J Comp Physiol A (1992) 171 : 105-109
Joum~ of
Physk ogy A
Neurat and
9 Springer-Verlag 1992
Auditory sensitivity of the cichlid fish Astronotus ocellatus (Cuvier) Hong Y. Yan and Arthur N. Popper Department of Zoology, The University of Maryland, College Park, MD 20742, USA Accepted April 16, 1992
Summary. Auditory sensitivity was determined for the oscar, Astronotus ocellatus, a cichlid fish that has no known structural specializations to enhance hearing. Trained A. ocellatus behaviorally responded to sound stimuli from 200 Hz to 800 Hz with best sensitivity of 18 dB (re: 1 ~tbar) to 21 dB for frequencies between 200 and 400 Hz. This is significantly poorer than hearing sensitivity for fish classified as hearing specialists, but well within the range of hearing capabilities reported for non-specialist teleost species.
Key words: Astronotus ocellatus - Audiogram - Acoustic sensitivity - Fish ear - Non-otophysan fish
Introduction Behavioral studies of hearing sensitivity of teleost fishes have demonstrated broad variability, both in absolute sensitivity and in bandwidth over which sounds can be detected (reviewed in Fay 1988). Best sensitivity has been found in species that have specialized structures to improve the mechanical coupling between a gasbladder, which serves as a pressure detector, and the inner ear. Best known of these enhancements are the Weberian ossicles of otophysan fishes (e.g., goldfish, catfish) which connect the swimbladder, a bubble of air in the abdominal cavity, with the fluid-filled chambers of the inner ear. Hearing capabilities are enhanced by other types of specializations such as anterior projections of the swimbladder that bring it into close proximity to the inner ear (e.g., some squirrelfishes, clupeids) (reviewed in Popper and Coombs 1982; Schellart and Popper 1992) or the presence of small air bubbles very close to the ear (e.g., mormyrids, anabantids) (McCormick and Popper 1984; Saidel and Popper 1987). Morphological studies of the peripheral auditory system of fishes have demonstrated substantial differences Correspondence to: H.Y. Yan
in the relationship between the swimbladder and the ear, and in the structure of the auditory regions of the ear (e.g., Popper and Coombs 1982; Schellart and Popper 1992). Data on hearing capabilities to correlate with these structural differences are limited, and the existing data for about 52 species represents a very limited taxon (reviewed in Fay 1988). Therefore, much more needs to be known about hearing capabilities before more thorough structure-function relationships can be elucidated. One of the problems in obtaining data on hearing capabilities is that the methods used in past investigations may not always be amenable for use with a wide range of species. It is always suggested that a valuable behavioral technique used to measure hearing should take advantage of the naturalistic behavior of an individual species. When the species does not naturally respond in the appropriate way for an experiment, it is virtually impossible to use a particular approach for any behavioral measure. For example, respiration conditioning only works if a species changes respiration rate in response to a noxious stimulus such as electric shock (Fay 1969). The avoidance conditioning paradigm is only useful for species that show escape behavior (Jacobs and Tavolga 1967). Recently, we developed a positive reward technique that is useful for measuring fish hearing (Yan and Popper 1991). The technique uses a food reward and a simple paddle press which mimics food/prey item picking behavior of a fish. The technique, with small modifications in the training paradigm, is applicable to a wide range of species since no aversive treatment is used in the procedure, and most species will come to a spot in a tank in response to a food reward. The oscar (Astronotus oeellatus), a teleost fish of the family Cichlidae, is a hardy aquarium species with a rapid growth rate even in captivity. The oscar has been used extensively in our laboratory as a model animal for the study of functional morphology and physiology of sensory hair cells of its inner ear including sensory hair cell growth (Popper and Hoxter 1984, 1990; Presson and Popper 1990a, b), hair cell innervation (Saidel et al.
106
H.Y. Yan and A.N. Popper: Audiogram of the oscar
1990a; Popper and Saidel 1990), immunocytochemical reactions to calcium binding protein (Saidel et al. 1990b), differential responses to ototoxic agent (Yan et al. 1991) and ultrastructure of cellular organelles (Chang and Popper 1991). To enhance our understanding about the relationships of functional morphology of sensory hair cells of the inner ear and auditory sensitivity of the oscar, the newly validated operant conditioning paradigm developed in our laboratory (Yan and Popper 1991) was used to evaluate pure tone auditory sensitivity of the oscar. Materials and methods Experimental animals. Three oscars (A. ocellatus), about 6 cm in standard length (measured from tip of snout to the end of the vertebral column) were obtained from a local aquarium supplier. Fish were housed as a group in a 40-1 glass-wall aquarium (50 x 30 x 26.6 era) and were fed daily with food pellets. The water was continuously aerated, filtered and water temperature was maintained at about 26.5 ( + 1) ~
Apparatus. A detailed description of the test apparatus including all the electronic modules, digital input/output board and computer interface has been reported (Yan and Popper 1991), so only a brief description is given here (Fig. 1). The test apparatus sat on a Plexiglass platform attached to a 4-legged support which could be transferred easily from one tank to another. A solenoid-controlled automatic feeder was attached to the top of the platform. A tube from the feeder contacted the water surface and was used to dispense floating food pellets (diameter 0.5 mm, average weight 2 mg). The pellets were confined within the tube and this served as a fixed feeding station which could be easily recognized by the fish. A 1 0 W ceiling light (C-light) was attached at the rear of the platform to illuminate the tank during the experiment as needed. A response panel attached to the underside of the platform. The panel consisted of two stainless steel rods about 6 cm apart. Each held a sealed clear plastic tube (diameter 1 cm, length 5 cm) containing a miniature 1 0 W light bulb inside the tube. A piece of pressuresensitive piezo film was attached outside each tube to form an observation paddle (O-paddle) and a report paddle (R-paddle). Each paddle was used as a transducer to transmit responses from fish to the computer. Acoustic signals were generated with a signal generator (Coulbourn Instrument, CI $81~)6) coupled with an attenuator (CI $85--08) and an amplifier (CI $82-24). An underwater speaker
t I
I =
9
I;;F
!
