Located in the forelegs, katydid ears are unique among arthropods in having outer, middle and inner component, analogous to the mammalian ear. Unlike mammals, sound is received externally, and internally via a narrow ear canal (EC) derived from the respiratory tracheal system. Inside the EC sound travels slower than in free air, causing temporal and pressure differences between external and internal inputs. The delay is suspected to arise as sound propagation changes from adiabatic to isothermal, imposed by EC geometry. If true, a reduction in sound velocity should persist independently of the gas composition in the EC. Integrating experimental (laser Doppler vibrometry, micro-CT) and numerical methods, we demonstrate that the narrow radius of the EC is the major cause of the signal time delay. Results imply that the EC is asymmetrically bifurcated, creating four notable auditory paths for each ear. Implication of methods and findings in avian hearing are discussed.

This research combines experimental and numerical approaches to infer the cause of sound velocity reduction inside the acoustic trachea of katydids. The experimental aspect involves measuring the time for an acoustic signal to transmit through the EC of a katydid and determining the length of the EC. This enables sound velocity to be calculated by dividing the length of the EC by the time it takes the sound stimulus to reach the TM. To achieve this a special platform that isolates the external and internal auditory inputs is utilised (6) (17), along with an array of probe-loudspeakers, microphones, function generators and an LDV. We record the effect of different gas compositions on the sound propagation velocity. Gas is added to the EC through the use of a customised delivery system.

This study used specimens of Copiphora gorgonensis that were 8th generation descendants of a colony collected in November 2015. Collection occurred in National Natural Park Gorgona, Colombia, an island to which the species is endemic. The island is located in the Pacific Ocean near south western Colombia (latitude 2_58’ 7.4208” N; longitude 78_11’ 10.4964” W). Insects were maintained in communal vivaria at the University of Lincoln, at a temperature between 22_C and 27_C. They were sustained on a diet of baby corn, cat biscuits and water. A total of 9 adult individuals (5 males and 4 females) were used in this study, with data collected from 17 ears.

In order to observe the effects of the internal pathway only, we needed to isolate the internal and external sound inputs. To achieve this the insect was mounted on a customised platform, resembling a medieval pillory (Fig. 2). The platform consists of two Perspex panels that fit together, the dorsal of which encompasses a central notch for the insects’ head, along with two adjacent holes for the forelegs. These panels are held together within a metal frame. Liquid latex (Magnacraft, Midhurst, UK) was applied between the panels, and around the neck and forefemora– this sealed any remaining gaps to form the continuous barrier necessary for preventing sound transmission. This design ensures acoustic isolation between the external, tympanal sound input and the internal sound input of EC. A 23 kHz pure tone is produced by a function generator (SDG1000 Series Function/Arbitrary Waveform Generator, Siglent Technologies Co. Ltd., Shenzhen, P.R. China) and emitted via a MF1-S 1 Multi Field Speaker (Tucker Davis Technologies, Alachua, FL, US) with a modified probe attachment.

Before the beginning of each experiment, a reference point was recorded in order to standardise the experimental results. The probe-loudspeaker was mounted onto a micromanipulator (World Precision Instruments, Inc., Sarasota, FL, US) to allow for extra control. A calibrated1/8” (3.2mm) microphone (Br¨uel & Kjær, 4138, Nærum, Denmark) was held in a clamp and the loudspeaker probe was positioned 2 mm away from the microphones protective cap. The time taken for sound to travel from the loudspeaker probe to the microphone was recorded as the reference time. The mounted insect was positioned in front of the LDV, and the laser point focused on a tympanic membrane. A gas delivery system (enabling the manipulation of the gas composition between experiments) was introduced behind the prothoracic side keel, directed towards the acoustic spiracle. The probe-loudspeaker was then placed 2 mm away from the spiracle (matching the distance of the microphone reference signal), allowing sound to travel through the acoustic trachea and vibrate the tympanum. For each ear, the LDV was used to record the time for the signal vibrations to reach the tympanum. The arrival of the signal at the tympanal organ was measured as tympanal displacement. Recordings were first acquired without any changes to the gas composition within the ear canal (normal conditions). The time taken for sound to travel from the loudspeaker to the tympanic membrane was noted and used as a benchmark, throughout the experiment, to indicate when the gas composition in the EC was normal. Then, over a period of five seconds, pure CO2(Genuine Innovations, CA, US) was gently introduced to the canal via the gas delivery system, and the time recorded again. It was important to add the CO2 slowly, to allow the naturally occurring gas to escape the trachea. The gas delivery system assisted with this since the small microcapillary opening helped to regulate the gas pressure.

The CO2 was then left to diffuse out of the trachea until the time taken for the signal to reach the tympanum had returned to normal. This was replicated a minimum of three times and then the entire procedure was repeated for the opposite foreleg. Experiments were conducted between ambient temperatures of 19.8_C and 25.0_C, with the body temperatures of the insects ranging from 23.6_ to 28.1.

Anatomical measurements of the trachea

The anatomy of the acoustic trachea was examined using X-ray micro-CT and 3D reconstruction, using standard biomedical imaging software following the published protocols (17). All the specimens (five males and four females) were scanned with a Bruker SkyScan 1272 (Bruker micro-CT, Kontich, Belgium) at 50 kV, 200 mA, with a voxel size ranging from 3.12 _m to 10.99 _m. Reconstruction and automated measurements of EC were carried out with AMIRA (v. 6.7, VSG, Berlin, Germany). The tracheal lengths of all individuals were measured from the 3D reconstructions using the Center Line Tree module in AMIRA.

Sound analysis and calculation of velocity

The first local minimum of the sine wave chirp was used to obtain time measurements. To ensure that we were measuring the same point in both the reference recording and the tympanal recording, we designed a wave that exhibited a marker on the first local maximum. The frequency of this theoretical wave was adjustable using the PSV 9.4 Acquisition Software (Polytec, Waldbronn, Germany). The wave was then played through the MF1-S 1 Multi Field Speaker, at 10 kHz, into the spiracle of the insect and a microphone, with both the tympanal and microphone response recorded. The marker was clearly visible in both of these recordings, revealing the correct wave to extract the time signal from. The time delay between the reference signal and the amplitude signal of the tympanum was obtained using Matlab (R2014a, The Math-Works, Inc., Natick, MA, US). This delay in time was then used to calculate the velocity of sound propagation through each acoustic trachea - dividing the tracheal lengths obtained from 3D segmentations by the respective mean time delay.