Multi-year soundscape recordings and automated call detection reveals varied impact of moonlight on calling activity of neotropical forest katydids.

Night-time light can have profound ecological effects, even when the source is natural moonlight. The impacts of light can, however, vary substantially by taxon, habitat and geographical region. We used a custom machine learning model built with the Python package Koogu to investigate the in situ effects of moonlight on the calling activity of neotropical forest katydids over multiple years. We prioritised species with calls that were commonly detected in human annotated data, enabling us to evaluate model performance. We focused on eight species of katydids that the model identified with high precision (generally greater than 0.90) and moderate-to-high recall (minimum 0.35), ensuring that detections were generally correct and that many calls were detected. These results suggest that moonlight has modest effects on the amount of calling, with the magnitude and direction of effect varying by species: half of the species showed positive effects of light and half showed negative. These findings emphasize the importance of understanding natural history for anticipating how biological communities respond to moonlight. The methods applied in this project highlight the emerging opportunities for evaluating large quantities of data with machine learning models to address ecological questions over space and time. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Levels of Airborne Sound And Substrate-borne Vibration Calling Are Negatively Related Across Neotropical False-leaf Katydids.

Animals often signal in multiple sensory modalities to attract mates, but the level of signaling investment in each modality can differ dramatically between individuals and across species. When functionally overlapping signals are produced in different modalities, their relative use can be influenced by many factors, including differences in signal active space, energetic costs, and predation risk. Characterizing differences in total signal investment across time can shed light on these factors, but requires long focal recordings of signal production. Neotropical pseudophylline katydids produce mate advertisement signals as airborne sound and substrate-borne vibration. Airborne calls, produced via stridulation, are extremely short, high-frequency, and longer-range signals. Conversely, substrate-borne calls produced via abdominal tremulation are longer, low-frequency, relatively more energetically costly, and shorter-range signals. To examine patterns of stridulation and tremulation across species and test hypotheses about the drivers of signal use in each modality, we recorded multimodal signaling activity over 24 hours for males from ten pseudophylline species from a single Panamanian community. We also collected data on demographic and morphological species characteristics, and acoustic features of airborne calls, such as bandwidth, peak frequency, and duration. Finally, we generated a molecular phylogeny for these species and used phylogenetic generalized least squares models to test for relationships between variables while controlling for evolutionary relationships. We found a negative relationship between sound and vibration calling, indicating that substrate-borne vibrational signaling may compensate for reduced airborne signaling in these species. Sound call bandwidth and the proportion of males collected at lights, a proxy for the amount of male movement, also explained a significant amount of variation in sound calling across species, indicating that the overall relationship between the two types of calling signals may be mediated by the specific characteristics of the signals as well as other species traits.

By a whisker: the sensory role of vibrissae in hovering flight in nectarivorous bats.

Whiskers are important tactile structures widely used across mammals for a variety of sensory functions, but it is not known how bats-representing about a fifth of all extant mammal species-use them. Nectar-eating bats typically have long vibrissae (long, stiff hairs) arranged in a forward-facing brush-like formation that is not present in most non-nectarivorous bats. They also commonly use a unique flight strategy to access their food-hovering flight. Here we investigated whether these species use their vibrissae to optimize their feeding by assisting fine flight control. We used behavioural experiments to test if bats' flight trajectory into the flower changed after vibrissa removal, and phylogenetic comparative methods to test whether vibrissa length is related to nectarivory. We found that bat flight trajectory was altered after vibrissae removal and that nectarivorous bats possess longer vibrissae than non-nectivorous species, providing evidence of an additional source of information in bats' diverse sensory toolkit.

Patterns of Herbivory in Neotropical Forest Katydids as Revealed by DNA Barcoding of Digestive Tract Contents.