I
L
c
R
0
Fig. 1. Schematic diagram of the test tank from a fish-eye view, showing the apparatus measuring hearing thresholds. C ceiling light; F automatic feeder; O observation paddle; P plexiglass platform; R report paddle; S underwater speaker; T food pellet delivery tube. Notice that both the O-light and the R-light are housed inside clear plastic waterproof tubes. See Yan and Popper (1991) for detailed description of the apparatus
(University Sound UW-30) was used to present these signals. The signal generation system was calibrated with a hydrophone (Celesco, LC-10) and a wave analyzer (Hewlett-Packard, 358 IA). Sound pressure levels (SPLs) of various frequencies and attenuations were also measured and calibrated around the O-paddle. An IBM-AT computer with a 24-bit parallel digital input/ output board (Metrabyte PIO12) was used to control the experiment and data storage through programs written in Microsoft QuickBASIC (Version 4.5). The experiments were conducted inside an IAC soundproof chamber. Ambient noise inside the chamber was measured in a 10 Hz wide band centered at each test frequency. Noise never exceeded - 3 5 dB, re: 1 dyne/cm 2 (1 labar).
Trainino of animals. Although the basic training procedure for the oscar bears similarity to earlier training for the goldfish (Yan and Popper 1991), the general behavior of the oscar is sufficiently different from the goldfish that we had to significantly modify the training procedure to elicit the appropriate behaviors for this species. The training of oscars was divided into 5 stages. In stage I, food pellets were dropped into the feeding station at random intervals so the fish would recognize the feeding site and become familiar with the experimental apparatus. In stage II, the O-paddle light was turned on and a medium sized pellet (diameter 4 mm) was fastened to the outside of the O-paddle with a string of near transparent parafilm paper. When immersed in water, the visual and odor cue of the food pellet attracted a fish to peck on the O-paddle. However, the fastening of the pellet prevented it from immediately being taken by the fish. The piezo film attached to the O-paddle activated an analog signal to an analog/digital converter when pecked by the fish. This signal triggered the computer to send a signal to the automatic feeder which dropped a food pellet. The fish thus learned to associate pecking the paddle with obtaining a food (reward). After the fish displayed consistent pecking on the O-paddle even after the attached pellet was taken it was possible to start stage III of training. In this stage no food pellet was fastened to the Opaddle. Instead, after the fish pecked the O-paddle its light was turned off and the R-paddle light was turned on. If the R-paddle was pecked within a defined time limit (no limit in the beginning, which was gradually reduced to 30 s), the R-paddle light was turned off and the fish was rewarded with food. However, if the R-paddle was not pecked within the time limit, or the R-paddle was pecked before the O-paddle, the ceiling light was turned off and the fish were subjected to complete darkness for 3 min. The blackout deprived the fish of feeding opportunities and served to deter the fish from making mistakes or cheating. After a fish displayed consistent pecking of the R-paddle within 30 s of pecking the O-paddle (and onset of the R-paddle light), stage IV training was introduced. In this stage, the training was carried out with an almost identical protocol to that of stage III, except that the pecking of the O-paddle was followed by a 500 Hz tone (33 dB, re: 1 labar) instead of lighting of the R-paddle light. The fish had to peck the R-paddle when the sound signal was on. At the beginning, the sound duration was extended to 180 s long, and it was gradually reduced to 7 s. At stage V of training, blank trials were introduced in random order and in equal numbers with sound presentations. The pecking of the R-paddle during blank trials led to a blackout. The purpose of the blank trial was to train the fish not to peck the R-paddle when sound was not heard, such as when the sound was below threshold. Training at stage V continued until the fish constantly attained a combined level of 90% correct responses and a false alarm rate of less than 10% over a session of 32 trials. The false alarm rate was calculated by dividing the number of responses to the R-paddle when blank was presented by the total number of responses (Okanoya and Dooling 1987). Auditory sensitivitystudy. In order to maintain high motivation and not to satiate the fish within a few tests, fish were deprived of food for 17-23 h prior to a test. The test apparatus was loaded into the test tank and determinations of thresholds were carried out inside
H.Y. Yan and A.N. Popper: Audiogram of the oscar
107
the soundproof chamber. A total of 8 fish were used during various training stages. Only 3 were able to pass through the 5 stages of training with an acceptable false alarm rate (< 10%) to get into threshold determinations. Due to the physical limitations of the signal generator, only sound frequencies at or above 200 Hz were tested. Thresholds were determined from 200 to 1000 Hz in 100 Hz intervals using a modified method of constant stimuli (see Yan and Popper 1991, for details). Four to 6 SPLs were used at each frequency. For each frequency tested, the initial SPL was always the maximum output from the speaker and this was subsequently attenuated either at 5 or 10 dB intervals. In each test run, 5 replicates of a chosen SPL and 5 blank trials were randomly presented to the fish. If there were more than 2 false responses in a single test run, the data for that particular run was discarded and the test was repeated later. A minimum of 2 test runs were repeated for each fish at each SPL in order to calculate the response rate (R). A response rate was calculated by dividing the number of correct responses to the Rpaddle by the total number of sound trials presented. On the basis of the R of each SPL tested, a polynomial psychometric function was generated in the form of: SPL (dB) = a + b R (%)+cR z (%) to relate the proportion of the R value (in %) to the stimulus values (SPL) (in dB) (Richards 1976). The threshold for a particular frequency was obtained by calculating the SPL that would yield a 50% proportion of responses in the psychometric function. For each fish, threshold determinations for each frequency were repeated at least 3 times. Results
All oscars showed no response to pure tone signals at 900 a n d 1000 H z even to the m a x i m u m possible signal o u t p u t s (49 dB a n d 43 dB re: 1 I~bar, respectively). Therefore, it is likely that the u p p e r limit o f a u d i t o r y detection ability is below 900 H z for the oscar. The threshold SPLs for 7 frequencies tested are s h o w n in Fig. 2 and Table 1. A o n e - w a y A N O V A test s h o w e d that for each frequency tested, no significant difference in thresholds was observed between the 3 fish (Table 1). However, significant differences (F = 42.17, P < 0.001) in thresholds were observed a m o n g frequencies. D u n c a n ' s multiple range test on m e a n thresholds at each frequency
30
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Frequency (Hz)
Fig. 2. Audiograms of goldfish ( Carassius auratus) (solid square with dashed line) and oscar ( Astronotus ocellatus ) (solid circle with solid line) obtained from the same operant conditioning paradigm and test apparatus. Data of goldfish from Yan and Popper (1991)
1. Pure tone auditory threshold data for the oscar, Astronotus ocellatus showing mean (~) threshold in dB re: 1 dyne/cm, numbers in parentheses indicate range of thresholds, standard deviation (s.d.) of the mean and total number (N) of thresholds tested from each frequency. F F values of one-way ANOVA testing if threshold SPLs differed among 3 oscars for each frequency, ns no significant difference in thresholds for each frequency tested among 3 oscars (P>0.05)
Table
Frequency
~
s.d.
