Flight loss linked to faster molecular evolution in insects

The loss of flight ability has occurred thousands of times independently during insect evolution. Flight loss may be linked to higher molecular evolutionary rates because of reductions in effective population sizes (N_e) and relaxed selective constraints. Reduced dispersal ability increases population subdivision, may decrease geographical range size and increases (sub)population extinction risk, thus leading to an expected reduction in N_e. Additionally, flight loss in birds has been linked to higher molecular rates of energy-related genes, probably owing to relaxed selective constraints on energy metabolism. We tested for an association between insect flight loss and molecular rates through comparative analysis in 49 phylogenetically independent transitions spanning multiple taxa, including moths, flies, beetles, mayflies, stick insects, stoneflies, scorpionflies and caddisflies, using available nuclear and mitochondrial protein-coding DNA sequences. We estimated the rate of molecular evolution of flightless (FL) and related flight-capable lineages by ratios of non-synonymous-to-synonymous substitutions (dN/dS) and overall substitution rates (OSRs). Across multiple instances of flight loss, we show a significant pattern of higher dN/dS ratios and OSRs in FL lineages in mitochondrial but not nuclear genes. These patterns may be explained by relaxed selective constraints in FL ectotherms relating to energy metabolism, possibly in combination with reduced N_e.     

How can Asphalt Roads Extend the Range of In Situ Polarized Light Pollution? A Complex Ecological Trap of <Emphasis Type="Italic">Ephemera danica</Emphasis> and a Possible Remedy

When an artificial surface (e.g. an asphalt road) reflects strongly and horizontally polarized light as water bodies do in the nature, polarotactic aquatic insects, like the creek-dwelling Ephemera danica mayflies easily become deceived. After swarming above the creek surface, E. danica females begin their upstream compensatory flight and can be deflected at bridges with an asphalt road and continue their flight above the road surface. Thus, the water-mimicking optical signal of the road may deceive water-seeking polarotactic mayflies and lead them to distant, polarized-light-polluting surfaces, which elicit anomalous oviposition. On an asphalt road crossing a creek, we deployed polarizing insect traps at different distances from the bridge. The traps captured E. danica mayflies and their catch numbers indicated that these mayflies originated from the direction of the bridge, proving that they followed the track of the road. Our results suggest that distant polarized-light-polluting objects along an asphalt road can trap mayflies emerging from a creek crossing the road. The combination of an asphalt road and a man-made in situ (local) polarizing surface forms a complex ecological trap, being capable of luring aquatic insects from greater distances. To eliminate the oviposition of dangered polarotactic aquatic insects emerging from a creek onto the asphalt road crossing the creek, we suggest to deploy strongly and horizontally polarizing water-filled black trays along the edge of the road during the swarming period. Thus, the eggs of the deceived insects can be moved back to the creek in order to assist the conservation of the offspring-generation.     

Bridges as optical barriers and population disruptors for the mayfly <Emphasis Type="Italic">Palingenia longicauda</Emphasis>: an overlooked threat to freshwater biodiversity?

Freshwater biodiversity is declining faster than marine or terrestrial diversity, yet its drivers are much less known. Although dams were shown to negatively affect river habitats, fragmentation by bridges has received less attention and is not as well understood. We tested whether and how bridges present barriers to aquatic insects by studying mass swarmings of Palingenia longicauda mayflies on river Tisza (NE-Hungary). Behavioural observations showed that upon approaching the bridge, upstream-flying mayflies typically turned back and 86% of them never crossed the bridge. Lack of physical contact showed that the bridge was an optical, rather than a mechanical barrier for the polarotactic mayflies. Imaging polarimetry showed that the bridge disrupted the horizontally polarizing channel guiding the flight of mayflies above the river. Energy loss, demonstrated by calorimetry, and time constraints forced females to lay eggs only downstream from the bridge. Counts of larval skins shed by swarming individuals showed nearly 2 to 1 female per male downstream from the bridge, while sex ratio above the bridge was slightly male-biased. We suggest that the surplus of parthenogenetic females, that produce only female larvae, downstream from the bridge may have led to the observed sex-ratio bias since the construction of the bridge (1942). Our results demonstrate that bridges can be optical barriers for aquatic insects and can cause population-level impacts, such as biased sex ratios, in natural populations. Sex ratio biases due to bridges may decrease effective population size and genetic variability, which may have contributed to the recent extinction of this species from most of Europe.     

