A synthesis of deimatic behaviour.

Deimatic behaviours, also referred to as startle behaviours, are used against predators and rivals. Although many are spectacular, their proximate and ultimate causes remain unclear. In this review we aim to synthesise what is known about deimatic behaviour and identify knowledge gaps. We propose a working hypothesis for deimatic behaviour, and discuss the available evidence for the evolution, ontogeny, causation, and survival value of deimatic behaviour using Tinbergen's Four Questions as a framework. Our overarching aim is to direct future research by suggesting ways to address the most pressing questions in this field.

Octopamine receptor gene influences social grouping in the masked birch caterpillar.

OBJECTIVE: Group-living plays a key role in the success of many insects, but the mechanisms underlying group formation and maintenance are poorly understood. Here we use the masked birch caterpillar, Drepana arcuata, to explore genetic influences on social grouping. These larvae predictably transition from living in social groups to living solitarily during the 3rd instar of development. Our previous study showed a notable shift in the D. arcuata transcriptome that correlates with the transition from grouping to solitary behavior. We noted that one differentially regulated gene, octopamine receptor gene (DaOAR), is a prominent 'social' gene in other insect species, prompting us to test the hypothesis that DaOAR influences grouping behavior in D. arcuata. This was done using RNA interference (RNAi) methods by feeding second instar larvae synthetic dsRNAs. RESULTS: RT-qPCR analysis confirmed a significant reduction in DaOAR transcript abundance in dsRNA-fed larvae compared to controls. Behavioral trials showed that caterpillars with reduced transcript abundance of DaOAR remained solitary throughout the observation period compared to controls. These results provide evidence that regulation of the octopamine receptor gene influences social grouping in D. arcuata, and that specifically, a decrease in octopamine receptor expression triggers the larval transition from social to solitary.

What Does an Insect Hear? Reassessing the Role of Hearing in Predator Avoidance with Insights from Vertebrate Prey.

Insects have a diversity of hearing organs known to function in a variety of contexts, including reproduction, locating food, and defense. While the role of hearing in predator avoidance has been extensively researched over the past several decades, this research has focused on the detection of one type of predator-echolocating bats. Here we reassess the role of hearing in antipredator defense by considering how insects use their ears to detect and avoid the wide range of predators that consume them. To identify the types of sounds that could be relevant to insect prey, we first review the topic of hearing-mediated predator avoidance in vertebrates. Sounds used by vertebrate prey to assess predation risk include incidental sound cues (e.g., flight sounds, rustling vegetation, and splashing) produced by an approaching predator or another escaping prey, as well as communication signals produced by a predator (e.g., echolocation calls, songs) or nonpredator (e.g., alarm calls). We then review what is known, and what is not known, about such sounds made by the main predators and parasitoids of insects (i.e., birds, bats, terrestrial vertebrates, and invertebrates) and how insects respond to them. Three key insights emerged from our review. First, there is a lack of information on how both vertebrate and insect prey use passive sound cues produced by predators to avoid being captured. Second, while there are numerous examples of vertebrate prey eavesdropping on the calls and songs of predators and nonpredators to assess risk, there are currently no such examples for eared insect prey. Third, the hearing sensitivity of many insects, including those with ears considered to be dedicated to detecting bats or mates, overlaps with both sound cues and signals generated by nonbat predators. Sounds of particular relevance to insect prey include the flight sounds and calls of insectivorous birds, the flight sounds of insect predators and parasitoids, and rustling vegetation sounds of birds and terrestrial predators. We conclude that research on the role of insect hearing in predator avoidance has been disproportionally focused on bat-detection, and that acoustically-mediated responses to other predators may have been overlooked because the responses of prey may be subtle (e.g., ceasing activity, increasing vigilance). We recommend that researchers expand their testing of hearing-mediated risk assessment in insects by considering the wide range of sounds generated by predators, and the varied responses exhibited by prey to these sounds.

Transcriptome analysis of a social caterpillar, Drepana arcuata: De novo assembly, functional annotation and developmental analysis.

