Sniffing at the river bottom: Influence of olfactory organ morphology on the life habits of freshwater stingrays (Potamotrygoninae)

Sniffing at the river bottom: Influence of olfactory organ morphology on the life habits of freshwater stingrays (Potamotrygoninae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sniffing at the river bottom: Influence of olfactory organ morphology on the life habits of freshwater stingrays (Potamotrygoninae) Akemi Shibuya, Rubia Machado, Wallice Duncan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4509528/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Sep, 2024 Read the published version in Zoomorphology → Version 1 posted 7 You are reading this latest preprint version Abstract The olfaction in batoids have an important role for initial detection of the chemical stimulus produced by prey during the foraging activities. Herein, the morphological and histological description of primary lamellae and secondary folds of olfactory rosettes is given to four species from Rio Negro basin. A simpler structure of olfactory organs in Paratrygon sp. does not indicate a primary sensory role during the initial phase of its feeding behavior. In Potamotrygon wallacei , the largest surface area of primary lamellae suggests enhanced olfactory sensitivity related to its generalist feeding habits and complex substrate exploration. Histological analysis revealed differences in epithelial cell composition among species, with variations in the secondary folds shape and the distribution of mucous cells. The simplicity of secondary folds in both Paratrygon sp. and Potamotrygon orbignyi probably is related to their specialized feeding habits, requiring fewer adaptations to detect different types of chemical stimuli. A central muscular layer in primary lamellae was observed only to P. motoro and P. wallacei and indicates a capacity to expand the olfactory epithelium area. These findings provide insights into the functional morphology of olfactory organs in potamotrygonin stingrays and their ecological implications, evidencing the intricate sensory adaptations crucial for foraging success in diverse freshwater habitats. Additionally, it becomes necessary to take into account the contribution of all sensory systems to understand their foraging behavior. Nonetheless, the generalization of the morphological characteristics of olfactory organ in a potamotrygonin species requires caution, since morphological variations can be found, especially to widespread species. olfaction Batoidea potamotrygonin stingrays Rio Negro basin sensory cells. Figures Figure 1 Figure 2 Figure 3 Introduction Elasmobranchs use a complex arrangement of sensory systems (gustation, electro and mechanosensorial organs, vision and olfaction) which act jointly to locate prey, potential predators and the presence of conspecifics (McComb and Kajiura, 2008 ). Studies focusing on olfactory organs in elasmobranchs have seen a notable increase on the recent years (e.g. Cox 2013 ; Ferrando et al. 2019a ; Dymek et al. 2021 ), driven by the accuracy chemical detection abilities exhibited by apex predator species (Yopak et al. 2015 ). Elasmobranchs have two different arrangement of multilamellar olfactory structures, oval or elongated shapes (e.g. Theisen et al. 1986 ; Takami et al. 1994 ; Ferrando et al. 2019a ); however, there are a diversity related to the relative size, number and shape of primary lamellae and secondary folds. Independently of the morphological distinctions, the olfactory organs are mainly used to detect chemical stimulus in the water (Bleckmann and Hoffman, 1999). As most batoid species, potamotrygonin stingrays depend specially on the ventral sense systems to locate and capture the prey. Evidently, the prominent eyes in most Potamotrygon species provide a wide vision of the benthic environment; however, the accuracy of localization of prey is managed by the other sensory organs responsible to detect the prey. Shibuya et al. ( 2010 ; 2020 ) stand that the distribution of lateral line canals and the high density of neuromasts around the mouth in freshwater stingrays are related to the detect the prey, which most of them are formed by benthic species or commonly live buried in the substrate (e.g. Duncan et al. 2016 ; Shibuya 2022 ). Even the reduced electrosensory structure in comparison to marine species, the Lorenzini ampullae in short canals concentrated in the dermis near the mouth, the electroreception is likely employed on the final stage of prey strike (Szabo et al. 1972 ; Szamier and Bennett 1980 ; Harris et al. 2015 ). As the entire sensory systems have integrative actions, the olfaction in may play the role of the initial detection of the chemical stimulus just prior the capture of prey on the foraging activities on the bottom for benthic stingrays. Dymek et al. ( 2021 ) analyzed the olfactory organs in five elasmobranch species, including two potamotrygonin stingrays, and showed detailed morphology of the olfactory epithelium. However, the morphological data on the olfactory organs in freshwater stingrays is still scarce, in face of 38 valid species (Fontenelle et al. 2021 ). An important fact is that the generalization of the morphological characteristics of olfactory organ in a potamotrygonin species requires caution, since environmental factors can vary significantly depending on the river in which they occur. Thus, the aim of the present study is describe the morphological characteristics of lamellar surface of the olfactory organs of freshwater stingrays from Rio Negro basin. The blackwater of Rio Negro presents high acidity (pH: 3.89–6.07), low conductivity (8.8 to 28.6 µS cm − 1 ) and lack of suspended sediments (Küchler et al. 2000 ). Potamotrygonin stingrays usually are found associated with the substrate, which is covered by of sand and the accumulation leaf litter from the flooded forest (Shibuya 2022 ). In order to compare the area of lamellae surface, the sensory area and densities of secondary folds were estimated. Histological comparisons can provide new insights related to their foraging and feeding habits. These morphological parameters were related to the habitat use and feeding habits previously investigated (Shibuya et al. 2009 ; Duncan et al. 2016 ; Shibuya 2022 ). Material and Methods Samples collections were carried out during the low (dry season) period of hydrologic cycle in 2019–2020 in the middle Rio Negro (Barcelos Municipality, Amazonas State, Brazil). Individuals were collected using a dip net by local fishermen, comprising four species: juveniles of Paratrygon sp. (n = 4), Potamotrygon motoro (n = 1) and P. orbignyi (n = 2) and adults of P. wallacei (n = 5). Disc width (DW, mm) of the specimens, as well as length and width of the olfactory rosettes into capsules (mm) were taken prior to dissection (in order to preserve the original shape) to calculate the relative size for each analyzed species. Olfactory capsules were removed, fixed in cold and buffered glutaraldehyde 2.5% solution (pH 7.4) for 24 hours and then preserved in 70% ethanol solution. Specimens were preserved in 10% buffered-formalin solution and stored in 75% ethanol solution. Schematic diagrams of the morphology of primary lamella structures were drawn from dissected specimens with the aid of a stereoscopic microscope (Figs. 1 a-d). The olfactory rosettes were removed from the right side (assuming the bilateral symmetry). The primary lamellae were counted and each of them was dissected, separated, labeled and stored into 0.5 ml microcentrifuge tubes. The secondary folds were counted of one side of contact surface in both anterior and posterior portion of each primary lamella and photographed with a millimetric scale. Width (W PL ) and total area (A PL ) of primary lamella and morphological measures of the anterior (AP) and posterior (PP) lamellar portions were obtained using ImageJ software (Schneider et al. 2012 ) (Fig. 1 b). The lamellar area was calculated only for the region comprising secondary folds (Fig. 1 c) and the W PL and A PL are the sum of measurements of the anterior and posterior portions of the primary lamella. The terminology of olfactory structures followed Ferrando et al. ( 2017b , 2019a ). Calculation of the total lamellar area was based on Ferrando et al. ( 2019a ), with few modifications in order to have an accuracy of the total area. The gross surface area (GSA) was calculated as the sum of all total area of each primary lamella as following: GSA total = A PL 1 + A PL 2 + A PL 3 +…+ A PL n . where: A PL is the total area of each primary lamella. The total area was calculated by the sum of the AP and PP areas, previously taken using ImageJ; n is the number of primary lamellae of the olfactory organ. To characterize the microscopical morphology, the olfactory rosette from the left side of each specimen was initially decalcified in 5% formic acid for 24 h, dehydrated in a graded ethanol solution series (96–100%) and infused in ethanol + resin solution for a minimum of 24 hours. Pieces of the organ were embedded in methacrylate resin (Historesin, Leica) in sagittal and transversal positions and sectioned into 3–5 µm-thickness slices. The slides were stained with Toluidine blue for the delineation of the shape of secondary folds. Additionally, Alcian Blue (AB) counterstained with safranin and Periodic Acid Schiff (PAS) counterstained with Hemotoxilin were applied separately, thus facilitating the discernment of distinct positive reaction of each dye. Since potamotrygonin stingrays have simple secondary folds (compared to most elasmobranchs examined by Ferrando et al. ( 2019a )), the total surface was not estimated according to the equation recommended by Ferrando et al.’s methodology. Thus, calculation of the total contact area of primary lamellae (A TC ) was taken by estimating the area of a 1 x 1 mm square, as follow: A TC = A PL x A Square , where A PL is the total area of primary lamella and A Square is the relative area of 2D perimeter of the silhouette (P SIL ) of sagittal slices (in mm 2 ). The perimeter of the silhouette was taken using subsampled areas of the histological slides (Fig. 1 d). Measurements of slices were taken with a histological slide with micrometric scale. The increase rate of total surface of the set of secondary folds is presented in relative percentage (%) of the total linear area of primary lamellae. Lamellae of the central region of the capsule were used to calculate the depth range at which water can reach the olfactory organ. For this purpose, the mean length of secondary folds was measured of both anterior and posterior lamellar portions of the larger primary lamella: (1) external, that comprise the first small fold; (2) median, which is located in the middle of the lamellae and (3) internal, that is the last fold near the raphe (Fig. 1 c). The central region of rosette was considered as the middle one third of the total number of primary lamellae (Fig. 1 b). Mean, standard deviation, minimum and maximum of measurements of primary lamellae and secondary folds were presented as mean ± SD and min-max. Voucher specimens were catalogued at Fish Collection of the Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus. Brazil: Paratrygon sp., #34959; Potamotrygon motoro , # 27091; P. orbignyi , # 27088 and P. wallacei , # 34960. Results Gross Morphology of olfactory rosettes The olfactory organs of potamotrygonin species comprise a pair of encapsuled rosettes positioned anteriorly to the mouth. The olfactory rosettes are categorized by an elongated shape which have the external edge thinner to the midline and internal edge of the raphe (see Fig. 1 b). The water current goes sinuously into the nostrils, flows through the olfactory rosettes (through both anterior and posterior lamellar portions) and drains off posteriorly to the nasal flap. The olfactory organs are covered by a pair of inner folded edge positioned just under the nasal flap, possibly assisting the water flow into the nostrils (Figs. 2 a and b). Each olfactory rosette is formed by a central raphe that separated in anterior and posterior lamellar portions. Overall, the anterior portion was larger lamellar side. Paratrygon sp. presents roundish lamellae and more concave shape, with the posterior portion slightly short (Fig. 2 c), while Potamotrygon species have an elongated and curved lamellae (Fig. 2 d), with a visibly shorter posterior portion. All species had a pair of process in both anterior and posterior lamellar portions, with most prominent and pointed shape to Potamotrygon species (Figs. 2 c and d). Morphological measurements of the olfactory structures are presented in the Table 1 . High number of primary lamellae was observed to Potamotrygon motoro (n = 54), while Paratrygon sp. presented the lowest number (min-max: 27–29) among analyzed species. Each primary lamella presents secondary folds positioned parallel to each other in anterior and posterior portions on both sides (see Figs. 2 c and d). The length and number of secondary folds varies according to the size of primary lamellae. Higher numbers of secondary folds were found to P. motoro and P. orbignyi comparing the larger primary lamellae, while the length of the secondary folds of the largest primary lamella was greater for P. wallacei (see Table 1 ). Table 1 Measures of olfactory organs of potamotrygonin species. The relative size of olfactory organs was calculated using the disc width and rosette width. The total area, number, and length of secondary folds were determined for the largest primary lamella of each examined specimen. AP and PP represent the anterior and posterior portions of primary lamella (PL), respectively. Length-1, Length-2 and Length-3 correspond to specific segments of PL, measured according to the methodology outlined in Fig. 1 C. Species Disc width (mm) Olf. rosette width (mm) Mean and Relative size (%) Olf. rosette length (mm) No. PL Total area of the largest lamella (mm 2 ) Secondary folds (largest lamella) No. AP Length-1 Length-2 Length-3 No. PP Length-3 Length-2 Length-1 Paratrygon sp. (n = 4) 274–302 7.9–10.5 9.1 (3.2) 3.0-3.6 27–29 9.45 ± 1.64* 24.6 ± 4.1* 0.42 ± 0.04* 1.08 ± 0.05* 1.33 ± 0.08* 20.0 ± 2.0* 1.24 ± 0.08* 1.00 ± 0.1* 0.33 ± 0.03 Potamotrygon motoro (n = 1) 180 11.2 6.2 4.8 52 12.21 36.0 0.32 1.23 1.17 28.0 1.30 1.14 0.33 P. orbignyi (n = 2) 152–241 8.6–13.9 5.7 4.2–6.8 45–48 20.30** 40.0** 0.63** 1.69** 1.42** 26.0** 1.15** 1.58** 0.57 P. wallacei (n = 5) 160–240 11.0-14.9 12.9 (6.6) 4.6–7.7 32–39 24.69 ± 3.86 28.0 ± 1.4 0.53 ± 0.14 1.66 ± 0.23 1.83 ± 0.28 20.4 ± 0.9 1.73 ± 0.22 1.68 ± 0.14 0.55 ± 0.10 * result of three specimens ** result of one specimen Total lamellar area The gross surface area was considered as the sum of the area of anterior and posterior portions of both side of primary lamellae. Paratrygon sp. presents the smallest total area (Table 2 ). Despite the very few differences of total area among Potamotrygon species, the result of P. orbignyi should be considered carefully, in order to the damage of primary lamellae located on the edges of olfactory rosettes, thus, the area might be underestimated. The largest lamellar surface was calculated to P. wallacei , even the low number of primary lamellae compared to P. motoro . Table 2 shows differences between the gross lamellar area (linear area) and the contact surface area, considering the perimeter of secondary folds of each species (Figs. 2 e-h). Paratrygon sp. presents slightly waved secondary folds (Fig. 2 e), with an increase of 9.0% of total area of primary lamellae. Despite Potamotrygon species have similar morphology of the primary lamellae, their secondary folds have distinct forms. Potamotrygon motoro presents a triangular shape of secondary folds (Fig. 2 f), which increases 42.5% of total surface area of olfactory lamellae. Likewise Paratrygon sp., P. orbignyi has wave-shaped secondary folds (Fig. 2 g) that provide a smaller increase of lamellar contact surface (5.7%). Conversely, the wide and flattened surface of secondary folds of P. wallacei (Fig. 2 h) has a largest expansion of lamellar contact comparing to the other examined species (115.4%). Table 2 Measures of the gross surface area of primary lamellae, defined as the sum of areas of all lamellae within one rosette, considering a 1 x 1 mm square linear area. Ratio values represent the increase in surface area using the perimeter of the silhouette of the secondary folds. The estimated area and its percentage were calculated based on the total area of all primary lamellae within one olfactory rosette. Species Gross surface area Ratio Estimated total area Paratrygon sp. 163.10 ± 2.38* 1.09 ± 0.04 a 177.78 (9.0%) Potamotrygon motoro 244.23** 1.42 ± 0.08 b 348.03 (42.5%) P. orbignyi 525.51*** 1.06 ± 0.02 a 555.46 (5.7%) P. wallacei 548.94 ± 5.51 2.15 ± 0.24 c 1,182.42 (115.4%) * result of three specimens ** result of one specimen *** sum of surface area of 34 lamellae of one specimen. a based on one specimen b based on three specimens c based on four specimens Histological analyses of secondary folds The primary lamellae are characterized by having secondary folds on both sides. These secondary folds exhibit morphological differences among the examined species, both in terms of shape and composition of epithelial cells. The epithelium is a sensory region covered by ciliary supporting cells placed on the sensory receptor cells. The apical zone of secondary folds is a non-sensory region presenting mucous cells and non-ciliary cells (Figs. 3 a-h). The wavy-shaped secondary folds of Paratrygon sp. (Fig. 3 a) has a group of PAS-positive mucus cells (Fig. 3 b), while Potamotrygon species have goblet cells secreting AB-positive and PAS-positive substances. Goblet cells are distributed among non-ciliary supporting cells which is covered by a mucus granules, except to P. motoro that has goblet cells in the sensory region (Fig. 3 d). In all examined species, the middle zone of secondary folds is filling by connective tissue that originated from the center of primary lamella. Secondary folds in Paratrygon sp., Potamotrygon motoro and P. orbignyi have similar size along the primary lamellae. In contrast, distinct width and size of secondary folds are found on the primary lamellae of P. wallacei (Fig. 3 f). The connective tissue in the middle of larger secondary folds of P. wallacei expands under the support and basal cells of epithelium in both sensory and non-sensory regions. The wall of basal portion among primary lamellae presents a PAS-positive mucus cells apically positioned on the epithelium and extending to near the secondary folds (Fig. 3 e). The primary lamellae of Paratrygon sp. and Potamotrygon orbignyi have a thick connective tissue that support secondary folds on both sides. Potamotrygon motoro and P. wallacei present thin layer of connective tissue in both sides of primary lamellae. In these species, the central portion of primary lamella is filled by smooth muscle longitudinally arranged, which is thicker in P. wallacei (Figs. 3 c and f). Discussion The investigation of olfactory organ revealed a morphological diversity in potamotrygonin stingrays. However, the delicate structure of the olfactory epithelium and the small size of the organs turned a challenge in maintaining adequate fixation to prevent degradation of lamellar structures. Additionally, the difficult handling of olfactory rosettes during the dissection of primary lamellae (often damaged during the individualization process) impeded a complete examination and the quantification of secondary lamellae. Nonetheless, the current results provide relevant information that, combined with knowledge of their life habits (Shibuya et al. 2009 ; Duncan et al. 2016 ; Shibuya et al. 2016 ; Shibuya 2022 ), can elucidate the functional role of the olfactory system. The bottom of the river may present different types of obstacles such as litter and tree trunks alongside sandy substrate, making the benthic zone an excellent refugee for insects larvae and crustaceans (e.g. Goulding and Ferreira 1983; Nessimian et al. 1998 ). Therefore, the behavior of stirring up the substrate becomes essential for stingrays to uncover the hidden prey (Garrone-Neto and Sazima 2009 ; Shibuya et al. 2012 ). Only young individuals of Paratrygon sp. were examined, and during this life stage, this species is associated with sandy beaches, feeding on small fish (Shibuya et al., 2009 ). Comparing to the Potamotrygon species, Paratrygon sp. exhibited smaller nostril size, lower number of total lamellae, and reduced lamellar area, even considering the silhouette of secondary folds. Paratrygon sp. is a large-sized species, reaching up to 93.0 cm DW in the Rio Negro (Sánchez-Duarte et al. 2013 ), and the morphological simplicity of its olfactory organs, compared to Potamotrygon species, may indicate lower olfactory efficiency. However, it is necessary to consider two important factors. Firstly, Paratrygon sp. inhabit river channels as adults (e.g. Shibuya 2022 ). The high water flow in such environments may hinder the detection of chemical stimuli released by their prey. Furthermore, fish living in river channel are not small in size (compared to those living on the beaches) (Shibuya et al. 2009 ) and live on the substrate, indicating that Paratrygon sp. does not primarily rely on chemical cues for foraging. Additionally, this species exhibits a widespread distribution of lateral line canals and a high number of pores on the dorsal surface that may play a primary role in locating mechanical stimuli generated by prey near ray’s body (Shibuya et al. 2010 , 2020 ). Thus, it becomes necessary to take into account the contribution of all sensory systems to understand how this species obtains its food. Despite examining few specimens, P. motoro exhibited a larger lamellar area than P. orbignyi ; however, the opposite was observed when considering the silhouette of the secondary folds. Although both species have high numbers of primary lamellae and secondary folds, the difference in the increment of lamellar surface area may be related to their respective life habits. Potamotrygon motoro is an active species that constantly explores different types of habitats, often found associated with various types of substrates in search of prey (Shibuya 2022 ). This species primarily consumes crabs and small fish (Shibuya et al., 2009 ). In contrast, P. orbignyi has a specialist