Outsourced hearing in an orb-weaving spider that uses its web as an auditory sensor.

SignificanceThe sense of hearing in all known animals relies on possessing auditory organs that are made up of cellular tissues and constrained by body sizes. We show that hearing in the orb-weaving spider is functionally outsourced to its extended phenotype, the proteinaceous self-manufactured web, and hence processes behavioral controllability. This finding opens new perspectives on animal extended cognition and hearing-the outsourcing and supersizing of auditory function in spiders. This study calls for reinvestigation of the remarkable evolutionary ecology and sensory ecology in spiders-one of the oldest land animals. The sensory modality of outsourced hearing provides a unique model for studying extended and regenerative sensing and presents new design features for inspiring novel acoustic flow detectors.

Tiny spies: mosquito antennae are sensitive sensors for eavesdropping on frog calls.

Most mosquito and midge species use hearing during acoustic mating behaviors. For frog-biting species, however, hearing plays an important role beyond mating as females rely on anuran calls to obtain blood meals. Despite the extensive work examining hearing in mosquito species that use sound in mating contexts, our understanding of how mosquitoes hear frog calls is limited. Here, we directly investigated the mechanisms underlying detection of frog calls by a mosquito species specialized on eavesdropping on anuran mating signals: Uranotaenia lowii. Behavioral, biomechanical and neurophysiological analyses revealed that the antenna of this frog-biting species can detect frog calls by relying on neural and mechanical responses comparable to those of non-frog-biting species. Our findings show that in Ur. lowii, contrary to most species, males do not use sound for mating, but females use hearing to locate their anuran host. We also show that the response of the antennae of this frog-biting species resembles that of the antenna of species that use hearing for mating. Finally, we discuss our data considering how mosquitoes may have evolved the ability to tap into the communication system of frogs.

Attention and distraction in the modular visual system of a jumping spider.

Animals must selectively attend to relevant stimuli and avoid being distracted by unimportant stimuli. Jumping spiders (Salticidae) do this by coordinating eyes with different capabilities. Objects are examined by a pair of high-acuity principal eyes, whose narrow field of view is compensated for by retinal movements. The principal eyes overlap in field of view with motion-sensitive anterior-lateral eyes (ALEs), which direct their gaze to new stimuli. Using a salticid-specific eyetracker, we monitored the gaze direction of the principal eyes as they examined a primary stimulus. We then presented a distractor stimulus visible only to the ALEs and observed whether the principal eyes reflexively shifted their gaze to it or whether this response was flexible. Whether spiders redirected their gaze to the distractor depended on properties of both the primary and distractor stimuli. This flexibility suggests that higher-order processing occurs in the management of the attention of the principal eyes.

Reducing the Cost of Electrophysiology in the Teaching Laboratory.

Electrophysiology is a fundamental part of neuroscience and there are many published laboratory exercises suitable for undergraduates. However, the cost of equipping a lab is often a barrier to implementing these exercises. In this paper, we outline lab needs, suggest strategies for building a lab incrementally by adding equipment as budgets permit, and suggest specific areas for cost-cutting. We also point out instances in which it makes most sense to purchase or borrow research-grade equipment. A linked Google document lists specific items, prices, and purchase links.

Mosquito (Aedes aegypti) flight tones: frequency, harmonicity, spherical spreading, and phase relationships.

Mosquito flight produces a tone as a side effect of wing movement; this tone is also a communication signal that is frequency-modulated during courtship. Recordings of tones produced by tethered flying male and female Aedes aegypti were undertaken using pairs of pressure-gradient microphones above and below, ahead and behind, and to the left and right over a range of distances. Fundamental frequencies were close to those previously reported, although amplitudes were lower. The male fundamental frequency was higher than that of the female and males modulated it over a wider range. Analysis of harmonics shows that the first six partials were nearly always within 1 Hz of integer multiples of the fundamental, even when the fundamental was being modulated. Along the front-back axis, amplitude attenuated as a function of distance raised to the power 2.3. Front and back recordings were out of phase, as were above and below, while left and right were in phase. Recordings from ahead and behind showed quadratic phase coupling, while others did not. Finally, two methods are presented for separating simultaneous flight tones in a single recording and enhancing their frequency resolution. Implications for mosquito behavior are discussed.