The masked birch caterpillar, Drepana arcuata, provides an excellent opportunity to study mechanisms mediating developmental changes in social behaviour. Larvae transition from being social to solitary during the 3rd instar, concomitant with shifts in their use of acoustic communication. In this study we characterize the transcriptome of D. arcuata to initiate sociogenomic research of this lepidopteran insect. We assembled and annotated the combined larval transcriptome of "social" early and "solitary" late instars using next generation Illumina sequencing, and used this transcriptome to conduct differential gene expression analysis of the two behavioural phenotypes. A total of 211,012,294 reads generated by RNA sequencing were assembled into 231,348 transcripts and 116,079 unigenes for the functional annotation of the transcriptome. Expression analysis revealed 3300 transcripts that were differentially expressed between early and late instars, with a large proportion associated with development and metabolic processes. We independently validated differential expression patterns of selected transcripts using RT-qPCR. The expression profiles of social and solitary larvae revealed differentially expressed transcripts coding for gene products that have been previously reported to influence social behaviour in other insects (e.g. cGMP- and cAMP- dependent kinases, and bioamine receptors). This study provides the first transcriptomic resources for a lepidopteran species belonging to the superfamily Drepanoidea, and gives insight into genetic factors mediating grouping behaviour in insects.

Hearing in caterpillars of the monarch butterfly (Danaus plexippus).

Many species of caterpillars have been reported to respond to sound, but there has been limited formal study of what sounds they hear, how they hear them and how they respond to them. Here, we report on hearing in caterpillars of the monarch butterfly (Danaus plexippus). Fourth and fifth instar caterpillars respond to sounds by freezing, contracting, and flicking their thorax in a vertical direction. Behavioural responses were evoked by sound frequencies between 50 and 900 Hz, with best sensitivity at 100-200 Hz. The lowest mean threshold was 79 dB SPL (particle velocity 605 µm s-1) at 150 Hz. When presented with a repeated 200 Hz sound tone, caterpillars habituate by no longer responding. A series of ablation experiments confirmed that the primary sensory receptors are a pair of long hairs, called trichoid sensilla, located on the upper prothorax. These sensilla are ∼450 µm long, rest in a socket and are innervated by a single bipolar sensory neuron. Removal of these setae reduced responses significantly compared with controls. Other setae contributed minimally to hearing in response to 200 Hz tones, and tubercles and prothoracic shields played no apparent role in sound reception. We propose that hearing functions to prevent attacks by aerial insect predators and parasitoids, which produce flight sounds in the frequency range to which the caterpillars are sensitive. This research lays the foundation for further investigations on the function and evolution of hearing in caterpillars, and has significance for the conservation of threatened monarch butterfly larvae living near noisy urban environments and roadways.

Phylogenomics reveals the evolutionary timing and pattern of butterflies and moths.

Butterflies and moths (Lepidoptera) are one of the major superradiations of insects, comprising nearly 160,000 described extant species. As herbivores, pollinators, and prey, Lepidoptera play a fundamental role in almost every terrestrial ecosystem. Lepidoptera are also indicators of environmental change and serve as models for research on mimicry and genetics. They have been central to the development of coevolutionary hypotheses, such as butterflies with flowering plants and moths' evolutionary arms race with echolocating bats. However, these hypotheses have not been rigorously tested, because a robust lepidopteran phylogeny and timing of evolutionary novelties are lacking. To address these issues, we inferred a comprehensive phylogeny of Lepidoptera, using the largest dataset assembled for the order (2,098 orthologous protein-coding genes from transcriptomes of 186 species, representing nearly all superfamilies), and dated it with carefully evaluated synapomorphy-based fossils. The oldest members of the Lepidoptera crown group appeared in the Late Carboniferous (∼300 Ma) and fed on nonvascular land plants. Lepidoptera evolved the tube-like proboscis in the Middle Triassic (∼241 Ma), which allowed them to acquire nectar from flowering plants. This morphological innovation, along with other traits, likely promoted the extraordinary diversification of superfamily-level lepidopteran crown groups. The ancestor of butterflies was likely nocturnal, and our results indicate that butterflies became day-flying in the Late Cretaceous (∼98 Ma). Moth hearing organs arose multiple times before the evolutionary arms race between moths and bats, perhaps initially detecting a wide range of sound frequencies before being co-opted to specifically detect bat sonar. Our study provides an essential framework for future comparative studies on butterfly and moth evolution.