N
F
200
18.4 (14.0-22.1) 20.5 (15.4-25.1) 20.7 (12.3-25.8) 25.1 (18.8-29.4) 29.6 (27.6-33.1) 31.4 (28.3-35.0) 34.0 (30.8 36.9)
2.8
9
1.18
ns
3.0
9
0.46
ns
3.9
9
0.11
ns
3.5
9
0.79
ns
1.8
9
0.13
ns
2.1
9
0.08
ns
1.9
9
2.11
ns
300 400 500 600 700 800
indicated that there were n o differences in thresholds for 200 Hz, 300 Hz, and 400 Hz, the 3 frequencies also h a d the lowest thresholds. The highest m e a n threshold o f 34 dB was at 800 Hz. In general, as frequency increased f r o m 200 H z to 800 H z so did threshold (Fig. 2; Table 1). Discussion
O u r d a t a for the oscar are the first a u d i t o r y thresholds obtained for a n o n - o t o p h y s a n fish using a f o o d reward system. O u r results indicate that the hearing capabilities o f the oscar are generally similar to d a t a o b t a i n e d for other non-specialist species. The m o s t closely related species to the oscar that has been studied is a n o t h e r cichlid, an African m o u t h - b r e e d e r , Tilapia macrocephala (Tavolga 1974). As oscar, T. macrocephala has a n a r r o w a u d i t o r y b a n d w i d t h and p o o r sensitivity. Thresholds for T. macrocephala were - 9.2 dB, - 8.4 dB, a n d 6.6 dB for 100 Hz, 300 Hz, a n d 500 Hz, respectively a n d d a t a were not obtained above 500 H z (Tavolga 1974). These thresholds are s o m e w h a t lower than o u r thresholds for oscar. In addition, preliminary d a t a for m i c r o p h o n i c potentials confirm the p o o r sensitivity and relatively n a r r o w b a n d w i d t h o f this species (Saidel W M , unpublished data). These data, recorded f r o m the saccule (one o f the inner ear e n d o r g a n s involved with hearing) s h o w e d thresholds o f 18.8 dB, 25.0 dB, 23.3 dB, 40 dB, 43 dB, 41 dB a n d 49 dB for 200 Hz, 300 Hz, 400 Hz, 500 Hz, 600 Hz, 700 H z and 800 Hz, respectively (Saidel W M , unpublished data). The higher physiological thresholds o f m i c r o p h o n i c s d a t a f r o m 500 H z to 800 H z t h a n behavioral thresholds could be explained by the fact that m i c r o p h o n i c recordings are taken only f r o m one endorgan, the saccule. I n contrast, the behavioral thresholds are the results o f s u m m a t i o n o f processing b y the whole nervous system. Such differences (14 dB to 43 dB, depending on frequencies) between m i c r o p h o n i c s a n d be-
108 havioral hearing thresholds have also been demonstrated in goldfish (Fay and Popper 1974). Despite the difference, the overall trend of change of thresholds with frequencies in microphonic recordings was similar to that of behavioral threshold data in the oscar. Therefore, the overall hearing ability of A. ocellatus is likely to be below 900 Hz. Clearly, there are significant differences in auditory sensitivities between the goldfish and the oscar (Fig. 2). The differences in bandwidth and sensitivity between these two species could be attributed to the fact that goldfish is a hearing specialist (otophysan) having auxiliary structures such as Weberian ossicles connecting the inner ear and gasbladder (Tavolga 1971) to transmit the pressure component of the sound to the ear. Without any connection between the gasbladder and the inner ear in the oscar, it is not surprising to find limited auditory sensitivity in this cichlid species. Our extensive observations on the response of the oscar to the experimental apparatus showed that a single oscar would hide in corners o f tank for an extended time (sometimes up to 10 days) before showing normal swimming activity again. This type o f behavior presumably due to social isolation has also been reported in convict cichlids (Cichlasoma nigrofasciatum) (Gallagher et al. 1972). However, when 2 or 3 fish were housed in the same tank it took less than 3 h for them to resume normal activities. Therefore, training for each individual fish used in this experiment was conducted so that the experimental animal could see two other fish but separated from the experimental animal by a transparent plexiglass divider. On the contrary, this type of training paradigm was not needed for goldfish (Yan and Popper 1991). Corwin (1983) showed an increase in sensory hair cells of the macula neglecta o f the ray Raja clavata from about 500 at birth to 6000 at 7 years o f age. Electrophysiological data from the same study also demonstrated that the macula neglecta's vibration sensitivity increased with the age and size. When comparing effects of size on auditory sensitivity of goldfish, Popper (1971) found no significant differences between fish of 45-48 m m and 110-120 mm even when a larger goldfish is known to have more sensory hair cells than a smaller one (Platt 1977). Popper and Hoxter (1984) demonstrated that sensory hair cells of saccule of the oscar continues to increase as fish grow from 2.0 cm to 19 cm. In the present study, the auditory threshold data come from fish of about same size (6 cm in standard length), it would be interesting in the future to determine whether a larger oscar could have a higher auditory sensitivity than a smaller fish. The oscar is not known to produce sounds for communication. Since it has limited auditory bandwidth and poor sensitivity, it is now imperative to ask about the role and function o f the auditory system in the life history of wild oscars. It is observed that oscar in the wild swims slowly with smooth, seemingly uninterrupted movements using its large eyes to examine roots of floating aquatic macrophytes for cryptic insects and fishes (Winemiller 1990). At Carlo Maraca, Venezuela, the fish prey items consumed by A. ocellatus are all highly cryptic, relatively sedentary catfishes, and 3 species of these small catfishes
H.Y. Yan and A.N. Popper: Audiogram of the oscar are commonly captured from the roots of floating aquatic plants (Winemiller 1990). Therefore, it seems unlikely that oscars would use acoustic cues to search out these catfishes, not to mention that these catfishes would have to produce very loud sounds to be heard by oscars. However, Miinz (1985) demonstrated that superficial and canal neuromasts of lateral lines of a cichlid fish (Sarotherodon niloticus) have a range of frequencies o f the maximal displacement sensitivity limited to below 200 Hz. Given the fact that oscars also consumed large quantities (69% of total stomach contents) of aquatic, terrestrial insects and Crustacea (Winemiller 1990) which are capable of producing low frequency vibrations from about 5 Hz up to 140 Hz (Lang 1980). It seems likely that visual discrimination and lateral line detection o f struggling movements of a terrestrial insect trapped on the water surface (Bleckmann 1986) and subsurface waves (frequency components up to 100 Hz) caused by moving fish and crustaceans (Bleckmann et al. 1991) are used by oscars to detect the presence of prey. It leaves the possibility that the very limited auditory sensitivity of the oscar is perhaps used for the detection of potential predators in its natural habitats. This view is supported by field observations that many predatory characids (piranhas), sciaenids, and catfishes that coexist with the oscar emit sounds loud enough to be detected by human observers located above the water's surface (Winemiller KO, unpublished data).
