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Evidence for hearing loss in amblyopsid cavefishes




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.;Attachment Preview;Niemiller_2013_Biology_Letters.pdf Download Attachment;Downloaded from 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;;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:;Downloaded from on May 28, 2013;Neurobiology;;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.;;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:;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 on May 28, 2013;(a);2;(b) 50;hair cell density (units 100 mm2);40;30;Biol Lett 9: 20130104;(ii);;(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 15.3, p 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 & Kjr). 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 Jarrells 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 2 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 0.437, p 0.873), so the density;estimates were averaged across epithelial areas. ANOVA was;Downloaded from 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 15.3, p 0.0007), with the two cave species having;lower hair cell densities (mean 34 and 29 hair cells/;2500 mm2) than the surface species (mean 45 hair cells/;2500 mm2, figure 1). There was no difference in threshold;between species below 800 Hz (F2,15 1.087, p 0.342;figure 2), and thresholds increased with frequency (F11,15;25.9, p, 0.001) with no significant frequencyspecies interaction;(F15,95 47.9, p 0.702). All three amblyopsid species were;most sensitive at 100 Hz (mean threshold range 112122 dB re;1 mPa), and thresholds increased between 100 and 800 Hz.;;160;In the two cave species, only one Typhlichthys responded to;tones 7001000 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 (10003000 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 on May 28, 2013;All procedures followed IACUC guidelines dictated by the University of Windsor. All data are available in;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, 215217. (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;552562. (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, 137148. (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, 732748.;(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;213221. (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, 309320.;15. Amoser S, Ladich F. 2005 Are hearing sensitivities of;freshwater fish adapted to the ambient noise in;their habitats? J. Exp. Biol. 208, 35333542.;(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.;;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


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