Answer the questions about the attached article. It needs to be 2 pages double-spaced. 1. What question did the authors seek to answer in their paper? 2. What types of data did they collect to answer their question,and how did they collect their data? 3. What did they find? 4. Did the authors answer the question they originally posed? 5. Finally, name one critique or flaw of the paper. Niemiller_2013_Biology_Letters.pdf Download Attachment This is an unformatted preview. Please download the attached document for the original format. Downloaded from rsbl.royalsocietypublishing.org on May 28, 2013 Evidence for hearing loss in amblyopsid cavefishes Matthew L. Niemiller, Dennis M. Higgs and Daphne Soares Biol. Lett. 2013 9, 20130104, published 27 March 2013 References This article cites 13 articles, 1 of which can be accessed free Subject collections Articles on similar topics can be found in the following collections http://rsbl.royalsocietypublishing.org/content/9/3/20130104.full.html#ref-list-1 ecology (629 articles) evolution (642 articles) neuroscience (75 articles) Email alerting service Receive free email alerts when new articles cite this article - sign up in the box at the top right-hand corner of the article or click here To subscribe to Biol. Lett. go to: http://rsbl.royalsocietypublishing.org/subscriptions Downloaded from rsbl.royalsocietypublishing.org on May 28, 2013 Neurobiology rsbl.royalsocietypublishing.org Evidence for hearing loss in amblyopsid cavefishes Matthew L. Niemiller1, Dennis M. Higgs2 and Daphne Soares3 1 Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06520, USA Department of Biological Sciences, University of Windsor, Windsor, Ontario, Canada N9B 3P4 3 Department of Biology, University of Maryland, College Park, MD 20742, USA 2 Research Cite this article: Niemiller ML, Higgs DM, Soares D. 2013 Evidence for hearing loss in amblyopsid cavefishes. Biol Lett 9: 20130104. http://dx.doi.org/10.1098/rsbl.2013.0104 Received: 1 February 2013 Accepted: 5 March 2013 Subject Areas: evolution, neuroscience, ecology Keywords: auditory, evolution, fish, subterranean Author for correspondence: Daphne Soares e-mail: daph@umd.edu The constant darkness of caves and other subterranean habitats imposes sensory constraints that offer a unique opportunity to examine evolution of sensory modalities. Hearing in cavefishes has not been well explored, and here we show that cavefishes in the family Amblyopsidae are not only blind but have also lost a significant portion of their hearing range. Our results showed that cave and surface amblyopsids shared the same audiogram profile at low frequencies but only surface amblyopsids were able to hear frequencies higher than 800 Hz and up to 2 kHz. We measured ambient noise in aquatic cave and surface habitats and found high intensity peaks near 1 kHz for streams underground, suggesting no adaptive advantage in hearing in those frequencies. In addition, cave amblyopsids had lower hair cell densities compared with their surface relative. These traits may have evolved in response to the loud high-frequency background noise found in subterranean pools and streams. This study represents the first report of auditory regression in a subterranean organism. 1. Introduction Animals that live in continual darkness are faced with unique challenges in order to locate and identify food, predators and each other [1]. Without visual information, independent lineages of obligate cave-dwelling organisms have evolved regressive features, such as the loss or reduction of eyes and pigmentation and constructive traits, such as longer appendages and hypertrophy of non-visual sensory systems [2]. Aside from darkness being common to all subterranean habitats, several other abiotic factors influence subterranean organisms, such as relatively stable temperature, high humidity and hydrological factors (for example, periodic flooding) [2]. However, little to nothing is known about how the diverse