The New Zealand bigeye, Pempheris adspersa, is a nocturnal planktivore and has recently been found to be an active sound producer. The rostral end of the swim bladder lies adjacent to Baudelot's ligament which spans between the bulla and the cleithrum bone of the pectoral girdle. The aim of this study was to use the auditory evoked potential technique to physiologically test the possibility that this structure provides an enhanced sensitivity to sound pressure in the bigeye. At 100 Hz, bigeye had hearing sensitivity similar to that of goldfish (species with a mechanical connection between the swim bladder and the inner ear mediated by the Weberian ossicles) and were much more sensitive than other teleosts without ancillary hearing structures. Severing Baudelot's ligament bilaterally resulted in a marked decrease in hearing sensitivity, as did swim bladder puncture or lateral line blockage. These results show that bigeye have an enhanced sensitivity to sound pressure and provide experimental evidence that the functional basis of this sensitivity represents a novel hearing specialization in fish involving the swim bladder, Baudelot's ligament and the lateral line.
Sound detection is a basal vertebrate capability, first evolving in the aquatic environment, and having a primary use in auditory scene analysis [1–4]. Underwater acoustic stimuli have two overlapping components that provide useful information. Hydrodynamic flow that dominates in the so-called ‘near-field’, and the propagated pressure wave that dominates in the ‘far-field’ [5,6]. The hydrodynamic component can be detected by both the ear and the lateral line hair cells directly [5,7,8], whereas the pressure component can only be detected after pressure-induced oscillations of an air-pocket, such as the swim bladder, are transferred as displacements to hair cells . Detection of the pressure component of sound waves typically results in enhanced sensitivity and/or bandwidth of hearing and may play a role in sound localization . Pressure sensitivity is most well known from otophysic connections which mechanically link vibrations of the swim bladder to the inner ear . With the large diversity of extant fish species, and variability of fish habitats, there is a corresponding diversity of acoustic systems and their evolution. The known variation in both auditory morphology and hearing bandwidth exceed that of any other vertebrate group , but the fish species tested to date represent only a fraction of the total known species . Expanding investigations to less well-studied groups, therefore, has great potential for identifying novel evolutionary adaptations and for better understanding the evolution of the diversity of acoustic abilities across vertebrates.
Here, we describe the hearing ability in the New Zealand bigeye (Pempheris adspersa; F. Pempheridae) a small (15 cm total length adult) nocturnal planktivorous species endemic to New Zealand that has recently been found to produce sound . This species has a prominent Baudelot's ligament, that in P. adspersa is particularly robust and is in contact with the swim bladder. Baudelot's ligament, situated between pectoral girdle and either skull base or first vertebra, is plesiomorphic for actinopterygian fish, and may be ossified or doubled . In P. adspersa, one end of the ligament attaches to the otic capsule and the other to the pectoral girdle at a point immediately internal to the lateral line. This arrangement may mechanically connect the swim bladder, otic capsule and lateral line and play a role in enhancing sensitivity to sound pressure. Using auditory evoked potentials (AEP), we tested the hypothesis that this represents a novel hearing specialization in teleosts.
2. Material and methods
(a) Anatomical description
Pempheris adspersa were examined by dissection, microCT and MRI scanning (see the electronic supplementary material for scanning procedures) to detail the relationship between Baudelot's ligament, the swim bladder and the otic capsule.
(b) Auditory evoked potentials
Auditory abilities of 30 bigeye were determined using AEP, a measure of neural activity in auditory periphery, nerve and brainstem in response to sound . For detailed description on the AEP technique used here, see Radford et al  and the electronic supplementary material. Following each baseline measurement, fish were subjected to one of five treatments: (i) severing the Baudelot's ligament (n = 5), with two small incisions through the membranous medial wall of the opercula cavity, medial to the pectoral girdle (figure 1); (ii) swim bladder deflation via a 27-gauge needle inserted through the body wall (n = 5); (iii) bilateral filling of lateral line canals with a polyurethane casting system (EASYCAST; n = 5); (iv) an incision sham, with incisions through the medial wall of the opercula cavity but the ligament not cut (n = 5); and (v) a lateral line sham, filling the lateral line canals with saline (n = 5). Each procedure lasted less than 5 min under anaesthesia and AEP was again measured following recovery. Goldfish (Carassius auratus, n = 5) were tested in the same system for comparison purposes. At the end of each trial, fish were euthanized and dissected to assess the morphological effects of the intervention. Dissections indicated that the ligament was fully severed and the swim bladder was fully deflated for both interventions.
(c) Data analysis
Two-way repeated-measures ANOVA was used to test the effect of treatments and frequency on bigeye, and a separate two-way ANOVA was run to examine differences between control bigeye and goldfish. To test hearing differences for multiple species at 100 Hz, a one-way ANOVA was run. For all ANOVAs, where a significant difference was observed, Tukey's HSD post hoc tests were conducted. All tests meet normality and homogeneity assumptions. For all tests, the significance level was at α = 0.05.