Many well-studied animal species use conspicuous, repetitive signals that attract both mates and predators. Orthopterans (crickets, katydids, and grasshoppers) are renowned for their acoustic signals. In Neotropical forests, however, many katydid species produce extremely short signals, totaling only a few seconds of sound per night, likely in response to predation by acoustically orienting predators. The rare signals of these katydid species raises the question of how they find conspecific mates in a structurally complex rainforest. While acoustic mechanisms, such as duetting, likely facilitate mate finding, we test the hypothesis that mate finding is further facilitated by colocalization on particular host plant species. DNA barcoding allows us to identify recently consumed plants from katydid stomach contents. We use DNA barcoding to test the prediction that katydids of the same species will have closely related plant species in their stomach. We do not find evidence for dietary specialization. Instead, katydids consumed a wide mix of plants within and across the flowering plants (27 species in 22 genera, 16 families, and 12 orders) with particular representation in the orders Fabales and Laurales. Some evidence indicates that katydids may gather on plants during a narrow window of rapid leaf out, but additional investigations are required to determine whether katydid mate finding is facilitated by gathering at transient food resources.

Daily Signaling Rate and the Duration of Sound per Signal are Negatively Related in Neotropical Forest Katydids.

Researchers have long examined the structure of animal advertisement signals, but comparatively little is known about how often these signals are repeated and what factors predict variation in signaling rate across species. Here, we focus on acoustic advertisement signals to test the hypothesis that calling males experience a tradeoff between investment in the duration or complexity of individual calls and investment in signaling over long time periods. This hypothesis predicts that the number of signals that a male produces per 24 h will negatively correlate with (1) the duration of sound that is produced in each call (the sum of all pulses) and (2) the number of sound pulses per call. To test this hypothesis, we measured call parameters and the number of calls produced per 24 h in 16 species of sympatric phaneropterine katydids from the Panamanian rainforest. This assemblage also provided us with the opportunity to test a second taxonomically specific hypothesis about signaling rates in taxa such as phaneropterine katydids that transition from advertisement calls to mating duets to facilitate mate localization. To establish duets, male phaneropterine katydids call and females produce a short acoustic reply. These duets facilitate searching by males, females, or both sexes, depending on the species. We test the hypothesis that males invest either in calling or in searching for females. This hypothesis predicts a negative relationship between how often males signal over 24 h and how much males move across the landscape relative to females. For the first hypothesis, there was a strong negative relationship between the number of signals and the duration of sound that is produced in each signal, but we find no relationship between the number of signals produced per 24 h and the number of pulses per signal. This result suggests the presence of cross-taxa tradeoffs that limit signal production and duration, but not the structure of individual signals. These tradeoffs could be driven by energetic limitations, predation pressure, signal efficacy, or other signaling costs. For the second hypothesis, we find a negative relationship between the number of signals produced per day and proportion of the light trap catch that is male, likely reflecting males investing either in calling or in searching. These cross-taxa relationships point to the presence of pervasive trade-offs that fundamentally shape the spatial and temporal dynamics of communication.

Sheep in wolves' clothing: prey rely on proactive defences when predator and non-predator cues are similar.

Predation produces intense selection and a diversity of defences. Reactive defences are triggered by predator cues, whereas proactive defences are always in effect. We assess whether prey rely on proactive defences when predator cues do not correlate well with predation risk. Many bats use echolocation to hunt insects, and many insects have evolved to hear bats. However, in species-rich environments like Neotropical forests, bats have extremely diverse foraging strategies, and the presence of echolocation corresponds only weakly to the presence of predators. We assess whether katydids that live in habitats with many non-dangerous bat species stop calling when exposed to echolocation. For 11 species of katydids, we quantified behavioural and neural responses to predator cues, and katydid signalling activity over 24 h periods. Despite having the sensory capacity to detect predators, many Neotropical forest katydids continued calling in the presence of predator cues, displaying proactive defences instead (short, infrequent calls totalling less than 2 cumulative seconds of sound per 24 h). Neotropical katydid signalling illustrates a fascinating case where trophic interactions are probably mediated by a third group: bats with alternative foraging strategies (e.g. frugivory). Although these co-occurring bats are not trophically connected, their mere presence disrupts the correlation between cue and predation risk.

Predation risks of signalling and searching: bats prefer moving katydids.