abiotic characteristics of caves affect the sensory ecology of cave animals. Here, we examine the relationship between the acoustic environment of caves and hearing in amblyopsid cavefishes. Aquatic cave organisms, such as cavefishes, survive in perpetual darkness. An important sensory modality in such environments may be the sense of hearing. In above-ground aquatic habitats, hearing is important for many aspects of fish behaviour (reviewed in [3]) and is effective over relatively long distances owing to the nature of underwater sound travel. Sound may play an especially important role in subterranean habitats owing to the lack of visual signals yet the acoustic properties of these habitats have been largely ignored to date. Hypertrophy of hearing characteristics could be adaptive in caves for several reasons, including working in association with other non-visual senses to detect prey, conspecifics or predators. However, the degree to which hearing abilities are modified in cavefishes is largely unknown, as behavioural and neurophysiological studies on the acoustical biology of cavefishes are extremely limited. Popper [4] showed that the cave and surface forms of the characid Astyanax mexicanus do not differ in hearing. Similarly, no differences were found between cave and surface forms of the molly Poecilia mexicana [5]. & 2013 The Author(s) Published by the Royal Society. All rights reserved. Downloaded from rsbl.royalsocietypublishing.org on May 28, 2013 (a) 2 (b) 50 hair cell density (units 100 mm–2) 40 30 Biol Lett 9: 20130104 (ii) rsbl.royalsocietypublishing.org (i) 20 (iii) Chologaster Troglichthys (i) Speoplatyrhinus (ii) (iii) 10 0 A. spelaea T. subterraneus F. agassizii Figure 1. (a) The phylogenetic relationships of the two obligate cave species (white) (i) Typhlichthys and (ii) Amblyopsis and one surface species (black) (iii) Forbesichthys. (b) Cell density counts for the three species show fewer hair cells in the cavefishes (*F2,23 ¼ 15.3, p ¼ 0.0007). Inserts show photomicrograms of the ears stained with phalloidin. Scale bar, 100 mm. (Online version in colour.) Here, we show the first report of differences in hearing characteristics in a cavefish compared with its surface relative. We compared the auditory evoked potentials (AEPs) of three species in the family Amblyopsidae, as well as the acoustic profiles of their subterranean habitats in order to investigate whether a relationship exists between noise in cave habitats and cavefish hearing. Amblyopsid caveshes are a model system for studying the ecological and evolutionary processes of cave adaptation because the cave-restricted species in the family represent a range of troglomorphy that reflects variable durations of isolation in caves [6]. Cave amblyopsids are one of the most comprehensively studied caveshes, with six genera and eight species [7]. In this study, we examine the hearing characteristics of three related amblyopsids: the surface dwelling, Forbesichthys agassizii and two cave species, Typhlichthys subterraneus and Amblyopsis spelaea (figure 1a). pools with some current (4– 12 m2, 0.1– 0.8 m depth, 0 – 0.6 ms21 (low flow), cobble/bedrock substrate) in L&N Railroad Cave, Barren Co., KY, USA. (a) Auditory evoked potentials This method measures the compound electrical potential created by the eighth cranial nerve and auditory brainstem nuclei in response to sound [9,10]. We restrained submerged fish and played 10 msec tones, ranging from 0.1 to 2 kHz at 0.1 Hz intervals. We increased the sound level in 5 dB intervals until a stereotypical evoked potential waveform was detected (figure 2, insert). We determined auditory threshold to be the lowest intensity for which AEP traces were detected [11]. Sound output was measured with a hydrophone (model LC-10, Reson Inc; Calibration sensitivity of 2208.9 dB re 1V uPa21, 0 – 100 kHz) and an accelerometer (model 4524 cubic triaxial deltatron, ¨ Bruel & Kjær). We calibrated sound level and particle acceleration at the beginning of each trial. Thresholds were compared between species and frequencies with a two-way ANOVA. 