for lift support during wing flapping, scored its circulation during wing translation, and investigated its behaviour at the stroke reversals. The data were measured in robotic model wings flapping with a generic kinematic pattern at Reynolds number of ~ 3.4, while applying two-dimensional particle image velocimetry. We found that aerodynamic performance due to LEV circulation linearly decreases with increasing bristle spacing. The wings of Gynaikothrips ficorum might thus produce approximately 9% less aerodynamic force for flight than a solid membranous wing. At the stroke reversals, leading and trailing edge vortices dissipate quickly within no more than ~ 2% of the stroke cycle duration. This elevated dissipation makes vortex shedding obsolete during the reversals and allows a quick build-up of counter-vorticity when the wing reverses flapping direction. In sum, our findings highlight the flow conditions associated with bristled wing design in insects and are thus significant for assessing biological fitness and dispersal of insects flying in a viscosity-dominated fluid regime.

Hedgehog is relayed through dynamic heparan sulfate interactions to shape its gradient.

Cellular differentiation is directly determined by concentration gradients of morphogens. As a central model for gradient formation during development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in Drosophila wing and eye discs. What is not known is how extracellular Hh spread is achieved and how it translates into precise gradients. Here we show that two separate binding areas located on opposite sides of the Hh molecule can interact directly and simultaneously with two heparan sulfate (HS) chains to temporarily cross-link the chains. Mutated Hh lacking one fully functional binding site still binds HS but shows reduced HS cross-linking. This, in turn, impairs Hhs ability to switch between both chains in vitro and results in striking Hh gradient hypomorphs in vivo. The speed and propensity of direct Hh switching between HS therefore shapes the Hh gradient, revealing a scalable design principle in morphogen-patterned tissues.

Wing kinematics measurement and aerodynamics of hovering droneflies with wing damage.

In this study, we performed successive unilateral and bilateral wing shearing to simulate wing damage in droneflies (Eristalis tenax) and measured the wing kinematics using high-speed photography technology. Two different shearing types were considered in the artificial wing damage. The aerodynamic force and power consumption were obtained by numerical method. Our major findings are the following. Different shearing methods have little influence on the kinematics, forces and energy consumption of insects. Following wing damage, among the potential strategies to adjust the three Euler angles of the wing, adjusting stroke angle (φ) in isolation, or combing the adjustment of stroke angle (φ) with pitch angle (ψ), contributed most to the change in vertical force. The balance of horizontal thrust can be restored by the adjustment of deviation angle (θ) under the condition of unilateral wing damage. Considering zero elastic energy storage, the mass-specific power (P1) increases significantly following wing damage. However, the increase in mass-specific power with 100% elastic energy storage (P2) is very small. The extra cost of the unilateral wing damage is that the energy consumption of the damaged wing and intact wing is highly asymmetrical when zero elastic energy storage is considered. The insects may alleviate the problems of increasing power consumption and asymmetric power distribution by storage and reuse of the negative inertial work of the wing.

Phenotypic variability in traits related to flight dispersal in the wing dimorphic species Triatoma guasayana.

BACKGROUND: Triatoma guasayana is considered an emerging vector of Chagas disease in the Southern Cone of South America. The presence of a triatomine population with brachypterous individuals, in which both wings are reduced, has recently been reported for this species. The aim of the present study was to determine if flight-related traits varied across populations, if these traits could explain differences in flight capacity across populations and if flight-related traits are associated with geographic and/or climatic variation. METHODS: The study involved 66 male T. guasayana specimens from 10 triatomine populations. Digital images of wing, head and pronotum were used to estimate linear and geometric morphometric variables. Variations in size and shape were analysed using one-way analysis of variance and canonical variate analysis (CVA), respectively. Mantel tests were applied to analyse the relationship between morphometric and geographic distances, and the association between size measurements was analysed using Pearson's correlation. We explored covariation between size and shape variables using partial least square analyses (PLS). The association of geographic and climatic variables with size measurements was tested using linear regression analyses. We performed PLS analyses for shape measurements. RESULTS: Wing size differed significantly across triatomine populations. The CVA showed that wing shape of the brachypterous population is well discriminated from that of the other populations. The Mantel test showed a positive and significant association between wing shape and geographic distances. The heads of the brachypterous population were significantly larger than those of the other populations. Similar to wing shape, the head shape of the brachypterous population was well discriminated from those of the other populations. Pronotum width did not show significant differences across populations. Geographic and climatic factors were associated with size and shape of both the wing and head, but not with pronotum width. CONCLUSIONS: Most of the traits related to flight dispersal varied across populations. Wing shape and head shape were found to be better markers for differentiated morphological variation across populations. Head measurements also varied in accordance with this condition. Geographic and climatic variables were associated with most of the flight-related traits.

