unlike in most other vertebrates that possess numerous tiny otoconia in their inner ears , modern bony fishes ( teleostei ) have a single massive calcareous concretion  the so - called otolith  in all three end organs .
calcium carbonate is usually deposited as aragonite in the otolith of the saccule ( sagitta ) and utricle ( lapillus ) and in a crystallized , less dense form termed vaterite in the otolith of the lagena ( asteriscus ) ( oliveira and farina , 1996 ; falini et  al . , 2005 ) .
these otoliths , especially the saccular otolith , show a ( species-)specific morphology ( nolf , 1985 ) . in examining the diversity of otolith morphologies and its implications for physiological functions in teleost fishes , one interesting question at the interface between otolith research and studies on inner ear physiology
is whether and how otolith morphology may be related to inner ear physiology , such as hearing sensitivities ( popper et  al . , 2005 ; popper and schilt , 2008 ) .
( 2007 ) , for example , reported that juvenile chinook salmon ( oncorhynchus tshawytscha ) with aberrant ( vateritic ) sagittae displayed significantly poorer auditory sensitivity than individuals with normally developed ( aragonitic ) sagittae .
( 2004 ) showed that the sciaenid species bairdiella chrysoura has a unique otolith morphology , i.e. thick , large otoliths ( asteriscus and lapillus ) and a sagitta with a conspicuously deep sulcus acusticus which may be linked to better hearing abilities compared to other members of the family sciaenidae . in a preceding comparative study ( schulz - mirbach et  al . , 2008 ) on the sagittae of different ecotypes of the atlantic molly ( poecilia mexicana ) , we found pronounced contour differences in cave- versus surface - dwelling populations
. moreover , sagittae of cave mollies often had a deep sulcus , lacking in surface - dwellers .
, we therefore examine whether otolith morphology reflects inner ear physiology ( i.e. , hearing sensitivities ) .
popper ( 1970 ) found similar hearing sensitivities in the blind , cave - dwelling mexican tetra astyanax mexicanus ( characidae ) and its surface - dwelling conspecifics , but did not investigate otolith morphology .
we therefore tested whether ( 1 ) the cave form of p. mexicana and its surface - dwelling relatives have similar hearing sensitivities ( as shown for the mexican tetra ) or ( 2 ) whether they have different hearing sensitivities that are reflected by the changed sagitta morphology .
cave fishes are under strong selection pressure to develop and enhance non - visual communication channels ( burt de perera , 2004 ; montgomery et  al . , 2001 ; parzefall , 1970 , 2001 ) . to compensate for the lack of visual input
, they have evolved several modifications of their sensory systems such as a well - developed lateral line system ( burt de perera , 2004 ; montgomery et  al . , 2001 ) or improved the senses of taste and touch ( parzefall , 1970 , 2001 ) . a potentially altered sense of hearing and acoustic communication
we therefore studied cave and surface populations of p. mexicana with respect to hearing abilities and acoustical signaling .
cave and surface populations of the atlantic molly differ in their sagitta morphology , making them perfect model organisms to investigate the potential relationship between otolith morphology and hearing sensitivities .
first , we tested for potential differences between the two ecotypes in the morphology of all three otolith types ( asterisci , sagittae , lapilli ) , with special emphasis on details of the sulcus region of the sagittae and on the overall morphology of asterisci and lapilli .
second , we tested whether cave and surface - dwelling fish show similar hearing sensitivities , or whether the former display changed hearing sensitivities as an adaptation to perpetual darkness .
the atlantic molly , p. mexicana steindachner , 1863 ( poeciliidae ) is widespread in freshwater surface habitats along the atlantic versant of central america ( miller , 2005 ) .
the cave form of p. mexicana inhabits the cueva del azufre cave system , which is divided into 13 interconnected cave chambers ( gordon and rosen , 1962 ) .
a creek flows through the cave , forming several shallow pools that are partially divided by riffle passages .
while the front cave chambers receive some dim light , the inner parts of the cave are lightless , and the molly population from the innermost cave chamber xiii ( gordon and rosen , 1962 ) permanently lives in the dark . with the exception of chamber xiii ,
the water in all cave chambers is characterized by medium to high concentrations ( up to 300  m / l ) of naturally occurring hydrogen sulfide ( tobler et  al . , 2006 , 2009 ) .