2. Material and methods All procedures followed IACUC guidelines dictated by the University of Windsor. All data are available in http://datadryad. org under doi:10.5061/dryad.9sj49 [8]. Fishes were collected under scientific permits issued by the states of TN (no. 1605) and KY (no. SC1211135), USA. We collected nine individuals of Forbesichthys agassizii from a quiet pool (10 m2, mean depth 0.6 m, mud/silt substrate with abundant vegetation) of a spring run fed by Jarrell’s Spring, Coffee Co., TN, USA; seven individuals for each of the two cave-dwelling species: Amblyopsis spelaea from several quiet pools (20– 150 m2, 0.2– 2þ m depth, silt/sand/cobble substrate) in Under the Road Cave, Breckinridge Co., KY, USA and Typhlichthys subterraneus from several (b) Hair cell histology Fish were euthanized with an overdose of 2-phenoxy-ethanol and fixed in 4 per cent paraformaldehyde. Epithelia were dissected and stained with Oregon Green phalloidin (Invitrogen) followed by fluorescent imaging. Hair cells were manually counted across eight different regions of saccular epithelia and quantified as density (hair cells/2500 mm2) to correct for differences in epithelium size. There were no apparent differences in fluorescent intensity sufficient to affect manual counts. Within species, there were no significant density differences between epithelial areas (ANOVA F7,40 ¼ 0.437, p ¼ 0.873), so the density estimates were averaged across epithelial areas. ANOVA was Downloaded from rsbl.royalsocietypublishing.org on May 28, 2013 3 Typhlichthys 140 Amblyopsis Forbesichthys 120 100 Biol Lett 9: 20130104 threshold (dB) environment 80 5 mS 10 mV 60 Typhlichthys 40 Amblyopsis 20 Forbesichthys 0 0 500 1000 1500 frequency (Hz) 2000 2500 3000 Figure 2. Auditory thresholds of amblyopsid fishes. Values are means+standard errors. The suface fish Forbesichthys reaches up to 2 kHz while the cavefish Typhlichthys (1) and Amblyopsis (2) are limited to 1 kHz. Fast Fourier Transformation (FFT) of sound recorded in a Drowned Rat Cave pool. The pool was carved in bedrock by a small stream. The recording was made 0.5 m deep and approximately 1 m from the waterfall. The ceiling of the cave was also dripping onto the pool. Insert: auditory evoked potential traces of all species to a 400 Hz tone burst at 60 dB. used to assess differences in hair cell density, followed by a Tukey post-hoc test. (c) Environmental sound profiles We characterized aquatic environmental sound profiles in cave and surface habitats, using a hydrophone (type 10CT hydrophone, calibration sensitivity of 2195 dB re. 1 V mPa21;+3 dB, 0.02– 10 kHz, omnidirectional, G.R.A.S., Denmark) connected through a preamplifier (Spikerbox, Backyard Brains) to an iPad (Apple). Three recordings of 5 min were taken per site. Within caves, we obtained sound profiles from two habitat types: shallow stream riffles at depths of 0.05 –0.1 m and pools with no current at depths of 0.1– 2 m. We also recorded at the same depths in surface streams and pools inhabited by Forbesichthys. Characterization of sound spectra and corresponding SPLs was performed using AUDIOTOOLS software (Studio Six Digital). We matched cave and surface habitats profiles as much as possible (e.g. area, substrate and water flow), with the exception of vegetation in surface habitats. 