]>HEARES3641S0378-5955(01)00217-910.1016/S0378-5955(01)00217-9Elsevier Science B.V.Fig. 1Schematic drawing of the lateral (a) and medial (b) side of the right ear of the anabantoid T. vittata. (a) Anterior is to the right. (b) Anterior is to the left. The saccular and lagenar maculae and nerve branchelets are indicated for orientation purposes. AC, anterior semicircular canal; HC, horizontal semicircular canal; L, lagena; LM, lagenar macula; LO, lagenar otolith (sagitta); N, vestibular and auditory branches of the eighth nerve; PC, posterior semicircular canal; S, saccule; SM, saccular macula; SO, saccular otolith; U, utricle; UM, utricular macula; UO, utricular otolith.Fig. 2Saccular maculae from (a) B. splendens (1.6 g, 39 mm), (b) M. opercularis (1.87 g, 39 mm) and (c) T. vittata (2.00 g, 44 mm). In each case, the long dashed lines separate groups of ciliary bundles oriented in different directions while short dashed lines in the saccular epithelia separate the anterior portion of the macula from the ribbon-shaped portion. The arrows point in the direction of the kinocilia on the cells in a particular group.Fig. 3Scanning electron micrograph showing the posterior saccular epithelium of M. opercularis. Cells shown are divided into two groups that differ in their orientation (separated by a dashed line). All cells in these two groups are oriented in the same direction and have their kinocilia pointed in the same way (arrows). Scale bar=3 μm.Fig. 4Lagenar maculae from (a) B. splendens, (b) M. opercularis and (c) T. vittata. See Fig. 2 for further explanations.Fig. 5Utricular maculae from (a) B. splendens, (b) M. opercularis and (c) T. vittata. See Fig. 2 for further explanations. Lc, lacinia; St, striola.Fig. 6Scanning electron micrographs of the saccular sensory epithelia of (a) B. splendens, (b) M. opercularis and (c) T. vittata. These SEMs show F1 and F2 ciliary bundles at the ventral margin on the posterior part of the maculae. The bundles are oriented ventrally. The arrows point in the direction of the kinocilia. Note that the kinocilia (K) of the F2 bundles are several times longer than the kinocilia of F1 bundles compared to stereocilia. Scale bar=5 μm.Fig. 7Scanning electron micrograph of the ventral saccular sensory epithelium of T. vittata. Arrowheads point to the separated row of F2 ciliary bundles at the ventral margin. Scale bar=10 μm.Fig. 8Scanning electron micrographs of the lagenar and utricular maculae of T. vittata showing ciliary bundle types. (a) Caudal margin of the lagenar macula. (b) Lateral margin of the striola of the utricle near the lacinia. Scale bar=5 μm.Table 1Mean (±S.E.M.) body weights, standard lengths (SL) and length of the saccular maculae in the rostral caudal direction in the three species examinedB. splendensM. opercularisT. vittataANOVASignificanceWeight (g)1.67±0.12.06±0.21.03±0.3F2,15=1.55P=0.01SL (mm)39.0±1.340.4±1.634.2±3.3F2,14=1.25P=0.14Macula (mm)1.05±0.011.04±0.010.98±0.09F2,14=0.63P=0.55Significances of the differences between species were tested using one-way ANOVA.Table 2Mean (±S.E.M.) ciliary bundle densities per 1000 μm2 in the rostral, middle and caudal area of the saccular maculae in the three species examinedB. splendensM. opercularisT. vittataANOVASignificanceRostral53.1±7.450.9±2.148.4±4.7F2,13=0.25P=0.78Middle49.5±3.754.0±1.451.6±5.8F2,15=0.42P=0.67Caudal51.1±2.156.3±2.360.5±2.4F2,14=3.70P=0.051Significances of the differences between species were tested using one-way ANOVA.Comparison of the inner ear ultrastructure between teleost fishes using different channels for communicationFriedrichLadicha*friedrich.ladich@univie.ac.atArthur NPopperbaInstitute of Zoology, University of Vienna, Althanstrasse 14, A-1090 Vienna, AustriabDepartment of Biology, University of Maryland, College Park, MD 20742, USA*Corresponding author. Tel.: +43 (1) 4277 54227; Fax: +43 (1) 4277 9544AbstractThe anatomy and ultrastructure of the inner ear of three species of gouramis which differ widely in acoustic behavior were studied using scanning electron microscopy. Of the three species, Trichopsis possess a pectoral sound-producing mechanism while Macropodus and Betta lack sonic organs. The general structure of the inner ear and the shapes of the sensory epithelia are very similar, although they do differ on the posterior part of the saccular macula which is more S-shaped in Trichopsis and Macropodus than in Betta. The maculae on the three species do not differ either in ciliary bundle type (cells with long kinocilia on the periphery of the maculae and cells with short kinocilia in the central region) or in hair cell orientation pattern. Quantitative measurements of hair cell densities and the size of the sensory epithelia of the saccule did not show significant differences between species. Data presented correlate with physiological results from other investigators showing similar auditory sensitivity in Trichopsis and Macropodus. The similarity in structure and function of the inner ears of gouramis on one hand, and the occurrence of sound-generating organs in just one genus, suggests that hearing evolved prior to vocalization and thus acoustic communication in this taxon.KeywordsSound productionAcoustic communicationHearing specialistHair cellAnabantoidEvolutionAbbreviationsAC, anterior semicircular canalF1, F2, ciliary bundle typesHC, horizontal semicircular canalK, kinociliaL, lagenaLc, laciniaLM, lagenar maculaLO, lagenar