Transgenerational inheritance of wing development defects in Drosophila melanogaster induced by cadmium.

The transgenerational inheritance of phenotype induced by environmental factors is a new focus in epigenetic research. In this study, Drosophila melanogaster (F0) was cultured in the medium containing cadmium (Cd, 4.5 mg/kg) from eggs to adults, and offspring (F1-F4) were continuously kept in standard medium (without cadmium). The phenotype analysis showed that cadmium induced developmental defects on wings and apoptosis in the wing disc cells of Drosophila (F0). The wing defects were transmitted for at least four generations even without Cd afterwards. And the effect on the mRNA expression of wing development related genes (shg, omb, F-actin, Mekk1) can be maintained for at least two or three generations. More importantly, under cadmium stress, the post-translational modification (PTM) on the histones H3K4me3 in the third instar larvae and ovaries or testes of adult flies increased significantly, while the levels of H3K9me3 and H3K27me3 decreased significantly. The expression of histone methylation related genes (dSet-1, ash1, Lsd1) increased significantly and these changes can be transmitted to offspring from one or two generations in ovaries or testes. These results suggest that the phenotypic defects of wings caused by cadmium can be inherited to the offspring, and this transgenerational inheritance effect may be related to the epigenetic regulation of histone methylation. Therefore, the adaptability of offspring should be considered when evaluating the toxicity and environmental risk of cadmium.

Proliferative and preparative cell divisions in wing discs of the last larval instar are regulated by different hormones and determine the size and differentiation of the wing of Bombyx mori.

Through investigating the two different enhanced cell division stages, we tried to clarify the switch from the growth to differentiation in the wing disc of the last larval instar of Bombyx mori. The response to insulin and 20E in vitro was stage specific. Bmmyc expression in V1 wing discs showed differences after being cultured with and without insulin. Bmmyc expression in V5 wing discs also showed differences after being cultured with and without 20E. Cell cycle-related genes, BmE2F1 and BmcycE, were upregulated with insulin or 20E in cultured wing discs of V1 or V5, respectively. Bmwnt1 and Bmras1 showed upregulation with 20E in cultured wing discs. Bmwnt1 showed upregulation with insulin in cultured wing discs, but Bmras1 did not show clear upregulation with insulin treatment. In contrast, Bmdpp showed upregulation with insulin, but did not show clear upregulation with 20E. The addition of PI3K or TOR inhibitors inhibited the upregulation of Bmmyc expression that was upregulated with insulin or 20E. The upregulation of Bmmyc and Bmwnt1 with insulin or 20E was inhibited with the addition of Myc or Wnt inhibitors, respectively. Genes related to matrix metalloprotease showed upregulation with 20E, and the upregulation was inhibited by the addition of Myc or Wnt inhibitors. From the present results, we concluded that cell division during the feeding stage occurred through PI3K/TOR cascade, and that at the wandering stage occurred through ecdysone and PI3K/TOR cascade; the former is for growth and the latter for differentiation.

Patterns of morphological variation highlight the effect of natural selection on eyespots modularity in the butterfly Morpho telemachus.

