Correction: BatCount: A software program to count moving animals.

[This corrects the article DOI: 10.1371/journal.pone.0278012.].

Characteristics of wild moose (Alces alces) vocalizations.

Moose are a popular species with recreationists but understudied acoustically. We used publicly available videos to characterize and quantify the vocalizations of moose in New Hampshire separated by age/sex class. We found significant differences in peak frequency, center frequency, bandwidth, and duration across the groups. Our results provide quantification of wild moose vocalizations across age/sex classes, which is a key step for passive acoustic detection of this species and highlights public videos as a potential resource for bioacoustics research of hard-to-capture and understudied species.

Graded alarm call behavior in wild fox squirrels (Sciurus niger).

Distress or alarm calls are vocalizations made when animals are in stressful situations or faced with a predator. Squirrels (Sciuridae) are known for being very vocal; however, most studies on alarm vocalizations are limited to ground squirrels. We investigated the acoustic behavior of the arboreal fox squirrel (Sciurus niger) under different conditions. Specifically, we tested the hypothesis that fox squirrels modify acoustic alarm behavior in response to different perceived threat levels and that this response is affected by sex and individual experience. Squirrels were trapped, and acoustic data were collected during periods in which the squirrels were alone, approached by humans, manipulated in traps, and handled by humans. Calls were categorized based on acoustic features, and we quantified the call rate (calls/s) across conditions. Threat level significantly affected vocal rate, with squirrels producing more calls overall when alone but shifting the proportion of emitted call types as threat level increased. Sex, capture history, and individual had no effect on call rate. These results suggest that fox squirrels use a graded alarm call response system to respond to threatening situations.

Acoustic behavior in the northern short-tailed shrew (Blarina brevicauda): Ultrasonic click production in a novel environment.

Although echolocation is classically associated with bats and dolphins, many animals produce ultrasonic and/or click-like signals to potentially navigate their environment. Shrews (Soricidae) are thought to rely on ultrasonic signals for echo-orientation, producing both ultrasonic click-like signals and tonal twittering signals. Recently, the role of ultrasonic clicks in shrew echo-orientation has been questioned, as these clicks have been proposed to be artifacts of shrews moving throughout their environment. By combining infrared video and ultrasonic audio recordings, we investigated the acoustic signals made by shrews (Blarina brevicauda) during different periods of locomotion. We hypothesized that if shrews are using ultrasonic vocalizations as a sensory mechanism, they will display head scanning behavior while emitting clicks that share key characteristics with other known echolocating animals. Our results show that B. brevicauda predominantly emit ultrasonic clicks rather than ultrasonic twittering calls while exploring a new environment, with clicks having the following characteristics: less than 120 µs in duration, median peak frequency of 26.9 kHz, and median bandwidth of 22.9 kHz. Across individuals we found no significant difference in peak frequency. Our results indicate that shrews emit ultrasonic clicks in novel environments which further supports the hypothesis of echo-orientation or echolocation in shrews.

BatCount: A software program to count moving animals.

One of the biggest challenges with species conservation is collecting accurate and efficient information on population sizes, especially from species that are difficult to count. Bats worldwide are declining due to disease, habitat destruction, and climate change, and many species lack reliable population information to guide management decisions. Current approaches for estimating population sizes of bats in densely occupied colonies are time-intensive, may negatively impact the population due to disturbance, and/or have low accuracy. Research-based video tracking options are rarely used by conservation or management agencies for animal counting due to the perceived training and elevated operating costs. In this paper, we present BatCount, a free software program created in direct consultation with end-users designed to automatically count bats emerging from cave roosts (historical populations 20,000-250,000) with a streamlined and user-friendly interface. We report on the software package and provide performance metrics for different recording habitat conditions. Our analysis demonstrates that BatCount is an efficient and reliable option for counting bats in flight, with performance hundreds of times faster than manual counting, and has important implications for range- and species-wide population monitoring. Furthermore, this software can be extended to count any organisms moving across a camera including birds, mammals, fish or insects.

Variations in advertisement call modulations do not influence vocal interactions in bullfrog choruses.

Chorusing male bullfrogs naturally vary the number of modulations within their advertisement call notes. A field playback experiment investigated whether these variations affect males' evoked vocal responses. Vocal responses were quantified manually and automatically by quantifying acoustic energy. The numbers of calls, number of notes, latency of response, and detected-note acoustic energy did not vary significantly across playback stimuli for focal males or the entire chorus, suggesting that variations in modulation number do not carry relevant information to males. Future work can determine whether modulation cues may function in sexual selection and affect female response.

Estimating chorusing activity by quantifying total acoustic energy.