3. Results Density of saccular hair cells differed between species (F2,6 ¼ 15.3, p ¼ 0.0007), with the two cave species having lower hair cell densities (mean ¼ 34 and 29 hair cells/ 2500 mm2) than the surface species (mean ¼ 45 hair cells/ 2500 mm2; figure 1). There was no difference in threshold between species below 800 Hz (F2,15 ¼ 1.087, p ¼ 0.342; figure 2), and thresholds increased with frequency (F11,15 ¼ 25.9, p , 0.001) with no significant frequency–species interaction (F15,95 ¼ 47.9, p ¼ 0.702). All three amblyopsid species were most sensitive at 100 Hz (mean threshold range 112–122 dB re 1 mPa), and thresholds increased between 100 and 800 Hz. rsbl.royalsocietypublishing.org 160 In the two cave species, only one Typhlichthys responded to tones 700–1000 Hz and just two Amblyopsis responded to tone bursts above 600 Hz, with only one responding at 1000 Hz. The surface species showed clear evoked responses well above this limit, with defined responses detected up to 2000 Hz. Underwater sounds were variable depending on habitat. In cave streams with rock and sand substrate, there was a peak in background noise at about 1000 Hz followed by peaks at low frequencies (below 200 Hz; figure 2). Overall sound intensity was less prominent between 200 and 5000 Hz in pool habitats away from the small streams. Nonetheless, the same general profile was present but with a smaller, less defined 1000 Hz peak. Surface streams showed low-frequency noise (less than 100 Hz) and high-frequency noise (more than 8000 Hz) with a small peak at 1200 Hz, but the overall noise level was much higher at intermediate frequencies (1000–3000 Hz) in the cave streams than surface streams. 4. Discussion Adaptation to cave environments is often associated with hypertrophy of non-visual sensory modalities. Cave amblyopsids exhibited similar hearing sensitivities as their surfacedwelling relative at 800 Hz and below, consistent with previous findings in other cavefishes [5,6]. Surprisingly however, cave amblyopsids have lost a significant portion of their hearing range. Both Amblyopsis and Typhlichthys are unable to hear frequencies above 800 Hz, unlike their surface relative Forbesichthys, which can hear up to 2 kHz. In addition, both cave species had lower hair cell densities than Forbesichthys. To our knowledge, this is the first report of auditory regression in a subterranean organism. Downloaded from rsbl.royalsocietypublishing.org on May 28, 2013 All procedures followed IACUC guidelines dictated by the University of Windsor. All data are available in http://datadryad.org under doi:10.5061/dryad.9sj49 [8]. Fishes were collected under scientific permits issued by the states of TN (no. 1605) and KY (no. SC1211135), USA. We thank Daniel Escobar Camacho for help and Dr Gal Haspel and Dr Kim Hoke for comments. This work was supported by the Yale Institute of Biospheric Studies (M.L.N.) and by ADVANCE grant no. 1008117 to D.S. References 1. 2. 3. 4. 5. Jeffery W. 2001 Cavefish as a model system in evolutionary developmental biology. Dev. Biol. 231, 1– 12. (doi:10.1006/dbio.2000.0121) Culver D, Pipan T. 2009 The biology of caves and other subterranean habitats. Oxford, UK: Oxford University Press. Fay RR, Popper AN. 2012 Fish hearing: new perspectives from two ‘senior’ bioacousticians. Brain Behav. Evol. 79, 215–217. (doi:10.1159/ 000338719) Popper AN. 1970 Auditory capacities of the Mexican blind cave fish (Astyanax jordani) and its eyed ancestor (Astyanax mexicanus). Anim. Behav. 18, 552–562. (doi:10.1016/0003-3472(70)90052-7) Schulz-Mirbach T, Ladich F, Riesch R, Plath M. 2010 Otolith morphology and hearing abilities in caveand surface-dwelling ecotypes of the Atlantic molly, Poecilia mexicana (Teleostei: Poeciliidae). Hear. Res. 267, 137–148. (doi:10.1016/j.heares.2010.04.001) 6. Poulson TL. 1963 Cave adaptation in amblyopsid fishes. Am. Midland Nat. 70, 257– 290. (doi:10. 2307/2423056) 7. Niemiller ML, Fitzpatrick BM, Shah P, Schmitz L, Near TJ. 2013 Evidence for repeated loss of selective constraint in rhodopsin of amblyopsid cavefishes (Teleostei: Amblyopsidae). Evolution 67, 732–748. (doi:10.1111/j.1558-5646.2012.01822.x) 8. Niemiller ML, Higgs DM, Soares D. 2013 Data from: evidence for hearing loss in amblyopsid cavefishes. Dryad Digital Respository. (doi:10.5061/dryad.9sj49) 9. Corwin JT, Bullock TH, Schweitzer J. 1982 The auditory brain stem response in five vertebrate classes. Electroencephalogr. Clin. Neurophysiol. 