Bark beetles use a spring-loaded mechanism to produce variable song patterns.

Many insects vary their song patterns to communicate different messages, but the underlying biomechanisms are often poorly understood. Here, we report on the mechanics of sound production and variation in an elytro-tergal stridulator, male Dendroctonus valens bark beetles. Using ablation experiments coupled with high-speed video and audio recordings, we show that: (1) chirps are produced using a stridulatory file on the left elytron (forewing) and a protrusion (plectrum) on the seventh abdominal segment; (2) chirps are produced by 'spring stridulation', a catch-and-release mechanism whereby the plectrum catches on a file tooth and, upon release, springs forward along the file; and (3) variability in chirp types is caused by introducing multiple catch-and-release events along the file to create regular interruptions. These results provide experimental evidence for the mechanics of elytro-tergal stridulation, and provide insight into how an insect can incorporate variability into its acoustic repertoire using a spring-loaded mechanism.

In that vein: inflated wing veins contribute to butterfly hearing.

Insects have evolved a diversity of hearing organs specialized to detect sounds critical for survival. We report on a unique structure on butterfly wings that enhances hearing. The Satyrini are a diverse group of butterflies occurring throughout the world. One of their distinguishing features is a conspicuous swelling of their forewing vein, but the functional significance of this structure is unknown. Here, we show that wing vein inflations function in hearing. Using the common wood nymph, Cercyonis pegala, as a model, we show that (i) these butterflies have ears on their forewings that are most sensitive to low frequency sounds (less than 5 kHz); (ii) inflated wing veins are directly connected to the ears; and (iii) when vein inflations are ablated, sensitivity to low frequency sounds is impaired. We propose that inflated veins contribute to low frequency hearing by impedance matching.

What does a butterfly hear? Physiological characterization of auditory afferents in Morpho peleides (Nymphalidae).

Many Nymphalidae butterflies possess ears, but little is known about their hearing. The tympanal membrane of butterflies typically comprises distinct inner and outer regions innervated by auditory nerve branches NII and NIII and their respective sensory organs. Using the Blue Morpho butterfly (Morpho peleides) as a model, we characterized threshold and suprathreshold responses of NII and NIII. Both are broadly tuned to 1-20 kHz with best frequencies at 1-3 kHz, but NIII is significantly more sensitive than NII. The compound action potentials (CAPs) of both branches increase their first peak amplitudes and areas in response to higher sound levels. NII and NIII differed in their suprathreshold CAP responses to sound frequencies, with stronger responses to 1-3 and 4-6 kHz, for NIII and NII respectively; results that are consistent with tympanal membrane mechanics. These results indicate that butterflies are capable of amplitude and frequency discrimination. Both auditory branches responded to playbacks of the flight and calls of predatory birds. We propose that the ears of butterflies, like those of many vertebrate prey such as some rabbits and lizards, function primarily in predator risk assessment.

Vocalization in caterpillars: a novel sound-producing mechanism for insects.

Insects have evolved a great diversity of sound-producing mechanisms largely attributable to their hardened exoskeleton, which can be rubbed, vibrated or tapped against different substrates to produce acoustic signals. However, sound production by forced air, while common in vertebrates, is poorly understood in insects. We report on a caterpillar that 'vocalizes' by forcing air into and out of its gut. When disturbed, larvae of the Nessus sphinx hawkmoth (Sphingidae: Amphion floridensis) produce sound trains comprising a stereotyped pattern of long (370 ms) followed by multiple short-duration (23 ms) units. Sounds are emitted from the oral cavity, as confirmed by close-up videos and comparing sound amplitudes at different body regions. Numerical models using measurements of the caterpillar foregut were constructed to test hypotheses explaining sound production. We propose that sound is generated by ring vortices created as air flows through the orifice between two foregut chambers (crop and oesophagus), a mechanism analogous to a whistling kettle. As air flows past the orifice, certain sound frequencies are amplified by a Helmholtz resonator effect of the oesophagus chamber. Long sound units occur during inflation, while short sound units occur during deflation. Several other insects have been reported to produce sounds by forced air, but the aeroacoustic mechanisms of such sounds remain elusive. Our results provide evidence for this mechanism by showing that caterpillars employ mechanisms similar to rocket engines to produce sounds.

