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Inner ear morphology in the Atlantic molly Poecilia mexicana--first detailed microanatomical study of the inner ear of a cyprinodontiform species - PubMed

️Fri Nov 25 9994

Inner ear morphology in the Atlantic molly Poecilia mexicana--first detailed microanatomical study of the inner ear of a cyprinodontiform species

Tanja Schulz-Mirbach et al. PLoS One. 2011.

Abstract

Background: Fishes show an amazing diversity in hearing abilities, inner ear structures, and otolith morphology. Inner ear morphology, however, has not yet been investigated in detail in any member of the diverse order Cyprinodontiformes. We, therefore, studied the inner ear of the cyprinodontiform freshwater fish Poecilia mexicana by analyzing the position of otoliths in situ, investigating the 3D structure of sensory epithelia, and examining the orientation patterns of ciliary bundles of the sensory hair cells, while combining μ-CT analyses, scanning electron microscopy, and immunocytochemical methods. P. mexicana occurs in different ecotypes, enabling us to study the intra-specific variability (on a qualitative basis) of fish from regular surface streams, and the Cueva del Azufre, a sulfidic cave in southern Mexico.

Results: The inner ear of Poecilia mexicana displays a combination of several remarkable features. The utricle is connected rostrally instead of dorso-rostrally to the saccule, and the macula sacculi, therefore, is very close to the utricle. Moreover, the macula sacculi possesses dorsal and ventral bulges. The two studied ecotypes of P. mexicana showed variation mainly in the shape and curvature of the macula lagenae, in the curvature of the macula sacculi, and in the thickness of the otolithic membrane.

Conclusions: Our study for the first time provides detailed insights into the auditory periphery of a cyprinodontiform inner ear and thus serves a basis--especially with regard to the application of 3D techniques--for further research on structure-function relationships of inner ears within the species-rich order Cyprinodontiformes. We suggest that other poeciliid taxa, or even other non-poeciliid cyprinodontiforms, may display similar inner ear morphologies as described here.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

**Figure 1. Schematic drawings of the membranous labyrinths of a surface fish of *Poecilia mexicana* from Río Oxolotán.**
(A), right labyrinth with semicircular canals and end organs with otoliths shown in shaded gray and maculae shown in red in lateral view (A₁) and in medial view (A₂). (B), left and right end organs in dorsal view displaying the position of end organs with respect to the main axis of the fish. d, dorsal; L, lagena; la, lateral; r, rostral; S, saccule; U, utricle. Scale bar = 1 mm.

**Figure 2. Gross morphology of brain and inner ears.**
Brain and inner ears of fish from the surface habitat Tampico (A) and fish from the Cueva del Azufre (**B, C**). (A) female, SL = 54 mm; (B) subadult specimen, SL = 26 mm; (C) male, SL = 35 mm. (**A₁, C₁**): dorsal view; (**A₂, B, C₂**): ventral view. TE, telencephalon; C, cerebellum; MO, medulla oblongata; BO, olfactory bulb; TO, optic tectum; P, pituitary gland; NO, optic nerve; SC, spinal cord; LI, inferior lobe; asc, anterior semicircular canal; hsc, horizontal semicircular canal; psc, posterior semicircular canal; ap^asc, ampulla of the anterior semicircular canal; ap^hsc, ampulla of the horizontal semicircular canal; ap^psc, ampulla of the posterior semicircular canal; L, lagena; la, lateral; r, rostral; S, saccule; U, utricle. Scale bars = 1 mm.

**Figure 3. Position of otoliths *in situ*.**
Three-dimensionally reconstructed otoliths from a cave (SL = 35 mm) and a surface fish (SL = 38 mm, both females) based on μ-CT analyses. In (A₁) and (F₁) the left asteriscus and sagitta are shown in caudal view. (A₂) and (F₂): dorsal view of left and right lapilli (red), sagittae (blue), and asterisci (yellow). (A₃) and (F₃): brightest point projection of μ-CT sections of the neurocranium and otoliths *in situ* shown in rostral view. (**B–E**) and (**G–J**): SEM images of the left otoliths of the cave and the surface fish in medial (**B, D; G, I**), rostral (**C, H**), and ventral views (**E, J**), respectively. B–C, G–H, asterisci; D, I, sagittae; E, J, lapilli. d, dorsal; la, lateral; me, medial; r, rostral. Scale bars = 100 µm.