Visual attention and processing in jumping spiders.

Jumping spiders have extraordinary vision. Using multiple, specialized eyes, these spiders selectively gather and integrate disparate streams of information about motion, color, and spatial detail. The saccadic movements of a forward-facing pair of eyes allow spiders to inspect their surroundings and identify objects. Here, we discuss the jumping spider visual system and how visual information is attended to and processed.

Associative learning in the dengue vector mosquito, Aedes aegypti: avoidance of a previously attractive odor or surface color that is paired with an aversive stimulus.

Associative learning has been shown in a variety of insects, including the mosquitoes Culex quinquefasciatus and Anopheles gambiae. This study demonstrates associative learning for the first time in Aedes aegypti, an important vector of dengue, yellow fever and chikungunya viruses. This species prefers to rest on dark surfaces and is attracted to the odor of 1-octen-3-ol. After training in which a dark surface alone or a dark surface with odor was paired with electric shock, mosquitoes avoided the previously attractive area. The association was stronger when odor was included in training, was retained for at least 60 min but not for 24 h, and was equal for males and females. These results demonstrate the utility of a bulk-training paradigm for mosquitoes similar to that used with Drosophila melanogaster.

Adaptive auditory risk assessment in the dogbane tiger moth when pursued by bats.

Moths and butterflies flying in search of mates risk detection by numerous aerial predators; under the cover of night, the greatest threat will often be from insectivorous bats. During such encounters, the toxic dogbane tiger moth, Cycnia tenera uses the received intensity, duration and emission pattern of the bat's echolocation calls to determine when, and how many, defensive ultrasonic clicks to produce in return. These clicks, which constitute an acoustic startle response, act as warning signals against bats in flight. Using an integrated test of stimulus generalization and dishabituation, here we show that C. tenera is able to discriminate between the echolocation calls characteristic of a bat that has only just detected it versus those of a bat actively in pursuit of it. We also show that C. tenera habituates more profoundly to the former stimulus train ('early attack') than to the latter ('late attack'), even though it was initially equally responsive to both stimuli. Matched sensory and behavioural data indicate that reduced responsiveness reflects habituation and is not merely attributable to sensory adaptation or motor fatigue. In search of mates in the face of bats, C. tenera's ability to discriminate between attacking bats representing different levels of risk, and to habituate less so to those most dangerous, should function as an adaptive cost-benefit trade-off mechanism in nature.

Classical conditioning through auditory stimuli in Drosophila: methods and models.

The role of sound in Drosophila melanogaster courtship, along with its perception via the antennae, is well established, as is the ability of this fly to learn in classical conditioning protocols. Here, we demonstrate that a neutral acoustic stimulus paired with a sucrose reward can be used to condition the proboscis-extension reflex, part of normal feeding behavior. This appetitive conditioning produces results comparable to those obtained with chemical stimuli in aversive conditioning protocols. We applied a logistic model with general estimating equations to predict the dynamics of learning, which successfully predicts the outcome of training and provides a quantitative estimate of the rate of learning. Use of acoustic stimuli with appetitive conditioning provides both an alternative to models most commonly used in studies of learning and memory in Drosophila and a means of testing hearing in both sexes, independently of courtship responsiveness.

Quantitative skills in undergraduate neuroscience education in the age of big data.