Why do caterpillars whistle at birds? Insect defence sounds startle avian predators.

Many insects produce sounds when attacked by a predator, yet the functions of these signals are poorly understood. It is debated whether such sounds function as startle, warning or alarm signals, or merely serve to augment other defences. Direct evidence is limited owing to difficulties in disentangling the effects of sounds from other defences that often occur simultaneously in live insects. We conducted an experiment to test whether an insect sound can function as a deimatic (i.e. startle) display. Variations of a whistle of the walnut sphinx caterpillar (Amorpha juglandis) were presented to a predator, red-winged blackbirds (Agelaius phoeniceus), when birds activated a sensor while feeding on mealworms (Tenebrio molitor). Birds exposed to whistles played back at natural sound levels exhibited significantly higher startle scores (by flying away, flinching, and hopping) and took longer to return to the feeding dish than during control conditions where no sounds were played. Birds habituated to sounds during a one-hour session, but after two days the startling effects were restored. Our results provide empirical evidence that an insect sound alone can function as a deimatic display against an avian predator. We discuss how whistles might be particularly effective 'acoustic eye spots' on avian predators.

A Comparative Analysis of Sonic Defences in Bombycoidea Caterpillars.

Caterpillars have long been used as models for studying animal defence. Their impressive armour, including flamboyant warning colours, poisonous spines, irritating sprays, and mimicry of plant parts, snakes and bird droppings, has been extensively documented. But research has mainly focused on visual and chemical displays. Here we show that some caterpillars also exhibit sonic displays. During simulated attacks, 45% of 38 genera and 33% of 61 species of silk and hawkmoth caterpillars (Bombycoidea) produced sounds. Sonic caterpillars are found in many distantly-related groups of Bombycoidea, and have evolved four distinct sound types- clicks, chirps, whistles and vocalizations. We propose that different sounds convey different messages, with some designed to warn of a chemical defence and others, to startle predators. This research underscores the importance of exploring acoustic communication in juvenile insects, and provides a model system to explore how different signals have evolved to frighten, warn or even trick predators.

Shaking Youngsters and Shaken Adults: Female Beetles Eavesdrop on Larval Seed Vibrations to Make Egg-Laying Decisions.

Egg-laying decisions are critical for insects, and particularly those competing for limited resources. Sensory information used by females to mediate egg-laying decisions has been reported to be primarily chemical, but the role of vibration has received little attention. We tested the hypothesis that vibrational cues produced by feeding larvae occupying a seed influences egg-laying decisions amongst female cowpea beetles. This hypothesis is supported by three lines of evidence using two strains of the cowpea beetle (Callosobruchus maculatus), an Indian strain with choosy females and aggressively competing larvae and a Brazilian strain with less choosy females and larvae exhibiting an "accommodating" type of competition. First, in free-choice bioassays of seed selection, choosy Indian females selected control seeds (free of eggs, larvae, or egg-laying marker) over seeds with live larvae (free of eggs and egg-laying marker), but did not discriminate between control seeds and those with dead larvae. In contrast, less choosy Brazilian females showed no preference for seeds containing live or dead larvae over controls. Second, laser-doppler vibrometer recordings confirmed that larvae feeding inside seeds generate vibrations that are available to the female during egg-laying decisions. Third, during dichotomous choice experiments where artificial vibrations approximating those produced by feeding larvae were played back during seed selection, Indian females preferred immobile control seeds over vibrating seeds, but Brazilian females showed no preference. These results support the hypothesis that females use larval vibrations in their egg-laying decisions; whether these vibrations are passive cues exploited by the female, or active signals that 'steer' the behaviour of the female is unknown. We propose that vibration cues and signals could be important for host selection in insects, particularly those laying on substrates where visual or chemical cues may be unreliable. This seems to be the case with females of the cowpea beetle since visual cues are not important and chemical egg-marking does not last more than two weeks, allowing vibration cues to improve discrimination of egg-laying substrate particularly by choosy females.