54, 629 –641. (doi:10.1016/0013-4694(82)90117-1) 10. Kenyon TN, Ladich F, Yan HY. 1998 A comparative study of hearing ability in fishes: the auditory brainstem response approach. J. Comp. Physiol. A 182, 307 –318. (doi:10.1007/s003590050181) 11. Mann DA, Higgs DM, Tavolga WN, Souza MJ, Popper AN. 2001 Ultrasound detection by clupeiform fishes. J. Acoust. Soc. Am. 109, 3048– 3054. (doi:10.1121/1.1368406) 12. Fine ML, Lenhardt ML. 1983 Shallow-water propagation of the toadfish mating call. Comp. Biochem. Physiol. A. 76, 225 –231. (doi:10.1016/ 0300-9629(83)90319-5) 13. Popper AN, Fay RR. 1997 Evolution of the ear and hearing: issues and questions. Brain Behav. Evol. 50, 213–221. (doi:10.1159/000113335) 14. Lugli M, Yan HY, Fine ML. 2003 Acoustic communication in two freshwater gobies: the relationship between ambient noise, hearing thresholds and sound spectrum. J. Comp. Physiol. A. 189, 309–320. 15. Amoser S, Ladich F. 2005 Are hearing sensitivities of freshwater fish adapted to the ambient noise in their habitats? J. Exp. Biol. 208, 3533–3542. (doi:10.1242/jeb.01809) 4 Biol Lett 9: 20130104 adaptation in this group and suggests it may be due to different equilibrium demands. If the sensory epithelium is growing in pace with the otolith without concomitant increase in hair cells, a decrease in hair cell density would result. If, however, the loss of high-frequency hearing ability in cave species was due to selective loss of high-frequency hair cells, this could also lead to a decrease in overall hair cell density. There is no evidence for tonotopy in fish ears, but there is some evidence for differential frequency selectivity in hair cells across the epithelia [15]. More work needs to be done on frequency responses at the level of individual hair cells before this idea can be supported. Our study provides evidence that two cavefish species have evolved loss of high-frequency hearing and reduced hair cell densities compared with a surface-dwelling relative. These traits may have evolved in response to loud highfrequency background noise that mask acoustic signals in their aquatic subterranean habitats; however, the mechanism (i.e. neutral loss versus selection) underlying hearing loss remain to be understood. rsbl.royalsocietypublishing.org Like the loss of eyes, loss of hearing range in cave amblyopsids represents an example of regressive evolution in subterranean organisms. Audio recordings from native cave habitats of cave amblyopsids showed that flowing streams (riffles) and water droplets dripping from the ceiling of cave passages contribute to loud high-frequency background noise generally above 800 Hz (figure 2), although the precise contribution of all noise sources have not been characterized. Lower frequencies are not likely to propagate far in these shallow environments [12] but the higher frequency components would propagate further and contribute to the more to the high background noise levels of the caves. The apparent match between hearing ability and background noise profiles has been hypothesized to be an evolutionary driver of hearing ability across the Teleostei [13], and the hearing of two species of goby (Padogobius martensii and Gobius nigricans) living in noisy waterfall environments is most sensitive in a frequency range corresponding to a quiet window in these environments [14]. Noisy stream environments mask high-frequency hearing in ostariophysan fishes [15] but hearing specializations of closely related species in different acoustic environments have rarely been tested. Our findings raise the intriguing possibility that cave amblyopsids may have lost hearing at high frequencies in response to the noisy acoustic environments in which they live. The reduction in hair cell density indicates peripheral involvement in high-frequency hearing loss. Fewer hair cells provide fewer sites for signal transduction and also may lead to less relative stimulation upon relative motion of the otolith. Poulson [9] reports an increase in otolith size with increasing cave