For over a half-Century, the mathematics requirement for graduation at most undergraduate colleges and universities has been one year of calculus and a semester of statistics. Many universities and colleges offer a neuroscience major that may or may not add additional mathematics, statistics, or data science requirements. Today in the age of Big Data and Systems Neuroscience, many students are ill-equipped for the future without the tools of computational competency that are necessary to tackle the large data sets generated by contemporary neuroscience research. Required courses in statistics still focus on parametric statistics based on the normal distribution and do not provide the computational tools required to analyze big data sets. Undergraduates in STEM fields including neuroscience need to enroll in the Data Science courses that are required in the social sciences (e.g., economics, political science and psychology). Contemporary systems neuroscience is routinely done by interdisciplinary research teams of statisticians, engineers, and physical scientists. Emerging "NeuroX-omics" such as connectomics have emerged along with genomics, proteomics, and transcriptomics, all of which deploy systems analysis techniques based on mathematical graph theory. Connectomics is the 21st Century's functional neuroanatomy. Whole brain connectome research appears almost monthly in the Drosphila, zebra fish, and mouse literature, and human brain connectomics is not far behind. The techniques for connectomics rely on the tools of data science. Undergraduate neuroscience students are already squeezed for credit hours given the high-prescribed science curriculum for biology majors and premedical students, in addition to required courses in social sciences and humanities. However, additional training in mathematics, statistics, computer science, and/or data science is urgently needed for undergraduate neuroscience majors just to understand the contemporary research literature. Undoubtedly, the faculty who teach neuroscience courses are acutely aware of the problem and most of them freely acknowledge the importance of quantitative analytical skills for their students. However, some faculty members may feel that their own math and statistics knowledge or other analytical skills have atrophied beyond recall or were never fulfilled in the first place. In this commentary I suggest that this problem can be ameliorated, though not solved, through organizing workshops, journal clubs, or independent studies courses in which the students and the instructors learn and teach each other in short-course format. In addition, web-available teaching materials such as targeted video clips are plentifully available on the internet. To attract and maintain student interest, qauntitative instruction and learning should occur in neuroscience context.

Neural responses to one- and two-tone stimuli in the hearing organ of the dengue vector mosquito.

Recent studies demonstrate that mosquitoes listen to each other's wing beats just prior to mating in flight. Field potentials from sound-transducing neurons in the antennae contain both sustained and oscillatory components to pure and paired tone stimuli. Described here is a direct comparison of these two types of response in the dengue vector mosquito, Aedes aegypti. Across a wide range of frequencies and intensities, sustained responses to one- and two-tone stimuli are about equal in magnitude to oscillatory responses to the beats produced by two-tone stimuli. All of these responses are much larger than the oscillatory responses to one-tone stimuli. Similarly, the frequency range extends up to at least the fifth harmonic of the male flight tone for sustained responses to one- and two-tone stimuli and oscillatory responses at the beat frequency of two-tone stimuli, whereas the range of oscillatory response to a one-tone stimulus is limited to, at most, the third harmonic. Thresholds near the fundamental of the flight tone are lower for oscillatory responses than for sustained deflections, lower for males than for females, and within the behaviorally relevant range. A simple model of the transduction process can qualitatively account for both oscillatory and sustained responses to pure and paired tones. These data leave open the question as to which of several alternative strategies underlie flight tone matching behavior in mosquitoes.

Ogre-Faced, Net-Casting Spiders Use Auditory Cues to Detect Airborne Prey.

Prey capture behavior among spiders varies greatly from passive entrapment in webs to running down prey items on foot. Somewhere in the middle are the ogre-faced, net-casting spiders [1] (Deinopidae: Deinopis) that actively capture prey while being suspended within a frame web [2-5]. Using a net held between their front four legs, these spiders lunge downward to ensnare prey from off the ground beneath them. This "forward strike" is sensorially mediated by a massive pair of hypersensitive, night-vision eyes [5-7]. Deinopids can also intercept flying insects with a "backward strike," a ballistically rapid, overhead back-twist, that seems not to rely on visual cues [4, 5, 8]. Past reports have hypothesized a role of acoustic detection in backward strike behavior [4, 5, 8]. Here, we report that the net-casting spider, Deinopis spinosa, can detect auditory stimuli from at least 2 m from the sound source, at or above 60 dB SPL, and that this acoustic sensitivity is sufficient to trigger backward strike behavior. We present neurophysiological recordings in response to acoustic stimulation, both from sound-sensitive areas in the brain and isolated forelegs, which demonstrate a broad range of auditory sensitivity (100-10,000 Hz). Moreover, we conducted behavioral assays of acoustic stimulation that confirm acoustic triggering of backward net-casting by frequencies in harmony with flight tones of known prey. However, acoustic stimulation using higher frequency sounds did not elicit predatory responses in D. spinosa. We hypothesize higher frequencies are emitted by avian predators and that detecting these auditory cues may aid in anti-predator behavior. VIDEO ABSTRACT.

g-PRIME: A Free, Windows Based Data Acquisition and Event Analysis Software Package for Physiology in Classrooms and Research Labs.

