Ultrastructure and Development of the Olfactory Organ in Indigenous Pelophylax Ridibundus (Amphibia, Rana)

The olfactory placodes that found in the rostrolateral regions of the head are specialized areas of cranial ectoderm and give rise to the olfactory epithelium. The olfactory epithelial thickening invaginates to form the nasal pit and differentiate as either secretory cells or glial cells. In mice, the olfactory placodes are identified as epithelial thickenings as early as embryonic day 9 (E9) and the formation of the nasal pit occurs soon after the closure of the neural tube (Tran 2008). The nasal pit invaginate into a more complex nasal cavity and the nostrils are narrowed to small slits. In the medial wall of the newly formed nasal cavity, the vomeronasal organ invaginated further into a separate, distinct cavity (Balmer and LaMantia 2005; Bhattacharyya et al., 2008; Santos et al., 2015; Kumar et al., 2016). The epithelium of the main olfactory cavity of salamnder (Dicamtodon tenebrosus) is divided into a series of transverse valleys lined with olfactory epithelium and seperated by ridges of respiratory epithelium. In frog Rana chensinensis the dorsal portion of the buccal cavity may correspond to the ventral olfactory epithelium (Wang et al., 2008). The olfactory organ of all aquatic animals (larvae and neotenes) is similar in structure, forming a tube extending from the external naris to the choana. The epithelium of the main olfactory cavity is thrown into a series of transverse valleys and ridges. The ridges enlarge with growth, forming large flaps extending into the lumen in neotenes. The vomeronasal organ is a diverticulum off the ventrolateral side of the main olfactory cavity (Kralovec et al., 2013). The morphological substrate constitutes a separate organ system consisting of a vomeronasal duct equipped with chemosensory cells (Jungblut et al., 2010, Kralovec et al., 2013). During metamorphosis thyroid hormones initiate extensive changes in the olfactory system in Xenopus laevis including the origins of new regions of the olfactory epithelium and olfactory bulb and a change in olfactory projection patterns (Santos et al., 2015). The aim of this study was to provide a structural and ultrastructural description of the olfactory organ of Pelophylax ridibunda larvae with the aim to contribute to future ontogenetic, evolutionary and phylogenetic studies.

Ribbons of fertilized eggs from couples of the available Pelophylax ridibunda were collected from breeding sites from natural sites near the city of Al-Hassa (Easten  region, K.S.A.) using a close-mesh net. After hatching, the larvae were daily fed on a meal of boiled spinach, transported to the laboratory and kept in plastic bowls filled with water of the same pond from where they are collected, containing some aquatic plants. The water was renewed periodically and kept at room temperature under a natural light/dark cycle until they reached the desired stages needed for experimental work, according to the normal table of Sedra and Michael (1961).

The larval stages were used for the present study namely, stages number 18-27. Five larvae at each stage were selected for the experiment.
 
Scanning electron microscope preparation
 
For scanning electron microscope examination, the specimens were fixed in a 2-3% Glutaraldehyde solution for 3-4 h at room temperature, then washed in 0.1 M phosphate buffer for three 15 min. Next, specimens were dehydrated in a graded Ethanol series as follows: 35%, 50%, 70%, 80%, 95%; three changes at 100% for 15 min each and a final wash in acetone for 5 min. Specimens were dried in CO2, mounted on aluminium stubs and sputter coated with gold. Features of dorsal and ventral internal oral anatomy were examined and photographed using a scanning electron microscope (Jeol) attached to a computer. Terms used to describe features of the oral cavity are derived from (Altig 2007).

The sequences of development of the olfactory organ of the marsh frog, Pelophylax ridibundus, was studied by using scanning electron microscope ranged from stage 20 through 27.