Morphological correlations can not only stem from developmental constraints but also from selective pressures. Butterfly eyespots are repeated wing color pattern elements, widespread across species. As developmental serial homologs, they are controlled by similar developmental pathways imposing correlations among eyespots: selection on a single eyespot may induce correlated responses in all eyespots. We study the variations in the ventral eyespots of Morpho telemachus, where two different selective regimes are likely to act: while most eyespots are always-visible, two eyespots are conditionally displayed: hidden at rest, they can be exposed when the butterflies are threatened, or during sexual interactions. We investigate how such contrasted selection across eyespots can alter the covariations imposed by their shared developmental origin. We quantified eyespots covariations within a large population of M. telemachus and compared the observed patterns to those found in M. helenor, where all eyespots are always-visible and thus probably affected by a similar selection regime. We found that M. telemachus conditionally displayed eyespots are less variable than always-visible eyespots and that these two eyespots form a separate variational module in this species, in contrast to M. helenor. Our results suggest that eyespots covariations were shaped by selection, highlighting how natural selection may promote the evolution of modularity.

The morphogen Hedgehog is essential for proper adult morphogenesis in Bombyx mori.

The well-known morphogen Hedgehog (Hh) is indispensable for embryo patterning and organ development from invertebrates to vertebrates. The role of Hh signaling pathway has been extensively investigated in the model organism Drosophila melanogaster, whereas its biological functions are still poorly understood in non-drosophilid insects. In the current study, we describe comprehensive investigation of Hh biological roles in the model lepidopteran insect Bombyx mori by using both CRISPR/Cas9-mediated gene ablation and Gal4/UAS-mediated ectopic expression. Direct injection of Cas9 protein and Hh-specific sgRNAs into preblastoderm embryos induced complete lethality. In contrast, Hh mutants obtained by the binary transgenic CRISPR/Cas9 system showed no deleterious phenotypes during embryonic and larval stages. However, mutants showed abnormalities from the pupal stage and most of adult body appendages exhibited severe developmental defects. Molecular analysis focused on wing development reveal that Hh signaling, Imd signaling and Wnt signaling pathways were distorted in Hh mutant wings. Ectopic expression by using the binary Gal4/UAS system induce early larval lethality. On contrary, moderate overexpression of Hh by using a unitary transgenic system resulted in severe defects in adult leg and antenna development. Our data directly provide genetic evidence that Hh plays vital roles in imaginal discs development and proper adult morphogenesis in B. mori.

The effect of dynamic twisting on the flow field and the unsteady forces of a heaving flat plate.

Many marine animals can dynamically twist their pectoral fins while swimming. The effects of such dynamic twisting on the unsteady forces on the fin and its surrounding flow field are yet to be understood in detail. In this paper, a flat plate executing a heaving maneuver is subjected to a similar dynamic twisting. In particular, the effects of the direction of twist, non-dimensional heaving amplitude, and reduced frequency are studied using a force sensor and particle image velocimetry (PIV) measurements. Two reduced frequencies,k=0.105, and0.209, and two twisting modes are investigated. In the first twisting mode, the plate is twisted in the direction of the heave (forward-twist), and in the second mode, the plate is twisted opposite to the direction of the heave (backward-twist). Force sensor measurements show that the forward-twist recovers some of the lift that is usually lost during the upstroke of flapping locomotion. Additionally, the forward-twist maintains a near-constant lift coefficient during the transition between downstroke and upstroke, suggesting a more stable form of locomotion. PIV results show that forward-twist limits circulation and leading-edge vortex growth during the downstroke, keepingCd≈0at the cost of the reduced lift. By contrast, backward-twist increases the circulation during the downstroke, resulting in large increases in both lift and drag coefficients. Force sensor data also showed that this effect on the lift is reversed during the upstroke, where the backward-twist causes a negative lift. The effects of each twisting mode are mainly caused by the changes in the shear layer velocity that occur as a result of twisting about the spanwise axis along the mid-chord. The twisting performed by forward-twist reduces the effective angle of attack through the upstroke and downstroke, resulting in a reduced shear layer velocity and lower circulation. The twisting performed by backward-twist does the exact opposite, increasing the effective angle of attack through the upstroke and downstroke and consequently increasing the shear layer velocity and circulation.

Transcriptomic exploration of the Coleopteran wings reveals insight into the evolution of novel structures associated with the beetle elytron.