feeding habit, consuming aquatic insects in sandy beaches of all studied populations. Considering the need to explore a broader range of habitats and consuming a diverse types of prey, P. motoro requires an olfactory system capable of detecting a variety of chemical stimuli released by its prey. These ecological characteristics can explain the increase of lamellar surface given by the secondary folds’ silhouette. Potamotrygon wallacei stands out having high values of total surface area, especially considering the silhouette of secondary folds. The estimate of the total contact area exceeded 100% for P. wallacei , highlighting the morphological importance of secondary folds for enhancing the sensitivity of olfactory organs. The generalist feeding habits of this species (see Shibuya et al. 2009 ), along with its ability to explore complex substrate of flooded forest (Duncan et al. 2016 ) for locating prey, underscore the necessity of P. wallacei to identify chemical stimuli, including those released by smaller prey hidden in leaf litter, such as small shrimps and insects larvae. The presence of a pair of lamellar processes on primary lamellae may serve an important function in guiding water flow along the olfactory organ, due to their prominent and curved shape. In addition to freshwater stingrays, the lamellar process has also been observed in marine batoid species such as Aetobatus narinari , Aptychotrema rostrata , and Neotrygon kuhlii (Schluessel et al. 2008 ) and may indicate its role in maintaining water flow over the primary lamella to identify chemical cues from prey buried in the bottom (Kyne and Bennett 2002 ; Schluessel et al. 2010 ; Jacobsen and Bennett 2011). The increase in surface area provided by the secondary folds is notable for enhancing olfactory sensitivity. Although potamotrygonin stingrays do not exhibit the complex branching of secondary folds seen in marine elasmobranchs (Ferrando et al. 2017a , 2019a , b ; Dymek et al. 2021 ), morphological diversity may indicates an increase of sensitivity surface area. Dymek et al. ( 2021 ) previously described the morphology of olfactory organs in two Potamotrogon species, showing a different morphology to P. motoro when compared to that observed in the present study. Despite Dymek et al. ( 2021 ) having used specimens from the ornamental market, these morphological differences corroborate the importance of considering the populations being examined. Widely distributed species such as P. motoro may exhibit ecological and morphological distinctions, so information regarding the origin of the specimens and the characteristics of the habitat where these stingrays used to live are essential for inferring the functional role of the lamellar structure and can indicate adaptations and pressures to their particular habitats (Schluessel et al., 2008 ). Taxonomic revisions that have been carried out on widely distributed species of potamotrygonines (e.g. Loboda and Carvalho, 2013 ; Silva and Carvalho, 2015; Loboda et al. 2021 ), emphasize the need to distinguish populations in ecological and functional morphology studies. In contrast to observations by Dymek et al. ( 2021 ), the goblet cells of P. motoro and P. wallacei were AB and PAS-positive, producing mucus in the non-sensory lamellar region. Therefore, mucus granules were also found on both sensory and non-sensory surface of Paratrygon sp., which were only PAS positive, indicating an assistance to cilia movement. However, the presence of goblet cells on the top of secondary folds can be considered to have a protective function. The benthic-associated life habits of freshwater stingrays mean that olfactory lamellae are in direct contact with river bottom elements, resulting in constant friction with sand and leaf-litter fragments. Thus, the presence of goblet cells and mucus granules on the lamellar surface may serve as important protection against potential friction damage and infection to the olfactory epithelium. The reduced or absence of goblet cells likely mean a lack of mucus-propel function, a characteristic observed in many elasmobranch species (Cox 2013 ). Instead, the cilia movement may be driven by water flow, playing a significant hydrodynamic role in guiding chemical stimuli to sensory cells. Nonetheless, a minimal contribution of ciliary propulsion may exist, especially in P. motoro , which exhibits a few goblet cells in sensory region. According to the classification of primary lamella morphology by Ferrando et al. ( 2019a ), the secondary folds of all examined species was classified as non-branched and of short size. The presence of a central layer of smooth muscle into the primary lamellae was observed in both P. motoro and P. wallacei , but neither observed in Paratrygon sp. nor P. orbignyi , nor described in previously studied freshwater stingrays (Dymek et al. 2021 ). The longitudinal arrangement of smooth muscle may be related to the process of expanding the primary lamellae, optimizing the contact of the olfactory epithelium with the water current. This characteristic supports the relationship between increased surface area of the olfactory epithelium and the foraging capabilities of P. motoro and P. wallacei in various types of habitats (Duncan et al. 2016 ; Shibuya 2022 ), possibly regulating the expansion of primary lamellae based on the need for chemosensitivity accuracy. The integration of all sensory systems in freshwater stingrays is indeed an important characteristic for the success of this group in a diversity of freshwater habitats, such as lakes, streams, shallow and deep waters such as beaches and river channels (Duncan et al. 2015 ; Shibuya 2022 ). The synchronized performance of different sensory modalities plays an essential role in feeding behavior, with each sensory element having a primary function in different phases of foraging (search, location, and the phase prior to prey apprehension). Differences of the morphology of olfactory organs in potamotrygonin stingrays may determine the level of accuracy in prey detect as specialized adaptations corresponding to the life habits of each species. Further examination of other species may reveal whether morphological diversity of olfactory organs have phylogenetic inferences for the Potamotrygoninae subfamily. Declarations The authors have no conflict of interest to declare. Ethics approval The specimens were collected with permission of Instituto Chico Mendes de Conservação da Biodiversidade – ICMBio (ICMBio, Brazilian Environmental Agency, license #15068–6). All protocols involving the handling of animals were previously approved by the Ethics Committee for Animal Experimentation/Federal University of Amazonas (CEUA/UFAM, protocol nº 007/2019) in accordance with the guidelines of the Brazilian Committee for the Control of Animal Experimentation. Acknowledgments We are grateful to Eudis Soares and Adimo Carneiro for their efforts in collecting stingray specimens, as well as to Gabriel Verçosa and Danilo Castanho for their assistance during the fieldwork. We acknowledge Dr. Jansen Zuanon for providing insightful comments and contributions that enhanced the methodology of this manuscript. Funding AS received fellowships from Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (PCI-INPA #301778/2024-8), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES (PDPG-FAP #88887.702973/2022-00) and Fundação de Amparo à Pesquisa do Estado do Amazonas-FAPEAM (FIXAM #062.01520/2018), and a grant from FAPEAM-PAMEQ (#062.01108/2019). WPD received grants from FAPEAM(PPP #209/2012; UNIVERSAL #389/2012) and CNPq (UNIVERSAL #484374/2011-7). References Bleckmann H, Hofmann MH (1999) Special senses. 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Front Physiol 10:1e14. https://doi.org/10.3389/fphys.2019.00245 Ferrando S, Amaroli A, Gallus L, Aicardi S, Di Blasi D, Vacchi M, Ghigliotti L (2019b) The olfactory organ of Torpedo marmorata (Risso, 1810): morphology, histology, and nos-like immunoreactivity. Bull Environ Life Sci 1:9-16. https://doi.org/10.15167/2612-2960/BELS2019.1.1.1064 Fontenelle JP, Lovejoy NR, Kolmann MA, Marques FP (2021) Molecular phylogeny for the Neotropical freshwater stingrays (Myliobatiformes: Potamotrygoninae) reveals limitations of traditional taxonomy. Biol J Linn Soc 134:381-401 https://doi.org/10.1093/biolinnean/blab090 Garrone-Neto D, Sazima, I (2009) Stirring, charging, and picking: hunting tactics of potamotrygonid rays in the upper Paraná River. Neotrop Ichthyol 7: 113-116. https://doi.org/10.1590/S1679-62252009000100015 Goulding M, Ferreira EJG (1984) Shrimp-eating fishes and a case of prey-switching in Amazon rivers. Rev. Bras. Zool 2:85-97 https://doi.org/10.1590/S0101-81751983000300001 Harris LJ, Bedore CN, Kajiura SM (2015) Electroreception in the obligate freshwater stingray, Potamotrygon motoro . Mar Freshw Res 66:1027-1036 https://doi.org/10.1071/MF14354 Jacobsen IP, Bennett MB (2012) Feeding ecology and dietary comparisons among three sympatric Neotrygon (Myliobatoidei: Dasyatidae) species. J Fish Biol 80:1580-1594. https://doi.org/10.1111/j.1095-8649.2011.03169.x Küchler IL, Miekeley N, Forsberg B (2000) A contribution to the chemical characterization of rivers in the Rio Negro Basin, Brazil. J Braz Chem Soc 11:286-292. https://doi.org/10.1590/S0103-50532000000300015 Kyne PM, Bennett MB (2002) Diet of the eastern shovelnose ray, Aptychotrema rostrata (Shaw & Nodder, 1794), from Moreton Bay, Queensland, Australia. Mar Freshw Res 53:679-686. https://doi.org/10.1071/MF01040 Loboda TS, Carvalho MR (2013) Systematic revision of the Potamotrygon motoro (Müller & Henle, 1841) species complex in the Paraná-Paraguay basin, with description of two new ocellated species (Chondricthyes: Myliobatiformes: Potamotrygonidae). Neotrop Ichthyol 11:693-737. https://doi.org/10.1590/S1679-62252013000400001 Loboda TS, Lasso CA, Rosa RS, Carvalho MR (2021) Two new species of freshwater stingrays of the genus Paratrygon (Chondrichthyes: Potamotrygonidae) from the Orinoco basin, with comments on the taxonomy of Paratrygon aiereba . Neotrop Ichthyol 19:e200083. https://doi.org/10.1590/1982-0224-2020-0083 Silva JPCB, Carvalho MR (2015b) Systematics and morphology of Potamotrygon orbignyi (Castelnau, 1855) and allied forms (Chondrichthyes: Myliobatiformes: Potamotrygonidae). Zootaxa 3982:1–82. https://doi.org/10.11646/zootaxa.3982.1.1 McComb M, Kajiura SM (2008) Visual fields of four batoid fishes: a comparative study. J Exp Biol 211: 482-490. https://doi.org/10.1242/jeb.014506 Nessimian JL, Dorvillé LFM, Sanseverino AM, Baptista DF (1998) Relation between flood pulse and functional composition of the macroinvertebrate benthic fauna in the lower Rio Negro, Amazonas, Brazil. Amazoniana 15:35-50 Sánchez-Duarte P et al (2013) Paratrygon aiereba Cuenca del Amazonas. In: Lasso CA, Rosa RS, Sánchez-Duarte P, Morales-Betancourt MA, Agudelo-Córdoba E (ed) IX Rayas de água dulce de Suramérica, Parte I, Colombia, Venezuela, Ecuador, Perú, Brasil, Guyana, Surinam y Guayana Francesa: diversidad, bioecología, uso y conservación. Serie Editorial Recursos Hidrobiológicos y Pesqueros Continentales de Colombia, Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, Bogotá. pp 151-156 