otolithN, vestibular and auditory branches of the eighth nervePC, posterior semicircular canalS, sacculusSBC, suprabranchial chamberSL, standard lengthSM, saccular maculaSO, saccular otolithSt, striolaU, utriculusUM, utricular maculaUO, utricular otolith1IntroductionTeleost fishes have evolved a diverse array of sound-generating organs, and many species regularly vocalize during social interactions (Myrberg, 1981; Ladich, 1997; Ladich and Bass, 1998; Zelick et al., 1999). Interestingly, only a few groups of fishes are known in which sonic organs are taxonomic characteristics. One such a group is the ‘arioids’, a suborder of siluriforms which includes five families such as the African mochokids (upside down catfishes) and the neotropical doradids (thorny catfishes). This group is characterized by having an elastic spring mechanism that is used in sound production (Lundberg, 1993). In many other fish groups, such as gadids (cods and relatives) and pomacentrids (damselfishes), some genera are known to be vocal while others are not (Hawkins and Rasmussen, 1978; Hawkins and Myrberg, 1983). Furthermore, within mormyrids (elephant-nose fishes), sound generation is only described in the genus Pollimyrus (Crawford et al., 1997; Crawford and Huang, 1999), and within anabantoids (gouramis) in the genera Trichopsis and Colisa (Kratochvil, 1985; Schuster, 1986; Ladich et al., 1992; Bischof, 1996). Within the family Cyprinidae (minnows and carps), a group which comprises about 2000 species, only representatives of a few genera such as Cyprinella, Gobio and Pimephales are known sound producers (Winn and Stout, 1960; Ladich, 1988; Johnston and Johnson, 2000).This limitation of sound production to certain genera of particular families has several behavioral and physiological implications which are poorly understood. Non-vocal species may primarily utilize other channels for intraspecific communication such as vision or chemoreception. Nelissen (1978) analyzed acoustical behavior of cichlid species and found that the number of sounds produced is inversely correlated to the number of color patterns shown. Fish which do not display visually or acoustically during social interactions might communicate chemically via pheromones (goldfish, Sörensen et al., 1989), via the lateral line (salmon, Satou et al., 1994), or through use of electric signals (knifefishes, Hagedorn and Heiligenberg, 1985).Does the occurrence of sound-generating mechanisms and acoustical communication correlate with the structure and function of the inner ear? Correlations between the region of the greatest hearing sensitivity and sound energy spectra in the characid Serrasalmus nattereri, the toadfish Porichthys notatus, the pomacentrids Eupomacentrus spp., and the gourami Trichopsis vittata support this idea (Cohen and Winn, 1967; Myrberg and Spires, 1980; Stabentheiner, 1988; Ladich and Yan, 1998; McKibben and Bass, 1999). However, comparative studies which also included closely related non-vocal species failed to find vocalization-specific differences in auditory sensitivity among anabantoids and otophysans (Ladich and Yan, 1998; Ladich, 1999).In order to determine if structural differences exist in the inner ear between teleosts that use acoustical communication and those which exploit other communication channels, we examined the ear and hearing ability of several anabantoid species (gouramis or labyrinth fishes). These are a group of small perciforms from Southeast Asia and Africa which differ widely in their methods of intraspecific communication. Only representatives of the genus Trichopsis (croaking gouramis) possess an elaborate sound-producing mechanism based on modifications of the pectoral fins (Kratochvil, 1978). Both males and females of these species emit loud croaking sounds during agonistic behavior. Closely related genera, such as the Siamese fighting fish Betta splendens and the paradise fish Macropodus opercularis, lack sonic organs and do not vocalize except with pharyngeal teeth as an incidental by-product of feeding. Betta and Macropodus, however, produce intensive visual displays by erecting fins, opercula, and gill membranes and by pronounced color changes (Simpson, 1968; Bischof, 1996).Contrary to this variation in the presence of sonic organs, all of the labyrinth fishes are characterized by having a ‘labyrinth’ which is an air-filled cavity located dorsal to the gills (=suprabranchial chamber). These chambers are supplementary respiratory organs utilized for air breathing since these fishes live in poorly oxygenated waters. At the same time, the chambers are located lateral to the inner ears and thereby potentially serve to enhance the hearing ability of these fish (Schneider, 1941; Ladich and Yan, 1998; Yan, 1998). Prior comparison of the saccular sensory epithelia in the blue gourami, Trichogaster trichopterus, and the kissing gourami, Helostoma temmincki, representatives of two different anabantoid families (Belontiidae vs. Helostomatidae), revealed major differences in the size and number of sensory cells of the saccule, despite the two species having similar hearing capabilities (Saidel and Popper, 1987).The goal of the present study was to investigate the ultrastructure of the otic end organs of T. vittata, M. opercularis, and B. splendens. We have analyzed the shape of the sensory epithelia (maculae), distribution of the hair cell types, and the hair cell orientation