a total of 46 fish from the cave form and two surface populations of p. mexicana were investigated with regard to their otolith morphology and hearing sensitivity ( table 1 ) .
one of the surface populations originated from the ro oxolotn , a river with sulfide - free water near the cave ( tobler et  al . ,
the second population of surface - dwelling fish came from brackish coastal waters near tampico ( tamaulipas , eastern mexico ) .
large , randomly outbred stocks of the cave population and from ro oxolotn have been maintained in 200-l aquaria at the university of potsdam since 2004 ; stocks from tampico were founded using wild - caught fish in 1995 .
fish were transferred to the university of vienna in december 2008 and february 2009 for the auditory analyses .
they were kept in 120 to 160-l aquaria , which were equipped with a sand bottom , halved flower pots as hiding places , and external filters .
no internal filters or air stones were used in order to create a quiet acoustic environment for the test fish .
fish were kept under a 12:12  h l : d cycle at 25    1  c and were fed once daily with commercial flake food .
fish were given a habituation period of two to three days prior to the auditory experiments .
all hearing experiments were performed with the permission of the austrian federal ministry of science and research ( gz 66.006/0023-ii/10b/2008 ) . cave fish for the survey of the acoustical behavior originated from chamber v of the cueva del azufre ( gordon and rosen , 1962 ) , and surface fish came from the ro amatan , another sulfide - free river that merges with the ro oxolotn downstream , but close to the cave ( tobler et  al . ,
both populations were a mix of wild - caught and first generation laboratory - reared fish , originally collected and established in january 2009 .
fish were maintained at the university of oklahoma under 12:12  h l : d cycles , kept in 160-l tanks at 26  c with gravel bottom and internal filters , and were fed once daily with either commercially available flake food or mosquito larvae ( bloodworms ) .
following the measurements of hearing sensitivities , the standard length of the fish was measured to the nearest millimeter and animals were decapitated .
sex was determined by inspection of the gonopodium ( transformed anal fin ) of the males and by dissection of the ovary of the females .
the three otolith types  lapillus , sagitta , and asteriscus  were dissected from the left membranous labyrinth , cleaned of organic residues with 1% potassium hydroxide solution for 46  h and rinsed several times in distilled water . after cleaning ,
otoliths were stored dry at room temperature in small plastic cells ( krantz - cells ) .
fresh otoliths showed the same morphological features , e.g. development of the sulcus center in sagittae , as those after cleaning .
otolith weight was determined to the nearest 0.01  mg with a mettler - toledo at21 ( 0.001  mg , s.d . ) . to determine otolith area and otolith contour
, the macula - oriented side of all otoliths was investigated using a scanning electron microscope ( a leo vp 1430 at the zoological state collection in munich , germany ) at a magnification of 45- to 58-fold ( sagittae ) , 95- to 160-fold ( asterisci ) , and 140- to 220-fold ( lapilli ) . for sem investigation
these sem images were converted into images containing a white object on black background in adobe photoshop cs2 .
otolith contours were saved as raw ( x / y)-coordinates in tpsdig2 and subjected to the shape analysis software hshape ( crampton and haines , 1996 ; haines and crampton , 2000 ) consisting of the three programs hangle , hmatch , and hcurve .
the output of hangle and hmatch are fourier descriptors ( fds ) , describing the contour by combination of sine- and cosine - waves .
normalization of size was performed automatically in hangle ( 29 smoothing iterations ) , whereas normalization of rotation and starting point was ensured by applying hmatch .
twenty fds were used to analyze contours of lapilli and 32 fds for sagittae and asterisci , respectively . in order to visualize differences in otolith contours between the populations , averaged fds of each population and otolith type were back - calculated into ( x / y)-coordinates ( 1024 per contour ) applying hcurve . in some hatchery - reared fish stocks ,
aberrant otoliths are more abundant than in wild animals ( e.g. , oxman et  al . , 2007 ; sweeting et  al . , 2004 )
thus , in order to test for possible artifacts of lab conditions , we screened our samples for the relative abundance of aberrant otoliths and also compared otolith morphology of lab - reared animals with that of wild fish studied in schulz - mirbach et  al .
auditory thresholds were determined by applying the auditory evoked potential ( aep ) recording technique following the protocol developed by kenyon et  al .