Acknowledgements. We are grateful to Robert J. Dooling for the loan of some equipment used in the present study. Kirk O. Winemiller provided details of field observation on feeding behavior and stomach contents data of wild oscar. William M. Saidel kindly provided unpublished microphonics data of oscar. Kirk O. Winemiller, Christopher Platt, Peggy Edds, Carolyn Hue and William M. Saidel offered valuable comments on the manuscript. This study was supported by grants from the National Institutes of Health (NIDCD, DC~)0140) and the Office of Naval Research (N--00014-87-K-0604).
References Bleckmann H (1986) Role of the lateral line in fish behaviour. In: Pitcher TJ (ed) The behavior of teleost fishes. Johns Hopkins Univ Press, Baltimore, pp 177-202 Bleckmann H, Breithaupt T, Blickhan R, Tautz J (1991) The time course and frequency content of hydrodynamic events caused by moving fish, frogs, and crustaceans. J Comp Physiol A 168 : 749-757 Chang JS, Popper AN (1991) An ultrastructural comparison of striola and extrastriola hair cells of a fish utricle. Soc Neurosci Abstr 17:630 Corwin JT (1983) Postembryonic growth of the macula neglecta auditory detector in the ray, Raja clavata: continual increases in hair cell number, neural convergence, and physiological sensitivity. J Comp Neurol 217:345-356 Fay RR (1969) Behavioral audiogram for the goldfish. J Audit Res 9:112-121 Fay RR (1988) Hearing in vertebrates: A psychophysics databook. Hill-Fay Associates, Winnetka, Illinois Fay RR, Popper AN (1974) Acoustic stimulation of the ear of the goldfish (Carassius auratus). J Exp Biol 61:243-260 Gallagher TE, Herz MJ, Peeke HVS (1972) Habituation of aggression: the effects of visual social stimuli on behavior between adjacently territorial convict cichlids (Cichlasoma ni#rofascia~ turn). Behav Biol 7 : 359-368
H.Y. Yan and A.N. Popper: Audiogram of the oscar Jacobs DW, Tavolga WN (1967) Acoustic intensity limen in the goldfish. Anim Behav 15: 324-335 Lang HH (1980) Surface wave discrimination between prey and nonprey by the backswimmer Notonecta #lauca L. (Hemiptera, Heteroptera). Behav Ecol Sociobiol 6:233-246 McCormick CA, Popper AN (1984) Auditory sensitivity and psychophysical tuning curves in the elephant nose fish, Gnathonemus petersii. J Comp Physiol A 155:753-761 Miinz H (1985) Single unit activity in the peripheral lateral line system of the cichlid fish Sarotherodon niloticus L. J Comp Physiol A 157:555-568 Okanoya K, Dooling RJ (1987) Hearing in passerine and psittacine birds: A comparative study of absolute and masked auditory thresholds. J Comp Psychol 101:7-15 Platt C (1977) Hair cell distribution and orientation in goldfish otolith organs. J Comp Neurol 172:283-297 Popper AN (1971) The effects of size on auditory capacities of the goldfish. J Audit Res 11 : 239-247 Popper AN, Coombs S (1982) The morphology and evolution of the ear in actinopterygian fishes. Am Zool 22:311-328 Popper AN, Hoxter B (1984) Growth of a fish ear. I. Quantitative analysis of hair cell and ganglion cell proliferation. Hearing Res 15:133-142 Popper AN, Hoxter B (1990) Growth of a fish ear. II. Locations of newly proliferated sensory hair cells in the saccular epithelium of Astronotus ocellatus. Hearing Res 45:33~40 Popper AN, Saidel WM (1990) Variations in receptor cell innervation in the saccule of a teleost fish ear. Hearing Res 46: 211-228 Presson JC, Popper AN (1990a) Possible precursors to new hair cells, support cells, and Schwann cells in the ear of a postembryonic fish. Hearing Res 46:9 21
109 Presson JC, Popper AN (1990b) A ganglionic source of new eighth nerve neurons in a post-embryonic fish. Hearing Res 46:23-28 Richards AM (1976) Basic experimentation in psychoacoustics. University Park Press, Baltimore, 168 pp Saidel WM, Popper AN (1987) Sound reception in two anabantid fishes. Comp Biochem Physiol 88A:37-44 Saidel WM, Popper AN, Chang JS (1990a) Spatial and morphological differentiation of trigger zones in afferent fibers to the teleost utricle. J Comp Neurol 302:629-642 Saidel WM, Presson JC, Chang JS (1990b) S-100 immunoreactivity identifies a subset of hair cells in the utricle and saccule of a fish. Hearing Res 47:139-146 Schellart N AM, Popper AN (1992) Functional aspects of the evolution of the auditory system of actinopterygian fish. In : Webster DB, Fay RR, Popper AN (eds) The evolutionary biology of hearing. Springer, Berlin Heidelberg New York, pp 295-322 Tavolga WN (1971) Sound production and detection, in: Hoar WS, Randall DJ (eds) Fish physiology, vol V. Academic Press, New York, pp 135 205 Tavolga WN (1974) Signal/noise ratio and the critical band in fishes. J Acoust Soc Am 55:1323-1333 Winemiller KO (1990) Caudal eyespots as deterrents against fin predation in the neotropical cichlid Astronotus ocellatus. Copeia 1990: 665-673 Yan HY, Popper AN (1991) An automated positive reward method for measuring acoustic sensitivity in fish. Behav Res Meth Instru & Compu 23:351-356 Yan HY, Saidel WM, Chang JS, Presson JC, Popper AN (1991) Sensory hair cells of a fish ear: evidence of multiple types based on ototoxicity sensitivity. Proc R Soc Lond B 245:133 138
Joum~ of
Physk ogy A
Neurat and
9 Springer-Verlag 1992
Auditory sensitivity of the cichlid fish Astronotus ocellatus (Cuvier) Hong Y. Yan and Arthur N. Popper Department of Zoology, The University of Maryland, College Park, MD 20742, USA Accepted April 16, 1992
Summary. Auditory sensitivity was determined for the oscar, Astronotus ocellatus, a cichlid fish that has no known structural specializations to enhance hearing. Trained A. ocellatus behaviorally responded to sound stimuli from 200 Hz to 800 Hz with best sensitivity of 18 dB (re: 1 ~tbar) to 21 dB for frequencies between 200 and 400 Hz. This is significantly poorer than hearing sensitivity for fish classified as hearing specialists, but well within the range of hearing capabilities reported for non-specialist teleost species.