patterns on the saccular, lagenar and utricular sensory epithelia. In addition, we compared hair cell densities and the size of the saccular maculae in all three species since the saccule is generally considered to be the main hearing end organ in most fishes (Popper and Fay, 1999).2Materials and methodsSix specimen of B. splendens (1.27–1.9 g; 34–42 mm in standard length), eight of M. opercularis (1.41–2.65 g; 34–45 mm SL), and nine of T. vittata (0.29–2.0 g; 25–44 mm SL) obtained from aquarium stores were used for investigations. Fish were kept at 28°C in aerated and filtered aquaria and fed daily until used in the study. All work was done with the approval of the Institutional Animal Care and Use Committee of the University of Maryland.2.1Scanning electron microscopyFish were deeply anesthetized with tricaine methanosulfonate (MS 222, Sigma) and the trunk of the fish, opercula, and gills were dissected away. Cold fixative (2% glutaraldehyde in 0.1 M phosphate or cacodylate buffer) was put into the opened ear capsules. The head was then placed in fixative for at least 24 h. The tissue was then rinsed twice in buffer before the ears were dissected out and otoliths removed. The ears were post-fixed in 1–2% osmium tetroxide for 1 h, washed three times in double distilled water, and dehydrated through a graded series of alcohols to 100% ethanol. Sensory epithelia were then critical point dried with liquid carbon dioxide as the intermediate fluid. The samples were mounted flat on aluminum stubs, coated with gold-palladium alloy and viewed with an Amray 1820 or a Philips XL 20 SEM operating at 20 kV.2.2Density measurements and statisticsIn order to compare the density of ciliary bundles, low power scanning electron micrographs were taken perpendicular to the surface of the maculae of six B. splendens, seven M. opercularis and five T. vittata. Three areas were sampled from each saccule, one in the rostral region of the epithelium in which the ciliary bundles on the hair cells are oriented along the fish’s horizontal axis (see below), and one each in the middle and caudal (where the macula curves ventrally) regions in which ciliary bundles are oriented along the fish’s vertical axis. Although this procedure may not give precise density values because of the shrinkage of the tissue during fixation, it does not effect a general comparison since shrinkage would be about the same in all specimens. The same regions of each end organ were sampled in all specimens. In all cases, peripheral margins of the sensory epithelia were avoided because density increases on the periphery and due to the presence of otolithic membrane which, in these species, were almost impossible to remove from the very edges of the maculae with any consistency.The density (number of hair cells/1000 μm2) was calculated from each of the three sample areas in pictures taken at 3000×. Each picture was 31×24 μm. Approximately 2000 ciliary bundles were counted. Statistical analysis between species were calculated for each area as well as for the mean using one-way ANOVA. In addition, the total length of the saccular macula in the anterior–posterior axis was determined using the scanning electron micrograph (SEM) measuring device.The hair cell orientation patterns were mapped by examining the orientation of ciliary bundles along transects of the maculae. Orientation of transects generally followed the orientation of ciliary bundles. Dorsal–ventral transects were taken from the caudal part of the saccular epithelium while anterior–posterior transects were taken from the anterior part of the saccular macula. Radially oriented transects were taken from the utricular macula, and anterior–posterior transects from the lagena. At least 10 such transects were taken from each epithelium. The orientations of ciliary bundles in each region were marked on a low power picture of the whole epithelium. Orientation patterns were determined for at least two specimens for each species.3Results3.1Gross morphologyThe gross morphology of the ear of all three species is basically similar to that found in most other teleosts. There are three semicircular canals and associated cristae (for detection of angular acceleration) and three otolithic end organs (saccule, lagena, utricle), each containing a sensory epithelium and an overlying single otolith (Fig. 1a,b). No macula neglecta was found. The saccule is the largest otic end organ and the lagena the smallest. The utricle is anterior and dorsal to the saccule and the two regions are connected by a narrow opening. The lagena is located dorso-posteriorly to the saccule. The utricular macula lies on the animal’s horizontal plane, while the saccular and lagenar maculae lie in the vertical plane.Otoliths of the end organs differ considerably in size. The sagitta and the small asterisci fill almost all of the volume of the large saccular and the lagenar chambers respectively, whereas the lapilli do not fill the whole utricular chamber (Fig. 1). Sensory epithelia are smaller than the overlying otoliths especially within the saccule where the ovoid sagittae only contact the sensory macula at the sulcus, an L-shaped shallow indentation. However, despite the otolith’s size, the dorsal parts of the rostral region of the saccular maculae are not covered by the sagittae. In this region, the otolith membrane, which lies between the sensory epithelium and otolith, is the only contact