Males signalling their attractiveness to females are at risk from predators that exploit mating signals to detect and locate prey. Signalling, however, is not the only risky activity in sexual interactions: mate searching can incur risk as well. Male Neotropical pseudophylline katydids produce both acoustic and vibrational signals (tremulations). Females reply to male signals with tremulations of their own, and both sexes walk to find one another. We asked if movement increases predation risk, and whether tremulation or walking was more attractive to predators. We offered the Neotropical gleaning bat Micronycteris microtis a series of two-choice tests, presenting the bats with katydid models that were motionless or moved in a way to mimic either tremulating or walking. We found that prey movements do put prey at risk. Although M. microtis can detect motionless prey on leaves, they preferred moving prey. Our study shows that movement can put searching or signalling prey in danger, potentially explaining why silent female katydids are frequently consumed by gleaning bats.

Applying and refining DNA analysis to determine the identity of plant material extracted from the digestive tracts of katydids.

BACKGROUND: Feeding habits are central to animal ecology, but it is often difficult to characterize the diet of organisms that are arboreal, nocturnal, rare, or highly mobile. Genetic analysis of gut contents is a promising approach for expanding our understanding of animal feeding habits. Here, we adapt a laboratory protocol for extracting and sequencing plant material from gut contents and apply it to Neotropical forest katydids (Orthoptera: Tettigoniidae) on Barro Colorado Island (BCI) in Panama. METHODS: Our approach uses three chloroplast primer sets that were previously developed to identify vegetation on BCI. We describe the utility and success rate of each primer set. We then test whether there is a significant difference in the amplification and sequencing success of gut contents based on the size or sex of the katydid, the time of day that it was caught, and the color of the extracted gut contents. RESULTS: We find that there is a significant difference in sequencing success as a function of gut color. When extracts were yellow, green, or colorless the likelihood of successfully amplifying DNA ranged from ~30-60%. When gut extracts were red, orange, or brown, amplification success was exceptionally low (0-8%). Amplification success was also higher for smaller katydids and tended to be more successful in katydids that were captured earlier in the night. Strength of the amplified product was indicative of the likelihood of sequencing success, with strong bands having a high likelihood of success. By anticipating which samples are most likely to succeed, we provide information useful for estimating the number of katydids that need to be collected and minimizing the costs of purifying, amplifying, and sequencing samples that are unlikely to succeed. This approach makes it possible to understand the herbivory patterns of these trophically important katydids and can be applied more broadly to understand the diet of other tropical herbivores.

The influence of bat echolocation call duration and timing on auditory encoding of predator distance in noctuoid moths.

Animals co-occur with multiple predators, making sensory systems that can encode information about diverse predators advantageous. Moths in the families Noctuidae and Erebidae have ears with two auditory receptor cells (A1 and A2) used to detect the echolocation calls of predatory bats. Bat communities contain species that vary in echolocation call duration, and the dynamic range of A1 is limited by the duration of sound, suggesting that A1 provides less information about bats with shorter echolocation calls. To test this hypothesis, we obtained intensity-response functions for both receptor cells across many moth species for sound pulse durations representing the range of echolocation call durations produced by bat species in northeastern North America. We found that the threshold and dynamic range of both cells varied with sound pulse duration. The number of A1 action potentials per sound pulse increases linearly with increasing amplitude for long-duration pulses, saturating near the A2 threshold. For short sound pulses, however, A1 saturates with only a few action potentials per pulse at amplitudes far lower than the A2 threshold for both single sound pulses and pulse sequences typical of searching or approaching bats. Neural adaptation was only evident in response to approaching bat sequences at high amplitudes, not search-phase sequences. These results show that, for short echolocation calls, a large range of sound levels cannot be coded by moth auditory receptor activity, resulting in no information about the distance of a bat, although differences in activity between ears might provide information about direction.

Evolutionary escalation: the bat-moth arms race.