Surface-skimming stoneflies and mayflies: the taxonomic and mechanical diversity of two-dimensional aerodynamic locomotion

The best supported hypothesis for the evolutionary origin of insect wings is that they evolved from articulated, leg-derived respiratory structures of aquatic ancestors. However, there are no fossils of the immediate ancestors of winged insects, and it is difficult to imagine how a functional transition from gills to wings could have occurred. Recent studies of surface-skimming locomotion in stoneflies and mayflies offer a plausible solution by showing how rudimentary wings and muscle power can be used to accomplish two-dimensional aerodynamic locomotion on the surface of water. Here we extend that line of research by examining the phylogenetic distribution and mechanistic diversity of surface skimming in stoneflies, along with a limited examination of mayflies. These investigations reveal both a broad taxonomic occurrence and a fine gradation of mechanically distinct forms. Distinct forms of wing-flapping surface skimming include (1) stoneflies that flap their wings weakly while maintaining their body in contact with the water and undulating their abdomen laterally in a swimming-like motion, (2) stoneflies that skim while elevating their body above the water and maintaining all six legs on the surface, (3) stoneflies and mayflies that skim with only four legs on the water surface, (4) stoneflies that skim with only their two hind legs on the surface, and (5) stoneflies that, beginning with a series of leg motions nearly identical to hind-leg skimmers, use their hind legs to jump from the water into the air to initiate flapping flight. Comparisons across these forms of skimming show that wing-beat amplitude, horizontal velocity, and the verticality of aerodynamic force production increase as the body orientation becomes more upright and contact with the water is minimized. These behaviors illustrate a mechanical pathway by which flying insects could have evolved from swimming ancestors via a series of finely graded intermediate stages. The phylogenetic distribution of skimming and flight in stoneflies does not indicate any clear directionality toward either greater or lesser aerodynamic abilities; however, the broad and apparently basal phylogenetic distribution of skimming taxa supports the hypothesis that the common ancestor of stoneflies was a surface skimmer. This may also be true for the common ancestor of stoneflies and mayflies, that is, the first winged insects. We combine these data with fossil evidence to form a synthetic model for the evolution of flying insects from surface skimmers.     

Lamp-Lit Bridges as Dual Light-Traps for the Night-Swarming Mayfly,Ephoron virgo: Interaction of Polarized and Unpolarized Light Pollution

Ecological photopollution created by artificial night lighting can alter animal behavior and lead to population declines and biodiversity loss. Polarized light pollution is a second type of photopollution that triggers water-seeking insects to ovisposit on smooth and dark man-made objects, because they simulate the polarization signatures of natural water bodies. We document a case study of the interaction of these two forms of photopollution by conducting observations and experiments near a lamp-lit bridge over the river Danube that attracts mass swarms of the mayfly Ephoron virgo away from the river to oviposit on the asphalt road of the bridge. Millions of mayflies swarmed near bridge-lights for two weeks. We found these swarms to be composed of 99% adult females performing their upstream compensatory flight and were attracted upward toward unpolarized bridge-lamp light, and away from the horizontally polarized light trail of the river. Imaging polarimetry confirmed that the asphalt surface of the bridge was strongly and horizontally polarized, providing a supernormal ovipositional cue to Ephoron virgo, while other parts of the bridge were poor polarizers of lamplight. Collectively, we confirm that Ephoron virgo is independently attracted to both unpolarized and polarized light sources, that both types of photopollution are being produced at the bridge, and that spatial patterns of swarming and oviposition are consistent with evolved behaviors being triggered maladaptively by these two types of light pollution. We suggest solutions to bridge and lighting design that should prevent or mitigate the impacts of such scenarios in the future. The detrimental impacts of such scenarios may extend beyond Ephoron virgo.     