Schluessel V, Bennett MB, Bleckmann H (2008) Morphometric and Ultrastructural Comparison of the Olfactory System in Elasmobranchs: The Significance of Structure–Function Relationships Based on Phylogeny and Ecology. J Morphol 269:1365-1386. https://doi.org/10.1002/jmor.10661 Schluessel V, Bennett MB, Collin SP (2010) Diet and reproduction in the white-spotted eagle ray Aetobatus narinari from Queensland, Australia and the Penghu Islands, Taiwan. Mar Freshw Res 2010 61:1278–1289. https://doi.org/10.1071/MF09261 Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9:671–675. https://doi.org/10.1038/nmeth.2089 Shibuya A (2022) A review of the ecological role of the Neotropical freshwater stingrays (Chondrichthyes: Potamotrygoninae). Food Webs 32:e00244 https://doi.org/10.1016/j.fooweb.2022.e00244 Shibuya A, Araújo MLG, Zuanon JAS (2009) Analysis of stomach contents of freshwater stingrays (Elasmobranchii, Potamotrygonidae) from the middle Negro River, Amazonas, Brazil. Pan-Am J Aqua Sci 4:466–475 Shibuya A, Zuanon J, Araujo MLG, Tanaka S (2010) Morphology of lateral line canals in Neotropical freshwater stingrays (Chondrichthyes: Potamotrygonidae) from Negro River, Brazilian Amazon. Neotrop Ichthyol 8:867-76. https://doi.org/10.1590/S1679-62252010000400017 Shibuya A, Zuanon J, Carvalho MR (2016) Alimentação e comportamento predatório em raias Potamotrygonidae. In: Lasso CA, Rosa R, Morales-Betancourt MA, Garrone-Neto D, Carvalho MR (Ed) Rayas de agua dulce (Potamotrygonidae) de suramérica. Parte II. Colombia, Brasil, Perú, Bolivia, Paraguay, Uruguay y Argentina, Instituto Humboldt, Bogotá. pp 66-81 Shibuya A, Zuanon J, Carvalho MR (2020) Neuromast distribution and its relevance to feeding in Neotropical freshwater stingrays (Elasmobranchii: Potamotrygonidae). Zoomorphology. 139:61-69. https://doi.org/10.1007/s00435-019-00472-2 Shibuya A, Zuanon J, Tanaka S (2012) Feeding behavior of the Neotropical freshwater stingray Potamotrygon motoro (Elasmobranchii: Potamotrygonidae). Neotrop Ichthyol 10:189–196. https://doi.org/10.1590/S1679- 62252012000100018 Szabo T, Kalmijn AJ, Enger PS, Bullock TH (1972) Microampullary organs and a submandibular sense organ in the freshwater ray, Potamotrygon . J Comp Physiol 79:15–27 Szamier RB, Bennett MVL (1980) Ampullary electroreceptors in the freshwater ray, Potamotrygon . J Comp Physiol 138A:225–230 Takami S, Luer CA, Graziadei PPC (1994) Microscopic structure of the olfactory organ of the clearnose skate, Raja eglanteria . Anat Embryol 190, 211-230 Theisen B, Zeiske E, Breucker H (1986) Functional morphology of the olfactory organs in the spiny dogfish ( Squalus acanthias L.) and the small-spotted catshark ( Scyliorhinus canicula L.). Acta Zool Stockh 67:73–86 Yopak KE, Lisney TJ, Collin SP (2015) Not all sharks are “swimming noses”: variation in olfactory bulb size in cartilaginous fishes. Brain Struct Funct 220:1127-1143. https://doi.org/10.1007/s00429-014-0705-0 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 10 Sep, 2024 Read the published version in Zoomorphology → Version 1 posted Editorial decision: Revision requested 20 Jul, 2024 Reviews received at journal 23 Jun, 2024 Reviewers agreed at journal 03 Jun, 2024 Reviewers invited by journal 02 Jun, 2024 Editor assigned by journal 02 Jun, 2024 Submission checks completed at journal 02 Jun, 2024 First submitted to journal 31 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4509528","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":310123777,"identity":"39b289d0-7665-49e8-ba5d-eda8cfbca038","order_by":0,"name":"Akemi Shibuya","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYFACHobDYJqZ+QCQlJAhQQs7WwJICw9RWpjBND+PAYRLCPD3nz14uLDtsDwDM8/nVzdqLHgY2A8f3YBPi8SNvITDM9sOGzYw826zzjkGdBhPWtoNvNbc4DE4zNt2mBGkxTiHDahFgscMrxb582fAWuwbmHmeGef8I0KLwYEcsJZEoBbmx7ltRGgxvAHUMuNcenIbM5sZc26fBA8bIb/InT9j/LmgzNq2n//w48853+rk+NkPH8PvfQhoZmBjYGCTADHZiFAOAnUggvkDkapHwSgYBaNghAEA2TtET9chgjYAAAAASUVORK5CYII=","orcid":"","institution":"National Institute of Amazonian Research","correspondingAuthor":true,"prefix":"","firstName":"Akemi","middleName":"","lastName":"Shibuya","suffix":""},{"id":310123778,"identity":"90548467-653e-4277-a11d-ad3eb6b03037","order_by":1,"name":"Rubia Machado","email":"","orcid":"","institution":"Federal University of Amazonas","correspondingAuthor":false,"prefix":"","firstName":"Rubia","middleName":"","lastName":"Machado","suffix":""},{"id":310123779,"identity":"4e09054d-791c-4b4f-b14f-da1db38e138c","order_by":2,"name":"Wallice Duncan","email":"","orcid":"","institution":"Federal University of Amazonas","correspondingAuthor":false,"prefix":"","firstName":"Wallice","middleName":"","lastName":"Duncan","suffix":""}],"badges":[],"createdAt":"2024-05-31 14:11:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4509528/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4509528/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00435-024-00682-3","type":"published","date":"2024-09-10T15:57:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58347926,"identity":"b3769d89-6cf5-407b-8728-a2c17ef25b76","added_by":"auto","created_at":"2024-06-14 08:17:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":98473,"visible":true,"origin":"","legend":"\u003cp\u003eDiagram illustrating the measurements taken from the olfactory organ in Potamotrygoninae species. (a) The olfactory system consists of paired organs located on the ventral surface, positioned anteriorly to the mouth. (b) Width and length of each rosette were measured within the capsule before dissection to prevent disruption of the original shape. The olfactory rosette is composed of a set of primary lamellae, divided into anterior (AP) and posterior (PP) portions by a central raphe (detail of a rosette of the left side). (c) Width of each portion (W\u003csub\u003eAP\u003c/sub\u003e and W\u003csub\u003ePP\u003c/sub\u003e) for each primary lamella was measured. The secondary folds (in gray) were counted and lengths were taken from external (1), medial (2) and internal (3) folds. Surface areas of W\u003csub\u003eAP\u003c/sub\u003e and W\u003csub\u003ePP\u003c/sub\u003e were measured, considering only the region containing secondary folds (delimited by dashed lines). (d) To estimate the total sensory area, the perimeter of the silhouette of secondary folds (P\u003csub\u003eSIL\u003c/sub\u003e) was measured from histological slides in 1.0 mm\u003csup\u003e2\u003c/sup\u003e and extrapolated to the total area of primary lamellae. Scale bar: 0.5 cm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4509528/v1/2e7fd0a5d306ab285df4fa9b.png"},{"id":58347615,"identity":"99eab7b0-7a45-405e-bc04-91ff014dd0be","added_by":"auto","created_at":"2024-06-14 08:09:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":101885,"visible":true,"origin":"","legend":"\u003cp\u003eWater flow in the nostrils of potamotrygonin species. (a) the entrance occurs laterally to the nasal flaps (red dashed arrows), when the water current under the disc is directed posteriorly (black arrows). The water flows into the rosettes with the aid of the inner folded edge (in asterisk) (b) The water runs through the olfactory rosettes and drain off under the posterior region of nasal flap (red dashed arrows). Sagittal view of theprimary lamella of \u003cem\u003eParatrygon\u003c/em\u003e sp. (c) shows a roundish shape, while \u003cem\u003ePotamotrygon\u003c/em\u003e species (d) have similar shape of primary lamella. The silhouette of the secondary folds is slightly waved in \u003cem\u003eParatrygon\u003c/em\u003e sp. (e). Despite the similar shape of primary lamellae, the silhouettes of secondary folds are visibly distinct among \u003cem\u003ePotamotrygon motoro\u003c/em\u003e (f), \u003cem\u003eP. orbignyi\u003c/em\u003e (g) and \u003cem\u003eP. wallacei\u003c/em\u003e (h). Silhouettes of secondary folds on the primary lamella to each species are based on the histological slices in longitudinal position of the olfactory rosettes. AP, anterior lamellar portion; Lp, lamellar process; PP, posterior lamellar portion; R, raphe. Scales: a and b (1.0 cm); c and d (2.0 mm); e-h (100 μm).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4509528/v1/97401dc71ee9038fb7555443.png"},{"id":58347613,"identity":"6c40ab5d-6ce1-4267-bfa1-384b4c1d263e","added_by":"auto","created_at":"2024-06-14 08:09:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":645627,"visible":true,"origin":"","legend":"\u003cp\u003eHistological structure of the olfactory lamellae of potamotrygonin species. Transversal sections through one primary lamellae show the morphology of secondary folds. (a) The slightly wavy secondary fold of \u003cem\u003eParatrygon\u003c/em\u003e sp. presents the apical zone with PAS-positive goblet cells (in b). The triangular secondary folds of \u003cem\u003ePotamotrygon motoro\u003c/em\u003e(c) have AB- positive goblet cells (in d). (e) \u003cem\u003ePotamotrygon orbignyi\u003c/em\u003e has a dense short ciliary sensory region. (f) Different size and shape of secondary folds were found in \u003cem\u003eP. wallacei\u003c/em\u003e and goblet cells are AB and PAS positives (g and h, respectively). Note the presence of central smooth muscles in primary lamellae of \u003cem\u003eP. motoro\u003c/em\u003e and \u003cem\u003eP. wallacei\u003c/em\u003e. a, c, e and f are stained with Toluidine blue; b, in PAS; d and g, in Alcian blue counter staining with Safranin; h, in Periodic Acid Schiff counter staining with Hematoxilin. CT, connective tissue; SM, smooth muscles; GC, goblet cells; MG, mucus granules; SR, sensory region; NS, non-sensory region. Scale bars: 100 μm (A-F) and 50 μm (G and H).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4509528/v1/516f3d06f0e26d1d71e64c4e.png"},{"id":64619119,"identity":"99c8d423-ad56-4924-b334-254b724c0dd9","added_by":"auto","created_at":"2024-09-16 16:11:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1333074,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4509528/v1/e1505b1f-118f-4789-a659-22c2c4dd6467.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sniffing at the river bottom: Influence of olfactory organ morphology on the life habits of freshwater stingrays (Potamotrygoninae)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eElasmobranchs use a complex arrangement of sensory systems (gustation, electro and mechanosensorial organs, vision and olfaction) which act jointly to locate prey, potential predators and the presence of conspecifics (McComb and Kajiura, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Studies focusing on olfactory organs in elasmobranchs have seen a notable increase on the recent years (e.g. Cox \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ferrando et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Dymek et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), driven by the accuracy chemical detection abilities exhibited by apex predator species (Yopak et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Elasmobranchs have two different arrangement of multilamellar olfactory structures, oval or elongated shapes (e.g. Theisen et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Takami et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Ferrando et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e); however, there are a diversity related to the relative size, number and shape of primary lamellae and secondary folds. Independently of the morphological distinctions, the olfactory organs are mainly used to detect chemical stimulus in the water (Bleckmann and Hoffman, 1999).