**Figure 4. Relationship of the depth of the sulcus acusticus, thickness of the otolithic membrane, and curvature of the macula sacculi.**
Light micrographs of transversal semithin-sections (thickness: 1 µm) displaying the difference of the thickness of the otolithic membrane between cave (**A, B**) and surface fish (**C, D**). (A), cave fish female (SL = 51 mm), (B), cave fish male (SL = 37 mm), (C), surface fish from Río Oxolotán (female, SL = 44 mm), and (D), surface fish from Tampico (female, SL = 30 mm). (**B₁–D₁**), sections of the rostral part of the right macula sacculi not overlain by the sagitta. Black arrowheads indicate a dorsal swelling of the basal lamina flanking the macula which is distinctly developed in cave fish and in surface fish from Río Oxolotán. (**A, B₂–D₂**), sections in the central region of the sulcus acusticus as indicated by the black dashed line in (B₃). (**B₃–D₃**) show the medial face of the left otoliths of the same individuals of which the sections in (**B₁–D₁**) and (**B₂–D₂**) are presented. 1*, upper ‘compact’ part of the otolithic membrane; 2*, lower part of the otolithic membrane displaying numerous pores. BL, basal lamina; OM, otolithic membrane; SE, sensory epithelium (macula sacculi); VIII, part of the eighth cranial nerve. Sections were stained with Richardson's solution. Scale bars = 100 µm.

**Figure 5. Orientation patterns of ciliary bundles on the macula lagenae.**
(A) and (D), drawings of the asterisci and the overlying maculae lagenae showing the amount of the region of the sensory epithelium not overlain by the otolith in a cave fish (A, male; SL = 31 mm) and a surface fish from Río Oxolotán (D, female; SL = 34 mm). (B₁) the left macula lagenae (mirrored) of the cave fish and (E₁) the right macula lagenae of the surface fish of which the stereocilia of ciliary bundles were stained with TRITC-labelled phalloidin. The macula lagenae in the cave fish (B₁) is only slightly bent while it is boomerang-shaped in the specimen from the surface habitat (E₁). Note that the tip of the rostral arm in (E₁) is slightly inflated. (F) shows the shape of an intact tip of the rostral arm of the macula lagenae of another surface fish (female; SL = 40 mm). (B₂) and (E₂), drawings of the same maculae as shown in (B₁) and (E₁), respectively, displaying a similar orientation pattern of the ciliary bundles in cave (B₂) and surface fish (E₂). (C) and (G), SEM images of the right macula lagenae of another cave fish (male; SL = 30 mm) and of the left macula lagenae (mirrored) of another surface fish (female; SL = 40 mm). d, dorsal, r, rostral. Scale bars = 100 µm.

**Figure 6. Three-dimensionally reconstructed lagena and macula lagenae of cave fish and surface fish from Tampico.**
(A) and (C), lagenae in lateral view showing the macula, (the otolithic membrane), the non-sensory epithelium, and the basal lamina in a cave (A; male, SL = 35 mm) and a surface fish (C; female, SL = 52 mm). Note that the lagena in the surface fish (C) displays a distinct caudo-dorsal ‘edge’ (black arrowhead), while the lagena of the cave fish lacks this “edge” (A). This feature corresponds to the prominent posterodorsal edge of asterisci from surface fish (see Figure 5D *vs.* A). (B) and (D) display differences in curvature and especially shape of the macula lagenae of cave (B) and surface fish (D). The maculae lagenae are shown in lateral (B₁, D₁) and dorsal view (**B₂, D₂**). BL, basal lamina; d, dorsal; NSE, non-sensory epithelium; la, lateral; OM, otolithic membrane; r, rostral; VIII, part of the eighth cranial nerve innervating the lagena. Scale bars = 100 µm.

**Figure 7. Overview of ciliary bundle types.**
(A), ciliary bundle forming an even slope with a kinocilium only slightly longer than the stereocilia (open arrowhead) (see F3-type in ; see also : Figure 5F) and another ciliary bundle with a kinocilium approximately twice as long as the longest stereocilium (black arrowhead) (see : Figure 5E). (B), morphological polarization of hair cells: ciliary bundles are oriented in two opposing directions as indicated by black arrows. The star labels a ciliary bundle forming a short and even staircase array similar to the F1-type described in . (C), ciliary bundle with a kinocilium at least three times longer than the longest stereocilium (white arrowhead) (see F2-type in ; see also : Figure 5A). Scale bars = 1 µm.