What is the password? Female bark beetles (Scolytinae) grant males access to their galleries based on courtship song.

Acoustic signals are commonly used by insects in the context of mating, and signals can vary depending on the stage of interaction between a male and female. While calling songs have been studied extensively, particularly in the Orthoptera, much less is known about courtship songs. One outstanding question is how potential mates are differentiated by their courtship signal characteristics. We examined acoustic courtship signals in a new system, bark beetles (Scolytinae). In the red turpentine beetle (Dendroctonus valens) males produce chirp trains upon approaching the entrance of a female's gallery. We tested the hypotheses that acoustic signals are honest indicators of male condition and that females choose males based on signal characteristics. Males generated two distinct chirp types (simple and interrupted), and variability in their prevalence correlated with an indicator of male quality, body size, with larger males producing significantly more interrupted chirps. Females showed a significant preference for males who produced interrupted chirps, suggesting that females distinguish between males on the basis of their chirp performances. We suggest that interrupted chirps during courtship advertise a male's size and/or motor skills, and function as the proverbial 'passwords' that allow him entry to a female's gallery.

Hearing in the crepuscular owl butterfly (Caligo eurilochus, Nymphalidae).

Tympanal organs are widespread in Nymphalidae butterflies, with a great deal of variability in the morphology of these ears. How this variation reflects differences in hearing physiology is not currently understood. This study provides the first examination of hearing organs in the crepuscular owl butterfly, Caligo eurilochus. We examined the tuning and sensitivity of the C. eurilochus hearing organ, called Vogel's organ, using laser Doppler vibrometry and extracellular neurophysiology. We show that the C. eurilochus ear responds to sound and is most sensitive to frequencies between 1 and 4 kHz, as confirmed by both the vibration of the tympanal membrane and the physiological response of the associated nerve branches. In comparison to the hearing of its diurnally active relative, Morpho peleides, C. eurilochus has a narrower frequency range with higher auditory thresholds. Hypotheses explaining the function of hearing in this crepuscular butterfly are discussed.

Whistling in caterpillars (Amorpha juglandis, Bombycoidea): sound-producing mechanism and function.

Caterpillar defenses have been researched extensively, and, although most studies focus on visually communicated signals, little is known about the role that sounds play in defense. We report on whistling, a novel form of sound production for caterpillars and rare for insects in general. The North American walnut sphinx (Amorpha juglandis) produces whistle 'trains' ranging from 44 to 2060 ms in duration and comprising one to eight whistles. Sounds were categorized into three types: broadband, pure whistles and multi-harmonic plus broadband, with mean dominant frequencies at 15 kHz, 9 kHz and 22 kHz, respectively. The mechanism of sound production was determined by selectively obstructing abdominal spiracles, monitoring air flow at different spiracles using a laser vibrometer and recording body movements associated with sound production using high-speed video. Contractions of the anterior body segments always accompanied sound production, forcing air through a pair of enlarged spiracles on the eighth abdominal segment. We tested the hypothesis that sounds function in defense using simulated attacks with blunt forceps and natural attacks with an avian predator - the yellow warbler (Dendroica petechia). In simulated attacks, 94% of caterpillars responded with whistle trains that were frequently accompanied by directed thrashing but no obvious chemical defense. In predator trials, all birds readily attacked the caterpillar, eliciting whistle trains each time. Birds responded to whistling by hesitating, jumping back or diving away from the sound source. We conclude that caterpillar whistles are defensive and propose that they function specifically as acoustic 'eye spots' to startle predators.

The evolutionary origins of ritualized acoustic signals in caterpillars.

Animal communication signals can be highly elaborate, and researchers have long sought explanations for their evolutionary origins. For example, how did signals such as the tail-fan display of a peacock, a firefly flash or a wolf howl evolve? Animal communication theory holds that many signals evolved from non-signalling behaviours through the process of ritualization. Empirical evidence for ritualization is limited, as it is necessary to examine living relatives with varying degrees of signal evolution within a phylogenetic framework. We examine the origins of vibratory territorial signals in caterpillars using comparative and molecular phylogenetic methods. We show that a highly ritualized vibratory signal--anal scraping--originated from a locomotory behaviour--walking. Furthermore, comparative behavioural analysis supports the hypothesis that ritualized vibratory signals derive from physical fighting behaviours. Thus, contestants signal their opponents to avoid the cost of fighting. Our study provides experimental evidence for the origins of a complex communication signal, through the process of ritualization.