\u003c/p\u003e \u003cp\u003eAs most batoid species, potamotrygonin stingrays depend specially on the ventral sense systems to locate and capture the prey. Evidently, the prominent eyes in most \u003cem\u003ePotamotrygon\u003c/em\u003e species provide a wide vision of the benthic environment; however, the accuracy of localization of prey is managed by the other sensory organs responsible to detect the prey. Shibuya et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) stand that the distribution of lateral line canals and the high density of neuromasts around the mouth in freshwater stingrays are related to the detect the prey, which most of them are formed by benthic species or commonly live buried in the substrate (e.g. Duncan et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shibuya \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Even the reduced electrosensory structure in comparison to marine species, the Lorenzini ampullae in short canals concentrated in the dermis near the mouth, the electroreception is likely employed on the final stage of prey strike (Szabo et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1972\u003c/span\u003e; Szamier and Bennett \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Harris et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs the entire sensory systems have integrative actions, the olfaction in may play the role of the initial detection of the chemical stimulus just prior the capture of prey on the foraging activities on the bottom for benthic stingrays. Dymek et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) analyzed the olfactory organs in five elasmobranch species, including two potamotrygonin stingrays, and showed detailed morphology of the olfactory epithelium. However, the morphological data on the olfactory organs in freshwater stingrays is still scarce, in face of 38 valid species (Fontenelle et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). An important fact is that the generalization of the morphological characteristics of olfactory organ in a potamotrygonin species requires caution, since environmental factors can vary significantly depending on the river in which they occur. Thus, the aim of the present study is describe the morphological characteristics of lamellar surface of the olfactory organs of freshwater stingrays from Rio Negro basin. The blackwater of Rio Negro presents high acidity (pH: 3.89\u0026ndash;6.07), low conductivity (8.8 to 28.6 \u0026micro;S cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and lack of suspended sediments (K\u0026uuml;chler et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Potamotrygonin stingrays usually are found associated with the substrate, which is covered by of sand and the accumulation leaf litter from the flooded forest (Shibuya \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In order to compare the area of lamellae surface, the sensory area and densities of secondary folds were estimated. Histological comparisons can provide new insights related to their foraging and feeding habits. These morphological parameters were related to the habitat use and feeding habits previously investigated (Shibuya et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Duncan et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shibuya \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003eSamples collections were carried out during the low (dry season) period of hydrologic cycle in 2019\u0026ndash;2020 in the middle Rio Negro (Barcelos Municipality, Amazonas State, Brazil). Individuals were collected using a dip net by local fishermen, comprising four species: juveniles of \u003cem\u003eParatrygon\u003c/em\u003e sp. (n\u0026thinsp;=\u0026thinsp;4), \u003cem\u003ePotamotrygon motoro\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1) and \u003cem\u003eP. orbignyi\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2) and adults of \u003cem\u003eP. wallacei\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;5). Disc width (DW, mm) of the specimens, as well as length and width of the olfactory rosettes into capsules (mm) were taken prior to dissection (in order to preserve the original shape) to calculate the relative size for each analyzed species. Olfactory capsules were removed, fixed in cold and buffered glutaraldehyde 2.5% solution (pH 7.4) for 24 hours and then preserved in 70% ethanol solution. Specimens were preserved in 10% buffered-formalin solution and stored in 75% ethanol solution. Schematic diagrams of the morphology of primary lamella structures were drawn from dissected specimens with the aid of a stereoscopic microscope (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe olfactory rosettes were removed from the right side (assuming the bilateral symmetry). The primary lamellae were counted and each of them was dissected, separated, labeled and stored into 0.5 ml microcentrifuge tubes. The secondary folds were counted of one side of contact surface in both anterior and posterior portion of each primary lamella and photographed with a millimetric scale. Width (W\u003csub\u003ePL\u003c/sub\u003e) and total area (A\u003csub\u003ePL\u003c/sub\u003e) of primary lamella and morphological measures of the anterior (AP) and posterior (PP) lamellar portions were obtained using ImageJ software (Schneider et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The lamellar area was calculated only for the region comprising secondary folds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) and the W\u003csub\u003ePL\u003c/sub\u003e and A\u003csub\u003ePL\u003c/sub\u003e are the sum of measurements of the anterior and posterior portions of the primary lamella. The terminology of olfactory structures followed Ferrando et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). Calculation of the total lamellar area was based on Ferrando et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e), with few modifications in order to have an accuracy of the total area. The gross surface area (GSA) was calculated as the sum of all total area of each primary lamella as following: GSA\u003csub\u003etotal\u003c/sub\u003e = A\u003csub\u003ePL\u003c/sub\u003e1 + A\u003csub\u003ePL\u003c/sub\u003e2 + A\u003csub\u003ePL\u003c/sub\u003e3 +\u0026hellip;+ A\u003csub\u003ePL\u003c/sub\u003e\u003cem\u003en\u003c/em\u003e. where: A\u003csub\u003ePL\u003c/sub\u003e is the total area of each primary lamella. The total area was calculated by the sum of the AP and PP areas, previously taken using ImageJ; \u003cem\u003en\u003c/em\u003e is the number of primary lamellae of the olfactory organ.\u003c/p\u003e \u003cp\u003eTo characterize the microscopical morphology, the olfactory rosette from the left side of each specimen was initially decalcified in 5% formic acid for 24 h, dehydrated in a graded ethanol solution series (96\u0026ndash;100%) and infused in ethanol\u0026thinsp;+\u0026thinsp;resin solution for a minimum of 24 hours. Pieces of the organ were embedded in methacrylate resin (Historesin, Leica) in sagittal and transversal positions and sectioned into 3\u0026ndash;5 \u0026micro;m-thickness slices. The slides were stained with Toluidine blue for the delineation of the shape of secondary folds. Additionally, Alcian Blue (AB) counterstained with safranin and Periodic Acid Schiff (PAS) counterstained with Hemotoxilin were applied separately, thus facilitating the discernment of distinct positive reaction of each dye. Since potamotrygonin stingrays have simple secondary folds (compared to most elasmobranchs examined by Ferrando et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e)), the total surface was not estimated according to the equation recommended by Ferrando et al.\u0026rsquo;s methodology. Thus, calculation of the total contact area of primary lamellae (A\u003csub\u003eTC\u003c/sub\u003e) was taken by estimating the area of a 1 x 1 mm square, as follow: A\u003csub\u003eTC\u003c/sub\u003e = A\u003csub\u003ePL\u003c/sub\u003e x A\u003csub\u003eSquare\u003c/sub\u003e, where A\u003csub\u003ePL\u003c/sub\u003e is the total area of primary lamella and A\u003csub\u003eSquare\u003c/sub\u003e is the relative area of 2D perimeter of the silhouette (P\u003csub\u003eSIL\u003c/sub\u003e) of sagittal slices (in mm\u003csup\u003e2\u003c/sup\u003e). The perimeter of the silhouette was taken using subsampled areas of the histological slides (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Measurements of slices were taken with a histological slide with micrometric scale. The increase rate of total surface of the set of secondary folds is presented in relative percentage (%) of the total linear area of primary lamellae.\u003c/p\u003e \u003cp\u003eLamellae of the central region of the capsule were used to calculate the depth range at which water can reach the olfactory organ. For this purpose, the mean length of secondary folds was measured of both anterior and posterior lamellar portions of the larger primary lamella: (1) external, that comprise the first small fold; (2) median, which is located in the middle of the lamellae and (3) internal, that is the last fold near the raphe (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The central region of rosette was considered as the middle one third of the total number of primary lamellae (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Mean, standard deviation, minimum and maximum of measurements of primary lamellae and secondary folds were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and min-max. Voucher specimens were catalogued at Fish Collection of the Instituto Nacional de Pesquisas da Amaz\u0026ocirc;nia (INPA), Manaus. Brazil: \u003cem\u003eParatrygon\u003c/em\u003e sp., #34959; \u003cem\u003ePotamotrygon motoro\u003c/em\u003e, # 27091; \u003cem\u003eP. orbignyi\u003c/em\u003e, # 27088 and \u003cem\u003eP. wallacei\u003c/em\u003e, # 34960.