Echolocation in bats and high-frequency hearing in their insect prey make bats and insects an ideal system for studying the sensory ecology and neuroethology of predator-prey interactions. Here, we review the evolutionary history of bats and eared insects, focusing on the insect order Lepidoptera, and consider the evidence for antipredator adaptations and predator counter-adaptations. Ears evolved in a remarkable number of body locations across insects, with the original selection pressure for ears differing between groups. Although cause and effect are difficult to determine, correlations between hearing and life history strategies in moths provide evidence for how these two variables influence each other. We consider life history variables such as size, sex, circadian and seasonal activity patterns, geographic range and the composition of sympatric bat communities. We also review hypotheses on the neural basis for anti-predator behaviours (such as evasive flight and sound production) in moths. It is assumed that these prey adaptations would select for counter-adaptations in predatory bats. We suggest two levels of support for classifying bat traits as counter-adaptations: traits that allow bats to eat more eared prey than expected based on their availability in the environment provide a low level of support for counter-adaptations, whereas traits that have no other plausible explanation for their origination and maintenance than capturing defended prey constitute a high level of support. Specific predator counter-adaptations include calling at frequencies outside the sensitivity range of most eared prey, changing the pattern and frequency of echolocation calls during prey pursuit, and quiet, or 'stealth', echolocation.

Evolution of a Communication System by Sensory Exploitation of Startle Behavior.

New communication signals can evolve by sensory exploitation if signaling taps into preexisting sensory biases in receivers [1, 2]. For mate attraction, signals are typically similar to attractive environmental cues like food [3-6], which amplifies their attractiveness to mates, as opposed to aversive stimuli like predator cues. Female field crickets approach the low-frequency calling song of males, whereas they avoid high-frequency sounds like predatory bat calls [7]. In one group of crickets (Eneopterinae: Lebinthini), however, males produce exceptionally high-frequency calling songs in the range of bat calls [8], a surprising signal in the context of mate attraction. We found that female lebinthines, instead of approaching singing males, produce vibrational responses after male calls, and males track the source of vibrations to find females. We also demonstrate that field cricket species closely related to the Lebinthini show an acoustic startle response to high-frequency sounds that generates substrate vibrations similar to those produced by female lebinthine crickets. Therefore, the startle response is the most likely evolutionary origin of the female lebinthine vibrational signal. In field crickets, the brain receives activity from two auditory interneurons; AN1 tuned to male calling song controls positive phonotaxis, and AN2 tuned to high-frequency bat calls triggers negative phonotaxis [9, 10]. In lebinthine crickets, however, we found that auditory ascending neurons are only tuned to high-frequency sounds, and their tuning matches the thresholds for female vibrational signals. Our results demonstrate how sensory exploitation of anti-predator behavior can evolve into a communication system that benefits both senders and receivers.

Sensory-based niche partitioning in a multiple predator - multiple prey community.

Many predators and parasites eavesdrop on the communication signals of their prey. Eavesdropping is typically studied as dyadic predator-prey species interactions; yet in nature, most predators target multiple prey species and most prey must evade multiple predator species. The impact of predator communities on prey signal evolution is not well understood. Predators could converge in their preferences for conspicuous signal properties, generating competition among predators and natural selection on particular prey signal features. Alternatively, predator species could vary in their preferences for prey signal properties, resulting in sensory-based niche partitioning of prey resources. In the Neotropics, many substrate-gleaning bats use the mate-attraction songs of male katydids to locate them as prey. We studied mechanisms of niche partitioning in four substrate-gleaning bat species and found they are similar in morphology, echolocation signal design and prey-handling ability, but each species preferred different acoustic features of male song in 12 sympatric katydid species. This divergence in predator preference probably contributes to the coexistence of many substrate-gleaning bat species in the Neotropics, and the substantial diversity in the mate-attraction signals of katydids. Our results provide insight into how multiple eavesdropping predator species might influence prey signal evolution through sensory-based niche partitioning.

The simple ears of noctuoid moths are tuned to the calls of their sympatric bat community.

Insects with bat-detecting ears are ideal animals for investigating sensory system adaptations to predator cues. Noctuid moths have two auditory receptors (A1 and A2) sensitive to the ultrasonic echolocation calls of insectivorous bats. Larger moths are detected at greater distances by bats than smaller moths. Larger moths also have lower A1 best thresholds, allowing them to detect bats at greater distances and possibly compensating for their increased conspicuousness. Interestingly, the sound frequency at the lowest threshold is lower in larger than in smaller moths, suggesting that the relationship between threshold and size might vary across frequencies used by different bat species. Here, we demonstrate that the relationships between threshold and size in moths were only significant at some frequencies, and these frequencies differed between three locations (UK, Canada and Denmark). The relationships were more likely to be significant at call frequencies used by proportionately more bat species in the moths' specific bat community, suggesting an association between the tuning of moth ears and the cues provided by sympatric predators. Additionally, we found that the best threshold and best frequency of the less sensitive A2 receptor are also related to size, and that these relationships hold when controlling for evolutionary relationships. The slopes of best threshold versus size differ, however, such that the difference in threshold between A1 and A2 is greater for larger than for smaller moths. The shorter time from A1 to A2 excitation in smaller than in larger moths could potentially compensate for shorter absolute detection distances in smaller moths.