Variation on a theme: vibrational signaling in caterpillars of the rose hook-tip moth, Oreto rosea.

Vibrational communication in hook-tip moth caterpillars is thought to be widely used and highly variable across species, but this phenomenon has been experimentally examined in only two species to date. The purpose of this study is to characterize and describe the function of vibrational signaling in a species, Oreta rosea Walker 1855 (Lepidoptera: Drepanidae), that differs morphologically from previously studied species. Caterpillars of this species produce three distinct types of vibrational signals during territorial encounters with conspecifics--mandible drumming, mandible scraping and lateral tremulation. Signals were recorded using a laser-doppler vibrometer and characterized based on temporal and spectral components. Behavioural encounters between a leaf resident and a conspecific intruder were staged to test the hypothesis that signaling functions as a territorial display. Drumming and scraping signals both involve the use of the mandibles, being hit vertically on, or scraped laterally across, the leaf surface. Lateral tremulation involves quick, short, successive lateral movements of the anterior body region that vibrates the entire leaf. Encounters result in residents signaling, with the highest rates observed when intruders make contact with the resident. Residents signal significantly more than intruders and most conflicts are resolved within 10 minutes, with residents winning 91% of trials. The results support the hypothesis that vibrational signals function to advertise leaf occupancy. Signaling is compared between species, and evolutionary origins of vibrational communication in caterpillars are discussed.

Auditory mechanics and sensitivity in the tropical butterfly Morpho peleides (Papilionoidea, Nymphalidae).

The ears of insects exhibit a broad functional diversity with the ability to detect sounds across a wide range of frequencies and intensities. In tympanal ears, the membrane is a crucial step in the transduction of the acoustic stimulus into a neural signal. The tropical butterfly Morpho peleides has an oval-shaped membrane at the base of the forewing with an unusual dome in the middle of the structure. We are testing the hypothesis that this unconventional anatomical arrangement determines the mechanical tuning properties of this butterfly ear. Using microscanning laser Doppler vibrometry to measure the vibrational characteristics of this novel tympanum, the membrane was found to vibrate in two distinct modes, depending on the frequency range: at lower frequencies (1-5 kHz) the vibration was focused at the proximal half of the posterior side of the outer membrane, while at higher frequencies (5-20 kHz) the entire membrane contributed to the vibration. The maximum deflection points of the two vibrational modes correspond to the locations of the associated chordotonal organs, suggesting that M. peleides has the capacity for frequency partitioning because of the different vibrational properties of the two membrane components. Extracellular nerve recordings confirm that the innervating chordotonal organs respond to the same frequency range of 1-20 kHz, and are most sensitive between 2 and 4 kHz, although distinct frequency discrimination was not observed. We suggest that this remarkable variation in structure is associated with function that provides a selective advantage, particularly in predator detection.

What's the buzz? Ultrasonic and sonic warning signals in caterpillars of the great peacock moth (Saturnia pyri).

Caterpillars have many natural enemies and, therefore, have evolved a diversity of antipredator strategies. Most research focuses on those strategies (crypsis, countershading, and warning coloration) targeting visually guided predators. In contrast, defensive sounds, although documented for more than a century, have been poorly studied. We report on a novel form of sound production--chirping--in caterpillars of the common European Great Peacock moth (Saturnia pyri). Chirps are broadband, with dominant peaks ranging between the sonic (3.7 kHz) and ultrasonic (55.1 kHz) and are generated by a rapid succession of mandibular "tooth strikes." Chirp trains are induced by simulated predator attacks and precede or accompany the secretion of a defensive chemical from integumental bristles, supporting our hypothesis that these sounds function in acoustic aposematism. We propose that these caterpillars generate multimodal warning signals (visual, chemical, and acoustic) to target the dominant sensory modalities of different predators, including birds, bats, and invertebrates.