for the ciliary bundles.The otolithic end organs in all three species are innervated by four branches of the eighth cranial nerve, an anterior utricular, two saccular, and one posterior lagenar branch. The first saccular branch leaves the brain along with the utricular nerve and innervates the oval rostral region of the saccular maculae. This branch also goes to the cristae of the anterior and lateral semicircular canals. The posterior part of the saccular macula is innervated by a massive separate branch of the eighth nerve (Fig. 1b). The posterior branch innervates the lagena and the crista of the posterior semicircular canal.3.2Sensory epitheliaThe shapes of the sensory epithelia are very similar in all three species (Figs. 2, 4, 5). The saccular macula consists of two regions, the oval anterior region and a more ribbon-shaped posterior region. The posterior region differs slightly between species. It was rather stretched in B. splendens and more S-shaped in M. opercularis and T. vittata (Fig. 2). The lagenar macula is moon-shaped, while the utricular macula is almost round with a lateral finger-like projection pointing laterally (=lacinia) (Figs. 4, 5).Morphological comparisons revealed that species differed in weight but not in standard length or length of the saccular maculae in the rostro-caudal dimensions (Table 1). Saccular epithelia ranged from 0.66 mm to 1.15 mm long and the size was correlated significantly with body measures (standard length: r=0.68, n=17, P=0.003; weight: r=0.58, P=0.016).Hair cells on each macula are divided into groups, with all of the ciliary bundles in each group oriented in approximately the same direction as defined by having their kinocilia on the same side of the ciliary bundle (see Fig. 6). Saccular hair cells are divided into four orientation groups. The rostral region of the saccular macula has posteriorly oriented hair cells in front of, and directed towards, a group of cells that are oriented anteriorly. The posterior region of the saccular macula has a dorsally oriented hair cell group on the dorsal half and a ventrally oriented group on the ventral half. These cells generally change orientation to follow macula curvatures (Fig. 2). The transitions between groups of hair cells, such as that from dorsal to ventral groups, occur over one or two rows of hair cells (Fig. 3).Lagenar hair cells are oriented dorsally on the anterior side of the epithelium while the cells on the posterior side are oriented ventrally (Fig. 4). Both cell groups tend to curve as the maculae curves. The regions at which the two orientation groups overlap by a small amount is usually called the transition zone. Utricular maculae differed from the other epithelia in the size of the two orientation groups. The cells along the narrow anterior border are oriented medially while the cells of the large central groups are oriented peripherally. This pattern is also found within the lacinia (Fig. 5), a lateral extension of the anterior margin of the utricular epithelium. The actual transition occurs in a narrow region that has been called the striola (e.g. Werner, 1933). In this region, as discussed below, the lengths of the ciliary bundles are somewhat smaller than in surrounding regions (called the extrastriola region).3.3Hair cell types and densitiesSensory epithelia in all three species consist of different types of hair cell ciliary bundles. The most common ciliary bundle type is F1 (definitions according to Popper, 1977) and consists of a series of short graded stereocilia and a kinocilium that is no more than two times longer than the longest stereocilia. Type F2 bundles, on the other hand, are characterized by short graded stereocilia, and kinocilia that are approximately three times longer than the largest stereocilia. Ciliary bundle types are fairly discrete, but there are intermediary forms making it difficult to discriminate between various bundle types.F1 bundles are found in the central region of the saccular macula and F2 ciliary bundles at the periphery (Fig. 6) of all three species. A common feature of all three species is the presence of one row of isolated F2 cells at the ventral saccular epithelium. This row of ciliary bundles is only found at the ribbon-shaped posterior part of these epithelia (Fig. 7).Within the utricular macula, type F1 bundles are found in the central region of the striola and in the extrastriolar region. F2 bundles are found at the margins of the striola and within the lacinia. Similarly, the central part of the moon-shaped lagenar maculae is built up of type F1 bundles whereas the periphery consists of type F2 bundles (Fig. 8). Densities of ciliary bundles varied but are always smaller in the central region of the saccular, lagenar and striolar maculae. Particularly low densities were observed within the extrastriolar regions of the utricle.Densities of hair cells ranged from 48.5 to 60.4 bundles per 1000 μm2 (Table 2) on all three maculae in the three species. No significant differences were found between species in the mean densities or in the densities of the anterior, middle or posterior areas. Differences were close to significance in the caudal area (Table 2). In addition, densities did not differ between the rostral, middle and caudal areas within species (one-way ANOVA, F2,48=1.97, P=0.15).4Discussion4.1Gross and fine structure of anabantoid earsThe ears of B. splendens, M. opercularis and