Passive acoustics provides a powerful method for localizing vocalizing animals and estimating species abundance. A passive acoustics method previously used to census dense populations of flying bats is applied here to estimate chorusing activity of male bullfrogs vocalizing against anthropogenic noise. There are significant links between manual counts of the numbers of advertisement call notes and automatically detected notes and two measures of acoustic energy. These data provide a foundation for the use of acoustic energy measures to census vocal activity in different habitats.

Raptors avoid the confusion effect by targeting fixed points in dense aerial prey aggregations.

Collective behaviours are widely assumed to confuse predators, but empirical support for a confusion effect is often lacking, and its importance must depend on the predator's targeting mechanism. Here we show that Swainson's Hawks Buteo swainsoni and other raptors attacking swarming Mexican Free-tailed Bats Tadarida brasiliensis steer by turning towards a fixed point in space within the swarm, rather than by using closed-loop pursuit of any one individual. Any prey with which the predator is on a collision course will appear to remain on a constant bearing, so target selection emerges naturally from the geometry of a collision. Our results show how predators can simplify the demands on their sensory system by decoupling steering from target acquisition when capturing prey from a dense swarm. We anticipate that the same tactic will be used against flocks and schools across a wide range of taxa, in which case a confusion effect is paradoxically more likely to occur in attacks on sparse groups, for which steering and target acquisition cannot be decoupled.

Foundations of form and function: A synthesis-based curriculum for introductory-level organismal biology.

First-year majors organismal biology courses are frequently taught as survey courses that promote memorization rather than synthesis of biological concepts. To address the shortcomings of this approach, we redesigned the organismal portion of our introductory biology curriculum to create a "Foundations of Form and Function" course. Foundations of Form and Function introduces different organismal forms and focuses on the relationship between those forms and the execution of key physiological functions. Goals of our new course include the following: developing student recognition of common characteristics that unite living organisms as well as features that distinguish taxonomic groups, facilitating student understanding of how organisms accomplish similar functions through different forms, and reinforcing course themes with independent student research. In this paper, we describe course learning outcomes, organization, content, assessment, and laboratory activities. We also present student perspectives and outcomes of our course design based on data from four years of student evaluations. Finally, we explain how we modified our course to meet remote learning and social-distancing challenges presented by the COVID-19 pandemic in 2020 and 2021.

Aerial attack strategies of hawks hunting bats, and the adaptive benefits of swarming.

Aggregation can reduce an individual's predation risk, by decreasing predator hunting efficiency or displacing predation onto others. Here, we explore how the behaviors of predator and prey influence catch success and predation risk in Swainson's hawks Buteo swainsoni attacking swarming Brazilian free-tailed bats Tadarida brasiliensis on emergence. Lone bats including stragglers have a high relative risk of predation, representing ~5% of the catch but ~0.2% of the population. Attacks on the column were no less successful than attacks on lone bats, so hunting efficiency is not decreased by group vigilance or confusion. Instead, lone bats were attacked disproportionately often, representing ~10% of all attacks. Swarming therefore displaces the burden of predation onto bats outside the column-whether as isolated wanderers not benefitting from dilution through attack abatement, or as peripheral stragglers suffering marginal predation and possible selfish herd effects. In contrast, the hawks' catch success depended only on the attack maneuvers that they employed, with the odds of success being more than trebled in attacks involving a high-speed stoop or rolling grab. Most attacks involved one of these two maneuvers, which therefore represent alternative rather than complementary tactics. Hence, whereas a bat's survival depends on maintaining column formation, a hawk's success does not depend on attacking lone bats-even though their tendency to do so is sufficient to explain the adaptive benefits of their prey's aggregation behavior. A hawk's success instead depends on the flight maneuvers it deploys, including the high-speed stoop that is characteristic of many raptors. Swarming bats emerging from a massive desert roost reduce their predation risk by maintaining tight column formation, because the hawks that predate them attack peripheral stragglers and isolated wanderers disproportionately. Whereas a bat's predation risk depends on maintaining its position within the column, the catch success of a hawk depends on how it maneuvers itself to attack, and is maximized by executing a high-speed dive or rolling grab maneuver.

Separation of overlapping sources in bioacoustic mixtures.

Source separation is an important step to study signals that are not easy or possible to record individually. Common methods such as deep clustering, however, cannot be applied to signals of an unknown number of sources and/or signals that overlap in time and/or frequency-a common problem in bioacoustic recordings. This work presents an approach, using a supervised learning framework, to parse individual sources from a spectrogram of a mixture that contains a variable number of overlapping sources. This method isolates individual sources in the time-frequency domain using only one function but in two separate steps, one for the detection of the number of sources and corresponding bounding boxes, and a second step for the segmentation in which masks of individual sounds are extracted. This approach handles the full separation of overlapping sources in both time and frequency using deep neural networks in an applicable manner to other tasks such as bird audio detection. This paper presents method and reports on its performance to parse individual bat signals from recordings containing hundreds of overlapping bat echolocation signals. This method can be extended to other bioacoustic recordings with a variable number of sources and signals that overlap in time and/or frequency.