Tympanal mechanics and neural responses in the ears of a noctuid moth.

Ears evolved in many groups of moths to detect the echolocation calls of predatory bats. Although the neurophysiology of bat detection has been intensively studied in moths for decades, the relationship between sound-induced movement of the noctuid tympanic membrane and action potentials in the auditory sensory cells (A1 and A2) has received little attention. Using laser Doppler vibrometry, we measured the velocity and displacement of the tympanum in response to pure tone pulses for moths that were intact or prepared for neural recording. When recording from the auditory nerve, the displacement of the tympanum at the neural threshold remained constant across frequencies, whereas velocity varied with frequency. This suggests that the key biophysical parameter for triggering action potentials in the sensory cells of noctuid moths is tympanum displacement, not velocity. The validity of studies on the neurophysiology of moth hearing rests on the assumption that the dissection and recording procedures do not affect the biomechanics of the ear. There were no consistent differences in tympanal velocity or displacement when moths were intact or prepared for neural recordings for sound levels close to neural threshold, indicating that this and other neurophysiological studies provide good estimates of what intact moths hear at threshold.

An aerial-hawking bat uses stealth echolocation to counter moth hearing.

Ears evolved in many nocturnal insects, including some moths, to detect bat echolocation calls and evade capture [1, 2]. Although there is evidence that some bats emit echolocation calls that are inconspicuous to eared moths, it is difficult to determine whether this was an adaptation to moth hearing or originally evolved for a different purpose [2, 3]. Aerial-hawking bats generally emit high-amplitude echolocation calls to maximize detection range [4, 5]. Here we present the first example of an echolocation counterstrategy to overcome prey hearing at the cost of reduced detection distance. We combined comparative bat flight-path tracking and moth neurophysiology with fecal DNA analysis to show that the barbastelle, Barbastella barbastellus, emits calls that are 10 to 100 times lower in amplitude than those of other aerial-hawking bats, remains undetected by moths until close, and captures mainly eared moths. Model calculations demonstrate that only bats emitting such low-amplitude calls hear moth echoes before their calls are conspicuous to moths. This stealth echolocation allows the barbastelle to exploit food resources that are difficult to catch for other aerial-hawking bats emitting calls of greater amplitude.

Auditory-based defence against gleaning bats in neotropical katydids (Orthoptera: Tettigoniidae).

Neotropical katydids (Orthoptera: Tettigoniidae) are preyed on by gleaning bats, which are known to use male calling songs to locate them. At least one katydid species has been reported to stop singing in response to bat echolocation calls. To investigate the relationship between this behavioural defence and ecological and sensory factors, we surveyed calling song characteristics, song cessation in response to the echolocation calls of a sympatric gleaning bat (Trachops cirrhosus), and T-cell responses (an auditory interneuron sensitive to ultrasound) in five katydid species from Panamá. The two katydid species that stopped singing in response to bat calls (Balboa tibialis and Ischnomela gracilis, Pseudophyllinae) also had the highest T-cell spike number and rate in response to these stimuli. The third pseudophylline species (Docidocercus gigliotosi) did not reliably cease singing and had low T-cell spiking activity. Neoconocephalus affinis (Copiphorinae) produced continuous calling song, possibly preventing males from hearing the bat during singing, and did not show a behavioural response despite high T-cell activity in response to bat calls. Steirodon rufolineatum (Phaneropterinae) did not cease singing and differed in T-cell activity compared to the other species. T-cell function might not be conserved in katydids, and evidence for this idea is discussed.

Release from bats: genetic distance and sensoribehavioural regression in the Pacific field cricket, Teleogryllus oceanicus.