T. vittata resemble each other and the typical pattern of most teleosts other than for the otophysan fishes (goldfish and relatives) (Retzius, 1881; Popper, 1977, 1981; Popper and Coombs, 1982). The saccule is by far the largest end organ and the lagena the smallest.The acoustical coupling of the inner ears of labyrinth fishes to the lateral air-filled suprabranchial chambers (SBCs) is based on the reduction of otic bones surrounding the saccule and separating it from the SBC. According to Schneider (1941), the thin bony sheet may be replaced by a membraneous window which markedly improves hearing in some individuals of Macropodus. In essence, Schneider (1941) first proposed that this air-filled chamber could serve as an accessory hearing organ, a role that is similar to the swim bladder in fishes that have connections between this organ and the inner ear, or air bubbles that are closely associated with the ear, as in the mormyrid fishes (Stipetić, 1939).In contrast to the lagenar and utricular maculae, the saccular maculae do not follow the shape of the otolith. The ovoid sagittae are much larger than the corresponding J-shaped sensory epithelia.4.2Hair cell orientation patternsHair cell orientation patterns within the lagenar and utricular maculae are quite similar to those described in other teleosts (Popper, 1977, 1980, 1981). However, significant differences from many other teleost groups are found in the saccule. Five hair cell orientation patterns have been identified on saccular maculae in different teleosts (Popper, 1981; Popper and Coombs, 1982). Except for the otophysans (goldfish and relatives) and a few non-otophysan species, saccular maculae generally have two horizontally and two vertically oriented groups of ciliary bundles (Popper and Coombs, 1982). The anabantoids described here represent the ‘opposing’ pattern described by Popper and Coombs (1982) in that the fish have two opposing horizontally oriented ciliary bundles on the anterior saccule. Vertically oriented bundles are found on the posterior ribbon-shaped part. This pattern is encountered in other anabantoids (e.g. Popper and Hoxter, 1981) and in the unrelated deep-sea myctophids (lantern fishes) (Popper, 1977).Popper and Coombs (1982) argued that there is a close correlation between the presence of an air bubble near the ear or a connection between the swim bladder and the ear with specializations in the structure of the anterior end of the saccular macula. They also argued for a correlation between such saccular epithelial specializations and enhanced hearing capabilities as compared to fishes that have no connections to an air bubble and the ‘general’ saccular hair cell orientation pattern. The results from the three species of anabantoids reported here tend to support this hypothesis.Thus, based upon the morphological results we not only show a strong correlation between the saccular structure of these three species and the presence of the suprabranchial organ as a hearing structure, but we would predict that were hearing studies to be done on the Siamese fighting fish Betta, it could be shown that it too has a wide hearing bandwidth (ranging from 100 Hz to 5 kHz).4.3Sensory hair cellsThe ciliary bundle types of gouramis investigated are basically similar to those found in other fishes. In the central populations the ratio of kinocilium to stereocilia length is between 1:1 and 2:1 (F1 type). At the margins of the sensory epithelia cells have bundles with ratios of about 3:1 (F2 type). These cells have very short stereocilia. Platt and Popper (1981, 1984) described similar patterns in the blue gourami T. trichopterus.4.4Interspecific differences between labyrinth fishesOur results show that all three species studied have specialized saccular sensory epithelia, although the function of this type of specialization is still not understood (Popper and Fay, 1999). These results support the data in the literature (Ladich and Yan, 1998) showing that two of these species, Trichopsis and Macropodus, have an extended hearing bandwidth and increased sensitivity when compared to fishes without such specializations. Fishes without hearing specializations tend to have saccular maculae where the rostral region is not very much deeper (from dorsal to ventral margin) than the caudal region. While we have not done statistical comparisons, examination of the literature shows that the rostral end of the saccular epithelium in non-specialists is generally less than twice the depth of the caudal region (e.g. Popper, 1977, 1981). In these anabantoids, however, the rostral end of the saccular macula is three times as deep as the caudal macula and the caudally oriented cells are in front of the anteriorly oriented cells. Further supporting the hypothesis that hearing specializations are paralleled by changes in the shape and/or the hair cell orientation pattern on the rostral region of the saccular macula (Popper and Coombs, 1982) is the finding of Saidel and Popper (1987) which showed that the blue gourami T. trichopterus, a species with a saccular specialization similar to that reported here, had lower hearing thresholds than the kissing gourami H. temmincki that has a ‘general’ saccular pattern.Comparison of shape and size of sensory epithelia as well as hair cell orientation patterns did not reveal any significant differences between the species investigated. The J-shaped saccular epithelium is also