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eGross Morphology of olfactory rosettes\u003c/p\u003e \u003cp\u003eThe olfactory organs of potamotrygonin species comprise a pair of encapsuled rosettes positioned anteriorly to the mouth. The olfactory rosettes are categorized by an elongated shape which have the external edge thinner to the midline and internal edge of the raphe (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The water current goes sinuously into the nostrils, flows through the olfactory rosettes (through both anterior and posterior lamellar portions) and drains off posteriorly to the nasal flap. The olfactory organs are covered by a pair of inner folded edge positioned just under the nasal flap, possibly assisting the water flow into the nostrils (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and b). Each olfactory rosette is formed by a central raphe that separated in anterior and posterior lamellar portions. Overall, the anterior portion was larger lamellar side. \u003cem\u003eParatrygon\u003c/em\u003e sp. presents roundish lamellae and more concave shape, with the posterior portion slightly short (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), while \u003cem\u003ePotamotrygon\u003c/em\u003e species have an elongated and curved lamellae (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), with a visibly shorter posterior portion. All species had a pair of process in both anterior and posterior lamellar portions, with most prominent and pointed shape to \u003cem\u003ePotamotrygon\u003c/em\u003e species (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and d). Morphological measurements of the olfactory structures are presented in the Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. High number of primary lamellae was observed to \u003cem\u003ePotamotrygon motoro\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;54), while \u003cem\u003eParatrygon\u003c/em\u003e sp. presented the lowest number (min-max: 27\u0026ndash;29) among analyzed species. Each primary lamella presents secondary folds positioned parallel to each other in anterior and posterior portions on both sides (see Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and d). The length and number of secondary folds varies according to the size of primary lamellae. Higher numbers of secondary folds were found to \u003cem\u003eP. motoro\u003c/em\u003e and \u003cem\u003eP. orbignyi\u003c/em\u003e comparing the larger primary lamellae, while the length of the secondary folds of the largest primary lamella was greater for \u003cem\u003eP. wallacei\u003c/em\u003e (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMeasures of olfactory organs of potamotrygonin species. The relative size of olfactory organs was calculated using the disc width and rosette width. The total area, number, and length of secondary folds were determined for the largest primary lamella of each examined specimen. AP and PP represent the anterior and posterior portions of primary lamella (PL), respectively. Length-1, Length-2 and Length-3 correspond to specific segments of PL, measured according to the methodology outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"15\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDisc width\u003c/p\u003e \u003cp\u003e(mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOlf. rosette\u003c/p\u003e \u003cp\u003ewidth (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMean and Relative size (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOlf. rosette length (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNo. PL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTotal area of the\u003c/p\u003e \u003cp\u003elargest lamella\u003c/p\u003e \u003cp\u003e(mm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c15\" namest=\"c8\"\u003e \u003cp\u003eSecondary folds (largest lamella)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNo. AP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLength-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eLength-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eLength-3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eNo. PP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eLength-3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003eLength-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003eLength-1\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParatrygon\u003c/em\u003e sp.\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e274\u0026ndash;302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.9\u0026ndash;10.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.1 (3.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.0-3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27\u0026ndash;29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e24.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePotamotrygon\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003emotoro\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e36.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e28.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e1.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. orbignyi\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e152\u0026ndash;241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.6\u0026ndash;13.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.2\u0026ndash;6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45\u0026ndash;48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20.30**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e40.0**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.63**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.69**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.42**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e26.0**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e1.15**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e1.58**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. wallacei\u003c/em\u003e\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e160\u0026ndash;240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.0-14.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.9 (6.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.6\u0026ndash;7.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32\u0026ndash;39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24.69\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e28.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e20.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"15\"\u003e* result of three specimens\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"15\"\u003e** result of one specimen\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTotal lamellar area\u003c/p\u003e \u003cp\u003eThe gross surface area was considered as the sum of the area of anterior and posterior portions of both side of primary lamellae. \u003cem\u003eParatrygon\u003c/em\u003e sp. presents the smallest total area (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Despite the very few differences of total area among \u003cem\u003ePotamotrygon\u003c/em\u003e species, the result of \u003cem\u003eP. orbignyi\u003c/em\u003e should be considered carefully, in order to the damage of primary lamellae located on the edges of olfactory rosettes, thus, the area might be underestimated. The largest lamellar surface was calculated to \u003cem\u003eP. wallacei\u003c/em\u003e, even the low number of primary lamellae compared to \u003cem\u003eP. motoro\u003c/em\u003e. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows differences between the gross lamellar area (linear area) and the contact surface area, considering the perimeter of secondary folds of each species (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-h). \u003cem\u003eParatrygon\u003c/em\u003e sp. presents slightly waved secondary folds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), with an increase of 9.0% of total area of primary lamellae. Despite \u003cem\u003ePotamotrygon\u003c/em\u003e species have similar morphology of the primary lamellae, their secondary folds have distinct forms. \u003cem\u003ePotamotrygon motoro\u003c/em\u003e presents a triangular shape of secondary folds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), which increases 42.5% of total surface area of olfactory lamellae. Likewise \u003cem\u003eParatrygon\u003c/em\u003e sp., \u003cem\u003eP. orbignyi\u003c/em\u003e has wave-shaped secondary folds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg) that provide a smaller increase of lamellar contact surface (5.7%). Conversely, the wide and flattened surface of secondary folds of \u003cem\u003eP. wallacei\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh) has a largest expansion of lamellar contact comparing to the other examined species (115.4%).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMeasures of the gross surface area of primary lamellae, defined as the sum of areas of all lamellae within one rosette, considering a 1 x 1 mm square linear area. Ratio values represent the increase in surface area using the perimeter of the silhouette of the secondary folds. The estimated area and its percentage were calculated based on the total area of all primary lamellae within one olfactory rosette.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGross surface area\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRatio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEstimated total area\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParatrygon\u003c/em\u003e sp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e163.10\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e177.78 (9.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePotamotrygon motoro\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e244.23**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e348.03 (42.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. orbignyi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e525.51***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e555.46 (5.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. wallacei\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e548.94\u0026thinsp;\u0026plusmn;\u0026thinsp;5.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1,182.42 (115.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e* result of three specimens\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e** result of one specimen\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e*** sum of surface area of 34 lamellae of one specimen.