Key words: Astronotus ocellatus - Audiogram - Acoustic sensitivity - Fish ear - Non-otophysan fish
Introduction Behavioral studies of hearing sensitivity of teleost fishes have demonstrated broad variability, both in absolute sensitivity and in bandwidth over which sounds can be detected (reviewed in Fay 1988). Best sensitivity has been found in species that have specialized structures to improve the mechanical coupling between a gasbladder, which serves as a pressure detector, and the inner ear. Best known of these enhancements are the Weberian ossicles of otophysan fishes (e.g., goldfish, catfish) which connect the swimbladder, a bubble of air in the abdominal cavity, with the fluid-filled chambers of the inner ear. Hearing capabilities are enhanced by other types of specializations such as anterior projections of the swimbladder that bring it into close proximity to the inner ear (e.g., some squirrelfishes, clupeids) (reviewed in Popper and Coombs 1982; Schellart and Popper 1992) or the presence of small air bubbles very close to the ear (e.g., mormyrids, anabantids) (McCormick and Popper 1984; Saidel and Popper 1987). Morphological studies of the peripheral auditory system of fishes have demonstrated substantial differences Correspondence to: H.Y. Yan
in the relationship between the swimbladder and the ear, and in the structure of the auditory regions of the ear (e.g., Popper and Coombs 1982; Schellart and Popper 1992). Data on hearing capabilities to correlate with these structural differences are limited, and the existing data for about 52 species represents a very limited taxon (reviewed in Fay 1988). Therefore, much more needs to be known about hearing capabilities before more thorough structure-function relationships can be elucidated. One of the problems in obtaining data on hearing capabilities is that the methods used in past investigations may not always be amenable for use with a wide range of species. It is always suggested that a valuable behavioral technique used to measure hearing should take advantage of the naturalistic behavior of an individual species. When the species does not naturally respond in the appropriate way for an experiment, it is virtually impossible to use a particular approach for any behavioral measure. For example, respiration conditioning only works if a species changes respiration rate in response to a noxious stimulus such as electric shock (Fay 1969). The avoidance conditioning paradigm is only useful for species that show escape behavior (Jacobs and Tavolga 1967). Recently, we developed a positive reward technique that is useful for measuring fish hearing (Yan and Popper 1991). The technique uses a food reward and a simple paddle press which mimics food/prey item picking behavior of a fish. The technique, with small modifications in the training paradigm, is applicable to a wide range of species since no aversive treatment is used in the procedure, and most species will come to a spot in a tank in response to a food reward. The oscar (Astronotus oeellatus), a teleost fish of the family Cichlidae, is a hardy aquarium species with a rapid growth rate even in captivity. The oscar has been used extensively in our laboratory as a model animal for the study of functional morphology and physiology of sensory hair cells of its inner ear including sensory hair cell growth (Popper and Hoxter 1984, 1990; Presson and Popper 1990a, b), hair cell innervation (Saidel et al.
106
H.Y. Yan and A.N. Popper: Audiogram of the oscar
1990a; Popper and Saidel 1990), immunocytochemical reactions to calcium binding protein (Saidel et al. 1990b), differential responses to ototoxic agent (Yan et al. 1991) and ultrastructure of cellular organelles (Chang and Popper 1991). To enhance our understanding about the relationships of functional morphology of sensory hair cells of the inner ear and auditory sensitivity of the oscar, the newly validated operant conditioning paradigm developed in our laboratory (Yan and Popper 1991) was used to evaluate pure tone auditory sensitivity of the oscar. Materials and methods Experimental animals. Three oscars (A. ocellatus), about 6 cm in standard length (measured from tip of snout to the end of the vertebral column) were obtained from a local aquarium supplier. Fish were housed as a group in a 40-1 glass-wall aquarium (50 x 30 x 26.6 era) and were fed daily with food pellets. The water was continuously aerated, filtered and water temperature was maintained at about 26.5 ( + 1) ~
Apparatus. A detailed description of the test apparatus including all the electronic modules, digital input/output board and computer interface has been reported (Yan and Popper 1991), so only a brief description is given here (Fig. 1). The test apparatus sat on a Plexiglass platform attached to a 4-legged support which could be transferred easily from one tank to another. A solenoid-controlled automatic feeder was attached to the top of the platform. A tube from the feeder contacted the water surface and was used to dispense floating food pellets (diameter 0.5 mm, average weight 2 mg). The pellets were confined within the tube and this served as a fixed feeding station which could be easily recognized by the fish. A 1 0 W ceiling light (C-light) was attached at the rear of the platform to illuminate the tank during the experiment as needed. A response panel attached to the underside of the platform. The panel consisted of two stainless steel rods about 6 cm apart. Each held a sealed clear plastic tube (diameter 1 cm, length 5 cm) containing a miniature 1 0 W light bulb inside the tube. A piece of pressuresensitive piezo film was attached outside each tube to form an observation paddle (O-paddle) and a report paddle (R-paddle). Each paddle was used as a transducer to transmit responses from fish to the computer. Acoustic signals were generated with a signal generator (Coulbourn Instrument, CI $81~)6) coupled with an attenuator (CI $85--08) and an amplifier (CI $82-24). An underwater speaker
t I
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9
I;;F
!