found in the blue gourami (Popper and Hoxter, 1981; Saidel and Popper, 1987) and seems to be a characteristic of the family Belontiidae. Differences exist in the shape of the saccular maculae between belontiids and the kissing gourami H. temmincki, the only representative of the family Helostomatidae. The rostral portion of the saccular macula of this species is widened but lacks the characteristic ventral extension of belontiids. In addition, the total area of the saccular macula in Helostoma is approximately 40% larger as compared to similar-sized specimens of T. trichopterus.Differences in hair cell densities are minor among representatives of the belontiid family. (While we did not do a statistical analysis on cells at the very margins of the epithelia, examination of tissue from all of our animals leads to the impression that the density was similar between species.) No significant differences were found between B. splendens, M. opercularis and T. vittata and densities of ciliary bundles appear to be similar to those found in T. trichopterus. However, the densities of hair cells in T. trichopterus were slightly higher than in H. temmincki in each sample area, again indicating an interfamiliar difference between anabantoids. With the larger area and slightly higher hair cell densities, Helostoma had about 90% more hair cells than T. trichopterus (44 000 vs. 23 000, respectively – Saidel and Popper, 1987).Do these differences between families account for any difference in hearing abilities? Saidel and Popper (1987) observed that the number of hair cells in the saccules of these two anabantoid fishes inversely correlates with the saccular microphonic thresholds. The somewhat lower microphonic thresholds were almost always obtained from T. trichopterus which had fewer hair cells than Helostoma. In general, the auditory sensitivities of the fish resembled one another in that both species have very similar thresholds and bandwidth (Saidel and Popper, 1987; Yan, 1998). So far it is unknown if differences in hair cell densities result in any physiological differences such as sound localization abilities, frequency discrimination or detection of masked sound.4.5Behavioral and evolutionary considerationsOur findings, and the similarity in gross and fine structure of the inner ears of B. splendens, M. opercularis, T. vittata and T. trichopterus, suggest that no major differences in hearing sensitivities are to be expected among the belontiid family. Ladich and Yan (1998) measured auditory sensitivities by utilizing auditory brainstem response (ABR) recording techniques and observed the lowest thresholds in T. trichopterus (76 dB re 1 μPa at 800 Hz). The paradise fish and the croaking gourami were about 8 dB less sensitive and possessed higher most sensitive frequencies (MSFs) than the blue gourami but otherwise did not differ from one other. This difference in hearing capacities among belontiids might rather be attributed to the size differences of the peripheral suprabranchial chambers than to fine structural differences of the inner ears such as number of hair cells. One possibility is that smaller pharyngeal air bubbles in smaller species such as Macropodus and Trichopsis might have higher resonant frequencies than larger bubbles, but resonance properties of constrained air bubbles, such as these (and the swim bladder) are hard to predict based upon bubble size alone (Popper, 1971, 1974; Sand and Enger, 1973a,b; Clarke et al., 1975).Are differences in auditory sensitivities between the anabantoids investigated (Ladich and Yan, 1998) related to their acoustical behavior? Dominant frequencies of sounds correspond with best hearing bandwidth in T. vittata (1–2 kHz). However, B. splendens, M. opercularis and T. trichopterus which communicate primarily using visual signals, and only incidentally with sound, have similar or even better hearing sensitivities than T. vittata and did not differ in their inner ear morphology. These findings strongly suggest that enhanced sound-detecting abilities in anabantoids evolved prior to, or independently of, the evolution of pectoral sound-generating mechanisms in the genus Trichopsis. Most likely the SBC is an adaptation to waters poor in oxygen and the improvement of hearing a by-product of this process. A similar development obviously took place in otophysans. The evolution of the Weberian apparatus resulted in a pronounced improvement of hearing capacities in cypriniforms, catfishes, and characiforms. However, auditory sensitivities in vocalizing otophysans resemble those of non-vocalizing species and most sensitive frequencies lack a clear relationship to the main energies of sounds (Ladich, 1999). Interestingly, only a few representatives of cypriniforms, the most primitive otophysan group, are known to be vocal and in no case was there any vocal organ described.This again points to the fact that constraints other than optimization of acoustical communication is likely to have caused the ancestors of otophysans, and perhaps fishes in general, to develop and enhance their sound-detecting abilities. A major selective pressure might have been the analysis of the auditory scene which means separating sounds of different origins due to differences in their frequency content or temporal patterns (Bregman, 1990). This could have been a major advantage in avoiding predators and detecting prey (Popper and Fay, 1993; Ladich, 