Pimelodella Lateristriga (Lichtenstein, 1823) with Emphasis in Club Cells
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Morphology of the Epidermis of the Neotropical Catfish Pimelodella lateristriga (Lichtenstein, 1823) with Emphasis in Club Cells Eduardo Medeiros Damasceno, Juliana Castro Monteiro, Luiz Fernando Duboc, Heidi Dolder, Karina Mancini* Departamento de Cieˆncias Agra´rias e Biolo´gicas, Centro Universita´rio Norte do Espı´rito Santo, Universidade Federal do Espı´rito Santo, Sa˜o Mateus, Espı´rito Santo, Brasil Abstract The epidermis of Ostariophysi fish is composed of 4 main cell types: epidermal cells (or filament containing cells), mucous cells, granular cells and club cells. The morphological analysis of the epidermis of the catfish Pimelodella lateristriga revealed the presence of only two types of cells: epidermal and club cells. The latter were evident in the middle layer of the epidermis, being the largest cells within the epithelium. Few organelles were located in the perinuclear region, while the rest of the cytoplasm was filled with a non-vesicular fibrillar substance. Club cells contained two irregular nuclei with evident nucleoli and high compacted peripheral chromatin. Histochemical analysis detected prevalence of protein within the cytoplasm other than carbohydrates, which were absent. These characteristics are similar to those described to most Ostariophysi studied so far. On the other hand, the epidermal cells differ from what is found in the literature. The present study described three distinct types, as follows: superficial, abundant and dense cells. Differences among them were restricted to their cytoplasm and nucleus morphology. Mucous cells were found in all Ostariophysi studied so far, although they were absent in P. lateristriga, along with granular cells, also typical of other catfish epidermis. The preset study corroborates the observations on club cells’ morphology in Siluriformes specimens, and shows important differences in epidermis composition and cell structure of P. lateristriga regarding the literature data. Citation: Damasceno EM, Monteiro JC, Duboc LF, Dolder H, Mancini K (2012) Morphology of the Epidermis of the Neotropical Catfish Pimelodella lateristriga (Lichtenstein, 1823) with Emphasis in Club Cells. PLoS ONE 7(11): e50255. doi:10.1371/journal.pone.0050255 Editor: Michael Koval, Emory University School of Medicine, United States of America Received July 18, 2012; Accepted October 16, 2012; Published November 30, 2012 Copyright: ß 2012 Damasceno et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction attributed an antipathogenic function to these cells [14–16], or suggested a phagocytic function [17]. Chondroitin and keratin The animal epidermis is a tissue exposed on the body surface, were also found in some fish [12], suggesting a healing function, which is in direct contact with the surrounding environment. It thus helping on repair of damaged tissue [18]. acts in numerous functions related to the interface organism/ Among their various functions in most Ostariophysi, the club environment, being also involved in the protection mechanisms cells are related to production, storage and release of the alarm against physical, chemical and biological agents, such as patho- substance, leading to intra or interspecific alarm reaction in gens. In specimens of the Superorder Ostariophysi, epidermis is phylogenetically close species [16], [19–22]. In Ostariophysi, the composed of four cells types: epidermal, mucous, granular and alarm reaction is triggered when individuals are threatened or club cells [1–8]. preyed upon his injured epidermis. This event causes disruption of Epidermal cells, also known as filament containing cells, are the the club cells cytoplasmic membrane, resulting in exposition and smallest and most numerous cells, being the major epithelium releasing of cytoplasmic content into the water, which is detected covering cells, and are found all over the epiderm, from basal to by other individuals in the school [16], [21], [23–26]. superficial layers [1], [3]. Mucous and granular cells are Despite several studies have already described the club cells in conspicuous round cells with peripheral flattened nucleus, located fishes, their results were based on cell-function relations, charac- on the apical region of the epithelium [1]. They are important terizing ecological and behavioral studies [10], [21], [27–33]. functional constituents of fish epidermis producing a glycoprotein We choose the genus Pimelodella Eigenmann & Eigenmann 1888 that, when secreted, lubricates the skin and favors the animal’s due to the