the aep technique records far - field potentials in response to sound stimuli of the whole auditory pathway from the inner ear up to midbrain nuclei ( corwin , 1981 ) . for a comparison between aep and behavioral thresholds , and for aep thresholds gained in different laboratories ,
prior to each auditory experiment , individuals were chosen randomly from one of the three populations .
depending on body weight , the test subject was immobilized with flaxedil ( gallamine triethiodide ; sigma aldrich handels gmbh , vienna , austria ) at a concentration of 9.549.3  g  g body weight in order to reduce muscle noise .
all auditory measurements were carried out in a bowl - shaped plastic tub ( diameter 33  cm , water depth 13  cm , 1  cm layer of sand ) , which was lined inside with acoustically absorbent material ( air - filled packing wrap ) to minimize resonances and reflections . for a more detailed description see wysocki and ladich ( 2002 ; fig . 
the tub was positioned on an air table ( tmc micro - g 63 - 540 , technical manufacturing corporation , peabody , ma , usa ) , which rested on a vibration - isolated plate of concrete .
a sound - proof chamber , constructed as a faraday cage ( interior dimensions : 3.2  m    3.2  m    2.4  m ) , enclosed the whole set - up .
test subjects were positioned in the center of the tub , so that the nape of the head was just above the water surface ( 1  mm ) . for respiration
a pipette was inserted into the fish s mouth and respiration was facilitated by a simple , temperature - controlled ( 25    1  c ) , gravity - fed water circulation system .
the area of the head above the water surface was covered with a small piece of kimwipes tissue paper to keep it moist .
the recording electrode was placed in the midline of the skull over the region of the medulla , the reference electrode cranially between the nares .
tone - bursts were presented through two speakers ( fostex pm-0.5 sub and pm-0.5 mkii , fostex corporation , tokyo , japan ) positioned 50  cm above the animal s head .
the aep waveform recording was performed by a modular rack - mount system [ tucker - davis technologies ( tdt ) system 3 , gainesville , fl , usa ] controlled by a pc containing a tdt digital signal processing board and running tdt biosig rp software .
auditory thresholds were evaluated in random order for 100 , 200 , 300 , 500 , 1000 , 1500 , and 2000  hz , respectively , by decreasing the sound pressure level in 4-db steps .
several individuals were tested at 3000 , 4000 , and 5000  hz , but none of them showed any response beyond 2000  hz .
p. mexicana is not known to possess any hearing specializations ( hearing non - specialists or generalists ; see also popper and fay , in press ) .
audiograms are often expressed in terms of sound pressure levels , which may not be adequate for non - specialists ( wysocki et  al . , 2009 ) .
a hydrophone ( brel and kjaer 8101 , naerum , denmark ; frequency range : 1  hz80  khz    2  db ; voltage sensitivity : 184 re 1  v/pa ) was placed on the right side of the animals ( 1  cm away ) in order to control for absolute stimulus spls underwater in close proximity to the subjects during each experimental session . in order to compare sound pressure and particle acceleration levels for all frequencies tested ,
a calibrated underwater miniature acoustic pressure  acceleration ( p  a ) sensor ( s / n 2007 - 001 , applied physical sciences corp .
, groton , ct , usa ) was placed at the fish s position in the test tub .
this p  a sensor ( with a frequency bandwidth from 20  hz to 2  khz ) allows simultaneous recording of sound pressure and particle acceleration .
it consists of two built - in units : a piezoelectric , omni - directional hydrophone ( sensitivity : 173.7  db re : 1  v/pa ) and a bi - directional accelerometer ( sensitivity : 137.6  db re 1  v/m / s ) .
measurements of all stimulus frequencies at various levels , including the hearing threshold levels of the fish , were measured with the acceleration sensor subsequently oriented in all three orthogonal directions . in consistence with previous studies ( casper and mann , 2006 ; horodysky et  al .