I
L
c
R
0
Fig. 1. Schematic diagram of the test tank from a fish-eye view, showing the apparatus measuring hearing thresholds. C ceiling light; F automatic feeder; O observation paddle; P plexiglass platform; R report paddle; S underwater speaker; T food pellet delivery tube. Notice that both the O-light and the R-light are housed inside clear plastic waterproof tubes. See Yan and Popper (1991) for detailed description of the apparatus
(University Sound UW-30) was used to present these signals. The signal generation system was calibrated with a hydrophone (Celesco, LC-10) and a wave analyzer (Hewlett-Packard, 358 IA). Sound pressure levels (SPLs) of various frequencies and attenuations were also measured and calibrated around the O-paddle. An IBM-AT computer with a 24-bit parallel digital input/ output board (Metrabyte PIO12) was used to control the experiment and data storage through programs written in Microsoft QuickBASIC (Version 4.5). The experiments were conducted inside an IAC soundproof chamber. Ambient noise inside the chamber was measured in a 10 Hz wide band centered at each test frequency. Noise never exceeded - 3 5 dB, re: 1 dyne/cm 2 (1 labar).
Trainino of animals. Although the basic training procedure for the oscar bears similarity to earlier training for the goldfish (Yan and Popper 1991), the general behavior of the oscar is sufficiently different from the goldfish that we had to significantly modify the training procedure to elicit the appropriate behaviors for this species. The training of oscars was divided into 5 stages. In stage I, food pellets were dropped into the feeding station at random intervals so the fish would recognize the feeding site and become familiar with the experimental apparatus. In stage II, the O-paddle light was turned on and a medium sized pellet (diameter 4 mm) was fastened to the outside of the O-paddle with a string of near transparent parafilm paper. When immersed in water, the visual and odor cue of the food pellet attracted a fish to peck on the O-paddle. However, the fastening of the pellet prevented it from immediately being taken by the fish. The piezo film attached to the O-paddle activated an analog signal to an analog/digital converter when pecked by the fish. This signal triggered the computer to send a signal to the automatic feeder which dropped a food pellet. The fish thus learned to associate pecking the paddle with obtaining a food (reward). After the fish displayed consistent pecking on the O-paddle even after the attached pellet was taken it was possible to start stage III of training. In this stage no food pellet was fastened to the Opaddle. Instead, after the fish pecked the O-paddle its light was turned off and the R-paddle light was turned on. If the R-paddle was pecked within a defined time limit (no limit in the beginning, which was gradually reduced to 30 s), the R-paddle light was turned off and the fish was rewarded with food. However, if the R-paddle was not pecked within the time limit, or the R-paddle was pecked before the O-paddle, the ceiling light was turned off and the fish were subjected to complete darkness for 3 min. The blackout deprived the fish of feeding opportunities and served to deter the fish from making mistakes or cheating. After a fish displayed consistent pecking of the R-paddle within 30 s of pecking the O-paddle (and onset of the R-paddle light), stage IV training was introduced. In this stage, the training was carried out with an almost identical protocol to that of stage III, except that the pecking of the O-paddle was followed by a 500 Hz tone (33 dB, re: 1 labar) instead of lighting of the R-paddle light. The fish had to peck the R-paddle when the sound signal was on. At the beginning, the sound duration was extended to 180 s long, and it was gradually reduced to 7 s. At stage V of training, blank trials were introduced in random order and in equal numbers with sound presentations. The pecking of the R-paddle during blank trials led to a blackout. The purpose of the blank trial was to train the fish not to peck the R-paddle when sound was not heard, such as when the sound was below threshold. Training at stage V continued until the fish constantly attained a combined level of 90% correct responses and a false alarm rate of less than 10% over a session of 32 trials. The false alarm rate was calculated by dividing the number of responses to the R-paddle when blank was presented by the total number of responses (Okanoya and Dooling 1987). Auditory sensitivitystudy. In order to maintain high motivation and not to satiate the fish within a few tests, fish were deprived of food for 17-23 h prior to a test. The test apparatus was loaded into the test tank and determinations of thresholds were carried out inside
H.Y. Yan and A.N. Popper: Audiogram of the oscar
107
the soundproof chamber. A total of 8 fish were used during various training stages. Only 3 were able to pass through the 5 stages of training with an acceptable false alarm rate (< 10%) to get into threshold determinations. Due to the physical limitations of the signal generator, only sound frequencies at or above 200 Hz were tested. Thresholds were determined from 200 to 1000 Hz in 100 Hz intervals using a modified method of constant stimuli (see Yan and Popper 1991, for details). Four to 6 SPLs were used at each frequency. For each frequency tested, the initial SPL was always the maximum output from the speaker and this was subsequently attenuated either at 5 or 10 dB intervals. In each test run, 5 replicates of a chosen SPL and 5 blank trials were randomly presented to the fish. If there were more than 2 false responses in a single test run, the data for that particular run was discarded and the test was repeated later. A minimum of 2 test runs were repeated for each fish at each SPL in order to calculate the response rate (R). A response rate was calculated by dividing the number of correct responses to the Rpaddle by the total number of sound trials presented. On the basis of the R of each SPL tested, a polynomial psychometric function was generated in the form of: SPL (dB) = a + b R (%)+cR z (%) to relate the proportion of the R value (in %) to the stimulus values (SPL) (in dB) (Richards 1976). The threshold for a particular frequency was obtained by calculating the SPL that would yield a 50% proportion of responses in the psychometric function. For each fish, threshold determinations for each frequency were repeated at least 3 times. Results
All oscars showed no response to pure tone signals at 900 a n d 1000 H z even to the m a x i m u m possible signal o u t p u t s (49 dB a n d 43 dB re: 1 I~bar, respectively). Therefore, it is likely that the u p p e r limit o f a u d i t o r y detection ability is below 900 H z for the oscar. The threshold SPLs for 7 frequencies tested are s h o w n in Fig. 2 and Table 1. A o n e - w a y A N O V A test s h o w e d that for each frequency tested, no significant difference in thresholds was observed between the 3 fish (Table 1). However, significant differences (F = 42.17, P < 0.001) in thresholds were observed a m o n g frequencies. D u n c a n ' s multiple range test on m e a n thresholds at each frequency
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Fig. 2. Audiograms of goldfish ( Carassius auratus) (solid square with dashed line) and oscar ( Astronotus ocellatus ) (solid circle with solid line) obtained from the same operant conditioning paradigm and test apparatus. Data of goldfish from Yan and Popper (1991)
1. Pure tone auditory threshold data for the oscar, Astronotus ocellatus showing mean (~) threshold in dB re: 1 dyne/cm, numbers in parentheses indicate range of thresholds, standard deviation (s.d.) of the mean and total number (N) of thresholds tested from each frequency. F F values of one-way ANOVA testing if threshold SPLs differed among 3 oscars for each frequency, ns no significant difference in thresholds for each frequency tested among 3 oscars (P>0.05)
Table
Frequency
~
s.d.