**Figure 8. Orientation patterns of ciliary bundles on the macula sacculi.**
(A) and (E), drawings of the sagittae and the overlying maculae sacculi showing the amount of the region of the sensory epithelium not overlain by the otolith in a cave (male, SL = 30 mm) and a surface fish from Río Oxolotán (female, SL = 41 mm). (B) and (F), sulcus acusticus of the corresponding sagitta; the open arrowhead indicates the position of the part of the sulcus housing the dorsal ‘bulge’ of the macula sacculi. The white arrowhead indicates the small caudal sulcus impression rarely seen in sagitta of cave fish (B) while this feature is often present in sagittae of surface fish (F). The sulcus impression is filled with otolithic membrane (F). However, there does not exist a corresponding structure in the macula sacculi (C₁, G₁). (C₁) left macula sacculi (mirrored) of the cave fish and (G₁) right macula sacculi of the surface specimen of which the stereocilia of ciliary bundles were stained with TRITC-labelled phalloidin. (C₂) and (G₂), drawing of the same macula as shown in (C₁) and (G₁) displaying the ‘four quadrant’ orientation pattern of the ciliary bundles. (D) and (H), SEM images of the right macula sacculi of another cave fish (male; SL = 31 mm) and of the left macula sacculi (mirrored) of another surface fish (female; SL = 40 mm) showing the degree of tissue shrinkage with regard to the maculae in (C₁) and (G₁). d, dorsal, r, rostral. Scale bars = 100 µm.

**Figure 9. Three-dimensionally reconstructed macula sacculi of cave and surface fish (Tampico).**
(A) and (C), maculae sacculi in lateral view showing the macula and the non-sensory epithelium. Black arrowheads label the ventral and dorsal bulges of the macula. (B) and (D), maculae sacculi in caudal view displaying the different amount of three-dimensional curvature. The strong curvature of the macula sacculi of the surface fish correlates with a thick sagitta whereas the flat almost two-dimensional macula comes along with a rather flat sagitta of the cave fish specimen. BL, basal lamina; d, dorsal; la, lateral; NSE, non-sensory epithelium; r, rostral; v, ventral. Scale bars = 100 µm.

**Figure 10. Three-dimensionally reconstructed utricle and orientation pattern of ciliary bundles on the macula utriculi.**
(A), utricle of a cave fish (male, SL = 35 mm) in dorsal view displaying the lapillus, otolithic membrane, macula utriculi, non-sensory epithelium, and the basal lamina. (B), Utricle and rostral most part of the saccule in caudal view. The black arrowhead labels the thickened part of the basal lamina that dorsally surrounds the rostral most region of the macula sacculi. (C), macula utriculi and the rostral most part of the macula sacculi in lateral view illustrating the closeness of the two maculae to each other. (D), macula utriculi and lapillus in dorsal view illustrating the three parts of the macula utriculi, namely the striola, lacinia, and cotillus and the amount of the macula overlain by the otolith. (E), Orientation pattern of ciliary bundles on the macula utriculi of a surface fish female (Río Oxolotán, SL = 34 mm) and (F), and on the lacinia of the right macula utriculi of a cave fish male (SL = 30 mm). Note that the orientation pattern of the lacinia was inferred from other surface fish individuals. The dashed line in (E) and (F) shows the change of the orientation pattern with ciliary bundles revealing the opposing orientation of ciliary bundles in the striola region and labels the change of the orientation in the lacinia. BL, basal lamina; d, dorsal; la, lateral; lap, reconstruction based on organic remains of the lapillus; NSE, non-sensory epithelium; me, medial; Msa, macula sacculi; Mu, macula utriculi; OM, otolithic membrane; r, rostral. Scale bars = 100 µm.

**Figure 11. Confocal images of double labeling of a saccular epithelium.**
(A₁), stereocilia of ciliary bundles stained with TRITC-labeled phalloidin. (A₂), kinocilia of the same ciliary bundles stained with anti-bovine α-tubulin mouse monoclonal antibodies and Alexa Fluor 488 conjugated anti-mouse secondary antibodies. White arrows indicate the orientation of ciliary bundles based on the position of the kinocilium. Scale bars = 2 µm.

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Inner ear morphology in the Atlantic molly Poecilia mexicana--first detailed microanatomical study of the inner ear of a cyprinodontiform species - PubMed