Thermal biology of flight in a butterfly: genotype,flight metabolism,and environmental conditions

Knowledge of the effects of thermal conditions on animal movement and dispersal is necessary for a mechanistic understanding of the consequences of climate change and habitat fragmentation. In particular, the flight of ectothermic insects such as small butterflies is greatly influenced by ambient temperature. Here, variation in body temperature during flight is investigated in an ecological model species, the Glanville fritillary butterfly (Melitaea cinxia). Attention is paid on the effects of flight metabolism, genotypes at candidate loci, and environmental conditions. Measurements were made under a natural range of conditions using infrared thermal imaging. Heating of flight muscles by flight metabolism has been presumed to be negligible in small butterflies. However, the results demonstrate that Glanville fritillary males with high flight metabolic rate maintain elevated body temperature better during flight than males with a low rate of flight metabolism. This effect is likely to have a significant influence on the dispersal performance and fitness of butterflies and demonstrates the possible importance of intraspecific physiological variation on dispersal in other similar ectothermic insects. The results also suggest that individuals having an advantage in low ambient temperatures can be susceptible to overheating at high temperatures. Further, tolerance of high temperatures may be important for flight performance, as indicated by an association of heat‐shock protein (Hsp70) genotype with flight metabolic rate and body temperature at takeoff. The dynamics of body temperature at flight and factors affecting it also differed significantly between female and male butterflies, indicating that thermal dynamics are governed by different mechanisms in the two sexes. This study contributes to knowledge about factors affecting intraspecific variation in dispersal‐related thermal performance in butterflies and other insects. Such information is needed for predictive models of the evolution of dispersal in the face of habitat fragmentation and climate change.     

Critical Flight Time for Switch from Flight to Reproduction in the Wing Dimorphic Cricket Velarifictorus aspersus

Flight dimorphism has been considered to result from a balance of costs and benefits between flight capability and reproduction. The validity of the hypothesis, however, has been challenged recently. In this study, we examined the effect of flight time on trade-off between flight capability and reproductive development in Velarifictorus aspersus and we found that flight of 5 min did not promote reproductive development of long-winged (LW) adults, but flight of 30, 60, or 120 min could promote reproductive development both in female and male crickets. The results indicate that flight time may serve as a signal for LW V. aspersus to switch from migration to reproduction, and trade-off between flight ability and reproduction may be attenuated when flight time reaches a critical threshold. In addition, rapid reproductive development occurred before dealation when LW insects were allowed to fly for 30 min, which indicates that flight may influence reproductive development directly. Food consumption of short-winged adults was significantly higher than that of unflown LW adults or LW adults with 5 min flight, but similar to that of LW adults with 30, 60, or 120 min flight, suggesting that difference of reproductive development may be positively correlated with their food consumption.     

Electrical Stimulation of Coleopteran Muscle for Initiating Flight

Some researchers have long been interested in reconstructing natural insects into steerable robots or vehicles. However, until recently, these so-called cyborg insects, biobots, or living machines existed only in science fiction. Owing to recent advances in nano/micro manufacturing, data processing, and anatomical and physiological biology, we can now stimulate living insects to induce user-desired motor actions and behaviors. To improve the practicality and applicability of airborne cyborg insects, a reliable and controllable flight initiation protocol is required. This study demonstrates an electrical stimulation protocol that initiates flight in a beetle (Mecynorrhina torquata, Coleoptera). A reliable stimulation protocol was determined by analyzing a pair of dorsal longitudinal muscles (DLMs), flight muscles that oscillate the wings. DLM stimulation has achieved with a high success rate (> 90%), rapid response time (< 1.0 s), and small variation (< 0.33 s; indicating little habituation). Notably, the stimulation of DLMs caused no crucial damage to the free flight ability. In contrast, stimulation of optic lobes, which was earlier demonstrated as a successful flight initiation protocol, destabilized the beetle in flight. Thus, DLM stimulation is a promising secure protocol for inducing flight in cyborg insects or biobots.     