We present g-PRIME, a software based tool for physiology data acquisition, analysis, and stimulus generation in education and research. This software was developed in an undergraduate neurophysiology course and strongly influenced by instructor and student feedback. g-PRIME is a free, stand-alone, windows application coded and "compiled" in Matlab (does not require a Matlab license). g-PRIME supports many data acquisition interfaces from the PC sound card to expensive high throughput calibrated equipment. The program is designed as a software oscilloscope with standard trigger modes, multi-channel visualization controls, and data logging features. Extensive analysis options allow real time and offline filtering of signals, multi-parameter threshold-and-window based event detection, and two-dimensional display of a variety of parameters including event time, energy density, maximum FFT frequency component, max/min amplitudes, and inter-event rate and intervals. The software also correlates detected events with another simultaneously acquired source (event triggered average) in real time or offline. g-PRIME supports parameter histogram production and a variety of elegant publication quality graphics outputs. A major goal of this software is to merge powerful engineering acquisition and analysis tools with a biological approach to studies of nervous system function.

The Long and Short of Hearing in the Mosquito Aedes aegypti.

Mating behavior in Aedes aegypti mosquitoes occurs mid-air and involves the exchange of auditory signals at close range (millimeters to centimeters) [1-6]. It is widely assumed that this intimate signaling distance reflects short-range auditory sensitivity of their antennal hearing organs to faint flight tones [7, 8]. To the contrary, we show here that male mosquitoes can hear the female's flight tone at surprisingly long distances-from several meters to up to 10 m-and that unrestrained, resting Ae. aegypti males leap off their perches and take flight when they hear female flight tones. Moreover, auditory sensitivity tests of Ae. aegypti's hearing organ, made from neurophysiological recordings of the auditory nerve in response to pure-tone stimuli played from a loudspeaker, support the behavioral experiments. This demonstration of long-range hearing in mosquitoes overturns the common assumption that the thread-like antennal hearing organs of tiny insects are strictly close-range ears. The effective range of a hearing organ depends ultimately on its sensitivity [9-13]. Here, a mosquito's antennal ear is shown to be sensitive to sound levels down to 31 dB sound pressure level (SPL), translating to air particle velocity at nanometer dimensions. We note that the peak of energy of the first formant of the vowels of the human speech spectrum range from about 200-1,000 Hz and is typically spoken at 45-70 dB SPL; together, they lie in the sweet spot of mosquito hearing. VIDEO ABSTRACT.

Tissue remodeling: a mating-induced differentiation program for the Drosophila oviduct.

BACKGROUND: In both vertebrates and invertebrates, the oviduct is an epithelial tube surrounded by visceral muscles that serves as a conduit for gamete transport between the ovary and uterus. While Drosophila is a model system for tubular organ development, few studies have addressed the development of the fly's oviduct. Recent studies in Drosophila have identified mating-responsive genes and proteins whose levels in the oviduct are altered by mating. Since many of these molecules (e.g. Muscle LIM protein 84B, Coracle, Neuroglian) have known roles in the differentiation of muscle and epithelia of other organs, mating may trigger similar differentiation events in the oviduct. This led us to hypothesize that mating mediates the last stages of oviduct differentiation in which organ-specific specializations arise. RESULTS: Using electron- and confocal-microscopy we identified tissue-wide post-mating changes in the oviduct including differentiation of cellular junctions, remodeling of extracellular matrix, increased myofibril formation, and increased innervation. Analysis of once- and twice-mated females reveals that some mating-responsive proteins respond only to the first mating, while others respond to both matings. CONCLUSION: We uncovered ultrastructural changes in the mated oviduct that are consistent with the roles that mating-responsive proteins play in muscle and epithelial differentiation elsewhere. This suggests that mating triggers the late differentiation of the oviduct. Furthermore, we suggest that mating-responsive proteins that respond only to the first mating are involved in the final maturation of the oviduct while proteins that remain responsive to later matings are also involved in maintenance and ongoing function of the oviduct. Taken together, our results establish the oviduct as an attractive system to address mechanisms that regulate the late stages of differentiation and maintenance of a tubular organ.

A polyimide pressure-contact multielectrode array for implantation along a submillimeter neural process in small animals.