2009 ) , the x - axis was considered to be anterior  posterior along each subject s body , the y - axis was considered to be lateral ( right  left ) relative to the subject , and the z - axis to be vertical ( i.e. , up - down ) relative to the subject .
this approach yielded simultaneous measurements of sound pressure and particle acceleration in all three directions over the entire stimulus range .
sound pressure levels ( spl ) were calculated in db rms re 1 pa and particle acceleration levels ( la ) in db rms re 1  m / s .
these are the international units for sound pressure and particle acceleration according to iso standards ( iso 1683 , 1983 ) .
sound recordings were obtained from a variety of different laboratory settings in either 40-l or 70-l rectangular aquaria .
we used three different settings : ( 1 ) all - male , ( 2 ) all - female , and ( 3 ) mixed - sex tanks , and recordings were made in both light and darkness .
each setting consisted of approximately 12 fish ( all - male : 1012 males ; all - female : 12 females ; mixed - sex : 6 males and 6 females ) , and fish were acclimatized for at least 2  h in the test tanks prior to recording sessions .
finally , each setting was replicated with p. mexicana from a surface stream ( ro amatan ) and the cueva del azufre ( cave chamber v ) . due to physical , physiological , and morphological constraints on teleost
sound - producing ( sonic ) organs , most fish sounds are pulsed signals ranging in frequency from around 60  hz to more than 1  khz ( amorim , 2006 ) .
( cyprinodontidae ; like poeciliids members of the order cyprinodontiformes ) found the dominant frequency of calls ranging from 400  hz to 1500  hz ( johnson , 2000 ; nicoletto and linscomb , 2008 ) . in the majority of fishes investigated so far ,
main energies of sounds are found in the frequency range of highest auditory sensitivity ( wysocki , 2006 ) .
this led us to expect the main energies of potential acoustic signals of p. mexicana to be found below 500  hz . in order to detect low - level sounds and
because test tanks were properly aerated prior to testing , filters and aeration were switched off and removed for the duration of the acoustic survey in order to create a quiet acoustic environment .
the survey was conducted using the sq26-mt underwater recording system , which consists of a sensor technology sq26 - 08 hydrophone ( cetacean research technology , seattle , wa ) and an m - audio microtrack ii solid state digital recorder ( m - audio , irwindale , ca ) .
recordings were digitized and analyzed using raven version 1.2.1 ( cornell laboratory of ornithology , ithaca , ny ) , and spectrograms were generated using a hanning window , a fast fourier transformation size of 4096 samples , a filter bandwidth of 124  hz , and 75% overlap . to allow for a correlation of acoustics with the behavioral context , tanks in light were filmed during acoustic recording sessions using a canon dm - xl1a professional camcorder ( canon inc . ,
all statistical analyses were conducted in spss 17.0 ( spss inc . , 2009 ) and past 1.90 ( hammer et  al . , 2001 ) .
mexicana shows a pronounced sexual dimorphism in morphology and behavior ( e.g. , plath , 2008 ) , and sex was therefore included as factor in the analyses . 
standard length was included as covariate ( but see otolith weight ) because of the wide size range of fish investigated ( sl : 28 up to 55  mm ) . 
weight and  area of asterisci , sagittae , and lapilli were ln - transformed to account for the non - linear relationship between those variables .
the ln - transformed weight of the asterisci , sagittae , and lapilli were each subjected ( as dependent variable ) to a general linear model ( glm ) , in which ln - transformed otolith area was included as a covariate . as interactions between
 population and the covariate as well as between  sex and the covariate were not significant in all cases ( p    0.11 ) , those interactions were removed from the final model . in order to study differences of otolith contours between populations , a full factorial general linear model with the shape descriptors ( fourier descriptors ) as dependent variables was calculated .
moreover , fourier descriptors were subjected to a principal component analysis based on a variance  covariance matrix to illustrate differences between groups in an unconstrained space .
relevant principal components ( pcs ) explaining more variance than expected by chance alone were evaluated according to the  broken stick model sensu 
 jackson ( 1993 ) .
subsequently , the relevant pcs were used as input variables of a discriminant function analysis ( dfa ) that was conducted in order to estimate the amount of difference between the populations .
because of the small sample size in the case of the ro oxolotn population , datasets from both surface populations were pooled prior to the dfa .
hearing thresholds expressed in terms of sound pressure level ( spl ) or particle acceleration level ( mag a ) obtained from auditory measurements were analyzed by calculating a full factorial general linear model with 
frequency as repeated measurements ( rmglm ) and  thresholds as dependent variable . for post hoc comparisons concerning differences in hearing sensitivities ( spl ) related to body size ( standard length ) , we used multiple t - tests while comparing fish smaller than and larger than the mean body size for each frequency , separately .
alpha levels were corrected for multiple comparisons using the bonferroni correction ( rice , 1989 ) .
the most striking difference between cave- and surface - dwelling fish regarding the structure of the sagittae was in the sulcus acusticus region .
divergent features in this region are ( 1 ) the structure of the crista superior , ( 2 ) the surface structure of the area above the crista superior , ( 3 ) the arrangement of crystals in the center of the sulcus , and ( 4 ) the presence or absence of pits on the area beneath the crista inferior ( fig .  1 , table 2 ) . the crista superior of sagittae of cave fish steeply declines towards the sulcus , whereas in surface fish it gradually merges into the sulcus ( fig . 