The acquisition of novel traits is central to organismal evolution, yet the molecular mechanisms underlying this process are elusive. The beetle forewings (elytra) are evolutionarily modified to serve as a protective shield, providing a unique opportunity to study these mechanisms. In the past, the orthologs of genes within the wing gene network from Drosophila studies served as the starting point when studying the evolution of elytra (candidate genes). Although effective, candidate gene lists are finite and only explore genes conserved across species. To go beyond candidate genes, we used RNA sequencing and explored the wing transcriptomes of two Coleopteran species, the red flour beetle (Tribolium castaneum) and the Japanese stag beetle (Dorcus hopei). Our analysis revealed sets of genes enriched in Tribolium elytra (57 genes) and genes unique to the hindwings, which possess more "typical" insect wing morphologies (29 genes). Over a third of the hindwing-enriched genes were "candidate genes" whose functions were previously analyzed in Tribolium, demonstrating the robustness of our sequencing. Although the overlap was limited, transcriptomic comparison between the beetle species found a common set of genes, including key wing genes, enriched in either elytra or hindwings. Our RNA interference analysis for elytron-enriched genes in Tribolium uncovered novel genes with roles in forming various aspects of morphology that are unique to elytra, such as pigmentation, hardening, sensory development, and vein formation. Our analyses deepen our understanding of how gene network evolution facilitated the emergence of the elytron, a unique structure critical to the evolutionary success of beetles.

An at-scale tailless flapping wing hummingbird robot: II. Flight control in hovering and trajectory tracking.

Flight control such as stable hovering and trajectory tracking of tailless flapping-wing micro aerial vehicles is a challenging task. Given the constraint on actuation capability, flight control authority is limited beyond sufficient lift generation. In addition, the highly nonlinear and inherently unstable vehicle dynamics, unsteady aerodynamics, wing motion caused body oscillations, and mechanism asymmetries and imperfections due to fabrication process, all pose challenges to flight control. In this work, we propose a systematic onboard control method to address such challenges. In particular, with a systematic comparative study, a nonlinear flight controller incorporating parameter adaptation and robust control demonstrates the preferred performances. Such a controller is designed to address time-varying system uncertainty in flapping flight. The proposed controller is validated on a 12-g at-scale tailless hummingbird robot equipped with two actuators. Maneuver experiments have been successfully performed by the proposed hummingbird robot, including stable hovering, waypoint and trajectory tracking, and stabilization under severe wing asymmetries.

Experimental analysis of the sweepback angle effect on the thrust generation of a robotic penguin wing.

Penguins have evolved excellent swimming skills as diving birds, benefiting from their agile wings. This paper experimentally analyzes the effects of the wing sweepback angle on thrust generation using a robotic penguin wing. A developed wing mechanism that can realize penguin-like flapping and feathering motion was used for actuating five alternative wing models, with different sweepback angles ranging from 0° to 50°. Force measurements under a steady water flow were conducted for both fixed and flapping states for all wing models. The results showed that small sweepback angles of 30° or less in the fixed state caused a steep lift curve and a moderate sweepback angle of 30° produced the largest lift-to-drag ratio. In the flapping state, the smaller sweepback wings generated a larger net thrust for the same wing motion, whereas the larger-sweepback wings produced more thrust under the same Strouhal number. The findings also revealed that larger sweepback wings more easily achieve the maximum net thrust in terms of less angle-of-attack control. In contrast, the hydrodynamic efficiency was not greatly affected by the sweepback. Regardless of the sweepback, the trend of the efficiency increasing with increasing flow speed indicates that the penguin wings can be more suitable for high-speed locomotion for higher hydrodynamic efficiency.

Digest: Multiple selective pressures drive color diversity in moths.

How are color polymorphisms maintained within populations? De Pasqual et al. (2022) showed that female wood tiger moths (Arctia plantaginis) that were heterozygous for a wing color gene had higher fertility, hatching success, and offspring survival. These findings suggest that the complex interaction between heterozygote advantage and pleiotropic effects maintains color variability through a balance between multiple and competing selective pressures in wild populations.