We describe a microelectrode system for recordings from nerve bundles with diameters ranging from 20-200 microm. A novel polyimide structure allows for a planar microfabricated device to constrain a free neural process against several recording sites. This polyimide array contains multiple zigzag vias through which a small nerve process may be woven while remaining functionally intact in a live preparation. Our electrode construct features the benefits of nerve cuffs (evenly spaced electrodes in a polymer) and the functionality of a nerve hook (ability to connect to submillimeter processes). The device records extracellular action potentials in the red-swamp crayfish, Procambarus clarkii. Action potential propagation is monitored at several sites along a constrained nerve in this model organism's peripheral nervous system. Details of temporal accuracy and error sources in absolute conduction velocity measurements from microelectrode arrays are discussed.

An inexpensive sub-millisecond system for walking measurements of small animals based on optical computer mouse technology.

Stimuli from a broad spectrum of sensory modalities, including visual, auditory, thermal, and chemical, can elicit walking responses in animals, reflecting neural activity in sensorimotor pathways. We have developed an integrated walking measurement system with sub-millisecond temporal accuracy capable of detecting position changes on the order of 100 microm. This tracking system provides the experimenter with a means by which to map out the response spectrum of a tethered animal to any number of sensory inputs on time scales relevant to propagation in the nervous system. The data acquisition system consists of a modified optical computer mouse, a microcontroller with peripheral support circuitry, a binary stimulus sync line, and a serial (RS-232) data transfer interface. The entire system is constructed of relatively inexpensive components mostly converted from commercially available peripheral devices. We have acquired walking data synchronized with auditory stimuli at rates in excess of 2100 samples per second while applying this system to the walking phonotactic response of the parasitic fly Ormia ochracea.

Flexible multielectrodes can resolve multiple muscles in an insect appendage.

Research into the neuromechanical basis of behavior, either in biomechanics, neuroethology, or neuroscience, is frequently limited by methods of data collection. Two of the most pressing needs are for methods with which to (1) record from multiple neurons or muscles simultaneously and (2) perform this recording in intact, behaving animals. In this paper we present the fabrication and testing of flexible multielectrode arrays (fMEAs) that move us significantly towards these goals. The fMEAs were used to record the activity of several distinct units in the coxa of the cockroach Blaberus discoidalis. The devices fabricated here address the first goal in two ways: (1) their flexibility allows them to be inserted into an animal and guided through internal tissues in order to access distinct groups of neurons and muscles and (2) their recording site geometry has been tuned to suit the anatomy under study, yielding multichannel spike waveforms that are easily separable under conditions of spike overlap. The flexible nature of the devices simultaneously addresses the second goal, in that it is less likely to interfere with the natural movement of the animal.

Airborne Acoustic Perception by a Jumping Spider.

Jumping spiders (Salticidae) are famous for their visually driven behaviors [1]. Here, however, we present behavioral and neurophysiological evidence that these animals also perceive and respond to airborne acoustic stimuli, even when the distance between the animal and the sound source is relatively large (∼3 m) and with stimulus amplitudes at the position of the spider of ∼65 dB sound pressure level (SPL). Behavioral experiments with the jumping spider Phidippus audax reveal that these animals respond to low-frequency sounds (80 Hz; 65 dB SPL) by freezing-a common anti-predatory behavior characteristic of an acoustic startle response. Neurophysiological recordings from auditory-sensitive neural units in the brains of these jumping spiders showed responses to low-frequency tones (80 Hz at ∼65 dB SPL)-recordings that also represent the first record of acoustically responsive neural units in the jumping spider brain. Responses persisted even when the distances between spider and stimulus source exceeded 3 m and under anechoic conditions. Thus, these spiders appear able to detect airborne sound at distances in the acoustic far-field region, beyond the near-field range often thought to bound acoustic perception in arthropods that lack tympanic ears (e.g., spiders) [2]. Furthermore, direct mechanical stimulation of hairs on the patella of the foreleg was sufficient to generate responses in neural units that also responded to airborne acoustic stimuli-evidence that these hairs likely play a role in the detection of acoustic cues. We suggest that these auditory responses enable the detection of predators and facilitate an acoustic startle response. VIDEO ABSTRACT.