If a bird flies in the forest,does an insect hear it?

Birds are major predators of many eared insects including moths, butterflies, crickets and cicadas. We provide evidence supporting the hypothesis that insect ears can function as ‘bird detectors’. First, we show that birds produce flight sounds while foraging. Eastern phoebes (Sayornis phoebe) and chickadees (Poecile atricapillus) generate broadband sounds composed of distinct repetitive elements (approx. 18 and 20 Hz, respectively) that correspond to cyclic wing beating. We estimate that insects can detect an approaching bird from distances of at least 2.5 m, based on insect hearing thresholds and sound level measurements of bird flight. Second, we show that insects with both high and low frequency hearing can hear bird flight sounds. Auditory nerve cells of noctuid moths (Trichoplusia ni) and nymphalid butterflies (Morpho peleides) responded in a bursting pattern to playbacks of an attacking bird. This is the first study to demonstrate that foraging birds generate flight sound cues that are detectable by eared insects. Whether insects exploit these sound cues, and alternatively, if birds have evolved sound-reducing foraging tactics to render them acoustically ‘cryptic’ to their prey, are tantalizing questions worthy of further investigation.     

Water balance in Rhodnius prolixus during flight: Quantitative aspects of diuresis and its relation to changes in haemolymph and flight-muscle water

The amount of water voided by male Rhodnius prolixus which were flown to exhaustion varied from 0 to over 10% of the initial live weight. It accounted for nearly all of the body water lost during the flight period. Simultaneous measurements on the loss of haemolymph water and an estimate of the amount of faecal water in the excreta indicated that the source of the voided water was primarily the haemolymph. The total water content of the flight muscles changed very little in insects which flew to exhaustion. It is concluded that, despite the diuresis and loss of water, and the considerable reduction in haemolymph volume, dehydration of the flight muscles of male R. prolixus does not occur during these flight periods, and is not a factor contributing to ‘exhaustion’. The possibility that insufficient haemolymph is a factor limiting the duration of flight is discussed.     

Relationship between biomass and drift of river benthic invertebrates

In order to estimate the relationship between biomass and drift of benthic invertebrates (Gammarus lacustris Sars., larvae of mayflies and chironomids), the specific drift rate and the ratio of drift rate to their biomass has been used. The negative correlation between the specific drift rate and the biomass density has been obtained. It indicates that competition between hydrobionts for space and food is not important for the drift. It has been suggested that the decrease in the specific rate of amphipods and the increase in their biomass in bottom sediments are associated with the patterns of feeding and sexual behavior of crustaceans, and the larvae of insects undergo a similar decrease in their specific rate due to the formation of aggregative behavior in them for the mass emergence of imagoes.     

Essential fatty acid for the mosquito Culex pipiens: arachidonic acid.

A nutrient associated with animal-derived phospholipids has previously been found essential for newly-emerged adults of the mosquito Culex pipiens to fly and survive more than a few days. Pure arachidonic acid was completely effective in supporting the emergence of viable flying adults; in combination with synthetic dipalmitoyl lecithin, which slightly improves larval growth rate without inducing adult flight, it wholly adequately replaces animal phospholipids. Linoleic and linolenic acids, which have satisfied the needs of all insects hitherto shown to require an essential fatty acid, were ineffective for C. pipiens, with or without synthetic lecithin. An optimal effect on adult flight was obtained with 0.05 mg of arachidonic acid per 100 ml of dietary medium, a concentration much lower than the linoleic/linolenic concentrations needed by other insects with an essential fatty acid requirement. The relationship of this unique mosquito fatty acid requirement to the essential fatty acid needs of both vertebrates and insects in general is discussed.     