1a , f ) . moreover , the anterior part of the crista superior in cave fish sagittae displays several small to medium - sized pits ( fig . 
the sagitta of surface fish bear almost equally sized , rod - shaped crystals arranged in a circular pattern in the sulcus center ; sagittae of cave fish lack this pattern and have crystals of varying size ( fig . 
in addition , sagittae of cave fish show medium- to large - sized pits in the area beneath the crista inferior ( absent in sagittae of surface fish ; fig .  1e vs. i ) .
minor differences between the otoliths of cave- and surface - dwelling fish were also found in the fossa acustica region of the asterisci .
the crista medial of the asterisci is commonly more bulging and narrower in the former than in the latter ( fig .  2 ) .
in contrast , the macula - oriented face of the lapilli is more variable within than among the examined populations , i.e. , no distinction based on habitat type can be made .
the development of this part of the lapilli ranges from almost smooth to displaying large bundles of crystals ( fig .  3 ,
asterisci and lapilli of cave fish were significantly heavier at comparable area values ( i.e. , denser ) than those of surface fish ( f2,35    4.49 ; p    0.018 ) ( tables 3 and 4 ; fig . 
sex ( f1,35  =  9.707 ; p  =  0.004 ; table 4 ) because males had heavier ( i.e. , denser ) sagittae than females ( fig .  5 ) .
the contours of asterisci ( f8,64  =  6.223 ; p  =  0.005 ) and lapilli ( f8,64  =  4.474 ; p  =  0.015 ) differed significantly among populations ( table 5 ; fig . 
by contrast , mean shape differences were only weak in the case of sagittae , and no statistically significant difference among populations was detected ( f8,64  =  2.287 ; p  =  0.106 ; table 5 ) .
furthermore , neither standard length nor sex significantly affected the contours of the three otolith types ( p    0.069 ; table 5 ) .
the first two principal components ( pcs ) were relevant according to the  broken stick model for the contours of asterisci .
for the contours of sagittae , the first six pcs , and for the contours of lapilli the first three pcs , explained more variance than expected by chance .
the pcs accounted for 45% ( asterisci ) , 74% ( sagittae ) , and 55% ( lapilli ) of the overall variance .
the 2d plots of the pca ( pc1 versus pc2 ) revealed a separation of asterisci and sagittae contours in cave versus tampico fish ( fig .  6 , left ) especially along pc1 for asterisci and along pc2 for sagittae
. the principal component plot of lapilli contours showed less separation of the populations than those of asterisci and sagittae .
the contours of asterisci and sagittae from the three ro oxolotn specimens plot into the 95% probability ellipses of those from the tampico individuals , indicating similarity among fish from surface populations .
the lapilli contours of the ro oxolotn individuals , however , fell into the 95% probability ellipses of both the cave and tampico specimens . the discriminant function analysis based on asterisci contours classified 92.9% of individuals to the correct habitat type 
cave or  surface ( tampico and ro oxolotn specimens merged ) ; 97.6% correct classification were obtained for sagittae contours , 88.1% for lapilli contours
, the analyses revealed that the contours of the three otolith types differed markedly between ecotypes ( cave fish versus fish from the surface populations ) .
two out of 41 sagittae ( 4.9% ) from the lab - reared fish showed an aberrant morphology , whereas all asterisci and lapilli were normally developed .
none of the wild - caught individuals ( n  =  67 ) had aberrant otoliths .
sagittae of wild - caught specimens from the cave system ( including individuals from chamber xiii and several surface populations near the cave system ) showed the same differences in the macula - oriented side as the lab - reared animals .
the circular pattern of rod - shaped crystals in the sulcus center in specimens from the surface populations tampico and ro oxolotn ( fig .  3 , middle ) was also identified in wild - caught individuals from the surface - dwelling population arroyo cristal ( schulz - mirbach et  al .
the differences in the asterisci of lab - reared individuals were less pronounced than those in wild - caught specimens , i.e. asteriscus shape in some cave fish was similar to that of surface fish .
nevertheless , wild - caught cave fish also exhibited a trend towards more compact asterisci with a bulging crista medial and occasionally a deep fossa acustica .
lapilli of wild - caught fish displayed a similar variability within populations as those from laboratory - reared specimens and tended to be thickened in some wild - caught cave fish .
the sound pressure level ( spl ) audiograms of the three populations of p. mexicana showed a similar shape ( fig . 