N
F
200
18.4 (14.0-22.1) 20.5 (15.4-25.1) 20.7 (12.3-25.8) 25.1 (18.8-29.4) 29.6 (27.6-33.1) 31.4 (28.3-35.0) 34.0 (30.8 36.9)
2.8
9
1.18
ns
3.0
9
0.46
ns
3.9
9
0.11
ns
3.5
9
0.79
ns
1.8
9
0.13
ns
2.1
9
0.08
ns
1.9
9
2.11
ns
300 400 500 600 700 800
indicated that there were n o differences in thresholds for 200 Hz, 300 Hz, and 400 Hz, the 3 frequencies also h a d the lowest thresholds. The highest m e a n threshold o f 34 dB was at 800 Hz. In general, as frequency increased f r o m 200 H z to 800 H z so did threshold (Fig. 2; Table 1). Discussion
O u r d a t a for the oscar are the first a u d i t o r y thresholds obtained for a n o n - o t o p h y s a n fish using a f o o d reward system. O u r results indicate that the hearing capabilities o f the oscar are generally similar to d a t a o b t a i n e d for other non-specialist species. The m o s t closely related species to the oscar that has been studied is a n o t h e r cichlid, an African m o u t h - b r e e d e r , Tilapia macrocephala (Tavolga 1974). As oscar, T. macrocephala has a n a r r o w a u d i t o r y b a n d w i d t h and p o o r sensitivity. Thresholds for T. macrocephala were - 9.2 dB, - 8.4 dB, a n d 6.6 dB for 100 Hz, 300 Hz, a n d 500 Hz, respectively a n d d a t a were not obtained above 500 H z (Tavolga 1974). These thresholds are s o m e w h a t lower than o u r thresholds for oscar. In addition, preliminary d a t a for m i c r o p h o n i c potentials confirm the p o o r sensitivity and relatively n a r r o w b a n d w i d t h o f this species (Saidel W M , unpublished data). These data, recorded f r o m the saccule (one o f the inner ear e n d o r g a n s involved with hearing) s h o w e d thresholds o f 18.8 dB, 25.0 dB, 23.3 dB, 40 dB, 43 dB, 41 dB a n d 49 dB for 200 Hz, 300 Hz, 400 Hz, 500 Hz, 600 Hz, 700 H z and 800 Hz, respectively (Saidel W M , unpublished data). The higher physiological thresholds o f m i c r o p h o n i c s d a t a f r o m 500 H z to 800 H z t h a n behavioral thresholds could be explained by the fact that m i c r o p h o n i c recordings are taken only f r o m one endorgan, the saccule. I n contrast, the behavioral thresholds are the results o f s u m m a t i o n o f processing b y the whole nervous system. Such differences (14 dB to 43 dB, depending on frequencies) between m i c r o p h o n i c s a n d be-
108 havioral hearing thresholds have also been demonstrated in goldfish (Fay and Popper 1974). Despite the difference, the overall trend of change of thresholds with frequencies in microphonic recordings was similar to that of behavioral threshold data in the oscar. Therefore, the overall hearing ability of A. ocellatus is likely to be below 900 Hz. Clearly, there are significant differences in auditory sensitivities between the goldfish and the oscar (Fig. 2). The differences in bandwidth and sensitivity between these two species could be attributed to the fact that goldfish is a hearing specialist (otophysan) having auxiliary structures such as Weberian ossicles connecting the inner ear and gasbladder (Tavolga 1971) to transmit the pressure component of the sound to the ear. Without any connection between the gasbladder and the inner ear in the oscar, it is not surprising to find limited auditory sensitivity in this cichlid species. Our extensive observations on the response of the oscar to the experimental apparatus showed that a single oscar would hide in corners o f tank for an extended time (sometimes up to 10 days) before showing normal swimming activity again. This type o f behavior presumably due to social isolation has also been reported in convict cichlids (Cichlasoma nigrofasciatum) (Gallagher et al. 1972). However, when 2 or 3 fish were housed in the same tank it took less than 3 h for them to resume normal activities. Therefore, training for each individual fish used in this experiment was conducted so that the experimental animal could see two other fish but separated from the experimental animal by a transparent plexiglass divider. On the contrary, this type of training paradigm was not needed for goldfish (Yan and Popper 1991). Corwin (1983) showed an increase in sensory hair cells of the macula neglecta o f the ray Raja clavata from about 500 at birth to 6000 at 7 years o f age. Electrophysiological data from the same study also demonstrated that the macula neglecta's vibration sensitivity increased with the age and size. When comparing effects of size on auditory sensitivity of goldfish, Popper (1971) found no significant differences between fish of 45-48 m m and 110-120 mm even when a larger goldfish is known to have more sensory hair cells than a smaller one (Platt 1977). Popper and Hoxter (1984) demonstrated that sensory hair cells of saccule of the oscar continues to increase as fish grow from 2.0 cm to 19 cm. In the present study, the auditory threshold data come from fish of about same size (6 cm in standard length), it would be interesting in the future to determine whether a larger oscar could have a higher auditory sensitivity than a smaller fish. The oscar is not known to produce sounds for communication. Since it has limited auditory bandwidth and poor sensitivity, it is now imperative to ask about the role and function o f the auditory system in the life history of wild oscars. It is observed that oscar in the wild swims slowly with smooth, seemingly uninterrupted movements using its large eyes to examine roots of floating aquatic macrophytes for cryptic insects and fishes (Winemiller 1990). At Carlo Maraca, Venezuela, the fish prey items consumed by A. ocellatus are all highly cryptic, relatively sedentary catfishes, and 3 species of these small catfishes
H.Y. Yan and A.N. Popper: Audiogram of the oscar are commonly captured from the roots of floating aquatic plants (Winemiller 1990). Therefore, it seems unlikely that oscars would use acoustic cues to search out these catfishes, not to mention that these catfishes would have to produce very loud sounds to be heard by oscars. However, Miinz (1985) demonstrated that superficial and canal neuromasts of lateral lines of a cichlid fish (Sarotherodon niloticus) have a range of frequencies o f the maximal displacement sensitivity limited to below 200 Hz. Given the fact that oscars also consumed large quantities (69% of total stomach contents) of aquatic, terrestrial insects and Crustacea (Winemiller 1990) which are capable of producing low frequency vibrations from about 5 Hz up to 140 Hz (Lang 1980). It seems likely that visual discrimination and lateral line detection o f struggling movements of a terrestrial insect trapped on the water surface (Bleckmann 1986) and subsurface waves (frequency components up to 100 Hz) caused by moving fish and crustaceans (Bleckmann et al. 1991) are used by oscars to detect the presence of prey. It leaves the possibility that the very limited auditory sensitivity of the oscar is perhaps used for the detection of potential predators in its natural habitats. This view is supported by field observations that many predatory characids (piranhas), sciaenids, and catfishes that coexist with the oscar emit sounds loud enough to be detected by human observers located above the water's surface (Winemiller KO, unpublished data).