1999).If one accepts the idea that detection of the general auditory scene is of importance for fishes, and particularly for fishes that may live in dark or murky waters, the evolution of mechanisms to enhance detection of higher frequencies than found in most hearing non-specialists may have been a response to particular environments in which species ancestral to hearing specialists lived. Rogers and Cox (1988) pointed out that the transmission characteristics of sound in shallow water (up to several meters in depth) are quite different than in deeper water. In shallow waters, there is a very high attenuation rate for low frequencies that is correlated with depth. While this would not have any impact on fishes living in deeper waters, fishes living in shallow waters would only be able to detect sounds from very near-by sources, unless they have evolved hearing to higher frequencies (Schellart and Popper, 1992). We speculate that anabantoids, like otophysans, clupeids (herrings and relatives), and a number of other diverse teleost taxa may have arisen in shallow waters and evolved high frequency hearing. The fact that the specificities of the specializations are so different in various species argues for the evolution of high frequency hearing several times. For example, as the anabantoids evolved the ability to use the air bubble in the pharyngeal chamber for high frequency hearing, otophysans evolved Weberian ossicles while clupeids evolved a highly specialized utricle (e.g. Mann et al., 1997, 1998).In contrast, there are many other fishes currently living in relatively shallow waters that do not have hearing specializations. We suggest that living in shallow water is secondary to these species, and that ancestral species either evolved in deeper waters, or there were no other selective pressures on the ancestors that could have lead to higher frequency hearing.AcknowledgementsWe thank Marcy Souza, Tim Maugel, Christian Beisser and Anton Losert for their technical advice and assistance and Heidemarie Grillitsch for the line drawings. This research was supported by the Austrian Science Fund (FWF Grant No. 12411 to F.L. and grant DC03936-01 from the National Institute on Deafness and Other Communication Disorders of the NIH to A.N.P.).ReferencesBischof, 1996C.BischofDiversity in agonistic behavior of croaking gouramis (Trichopsis vittata, T. schalleri, and T. pumila; Anabantoidei) and the paradise fish (Macropodus opercularis; Anabantoidei)Aggr. Behav.221996447455Bregman, 1990Bregman, A., 1990. Auditory Scene Analysis: The Perceptual Organization of Sound. MIT Press, Cambridge, MA.Clarke et al., 1975N.L.ClarkeA.N.PopperJ.A.MannJr.Laser light scattering investigations of the teleost swimbladder response to acoustic stimuliBiophys. J.151975307318Cohen and Winn, 1967M.J.CohenH.F.WinnElectrophysiological observations on hearing and sound production in the fish Porichthys notatusJ. Exp. Zool.1651967355370Crawford et al., 1997J.D.CrawfordA.P.CookA.S.HeberleinBioacoustic behaviour of African fishes (Mormyridae): potential cues for species and individual recognition in PollimyrusJ. Acoust. Soc. Am.1021997113Crawford and Huang, 1999J.D.CrawfordX.HuangCommunication signals and sound production mechanism of mormyrid electric fishJ. Exp. Biol.202199914171426Hagedorn and Heiligenberg, 1985M.HagedornW.HeiligenbergCourt and spark: electric signals in the courtship and mating of gymnotoid fishAnim. Behav.331985254265Hawkins and Myrberg, 1983Hawkins, A.D., Myrberg, A.A., 1983. Hearing and sound communication underwater. In: Lewis, B. (Ed.), Bioacoustics, a Comparative Approach. Academic Press, London, pp. 347–405.Hawkins and Rasmussen, 1978A.D.HawkinsK.J.RasmussenThe calls of gadoid fishJ. Mar. Biol. Assoc. U.K.581978891911Johnston and Johnson, 2000C.E.JohnstonD.L.JohnsonSound production in Pimephales notatus (Rafinesque) (Cyprinidae)Copeia20002000567571Kratochvil, 1978H.KratochvilDer Bau des Lautapparates vom Knurrenden Gurami (Trichopsis vittatus Cuvier and Valenciennes) (Anabantidae, Belontiidae)Zoomorphologie9119789199Kratochvil, 1985H.KratochvilBeiträge zur Lautbiologie der Anabantoidei-Bau, Funktion und Entwicklung von lauterzeugenden SystemenZool. Jahrb. Physiol.891985203255Ladich, 1988F.LadichSound production by the gudgeon, Gobio gobio L., a common European freshwater fish (Cyprinidae, Teleostei)J. Fish. Biol.321988707715Ladich, 1997F.LadichAgonistic behaviour and significance of sounds in vocalizing fishMar. Freshw. Behav. Physiol.29199787108Ladich, 1999F.LadichDid auditory sensitivity and vocalization evolve independently in otophysan fishes?Brain Behav. Evol.531999288304Ladich and Bass, 1998F.LadichA.H.BassSonic/vocal motor pathways in catfishes: comparison with other teleostsBrain Behav. Evol.511998315330Ladich et al., 1992F.LadichC.BischofG.SchleinzerA.FuchsIntra- and interspecific differences in agonistic vocalization in croaking gouramis (Genus: Trichopsis, Anabantoidei, Teleostei)Bioacoustics41992131141Ladich and Yan, 1998F.LadichH.Y.YanCorrelation between auditory sensitivity and vocalization in anabantoid fishesJ. Comp. Physiol. A1821998737746Lundberg, 1993Lundberg, J.G., 1993. African-South American freshwater fish clades and continental drift: problem with a paradigma. In: Goldblatt, P. (Ed.), Biological Relationship between Africa and South America. Yale University Press, New Haven, CT, pp. 156–199.Mann et al., 1997D.A.MannZ.LuA.N.PopperA clupeid