7a ; table 7 ) and , overall , mean thresholds were not significantly different ( table 8) . also , no significant interaction effect of 
frequency by population was found , showing that populations did not differ in their specific response to different frequencies .
in general , fish were most sensitive at 200 and 300  hz , whereas thresholds increased abruptly above 300  hz ( fig . 
fish in all experiments responded to frequencies of up to 1500  hz , and most also responded at 2000  hz .
because not all individuals responded at 2000  hz , this frequency was excluded from the statistical analyses .
the particle acceleration level audiograms of the populations were similar in shape displaying lowest thresholds at 200 and 300  hz ( fig . 
7b ; table 7 ) , and the populations showed no significant differences in their mean thresholds ( table 8) . a significant effect of  frequency by standard length ( p  =  0.014 ; table 8) indicated that larger fish had slightly lower response auditory thresholds than smaller specimens at the highest frequency , namely at 1500  hz ( t28  =  3.499 ; p  =  0.002 ) ( fig . 
no significant differences between sexes and no significant interaction terms involving sex were found ( table 8) .
altogether , we surveyed more than 30  h of acoustic recordings for all different settings and populations .
p. mexicana from both populations exhibited the full range of typical behaviors ( e.g. , male  male aggression or male  female mating ) .
nonetheless , we found no evidence for sound production and , thus , acoustic communication in any population of p. mexicana .
our study is the first to compare a cave fish with its surface - dwelling relatives with respect to otolith morphology , hearing sensitivities , and the possible role of acoustic communication .
cave mollies differed distinctly from surface - dwelling conspecifics in the morphology of asterisci , sagittae , and lapilli , but they exhibited similar hearing sensitivities .
both ecotypes appeared to be non - vocal . discussing and explaining differences in otolith morphology first requires examining possible effects of rearing conditions .
as cave fish were light - reared , otolith morphology might have been affected to a certain degree by lab conditions . however , the relative abundance of aberrant otoliths in the lab - reared individuals was within the range reported for several fish species caught in the wild ( 0.414% : gauldie , 1993 ) and lower than the 7.813.9% for lab - reared herring ( clupea harengus ; tomas and geffen , 2003 ) .
in addition , otolith differences between lab - reared cave and surface fish were similar to those in wild - caught specimens .
thus , we conclude that otolith contours and features of the macula - oriented side of all three otolith types were at best marginally affected by rearing conditions .
we found distinct differences between cave and surface fish in all three otolith types , whereas both ecotypes had similar hearing sensitivities .
these similar sensitivities in p. mexicana agree with results on the mexican tetra ( astyanax mexicanus ; popper , 1970 ) . on the one hand , altered otolith morphology in cave fish might point to modified functions of the inner ear such as for detection of linear acceleration , sound source localization , or the vestibular sense .
little , however , is known about the relationships between otolith features and physiological functions , hampering interpretations ( especially concerning otolith contours ) . nonetheless , different contours of all three otolith types between cave- and surface - dwellers might point to a modified stimulation of the sensory epithelia .
( 2005 ) and popper and schilt ( 2008 ) noted that different contours might result in different centers of gravity of otoliths , potentially affecting the motion of the otolith relative to the sensory epithelium .
in addition , lychakov and rebane ( 1992 ) hypothesized  based on a mathematical analysis of otolith vibrations in an acoustic field  that otolith shape may be involved in the ability to perceive directional sound .
the differences in the surface structures of the asteriscus and sagitta s macula - oriented sides in cave fish might change the attachment of the otolithic membrane .
this , in turn , may result in a different mechanical transduction of shearing forces of the asteriscus or sagitta motion mediated via the otolithic membrane to the sensory epithelium .