lack of morphological studies of the epidermis of motion inside water. Neotropical fishes. Pimelodella is one of the most diverse genera of Club cells are distributed throughout the epidermal layer and the Heptapteridae family, with 82 described species until the possess cytoplasm filled with material to be secreted and one conclusion of this work [34], [35]. They are distributed from centered nucleus [1–4], [7], [9–11]. They are found in different southern South America to Panama and Central America [36]. fish groups associated with distinct functions [12]. Zaccone and The genus is popularly known as ‘‘mandi-chora˜o’’ (crying mandi) collaborators [13] demonstrated the presence of serotonin in these because of the sound it makes during his capture. Rhamdia and cells and suggested a pheromonal function. Other authors Pimelodella are among the most common Heptapteridae in South PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e50255 Epidermis of a Neotropical Catfish America, being endemic to Neotropical regions, however, its regions of the animals. Tissues were fixed by immersion in biology is poorly known [37]. Karnovsky solution (2.5% glutaraldehyde, 4% paraformaldehyde Therefore, this study aimed to describe the structure of the skin in 0.1M sodium phosphate buffer, pH 7.2) for 24 hours at 4uC. of the catfish Pimelodella lateristriga (Lichtenstein, 1823) with The material was washed, post-fixed with 1% osmium tetroxide, emphasis on club cell morphology. dehydrated in increasing series of acetone, infiltrated, and embedded in epoxy resin. The embedded material was sectioned Materials and Methods and collected on copper grids, thus contrasted with uranyl acetate and lead citrate solutions. The collections of Pimelodella lateristriga were performed in two Scanning Electron Microscopy: skin fragments with approxi- points of Sa˜o Mateus river basin (Espı´rito Santo state, Brazil) mately 1 cm3, were fixed by immersion in Karnovsky solution (PELD 1: 18u39900.80S and 40u05939.90W and PELD 2: (glutaraldehyde 2.5%, 4% paraformaldehyde in 0.1 M sodium 18u39902.20S and 40u07923.40W) during the months of August phosphate buffer, pH 7.2), washed and bathed in sucrose solutions and September of 2011. All Brazilian rivers are considered public (0.5M; 1M 1.5M, 2M, 3M, and 2.5M, in this order) for 24 hours area; however, we were authorized directly by all owners to have in each solution at 4uC. The fragments were criofractured in liquid access to the collection areas. Catches were made using a trawl nitrogen, post-fixed in 1% osmium tetroxide and dehydrated in net, under SISBIO license – permanent license to zoological increasing series of ethanol. Thus, the samples were critical point material sampling – number 19158-1; Prof. Dr. Luiz Fernando dried and coated with gold/palladium. For ultrastructural Duboc. characteristics, the micrographs were analyzed using measurement For the identification, the species were fixed in the field with tools from the imaging software Adobe Photoshop. formalin 10%. This identification was made by Prof. Dr. Stereological and morphometric analysis: Micrographs were Leonardo Ferreira da Silva Ingenito at the lowest taxonomic taken from different randomly chosen fields at a 4006 magnifi- level. Surplus copies were listed in the Zoological Collection of the cation. The volume density (%) of skin components (epithelial North Capixaba [CZNC - CEUNES/UFES: number CZNC 72 cells, club cells, connective and muscle tissues) was obtained by the (PELD 1, 3 ex.) and number CZNC 65 (PELD 2, 10 ex.)]. stereological methods described by [38]. Stereology was employed For light and transmission electron microscopies, the species using a test-system with 165-test-points over a known area. The were anesthetized with Benzocaine 0,5 g/l before dissection. All volume densities of the structures were estimated as Vv[struc- skin fragments were taken from the sacrificed individuals. ture] = Pp[structure]/PT, being Pp the number of points that were superimposed over the structure and PT the total number of test- Methods points contained in the area surrounded by the frame (grid). The Light Microscopy: P. lateristriga skin fragments with approxi- connective tissue height, nuclear and cellular area (epithelial and mately 1 cm3 were removed from the cranial and caudal regions club cells) were measured with a 4006 magnification, using the of the animals (asterisks, Fig. 1) and fixed with Bouin’s solution for software Image Pro-Plus. Fifteen micrographs of the anterior and 24 hours at 4uC. The fragments were washed, dehydrated in posterior regions were used for measurements, being all cells from