The auditory thresholds of the AN2 interneuron and the behavioural thresholds of the anti-bat flight-steering responses that this cell evokes are less sensitive in female Pacific field crickets that live where bats have never existed (Moorea) compared with individuals subjected to intense levels of bat predation (Australia). In contrast, the sensitivity of the auditory interneuron, ON1 which participates in the processing of both social signals and bat calls, and the thresholds for flight orientation to a model of the calling song of male crickets show few differences between the two populations. Genetic analyses confirm that the two populations are significantly distinct, and we conclude that the absence of bats has caused partial regression in the nervous control of a defensive behaviour in this insect. This study represents the first examination of natural evolutionary regression in the neural basis of a behaviour along a selection gradient within a single species.

Gleaning bat echolocation calls do not elicit antipredator behaviour in the Pacific field cricket, Teleogryllus oceanicus (Orthoptera: Gryllidae).

Bats that glean prey (capture them from surfaces) produce relatively inconspicuous echolocation calls compared to aerially foraging bats and could therefore be difficult predators to detect, even for insects with ultrasound sensitive ears. In the cricket Teleogryllus oceanicus, an auditory interneuron (AN2) responsive to ultrasound is known to elicit turning behaviour, but only when the cricket is in flight. Turning would not save a cricket from a gleaning bat so we tested the hypothesis that AN2 elicits more appropriate antipredator behaviours when crickets are on the ground. The echolocation calls of Nyctophilus geoffroyi, a sympatric gleaning bat, were broadcast to singing male and walking female T. oceanicus. Males did not cease singing and females did not pause walking more than usual in response to the bat calls up to intensities of 82 dB peSPL. Extracellular recordings from the cervical connective revealed that the echolocation calls elicited AN2 action potentials at high firing rates, indicating that the crickets could hear these stimuli. AN2 appears to elicit antipredator behaviour only in flight, and we discuss possible reasons for this context-dependent function.

The neuroethology of song cessation in response to gleaning bat calls in two species of katydids, Neoconocephalus ensiger and Amblycorypha oblongifolia.

We investigated whether the use of primary or secondary behavioural defences is related to prey sensory thresholds using two species of North American katydids, Neoconocephalus ensiger and Amblycorypha oblongifolia. Male katydids produce intense calling songs to attract mates, and many gleaning bat species are known to use these calls to locate them as prey. Low duty cycle calling (i.e. sporadic calls) is a primary defence against gleaning bats (prevents attacks), and song cessation is a secondary defence (enables survival of an attack), for which these two species show behavioural differences. Echolocation calls of Myotis septentrionalis, a sympatric gleaning bat species, were broadcast to singing katydids and to neural preparations of these katydids to test if differences in behavioural response were related to differences in auditory sensitivity. We measured thresholds and firing patterns of the T-cell, an auditory interneuron involved in predator detection. We hypothesized that low duty cycle calling is the best defence for species not sensitive enough to mount a secondary defence in response to predator cues; therefore, we predicted that N. ensiger (high duty cycle song) would have lower behavioural and T-cell thresholds than A. oblongifolia (low duty cycle song). Although more N. ensiger ceased singing than A. oblongifolia, the number and maximum firing rate of T-cell action potentials did not differ between species for echolocation call sequences. We suggest that the T-cell has divergent functions within the Tettigoniidae, including predator and mate detection, and the function could be context dependent in some species.

Nocturnal activity positively correlated with auditory sensitivity in noctuoid moths.

We investigated the relationship between predator detection threshold and antipredator behaviour in noctuoid moths. Moths with ears sensitive to the echolocation calls of insectivorous bats use avoidance manoeuvres in flight to evade these predators. Earless moths generally fly less than eared species as a primary defence against predation by bats. For eared moths, however, there is interspecific variation in auditory sensitivity. At the species level, and when controlling for shared evolutionary history, nocturnal flight time and auditory sensitivity were positively correlated in moths, a relationship that most likely reflects selection pressure from aerial-hawking bats. We suggest that species-specific differences in the detection of predator cues are important but often overlooked factors in the evolution and maintenance of antipredator behaviour.