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003ea\u003c/sup\u003e based on one specimen\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003eb\u003c/sup\u003e based on three specimens\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003ec\u003c/sup\u003e based on four specimens\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eHistological analyses of secondary folds\u003c/p\u003e \u003cp\u003eThe primary lamellae are characterized by having secondary folds on both sides. These secondary folds exhibit morphological differences among the examined species, both in terms of shape and composition of epithelial cells. The epithelium is a sensory region covered by ciliary supporting cells placed on the sensory receptor cells. The apical zone of secondary folds is a non-sensory region presenting mucous cells and non-ciliary cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-h). The wavy-shaped secondary folds of \u003cem\u003eParatrygon\u003c/em\u003e sp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) has a group of PAS-positive mucus cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), while \u003cem\u003ePotamotrygon\u003c/em\u003e species have goblet cells secreting AB-positive and PAS-positive substances. Goblet cells are distributed among non-ciliary supporting cells which is covered by a mucus granules, except to \u003cem\u003eP. motoro\u003c/em\u003e that has goblet cells in the sensory region (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In all examined species, the middle zone of secondary folds is filling by connective tissue that originated from the center of primary lamella. Secondary folds in \u003cem\u003eParatrygon\u003c/em\u003e sp., \u003cem\u003ePotamotrygon motoro\u003c/em\u003e and \u003cem\u003eP. orbignyi\u003c/em\u003e have similar size along the primary lamellae. In contrast, distinct width and size of secondary folds are found on the primary lamellae of \u003cem\u003eP. wallacei\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). The connective tissue in the middle of larger secondary folds of \u003cem\u003eP. wallacei\u003c/em\u003e expands under the support and basal cells of epithelium in both sensory and non-sensory regions. The wall of basal portion among primary lamellae presents a PAS-positive mucus cells apically positioned on the epithelium and extending to near the secondary folds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). The primary lamellae of \u003cem\u003eParatrygon\u003c/em\u003e sp. and \u003cem\u003ePotamotrygon orbignyi\u003c/em\u003e have a thick connective tissue that support secondary folds on both sides. \u003cem\u003ePotamotrygon motoro\u003c/em\u003e and \u003cem\u003eP. wallacei\u003c/em\u003e present thin layer of connective tissue in both sides of primary lamellae. In these species, the central portion of primary lamella is filled by smooth muscle longitudinally arranged, which is thicker in \u003cem\u003eP. wallacei\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe investigation of olfactory organ revealed a morphological diversity in potamotrygonin stingrays. However, the delicate structure of the olfactory epithelium and the small size of the organs turned a challenge in maintaining adequate fixation to prevent degradation of lamellar structures. Additionally, the difficult handling of olfactory rosettes during the dissection of primary lamellae (often damaged during the individualization process) impeded a complete examination and the quantification of secondary lamellae. Nonetheless, the current results provide relevant information that, combined with knowledge of their life habits (Shibuya et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Duncan et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shibuya et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shibuya \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), can elucidate the functional role of the olfactory system. The bottom of the river may present different types of obstacles such as litter and tree trunks alongside sandy substrate, making the benthic zone an excellent refugee for insects larvae and crustaceans (e.g. Goulding and Ferreira 1983; Nessimian et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Therefore, the behavior of stirring up the substrate becomes essential for stingrays to uncover the hidden prey (Garrone-Neto and Sazima \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Shibuya et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOnly young individuals of \u003cem\u003eParatrygon\u003c/em\u003e sp. were examined, and during this life stage, this species is associated with sandy beaches, feeding on small fish (Shibuya et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Comparing to the \u003cem\u003ePotamotrygon\u003c/em\u003e species, \u003cem\u003eParatrygon\u003c/em\u003e sp. exhibited smaller nostril size, lower number of total lamellae, and reduced lamellar area, even considering the silhouette of secondary folds. \u003cem\u003eParatrygon\u003c/em\u003e sp. is a large-sized species, reaching up to 93.0 cm DW in the Rio Negro (S\u0026aacute;nchez-Duarte et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and the morphological simplicity of its olfactory organs, compared to \u003cem\u003ePotamotrygon\u003c/em\u003e species, may indicate lower olfactory efficiency. However, it is necessary to consider two important factors. Firstly, \u003cem\u003eParatrygon\u003c/em\u003e sp. inhabit river channels as adults (e.g. Shibuya \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The high water flow in such environments may hinder the detection of chemical stimuli released by their prey. Furthermore, fish living in river channel are not small in size (compared to those living on the beaches) (Shibuya et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and live on the substrate, indicating that \u003cem\u003eParatrygon\u003c/em\u003e sp. does not primarily rely on chemical cues for foraging. Additionally, this species exhibits a widespread distribution of lateral line canals and a high number of pores on the dorsal surface that may play a primary role in locating mechanical stimuli generated by prey near ray\u0026rsquo;s body (Shibuya et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Thus, it becomes necessary to take into account the contribution of all sensory systems to understand how this species obtains its food.\u003c/p\u003e \u003cp\u003eDespite examining few specimens, \u003cem\u003eP. motoro\u003c/em\u003e exhibited a larger lamellar area than \u003cem\u003eP. orbignyi\u003c/em\u003e; however, the opposite was observed when considering the silhouette of the secondary folds. Although both species have high numbers of primary lamellae and secondary folds, the difference in the increment of lamellar surface area may be related to their respective life habits. \u003cem\u003ePotamotrygon motoro\u003c/em\u003e is an active species that constantly explores different types of habitats, often found associated with various types of substrates in search of prey (Shibuya \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This species primarily consumes crabs and small fish (Shibuya et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In contrast, \u003cem\u003eP. orbignyi\u003c/em\u003e has a specialist feeding habit, consuming aquatic insects in sandy beaches of all studied populations. Considering the need to explore a broader range of habitats and consuming a diverse types of prey, \u003cem\u003eP. motoro\u003c/em\u003e requires an olfactory system capable of detecting a variety of chemical stimuli released by its prey. These ecological characteristics can explain the increase of lamellar surface given by the secondary folds\u0026rsquo; silhouette. \u003cem\u003ePotamotrygon wallacei\u003c/em\u003e stands out having high values of total surface area, especially considering the silhouette of secondary folds. The estimate of the total contact area exceeded 100% for \u003cem\u003eP. wallacei\u003c/em\u003e, highlighting the morphological importance of secondary folds for enhancing the sensitivity of olfactory organs. The generalist feeding habits of this species (see Shibuya et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), along with its ability to explore complex substrate of flooded forest (Duncan et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) for locating prey, underscore the necessity of \u003cem\u003eP. wallacei\u003c/em\u003e to identify chemical stimuli, including those released by smaller prey hidden in leaf litter, such as small shrimps and insects larvae.\u003c/p\u003e \u003cp\u003eThe presence of a pair of lamellar processes on primary lamellae may serve an important function in guiding water flow along the olfactory organ, due to their prominent and curved shape. In addition to freshwater stingrays, the lamellar process has also been observed in marine batoid species such as \u003cem\u003eAetobatus narinari\u003c/em\u003e, \u003cem\u003eAptychotrema rostrata\u003c/em\u003e, and \u003cem\u003eNeotrygon kuhlii\u003c/em\u003e (Schluessel et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and may indicate its role in maintaining water flow over the primary lamella to identify chemical cues from prey buried in the bottom (Kyne and Bennett \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Schluessel et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Jacobsen and Bennett 2011). The increase in surface area provided by the secondary folds is notable for enhancing olfactory sensitivity. Although potamotrygonin stingrays do not exhibit the complex branching of secondary folds seen in marine elasmobranchs (Ferrando et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Dymek et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), morphological diversity may indicates an increase of sensitivity surface area. Dymek et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) previously described the morphology of olfactory organs in two \u003cem\u003ePotamotrogon\u003c/em\u003e species, showing a different morphology to \u003cem\u003eP. motoro\u003c/em\u003e when compared to that observed in the present study. Despite Dymek et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) having used specimens from the ornamental market, these morphological differences corroborate the importance of considering the populations being examined. Widely distributed species such as \u003cem\u003eP. motoro\u003c/em\u003e may exhibit ecological and morphological distinctions, so information regarding the origin of the specimens and the characteristics of the habitat where these stingrays used to live are essential for inferring the functional role of the lamellar structure and can indicate adaptations and pressures to their particular habitats (Schluessel et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Taxonomic revisions that have been carried out on widely distributed species of potamotrygonines (e.g. Loboda and Carvalho, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Silva and Carvalho, 2015; Loboda et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), emphasize the need to distinguish populations in ecological and functional morphology studies.\u003c/p\u003e \u003cp\u003eIn contrast to observations by Dymek et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), the goblet cells of \u003cem\u003eP. motoro\u003c/em\u003e and \u003cem\u003eP. wallacei\u003c/em\u003e were AB and PAS-positive, producing mucus in the non-sensory lamellar region. Therefore, mucus granules were also found on both sensory and non-sensory surface of \u003cem\u003eParatrygon\u003c/em\u003e sp., which were only PAS positive, indicating an assistance to cilia movement. However, the presence of goblet cells on the top of secondary folds can be considered to have a protective function. The benthic-associated life habits of freshwater stingrays mean that olfactory lamellae are in direct contact with river bottom elements, resulting in constant friction with sand and leaf-litter fragments. Thus, the presence of goblet cells and mucus granules on the lamellar surface may serve as important protection against potential friction damage and infection to the olfactory epithelium. The reduced or absence of goblet cells likely mean a lack of mucus-propel function, a characteristic observed in many elasmobranch species (Cox \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Instead, the cilia movement may be driven by water flow, playing a significant hydrodynamic role in guiding chemical stimuli to sensory cells. Nonetheless, a minimal contribution of ciliary propulsion may exist, especially in \u003cem\u003eP. motoro\u003c/em\u003e, which exhibits a few goblet cells in sensory region.