fish can detect ultrasoundNature3891997341Mann et al., 1998D.A.MannZ.LuM.HastingsA.N.PopperDetection of ultrasonic tones and simulated dolphin echolocation clicks by a teleost fish, the American shad (Alosa sapidissima)J. Acoust. Soc. Am.1041998562568McKibben and Bass, 1999J.R.McKibbenA.H.BassPeripheral encoding of behaviorally relevant acoustic signals in a vocal fish: single tonesJ. Comp. Physiol. A1841999563576Myrberg, 1981Myrberg, A.A. Jr., 1981. Sound communication and interception in fishes. In: Tavolga, W.N., Popper, A.N., Fay, R.R. (Eds.), Hearing and Sound Communication in Fishes. Springer, New York, pp. 395–425.Myrberg and Spires, 1980A.A.MyrbergJr.J.Y.SpiresHearing in damselfishes: an analysis of signal detection among closely related speciesJ. Comp. Physiol. A1401980135144Nelissen, 1978M.H.J.NelissenSound production by some tanganyikan cichlid fishes and a hypothesis for the evolution of the R communication mechanismsBehaviour641978137147Platt and Popper, 1981Platt, C., Popper, A.N., 1981. Fine structure and function of the teleost ear. In: Tavolga, W.N., Popper, A.N., Fay, R.R. (Eds.), Hearing and Sound Communication in Fishes. Springer, New York, pp. 3–36.Platt and Popper, 1984C.PlattA.N.PopperVariation in lengths of ciliary bundles on hair cells along the macula of the sacculus in two species of teleost fishesScann. Electr. Microsc.4198419151924Popper, 1971A.N.PopperThe effects of size on the auditory capacities of the goldfishJ. Audit. Res.111971239247Popper, 1974A.N.PopperThe response of the swimbladder of the goldfish (Carassius auratus) to acoustic stimuliJ. Exp. Biol.601974295304Popper, 1977A.N.PopperA scanning electron microscopic study of the sacculus and lagena in the ears of fifteen species of teleost fishesJ. Morphol.1531977397417Popper, 1980A.N.PopperScanning electron microscopic studies of the sacculus and lagena in several deep-sea fishesAm. J. Anat.1571980115136Popper, 1981A.N.PopperComparative scanning electron microscopic investigations of the sensory epithelia in the teleost sacculus and lagenaJ. Comp. Neurol.2001981357374Popper and Coombs, 1982A.N.PopperS.CoombsThe morphology and evolution of the ear in actinopterygian fishAm. Zool.221982311328Popper and Fay, 1993A.N.PopperR.R.FaySound detection and processing by fish: critical review and major research questionsBrain Behav. Evol.4119931438Popper and Fay, 1999Popper, A.N., Fay, R.R., 1999. The auditory periphery in fishes. In: Fay, R.R., Popper, A.N. (Eds.), Comparative Hearing: Fish and Amphibians. Springer, New York, pp. 43–100.Popper and Hoxter, 1981A.N.PopperB.HoxterThe fine structure of the sensory epithelia of the sacculus and lagena of a teleost fishHear. Res.51981245263Retzius, 1881Retzius, G., 1881. Über das Gehörorgan der Wirbelthiere: Morphologisch-histologische Studien, Vol. 1. Das Gehörorgan der Fische und Amphibien. Samson and Wallin, Stockholm.Rogers and Cox, 1988Rogers, P., Cox, M., 1988. Underwater sound as a biological stimulus. In: Atema, J., Fay, R.R., Popper, A.N., Tavolga, W.N. (Eds.), Sensory Biology of Aquatic Animals. Springer, New York, pp. 131–149.Saidel and Popper, 1987W.M.SaidelA.N.PopperSound reception in two anabantid fishesComp. Biochem. Physiol.8819873744Sand and Enger, 1973aO.SandP.S.EngerEvidence for an auditory function of the swimbladder in the codJ. Exp. Biol.591973405414Sand and Enger, 1973bSand, O., Enger, P.S., 1973b. Function of the swimbladder in fish hearing. In: Moller, A. (Ed.), Basic Mechanisms of Hearing. Academic Press, New York, pp. 893–910.Satou et al., 1994M.SatouH.A.TakeuchiJ.NishiiM.TanabeS.KitamuraN.OkumotoM.IwataBehavioral and electrophysiological evidences that the lateral line is involved in the inter-sexual vibrational communication of the hime salmon (Landlocked red salmon, Oncorhynchus nerka)J. Comp. Physiol. A1741994539549Schellart and Popper, 1992Schellart, N.A.M., Popper, A.N., 1992. Functional aspects of the evolution of the auditory system of actinopterygian fish. In: Webster, D.B., Fay, R.R., Popper, A.N. (Eds.), The Evolutionary Biology of Hearing. Springer, New York, pp. 295–322.Schneider, 1941H.SchneiderDie Bedeutung der Atemhöhle der Labyrinthfische für ihr HörvermögenZ. Vergl. Physiol.291941172194Schuster, 1986S.SchusterUntersuchungen zur Bioakustik des Zwergfisches Colisa lalia (Perciformes, Belontiidae)Zool. Beitr.291986295306Simpson, 1968M.J.ASimpsonThe display of the Siamese fighting fish Betta splendensAnim. Behav. Monogr.11968173Sörensen et al., 1989P.W.SörensenN.E.StaceyK.J.ChamberlainDiffering behavioral and endocrinological effects of two female sex pheromones on male goldfishHorm. Behav.231989317332Stabentheiner, 1988A.StabentheinerCorrelations between hearing and sound production in piranhasJ. Comp. Physiol. A16219886776Stipetić, 1939E.StipetićÜber das Gehörorgan der MormyridenZ. Vergl. Physiol.261939740752Werner, 1933C.F.WernerDie Differenzierung der Maculae im Labyrinth, insbesondere bei SäugetierenZ. Anat. Entwicklungsgesch.991933696709Winn and Stout, 1960H.E.WinnJ.F.StoutSound production by the satinfin shiner, Notropis analostanus, and related fishesScience1321960222223Yan, 1998H.Y.YanAuditory role of the suprabranchial chamber in gourami fishJ. Comp. Physiol. A1831998325333Zelick et al., 1999Zelick, R., Mann, D., Popper, A.N., 1999. Acoustic communication in fishes and frogs. In: Fay, R.R., Popper, A.N. (Eds.), Comparative Hearing: Fish and Amphibians. Springer, New York, pp. 363–411.