In P. adspersa, Baudelot's ligament is stretched between the pectoral girdle (supracleithrum and cleithrum) and skull base immediately caudal to the otic capsule. The rostral face of the swim bladder is in direct contact with Baudelot's ligament (figure 1a,b). In fresh specimens, the ligament can be tensioned by caudal pull of the hypaxial muscles on the pectoral girdle.
Intact bigeye detected sound up to 1000 Hz but were most sensitive at lower frequencies (100–400 Hz; figure 2a). Overall, this species’ hearing ability was as sensitive as goldfish at 100 Hz, although bigeye was significantly (F5,15 = 11.49, p < 0.001) less sensitive at higher frequencies (figure 2a). Severing the Baudelot's ligament while leaving the swim bladder intact caused a significant increase in threshold (F15,60 = 2.40, p < 0.001, figure 2b) with the greatest differences from 100 to 400 Hz, where the shift was up to 20 dB, as opposed to a shift of 8 dB at 600–1000 Hz (figure 2b). Deflating the swim bladder and blocking the lateral line also increased thresholds relative to controls but were not significantly different from severing Baudelot's ligament (figure 2b). Both sham experiments resulted in threshold differences from manipulated fish (F15,60 = 2.40, p < 0.001, figure 2b) and were similar to untreated fish.
When compared with goldfish and other species tested on the same AEP rig at 100 Hz, bigeye were as sensitive as goldfish and much more sensitive than other species without ancillary hearing structures (hapuka (Polyprion oxygeneios) 100 dB re 1 µPa; snapper (Pagrus auratus) 112 dB re 1 µPa; trevally (Caranx georgianus) 120 dB re 1 µPa; spotty (Notolabrus celidotus) 110 dB re 1 µPa; common triplefin (Forsterygion lapillum) 118 dB re 1 µPa [12,14,16]). Most known hearing specializations in fishes involve modification of the swim bladder in relation to the inner ear. Fluctuations in pressure owing to sound waves cause the swim bladder to oscillate in volume and the walls to pulsate , and fish have evolved a variety of ways to transfer this motion to the inner ear to enhance hearing . In addition, at least three families (Chaetodontidae , Clupeidae  and Loricariidae ) also have an association between the ear, swim bladder and lateral line that may enhance hearing. Bigeye's association between the swim bladder, ear, lateral line and Baudelot's ligament described here represents a possible new mechanism for the transfer of motion from the swim bladder to the ear. Bigeye were significantly more sensitive to sound pressure than fish that do not have ancillary hearing structures . Although bigeye hearing ability does not cover the same frequency bandwidth as otophysan fishes, they are as sensitive as goldfish at 100 Hz (figure 2a). The relationship between swim bladder/ear connections and bandwidth remains problematic in fishes. While otophysan and clupeiform fishes with contact between the swim bladder and ear do have expanded auditory bandwidth relative to other teleosts , experimental deflation of the swim bladder generally affects only sensitivity, not bandwidth, in contradiction of established hypotheses . Similar results were seen in this study after swim bladder deflation (figure 2b). Increases, either developmentally or evolutionarily, in the degree of swim bladder/ear connections [22–24] do drive increases in bandwidth but do not always correlate with increases in sensitivity [19,25]. In bigeye, there are no swim bladder horns, instead the anatomical evidence suggests the anterior part of the swim bladder is in direct contact with Baudelot's ligament which in turn connects with the otic capsule and the pectoral girdle. Disrupting this connection and/or the lateral line affects low frequency detection the most. We propose that Baudelot's ligament may act as a tensioned string that conducts sound better than the surrounding soft tissues, and forms an acoustic link that transmits vibration of the swim bladder to the otic capsule and to the lateral line as it passes through the pectoral girdle.
In conclusion, the current results show that bigeye have an enhanced sensitivity to sound pressure and provide direct experimental evidence that the functional basis of this sensitivity involves the swim bladder, Baudelot's ligament and the lateral line. We propose that the Baudelot's ligament transmits pressure-induced oscillations in the swim bladder to the ear and lateral line and that this complex represents a novel hearing specialization mechanism for enhanced pressure sensitivity. Our discovery also provides a qualitative addition to the evolutionary adaptations for hearing found in this most diverse group of vertebrates.
All procedures followed Animal Ethics guidelines of the University of Auckland. All data are available at doi:10.5061/dryad.6mj4t.
This collaborative research was supported by a Marsden Fast Start award from the Royal Society of New Zealand to C.A.R. and a Natural Sciences and Engineering Research Council of Canada Discovery grant to D.M.H.
- Received February 17, 2013.
- Accepted May 1, 2013.
- © 2013 The Author(s) Published by the Royal Society. All rights reserved.