Echolocation while drinking: Pulse-timing strategies by high- and low-frequency FM bats.

During nightly foraging activity, echolocating bats drink by flying low over the water surface and dipping the lower jaw while avoiding further bodily contact with the water. This task poses different sensorimotor challenges than flying in the open to forage for insects. Of interest is how bats adjust the timing of their echolocation pulses to accommodate the surrounding scene, from the progressively nearer water surface itself to objects at longer distances. Drinking behavior has been described in only a few of the roughly 1,000 echolocating bat species, and in none of the 110 species in the Indian subcontinent. Here, we describe how bats emitting frequency-modulated (FM) echolocation pulses behaved while drinking from a swimming pool in urban northeast India. At least two different bat species were present, using 1st-harmonic frequencies sweeping down to about 35 Hz ("low frequency") and down to about 50 kHz ("high frequency"), separable at a 40 kHz boundary. Over entire drinking maneuvers, intervals between broadcast pulses accommodate both the proximate task of registering the water surface while drinking and registering echoes from the farther reaches of the scene. During approach to the water, both low and high frequency bats emit longer, more stable interpulse intervals that matched the time interval covering echo arrival-times out to the frequency-dependent maximum operating range. High frequency bats use shorter interpulse intervals than low frequency bats, consistent with the shorter operating range at higher frequencies. Bats then accelerate their pulse rate to guide the dive down to drinking, with low frequency bats continuing to decrease pulse intervals and high frequency bats maintaining a more steady interval during the drinking buzz. The circumstance that both groups were engaged in the same task made this a natural experiment on the behavior during approach.

The effect of jamming stimuli on the echolocation behavior of the bottlenose dolphin, Tursiops truncatus.

Echolocating bats and odontocetes face the potential challenge of acoustic interference from neighbors, or sonar jamming. To counter this, many bat species have adapted jamming avoidance strategies to improve signal detection, but any such avoidance strategies in dolphins is unknown. This study provides an investigation into whether dolphins modify echolocation behavior during jamming scenarios. Recorded echolocation clicks were projected at different click repetition rates and at different aspect angles relative to two dolphins' heads while each dolphin was performing a target detection task. Changes in the timing, amplitude, and frequency of structure of the dolphin's emitted signals were compared to determine if and how dolphins modify echolocation when faced with potentially interfering conspecific echolocation signals. The results indicate that both dolphins demonstrated different responses when faced with jamming scenarios, which may reflect optimal strategies according to individual auditory perception abilities.

Filibacter tadaridae sp. nov., isolated from within a guano pile from a colony of Mexican free-tailed bats Tadarida brasiliensis.

A Gram-stain-positive, aerobic bacterium, TB-66T, was isolated from a pile of bat guano in a cave of New Mexico, USA. On the basis of 16S rRNA gene sequence similarity comparisons, strain TB-66Tgrouped together with Filibacter limicola showing a 16S rRNA gene sequence similarity of 98.5 % to the type strain. The quinone system of strain TB-66T consisted predominantly of menaquinone MK-7. The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylserine and three unidentified phospholipids. The peptidoglycan type was A4α l-Lys-d-Glu (A11.33). The major fatty acids were C15 : 0 anteiso, C16 : 0, and C16 : 1 ω7c. The G+C content of the genomic DNA was 37.6 (±1.8) mol%. On the basis of the genotypic and phenotypic properties it is clear that strain TB-66T represents a member of the genus Filibacter, but is distinct from the only other species in the genus, Filibacter limicola DSM 13886T. We propose a novel species with the name Filibacter tadaridae sp. nov. The type strain is TB-66T (= CIP 111629T= LMG 30660T= CCM 8866T).

Echolocation adaptations during high-speed roost re-entry for Brazilian free-tailed bats (Tadarida brasiliensis).

Some bats re-enter their cave while using echolocation at very high speeds, but this behavior is poorly studied. Thermal imaging and an array of ultrasonic microphones were used to investigate the acoustic adaptations made during high-speed re-entry for single bats entering a cave. There was a significant overall effect between bat, distance to the ground, and its flight speed on pulse duration and interpulse interval (IPI). The data imply that overall bats reduce pulse duration, IPI, and bandwidth as they approach the cave and slow down, but the behavior changes among individuals.

Variation in Bat Guano Bacterial Community Composition With Depth.