preliminary studies on histological sections of saccules from cave mollies show that the deep sulcus in some individuals is filled with a distinctly thickened otolithic membrane , whereas in sagittae with a flat sulcus the otolithic membrane consists of only a very thin layer ( t. schulz - mirbach , unpublished ) . assuming that saccule and sagitta play a key role in the acoustic sense ( popper and lu , 2000 ) , the similarity in sagitta weight of cave- and surface - dwellers
a recent study suggested that differences in the weight - to - area ratio and density of vateritic and aragonitic sagittae of oncorhynchus tshawytscha may be correlated with significant differences in hearing sensitivities ( oxman et  al . , 2007 ) .
those authors hypothesized that in less dense sagittae , the lag of sagitta movement relative to the motion of the sensory epithelium in response to a certain frequency is attenuated , thereby causing less stimulation than denser sagittae .
accordingly , the increased weight per area of asterisci and lapilli in cave fish may enhance the stimulation of the sensory hair cells .
in contrast to surface - dwelling fish from ro oxolotn and tampico , cave fish must spend much of their time and energy to cope with the hydrogen sulfide and the correlated hypoxia by showing aquatic surface respiration ( tobler et  al . , 2009 ) . although fish from chamber xiii do not have to cope with this toxin , they show a similarly poor body condition as fish from the other cave chambers . in general , cave fishes exhibit a different ( slower ) metabolism ( cave - dwelling amplyopsids : poulson , 1963 ; nemacheilus evezardi : pati and agrawal , 2002 ) , and an altered metabolism might affect otolith mineralization .
for example , oxygen consumption  used as a proxy for metabolic rate in juvenile atlantic salmon parr ( salmo salar ) and larval zebrafish ( danio rerio )  was positively correlated with sagitta size ( bang and grnkjr , 2005 ; wright , 1991 ) .
otolith mineralization and the role of proteins are still poorly understood ( allemand et  al .
, 2007 ; sllner and nicolson , 2004 ) . in the case of molly otoliths , future studies are required to determine the influence of the special water chemistry of the cave creek and the extent to which otolith morphology may be phenotypically plastic .
vocalizations may play an important role in intra - specific communication in fishes ( for reviews see ladich and myrberg , 2006 ; myrberg and lugli , 2006 ; parmentier and diogo , 2006 ; wysocki , 2006 )
. this could be particularly important in a lightless habitat where fish are unable to communicate visually .
interestingly , neither surface nor cave mollies appear to produce species - specific sounds , indicating that sounds might be less important in communicating with conspecifics in this teleost family .
parzefall , 1970 , 2001 ) compensates for the lack of visual communication in caves .
although we observed the full range of agonistic and mating behavior in mollies during our laboratory survey , we can not completely exclude the possibility that surface and/or cave fish might produce sounds in the field ( i.e. , in another social and/or environmental context ) , as has been reported in cyprinodontids ( johnson , 2000 ; nicoletto and linscomb , 2008 ) .
the observed differences in otolith shape , in the otoliths macula - oriented faces and in the weight of asterisci and lapilli in cave fish apparently do not affect hearing sensitivities , at least in terms of auditory thresholds . cave life and an altered metabolism
might have affected otolith morphology , but did not alter auditory sensitivity , which was maintained for acoustic orientation .
 cave fish have rarely been investigated with regard to their inner ear morphology , hearing abilities , and acoustic communication . 
 based on a previous study that revealed morphological differences in the saccular otolith between a cave and two surface populations of poecilia mexicana , we checked for additional differences in utricular and lagenar otoliths and tested whether different populations have similar hearing sensitivities . 
 we found pronounced differences in the shape of all three otoliths . 
 otoliths of the saccule and lagena from cave fish differed from those of surface fish in the features of the face oriented towards the sensory epithelium . 
 in addition , otoliths of the utricle and lagena were significantly heavier in cave fish . 
 auditory sensitivities were measured between 100 and 1500  hz , utilizing the auditory evoked potential recording technique . 
 we found similar hearing abilities in cave and surface fish , with greatest sensitivity between 200 and 300  hz . 
 an acoustic survey revealed that neither ecotype produced species - specific sounds . 
 our data indicate that cave dwelling altered the otolith morphology in atlantic mollies , probably due to metabolic differences . 
 different otolith morphology , however , did not affect general auditory sensitivity or acoustic behavior . 
Introduction
Materials and methods
Results
Discussion
Conclusions