Acknowledgements. We are grateful to Robert J. Dooling for the loan of some equipment used in the present study. Kirk O. Winemiller provided details of field observation on feeding behavior and stomach contents data of wild oscar. William M. Saidel kindly provided unpublished microphonics data of oscar. Kirk O. Winemiller, Christopher Platt, Peggy Edds, Carolyn Hue and William M. Saidel offered valuable comments on the manuscript. This study was supported by grants from the National Institutes of Health (NIDCD, DC~)0140) and the Office of Naval Research (N--00014-87-K-0604).
References Bleckmann H (1986) Role of the lateral line in fish behaviour. In: Pitcher TJ (ed) The behavior of teleost fishes. Johns Hopkins Univ Press, Baltimore, pp 177-202 Bleckmann H, Breithaupt T, Blickhan R, Tautz J (1991) The time course and frequency content of hydrodynamic events caused by moving fish, frogs, and crustaceans. J Comp Physiol A 168 : 749-757 Chang JS, Popper AN (1991) An ultrastructural comparison of striola and extrastriola hair cells of a fish utricle. Soc Neurosci Abstr 17:630 Corwin JT (1983) Postembryonic growth of the macula neglecta auditory detector in the ray, Raja clavata: continual increases in hair cell number, neural convergence, and physiological sensitivity. J Comp Neurol 217:345-356 Fay RR (1969) Behavioral audiogram for the goldfish. J Audit Res 9:112-121 Fay RR (1988) Hearing in vertebrates: A psychophysics databook. Hill-Fay Associates, Winnetka, Illinois Fay RR, Popper AN (1974) Acoustic stimulation of the ear of the goldfish (Carassius auratus). J Exp Biol 61:243-260 Gallagher TE, Herz MJ, Peeke HVS (1972) Habituation of aggression: the effects of visual social stimuli on behavior between adjacently territorial convict cichlids (Cichlasoma ni#rofascia~ turn). Behav Biol 7 : 359-368
H.Y. Yan and A.N. Popper: Audiogram of the oscar Jacobs DW, Tavolga WN (1967) Acoustic intensity limen in the goldfish. Anim Behav 15: 324-335 Lang HH (1980) Surface wave discrimination between prey and nonprey by the backswimmer Notonecta #lauca L. (Hemiptera, Heteroptera). Behav Ecol Sociobiol 6:233-246 McCormick CA, Popper AN (1984) Auditory sensitivity and psychophysical tuning curves in the elephant nose fish, Gnathonemus petersii. J Comp Physiol A 155:753-761 Miinz H (1985) Single unit activity in the peripheral lateral line system of the cichlid fish Sarotherodon niloticus L. J Comp Physiol A 157:555-568 Okanoya K, Dooling RJ (1987) Hearing in passerine and psittacine birds: A comparative study of absolute and masked auditory thresholds. J Comp Psychol 101:7-15 Platt C (1977) Hair cell distribution and orientation in goldfish otolith organs. J Comp Neurol 172:283-297 Popper AN (1971) The effects of size on auditory capacities of the goldfish. J Audit Res 11 : 239-247 Popper AN, Coombs S (1982) The morphology and evolution of the ear in actinopterygian fishes. Am Zool 22:311-328 Popper AN, Hoxter B (1984) Growth of a fish ear. I. Quantitative analysis of hair cell and ganglion cell proliferation. Hearing Res 15:133-142 Popper AN, Hoxter B (1990) Growth of a fish ear. II. Locations of newly proliferated sensory hair cells in the saccular epithelium of Astronotus ocellatus. Hearing Res 45:33~40 Popper AN, Saidel WM (1990) Variations in receptor cell innervation in the saccule of a teleost fish ear. Hearing Res 46: 211-228 Presson JC, Popper AN (1990a) Possible precursors to new hair cells, support cells, and Schwann cells in the ear of a postembryonic fish. Hearing Res 46:9 21
109 Presson JC, Popper AN (1990b) A ganglionic source of new eighth nerve neurons in a post-embryonic fish. Hearing Res 46:23-28 Richards AM (1976) Basic experimentation in psychoacoustics. University Park Press, Baltimore, 168 pp Saidel WM, Popper AN (1987) Sound reception in two anabantid fishes. Comp Biochem Physiol 88A:37-44 Saidel WM, Popper AN, Chang JS (1990a) Spatial and morphological differentiation of trigger zones in afferent fibers to the teleost utricle. J Comp Neurol 302:629-642 Saidel WM, Presson JC, Chang JS (1990b) S-100 immunoreactivity identifies a subset of hair cells in the utricle and saccule of a fish. Hearing Res 47:139-146 Schellart N AM, Popper AN (1992) Functional aspects of the evolution of the auditory system of actinopterygian fish. In : Webster DB, Fay RR, Popper AN (eds) The evolutionary biology of hearing. Springer, Berlin Heidelberg New York, pp 295-322 Tavolga WN (1971) Sound production and detection, in: Hoar WS, Randall DJ (eds) Fish physiology, vol V. Academic Press, New York, pp 135 205 Tavolga WN (1974) Signal/noise ratio and the critical band in fishes. J Acoust Soc Am 55:1323-1333 Winemiller KO (1990) Caudal eyespots as deterrents against fin predation in the neotropical cichlid Astronotus ocellatus. Copeia 1990: 665-673 Yan HY, Popper AN (1991) An automated positive reward method for measuring acoustic sensitivity in fish. Behav Res Meth Instru & Compu 23:351-356 Yan HY, Saidel WM, Chang JS, Presson JC, Popper AN (1991) Sensory hair cells of a fish ear: evidence of multiple types based on ototoxicity sensitivity. Proc R Soc Lond B 245:133 138