Bats are known to be reservoirs for a variety of mammalian pathogens, including viruses, fungi, and bacteria. Many of the studies examining the microbial community inhabiting bats have investigated bacterial taxa found within specific bat tissues and isolated bat guano pellets, but relatively few studies have explored bacterial diversity within bat guano piles. In large bat caves, bat guano can accumulate over time, creating piles several meters deep and forming complex interactions with coprophagous organisms in a habitat with low light and oxygen. As the guano decays, the nutrient composition changes, but the bacterial communities deep within the pile have not been characterized. Here, we assess the bacterial communities across varying depths within the guano pile using both culture-independent and culture-dependent methods. We found that although similar taxa are found throughout the guano pile, the relative abundances of taxa within the pile shift, allowing certain taxa to dominate the bacterial community at varying depths. We also identified potential bacterial functions being performed within the bat guano as various depths within the pile and found little variation in terms of the dominant predicted functions, suggesting that although the relative abundances of bacterial taxa are changing, the functions being performed are similar. Additionally, we cultured 15 different bacterial species, including 2 not present in our culture-independent analysis, and discuss the pathogenicity potential of these taxa. This study represents the first characterization of the bacterial community from the extreme environment within a bat guano pile and demonstrates the potential for bat caves as resources for identifying new bacterial species.

Recording animal vocalizations from a UAV: bat echolocation during roost re-entry.

Unmanned aerial vehicles (UAVs) are rising in popularity for wildlife monitoring, but direct recordings of animal vocalizations have not yet been accomplished, likely due to the noise generated by the UAV. Echolocating bats, especially Tadarida brasiliensis, are good candidates for UAV recording due to their high-speed, high-altitude flight. Here, we use a UAV to record the signals of bats during morning roost re-entry. We designed a UAV to block the noise of the propellers from the receiving microphone, and report on the characteristics of bioacoustic recordings from a UAV. We report the first published characteristics of echolocation signals from bats during group flight and cave re-entry. We found changes in inter-individual time-frequency shape, suggesting that bats may use differences in call design when sensing in complex groups. Furthermore, our first documented successful recordings of animals in their natural habitat demonstrate that UAVs can be important tools for bioacoustic monitoring, and we discuss the ethical considerations for such monitoring.

A systematic method for isolating, tracking and discriminating time-frequency components of bat echolocation calls.

Echolocating bats can rapidly modify frequency modulation (FM) curvatures of their calls when facing challenging echolocation tasks. Frequency parameters, such as start/end/peak frequency, have often been extracted from the time-frequency domain to study the call variation. Even though this kind of signal investigation method reveals important findings, these approaches to analyze bat echolocation calls use bulk parameters, which hide subtleties in the call structure that may be important to the bat. In some cases, calls can have the same start and end frequencies but have different FM curvatures, and subsequently may influence the sensory task performance. In the present study, the authors demonstrate an algorithm using a combination of digital filters, power limited time-frequency information, derivative dynamic time warping, and agglomerative hierarchical clustering to extract and categorize the time-frequency components (TFCs) of 21 calls from Brazilian free-tailed bat (Tadarida brasiliensis) to quantitatively compare FM curvatures. The detailed curvature analysis shows an alternative perspective to look into the TFCs and hence serves as the preliminary step to understand the adaptive call design of bats.

Off-axis targets maximize bearing Fisher Information in broadband active sonar.

Broadband active sonar systems estimate range from time delay and velocity from Doppler shift. Relatively little attention has been paid to how the received echo spectrum encodes information about the bearing of an object. This letter derives the bearing Fisher Information encoded in the frequency dependent transmitter beampattern. This leads to a counter-intuitive result: directing the sonar beam so that a target of interest is slightly off-axis maximizes the bearing information about the target. Beam aim data from a dolphin biosonar experiment agree closely with the angle predicted to maximize bearing information.

Estimating colony sizes of emerging bats using acoustic recordings.

The decline of bats demands more widespread monitoring of populations for conservation and management. Current censusing methods are either prone to bias or require costly equipment. Here, we report a new method using passive acoustics to determine bat count census from overall acoustic amplitude of the emerging bat stream. We recorded the video and audio of an emerging colony of Mexican free-tailed bats from two cave locations across multiple nights. Instantaneous bat counts were calculated from the video frames, and the bat stream's acoustic amplitude corresponding to each video frame was determined using three different methods for calculating acoustic intensity. We found a significant link between all three acoustic parameters and bat count, with the highest R (2) of 0.742 linking RMS pressure and bat count. Additionally, the relationship between acoustics and population size at one cave location could accurately predict the population size at another cave location. The data were gathered with low-cost, easy-to-operate equipment, and the data analysis can be easily accomplished using automated scripts or with open-source acoustic software. These results are a potential first step towards creating an acoustic model to estimate bat population at large cave colonies worldwide.