The olfactory organ originates from an anlage formed by the ectoderm on the ventrolateral part of the head during stage 19. Starting at stage 20 by formation of two olfactory placodes, the olfactory placodes become ciliated early in embryonic development. Our result explained that the first appearance of the olfactory placode is sign of differentiation in the developing olfactory system. The olfactory placode composed of a superficial layer, which give rise to the olfactory supporting cells and a deep layer that give rise to the olfactory receptor cells, this is coincide with that reported with (Hansen et al., 1998, Hansen and Zeiske 2005). Embryologically, the olfactory cells of early stages were indistinguishable from the supporting cells in the olfactory placode lining the nasal pit, at hatch this is agree with (Taniguchi et al., 2008). The superficial epidermal ciliation of the placode can easily be distinguished from surrounding epidermal cilia. Each placode has thickened and begins to invaginate; this is known as olfactory (nasal) pit (Fig 1a). At stage 22, the nasal pits (NP) are shallow depressions lies just dorsolateral to the stomodael depression, the epithelial surface of the shallow depression is now less abundant with ciliated (Fig 1b). At stages 23, 24 the depression begun to invaginate further, forming distinct marginal rims and become sharply demarcated from the surrounding epidermis. At the ultrastructural level, in Rana ridibunda, the main olfactory cavity is the principle cavity. There are three different types of receptor cells: ciliated, microvillous and secretory cells. ciliation of  receptor cells of can have cilia, short microvilli, a mix of the two, or long microvilli. Supporting cells are of two types: non ciliated secretory supporting cells with small, electron-dense secretory granules and ciliated supporting cells. The anterior part of the principal cavity contains a “larval type” epithelium that has both microvillar and ciliated receptor cells and both microvillar and ciliated supporting cells, whereas the posterior part is lined with an “adult-type” epithelium that has only ciliated receptor cells and microvillar supporting cells. The middle cavity is nonsensory (Benzekri and Reiss. 2012). The development of vomernasal organ is present in both larval and adult anurans and occurs early in most anurans (Khalil 1978a). However, in Bufo Americana and Bufo regularis it formed later nearer to metamorphic stages than larval stages. (Hansen et al., 1998, Sansone et al., 2015). At stage 24, the nasal pits has invaginated into more complex an internal lumen (olfactory cavity) as primordium of the future olfactory organ. The olfactory cavity of tadpoles of Pelophylax ridibundus consists of a principal cavity which is composed of a series of three interconnected chambers: the outer, middle and inner chambers (Fig 1c). Embryologically, at this stage, the ciliated and supporting cells of the nasal pit became distinguishable from each other. The ciliation of the principal cavity are of two types, receptor cells and supporting cells lining the nasal pit. There are two types of receptor cells either elongated ciliated (CC) or microvillated cells (MC), also supporting cells are of two types: ciliated (CS) and non ciliated secretory cells (NCS) (Fig 1d). The outer chamber contains only ciliated secretory cells (CS), the middle chamber contains ciliated secretory supporting cells. In the inner chamber, very long ciliated receptor cells are abundant and shows a pattern of cilia with definite orientation. The arrangment of the cilia in ridges repeats the wave -like arrangment of theses cilia that in vivo have a metachronal rhythm .the distal ends of ciliated cells arranged in parallel rows near the surface of the mucus and alternative with microvillated cells (Fig 1d). At stage 25, loss of epidermal body surface is visible, otherwise around the olfactory organ (Fig 1e) (Kalita and. Kalita 2004).

The variation of shape, number and distribution of cells specially in the middle and inner chambers are visible in the late stages than that of early stages (Fig 1c and f). Also the density of cells on the inner chamber is increased (Fig 1f). At stage 27 the ciliated and supporting cells of the nasal pit became distinguishable from each other, the cells of the outer chamber made of flattened non secretory cells, the middle chamber is a neuroepithelium that comprises multiple ciliated secretory cells layers that extend vertically from the epithelial surface they are vary in their morphology. Each supporting cell contacs the neighbouring ones and the surrounding receptors by means of a series of specialized structures and intercellular junctions. The inner chamber were equipped with long cilia, but supporting cells with microvilli (Fig 1h, black small arrow heads). By high resolution observation, we found small cells, finger like structures, subpopultion of ciliated secretory cells (Fig 1g, white small arrow heads). The olfactory placodes are laterally located and as they invaginate and nasal pits form, the orientation of the developing nasal cavity gradually orientes and moves from rostrolaterally to rostrally until the olfactory organ form at the most rostral tip of the head. By higher resolution, we observed a tiny projections of small secretory supporting cells in the middle chamber of the principle cavity, arranged in row between the main secretory supporting cells and known as Bowman’s glands. The appearance of Bowman’s glands in the olfactory epithelium at this time suggests that the nose first begins to detect odorants in the air and this is thus also a metamorphic event. The Bowman’s glands together with the surrounding supporting cells, are presumply responsible for a thick layer of mucus that now coats the epithelium of the principle cavity this result is coincide withtha same result done on frog by (Taniguchi et al., 2008; Hensen et al., 1998; Sansone et al., 2015) on goat (Kumar et al., 1992), on camel (Nagpal et al., 1988). In contrast, noetic salamanders necturus and caelians. lack Bowman’s glands  in the olfactory epithelium (Benzekri  and Reiss. 2012).

Development of the olfactory organ of vertebrates has been studied in some taxa for more than one century. Our results described the morphological components and the basic principles of developmental patterns of the olfactory organ as well as cellular differentiation of receptors in Rana ridibunda by electron micrographs. As the complex sensory organ, its ontogenetic development has often been a matter of discussion on higher vertebrates evolution. 

The author acknowledge the Deanship of Scientific Research At King Faisal University for the financial support under Nasher Track (Grant No.186369).