Flight duration and flight muscle ultrastructure of unfed hawk moths

Flight muscle breakdown has been reported for many orders of insects, but the basis of this breakdown in insects with lifelong dependence on flight is less clear. Lepidopterans show such muscle changes across their lifespans, yet how this change affects the ability of these insects to complete their life cycles is not well documented. We investigated the changes in muscle function and ultrastructure of unfed aging adult hawk moths (Manduca sexta). Flight duration was examined in young, middle-aged, and advanced-aged unfed moths. After measurement of flight duration, the main flight muscle (dorsolongitudinal muscle) was collected and histologically prepared for transmission electron microscopy to compare several measurements of muscle ultrastructure among moths of different ages. Muscle function assays revealed significant positive correlations between muscle ultrastructure and flight distance that were greatest in middle-aged moths and least in young moths. In addition, changes in flight muscle ultrastructure were detected across treatment groups. The number of mitochondria in muscle cells peaked in middle-aged moths. Many wild M. sexta do not feed as adults; thus, understanding the changes in flight capacity and muscle ultrastructure in unfed moths provides a more complete understanding of the ecophysiology and resource allocation strategies of this species.     

Evolutionary relationships among food habit, loss of flight, and reproductive traits: life-history evolution in the Silphinae (Coleoptera: Silphidae)

Flightlessness in insects is generally thought to have evolved due to changes in habitat environment or habitat isolation. Loss of flight may have changed reproductive traits in insects, but very few attempts have been made to assess evolutionary relationships between flight and reproductive traits in a group of related species. We elucidated the evolutionary history of flight loss and its relationship to evolution in food habit, relative reproductive investment, and egg size in the Silphinae (Coleoptera: Silphidae). Most flight-capable species in this group feed primarily on vertebrate carcasses, whereas flightless or flight-dimorphic species feed primarily on soil invertebrates. Ancestral state reconstruction based on our newly constructed molecular phylogenetic tree implied that flight muscle degeneration occurred twice in association with food habit changes from necrophagy to predatory, suggesting that flight loss could evolve independently from changes in the environmental circumstances per se. We found that total egg production increased with flight loss. We also found that egg size increased with decreased egg number following food habit changes in the lineage leading to predaceous species, suggesting that selection for larger larvae intensified with the food habit change. This correlated evolution has shaped diverse life-history patterns among extant species of Silphinae.     

The influence of predatory fish on mayfly drift: extrapolating from experiments to nature

1. A knowledge of how individual behaviour affects populations in nature is needed to understand many ecologically important processes, such as the dispersal of larval insects in streams. The influence of chemical cues from drift‐feeding fish on the drift dispersal of mayflies has been documented in small experimental channels (i.e. < 3 m), but their influence on dispersal in natural systems (e.g. 30 m stream reaches) is unclear. 2. Using surveys in 10 Rocky Mountain streams in Western Colorado we examined whether the effects of predatory brook trout (Salvelinus fontinalis) on mayfly drift, that were apparent in stream‐side channels, could also be detected in natural streams. 3. In channel experiments, the drift of Baetis bicaudatus (Baetidae) was more responsive to variation in the concentration of chemical cues from brook trout than that of another mayfly, Epeorus deceptivus (Heptageniidae). The rate of brook trout predation on drifting mayflies of both species in a 2‐m long observation tank was higher during the day (60–75%) but still measurable at night (5–10%). Epeorus individuals released into the water column were more vulnerable to trout predation by both day and night than were Baetis larvae treated similarly. 4. Drift of all mayfly taxa in five fishless streams was aperiodic, whereas their drift was nocturnal in five trout streams. The propensity of mayflies to drift was decreased during the day and increased during the night in trout streams compared with fishless streams. In contrast to the channel experiments, fish biomass and density did not alter the nocturnal nature nor magnitude of mayfly drift in natural streams. 5. In combination, these results indicate that mayflies respond to subtle differences in concentration of fish cues in experimental channels. However, temporal and spatial variation in fish cues available to mayflies in natural streams may have obscured our ability to detect responses at larger scales.     