\u003c/p\u003e \u003cp\u003eAccording to the classification of primary lamella morphology by Ferrando et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e), the secondary folds of all examined species was classified as non-branched and of short size. The presence of a central layer of smooth muscle into the primary lamellae was observed in both \u003cem\u003eP. motoro\u003c/em\u003e and \u003cem\u003eP. wallacei\u003c/em\u003e, but neither observed in \u003cem\u003eParatrygon\u003c/em\u003e sp. nor \u003cem\u003eP. orbignyi\u003c/em\u003e, nor described in previously studied freshwater stingrays (Dymek et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The longitudinal arrangement of smooth muscle may be related to the process of expanding the primary lamellae, optimizing the contact of the olfactory epithelium with the water current. This characteristic supports the relationship between increased surface area of the olfactory epithelium and the foraging capabilities of \u003cem\u003eP. motoro\u003c/em\u003e and \u003cem\u003eP. wallacei\u003c/em\u003e in various types of habitats (Duncan et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shibuya \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), possibly regulating the expansion of primary lamellae based on the need for chemosensitivity accuracy.\u003c/p\u003e \u003cp\u003eThe integration of all sensory systems in freshwater stingrays is indeed an important characteristic for the success of this group in a diversity of freshwater habitats, such as lakes, streams, shallow and deep waters such as beaches and river channels (Duncan et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Shibuya \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The synchronized performance of different sensory modalities plays an essential role in feeding behavior, with each sensory element having a primary function in different phases of foraging (search, location, and the phase prior to prey apprehension). Differences of the morphology of olfactory organs in potamotrygonin stingrays may determine the level of accuracy in prey detect as specialized adaptations corresponding to the life habits of each species. Further examination of other species may reveal whether morphological diversity of olfactory organs have phylogenetic inferences for the Potamotrygoninae subfamily.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors have no conflict of interest to declare.\u003c/p\u003e\n\u003cp\u003eEthics approval\u003c/p\u003e\n\u003cp\u003eThe specimens were collected with permission of Instituto Chico Mendes de Conservação da Biodiversidade – ICMBio (ICMBio, Brazilian Environmental Agency, license #15068–6). All protocols involving the handling of animals were previously approved by the Ethics Committee for Animal Experimentation/Federal University of Amazonas (CEUA/UFAM, protocol nº 007/2019) in accordance with the guidelines of the Brazilian Committee for the Control of Animal Experimentation.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe are grateful to Eudis Soares and Adimo Carneiro for their efforts in collecting stingray specimens, as well as to Gabriel Verçosa and Danilo Castanho for their assistance during the fieldwork. We acknowledge Dr. Jansen Zuanon for providing insightful comments and contributions that enhanced the methodology of this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eAS\u0026nbsp;received fellowships from\u0026nbsp;Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (PCI-INPA #301778/2024-8), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES (PDPG-FAP #88887.702973/2022-00)\u0026nbsp;and\u0026nbsp;Fundação de Amparo à Pesquisa do Estado do Amazonas-FAPEAM (FIXAM #062.01520/2018),\u0026nbsp;and a grant from\u0026nbsp;FAPEAM-PAMEQ (#062.01108/2019). WPD received grants from FAPEAM(PPP #209/2012; UNIVERSAL #389/2012) and CNPq (UNIVERSAL #484374/2011-7).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBleckmann H, Hofmann MH (1999) Special senses. 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J Braz Chem Soc 11:286-292.\u0026nbsp;https://doi.org/10.1590/S0103-50532000000300015\u003c/li\u003e\n \u003cli\u003eKyne PM, Bennett MB (2002) Diet of the eastern shovelnose ray, \u003cem\u003eAptychotrema rostrata\u003c/em\u003e (Shaw \u0026amp; Nodder, 1794), from Moreton Bay, Queensland, Australia. Mar Freshw Res 53:679-686. https://doi.org/10.1071/MF01040\u003c/li\u003e\n \u003cli\u003eLoboda TS, Carvalho MR (2013) Systematic revision of the \u003cem\u003ePotamotrygon motoro\u003c/em\u003e (M\u0026uuml;ller \u0026amp; Henle, 1841) species complex in the Paran\u0026aacute;-Paraguay basin, with description of two new ocellated species (Chondricthyes: Myliobatiformes: Potamotrygonidae). Neotrop Ichthyol 11:693-737. https://doi.org/10.1590/S1679-62252013000400001\u003c/li\u003e\n \u003cli\u003eLoboda TS, Lasso CA, Rosa RS, Carvalho MR (2021) Two new species of freshwater stingrays of the genus \u003cem\u003eParatrygon\u003c/em\u003e (Chondrichthyes: Potamotrygonidae) from the Orinoco basin, with comments on the taxonomy of \u003cem\u003eParatrygon aiereba\u003c/em\u003e. Neotrop Ichthyol 19:e200083. https://doi.org/10.1590/1982-0224-2020-0083\u003c/li\u003e\n \u003cli\u003eSilva JPCB,\u0026nbsp;Carvalho MR (2015b) Systematics and morphology of \u003cem\u003ePotamotrygon orbignyi\u003c/em\u003e (Castelnau, 1855) and allied forms (Chondrichthyes: Myliobatiformes: Potamotrygonidae). 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Neotrop Ichthyol 10:189\u0026ndash;196. https://doi.org/10.1590/S1679- 62252012000100018\u003c/li\u003e\n \u003cli\u003eSzabo T, Kalmijn AJ, Enger PS, Bullock TH (1972) Microampullary organs and a submandibular sense organ in the freshwater ray, \u003cem\u003ePotamotrygon\u003c/em\u003e.\u0026nbsp;J Comp Physiol 79:15\u0026ndash;27\u003c/li\u003e\n \u003cli\u003eSzamier RB, Bennett MVL (1980) Ampullary electroreceptors in the freshwater ray, \u003cem\u003ePotamotrygon\u003c/em\u003e.\u0026nbsp;J Comp Physiol 138A:225\u0026ndash;230\u003c/li\u003e\n \u003cli\u003eTakami S, Luer CA, Graziadei PPC (1994) Microscopic structure of the olfactory organ of the clearnose skate, \u003cem\u003eRaja eglanteria\u003c/em\u003e. Anat Embryol 190, 211-230\u003c/li\u003e\n \u003cli\u003eTheisen B, Zeiske E, Breucker H (1986) Functional morphology of the olfactory organs in the spiny dogfish (\u003cem\u003eSqualus acanthias\u003c/em\u003e L.) and the small-spotted catshark (\u003cem\u003eScyliorhinus canicula\u003c/em\u003e L.). Acta Zool Stockh 67:73\u0026ndash;86\u003c/li\u003e\n \u003cli\u003eYopak KE, Lisney TJ, Collin SP (2015) Not all sharks are \u0026ldquo;swimming noses\u0026rdquo;: variation in olfactory bulb size in cartilaginous fishes. Brain Struct Funct 220:1127-1143. https://doi.org/10.1007/s00429-014-0705-0\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"zoomorphology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"zomo","sideBox":"Learn more about [Zoomorphology](http://link.springer.com/journal/435)","snPcode":"435","submissionUrl":"https://submission.nature.com/new-submission/435/3","title":"Zoomorphology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"olfaction, Batoidea, potamotrygonin stingrays, Rio Negro basin, sensory cells.","lastPublishedDoi":"10.21203/rs.3.rs-4509528/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4509528/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe olfaction in batoids have an important role for initial detection of the chemical stimulus produced by prey during the foraging activities. Herein, the morphological and histological description of primary lamellae and secondary folds of olfactory rosettes is given to four species from Rio Negro basin. A simpler structure of olfactory organs in \u003cem\u003eParatrygon\u003c/em\u003e sp. does not indicate a primary sensory role during the initial phase of its feeding behavior. In \u003cem\u003ePotamotrygon wallacei\u003c/em\u003e, the largest surface area of primary lamellae suggests enhanced olfactory sensitivity related to its generalist feeding habits and complex substrate exploration. Histological analysis revealed differences in epithelial cell composition among species, with variations in the secondary folds shape and the distribution of mucous cells. The simplicity of secondary folds in both \u003cem\u003eParatrygon\u003c/em\u003e sp. and \u003cem\u003ePotamotrygon orbignyi\u003c/em\u003e probably is related to their specialized feeding habits, requiring fewer adaptations to detect different types of chemical stimuli. A central muscular layer in primary lamellae was observed only to \u003cem\u003eP. motoro\u003c/em\u003e and \u003cem\u003eP. wallacei\u003c/em\u003e and indicates a capacity to expand the olfactory epithelium area. These findings provide insights into the functional morphology of olfactory organs in potamotrygonin stingrays and their ecological implications, evidencing the intricate sensory adaptations crucial for foraging success in diverse freshwater habitats. Additionally, it becomes necessary to take into account the contribution of all sensory systems to understand their foraging behavior. Nonetheless, the generalization of the morphological characteristics of olfactory organ in a potamotrygonin species requires caution, since morphological variations can be found, especially to widespread species.\u003c/p\u003e","manuscriptTitle":"Sniffing at the river bottom: Influence of olfactory organ morphology on the life habits of freshwater stingrays (Potamotrygoninae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-14 08:09:09","doi":"10.21203/rs.3.rs-4509528/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-20T16:29:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-23T16:08:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113101415892330923719807801629058501146","date":"2024-06-03T15:54:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-02T15:41:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-02T14:12:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-02T14:12:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Zoomorphology","date":"2024-05-31T14:10:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"zoomorphology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"zomo","sideBox":"Learn more about [Zoomorphology](http://link.springer.com/journal/435)","snPcode":"435","submissionUrl":"https://submission.nature.com/new-submission/435/3","title":"Zoomorphology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dd4d3d89-2a77-4bed-9a3d-9e629a0bfa74","owner":[],"postedDate":"June 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-16T16:03:01+00:00","versionOfRecord":{"articleIdentity":"rs-4509528","link":"https://doi.org/10.1007/s00435-024-00682-3","journal":{"identity":"zoomorphology","isVorOnly":false,"title":"Zoomorphology"},"publishedOn":"2024-09-10 15:57:50","publishedOnDateReadable":"September 10th, 2024"},"versionCreatedAt":"2024-06-14 08:09:09","video":"","vorDoi":"10.1007/s00435-024-00682-3","vorDoiUrl":"https://doi.org/10.1007/s00435-024-00682-3","workflowStages":[]},"version":"v1","identity":"rs-4509528","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4509528","identity":"rs-4509528","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}