Kinematic compensation for wing loss in flying damselflies

Flying insects can tolerate substantial wing wear before their ability to fly is entirely compromised. In order to keep flying with damaged wings, the entire flight apparatus needs to adjust its action to compensate for the reduced aerodynamic force and to balance the asymmetries in area and shape of the damaged wings. While several studies have shown that damaged wings change their flapping kinematics in response to partial loss of wing area, it is unclear how, in insects with four separate wings, the remaining three wings compensate for the loss of a fourth wing. We used high-speed video of flying blue-tailed damselflies (Ischnura elegans) to identify the wingbeat kinematics of the two wing pairs and compared it to the flapping kinematics after one of the hindwings was artificially removed. The insects remained capable of flying and precise maneuvering using only three wings. To compensate for the reduction in lift, they increased flapping frequency by 18 ± 15.4% on average. To achieve steady straight flight, the remaining intact hindwing reduced its flapping amplitude while the forewings changed their stroke plane angle so that the forewing of the manipulated side flapped at a shallower stroke plane angle. In addition, the angular position of the stroke reversal points became asymmetrical. When the wingbeat amplitude and frequency of the three wings were used as input in a simple aerodynamic model, the estimation of total aerodynamic force was not significantly different (paired t-test, p = 0.73) from the force produced by the four wings during normal flight. Thus, the removal of one wing resulted in adjustments of the motions of the remaining three wings, exemplifying the precision and plasticity of coordination between the operational wings. Such coordination is vital for precise maneuvering during normal flight but it also provides the means to maintain flight when some of the wings are severely damaged.     

Why do adult insects not moult?

Hypotheses are presented concerning why mayflies moult after functional wings develop and why most insects cease to moult at this time. The pattern of retention or loss of the subimaginal moult in extant mayflies suggests that this moult may be necessary to complete elongation of caudal filaments and forelegs of adults. It is then analogous to the pupal moult of holometabolous insects. I propose that selection for wing efficiency has normally confined the presence of functional wings to one instar only, which is also the last and reproductive stage. Selection for light wings has generally caused the epidermis of membranous insect wings to degenerate, thereby precluding otherwise advantageous adult moults.     

Natural variation in the regulation of neurodevelopmental genes modifies flight performance in Drosophila

The winged insects of the order Diptera are colloquially named for their most recognizable phenotype: flight. These insects rely on flight for a number of important life history traits, such as dispersal, foraging, and courtship. Despite the importance of flight, relatively little is known about the genetic architecture of flight performance. Accordingly, we sought to uncover the genetic modifiers of flight using a measure of flies’ reaction and response to an abrupt drop in a vertical flight column. We conducted a genome wide association study (GWAS) using 197 of the Drosophila Genetic Reference Panel (DGRP) lines, and identified a combination of additive and marginal variants, epistatic interactions, whole genes, and enrichment across interaction networks. Egfr, a highly pleiotropic developmental gene, was among the most significant additive variants identified. We functionally validated 13 of the additive candidate genes’ (Adgf-A/Adgf-A2/CG32181, bru1, CadN, flapper (CG11073), CG15236, flippy (CG9766), CREG, Dscam4, form3, fry, Lasp/CG9692, Pde6, Snoo), and introduce a novel approach to whole gene significance screens: PEGASUS_flies. Additionally, we identified ppk23, an Acid Sensing Ion Channel (ASIC) homolog, as an important hub for epistatic interactions. We propose a model that suggests genetic modifiers of wing and muscle morphology, nervous system development and function, BMP signaling, sexually dimorphic neural wiring, and gene regulation are all important for the observed differences flight performance in a natural population. Additionally, these results represent a snapshot of the genetic modifiers affecting drop-response flight performance in Drosophila, with implications for other insects.