Zoological Studies 46(2): 203-215 (2007) Effects of Hypo- and Hypersaline Seawater on the Microanatomy and Ultrastructure of Epithelial Tissues of Echinometra lucunter (Echinodermata: Echinoidea) of Intertidal and Subtidal Populations Ivonete A. Santos-Gouvea and Carolina A. Freire* Departamento de Fisiologia, Setor de Ciências Biológicas, Universidade Federal do Paraná, Centro Politécnico, Curitiba, Paraná, 81531-990 Brazil (Accepted May 9, 2006) Ivonete A. Santos-Gouvea and Carolina A. Freire (2007) Effects of hypo- and hypersaline seawater on the microanatomy and ultrastructure of epithelial tissues of Echinometra lucunter (Echinodermata, Echinoidea) of intertidal and subtidal populations. Zoological Studies 46(2): 203-215. Echinoderms are widely distributed in intertidal zones and are thus subject to wide salinity variations and even air exposure. Physiological studies have shown them to be osmoconformers, but also to specifically, although moderately, regulate certain ions. Morphological studies associated with salinity challenges were not found in a literature search. Two popula- tions of the sea urchin, Echinometra lucunter Linnaeus 1758, were studied: 1 intertidal and 1 subtidal popula- tion. Urchins from both populations were exposed for 5 d to hyposaline seawater (SW) of 25 ppt, or for 40 h to hypersaline SW of 45 ppt, and were compared to control urchins kept in full-strength SW of 35 ppt. Two exter- nal tissues, bathed by SW, the peristomial gills (PG) and ambulacral feet (AF), and 2 internal tissues, the coelomic wall of the ambulacral system (CWAS) and the intestinal rectum (IR), were investigated using trans- mission electron microscopy. With respect to the effect of salinity, there was more tissue damage in 45 ppt than in 25 ppt, coherent with the more-frequent SW dilution than concentration in marine shore habitats. Damage detected by electron microscopy included tissue disruption, destruction of microvillae, or evidence of fragment- ing or peeling off of cells. Tissues of subtidal urchins were as sensitive to salinity stress as were those of inter- tidal urchins. PG, AF, and the IR presented evidence of an excretory function by the presence of large morula cells within these tissues. Only the IR displayed an ultrastructure compatible with metabolically active epithelia, in that it possessed numerous mitochondria. http://zoolstud.sinica.edu.tw/Journals/46.2/203.pdf Key words: Ambulacral feet, Hypersaline seawater, Intestinal rectum, Peristomial gills, Water vascular system. All members of the phylum Echinodermata 1977, Stickle and Diehl 1987). However, despite are marine, considered stenohaline, and osmo- the fact that ionic regulation might not be a major conformers. Although they do not actively engage feature of echinoderm physiology, these marine in osmoregulation, these invertebrates may regu- invertebrates are common dwellers of intertidal late specific ions (Robertson 1949, Binyon 1962, zones (McPherson 1969, Diehl 1986). As these Prusch 1977, Diehl 1986, Bishop et al. 1994). For habitats offer wide daily salinity fluctuations, at example, transport of Cl- has been related to nutri- least some species must attain some degree of ent uptake in the digestive tube of the sea urchin, euryhalinity (e.g., Stickle and Diehl 1987). Lytechinus variegatus (Bishop et al. 1994), and Echinoderms also apparently do not possess transport of K+ to the lumen of ambulacral feet in any specific excretory organ (Hyman 1955, starfishes has been related to the maintenance of Boolootian 1966, Diehl 1986, Cavey and Märkel internal pressure and volume through osmotic 1994); ammonia easily crosses the body wall, water flow (Prusch and Whoriskey 1976, Prusch being the essential method of nitrogen excretion, *To whom correspondence and reprint requests should be addressed. Tel: 55-41-33611712. Fax: 55-41-32662042. E-mail:[email protected] 203 204 Zoological Studies 46(2): 203-215 (2007) as is typical of most aquatic animals. Ammonia MATERIALS AND METHODS diffusion occurs in the ambulacral feet and in the papullae of asteroids, as well as in the respiratory Animals trees of holothuroids (Boolootian 1966). Non-dif- fusible metabolic wastes are actively secreted out An intertidal population of E. lucunter of of the body cavity, crossing rectal cecum cells and Quilombo Beach, Penha (26 46'S, 48 38'W), enterocytes into the intestinal lumen in echinoids, Santa Catarina State, Brazil, was° manually° sam- holothuroids, and asteroids (Warnau et al. 1998, pled from crevices and under rocks in tidal pools Warnau and Jangoux 1999). These enterocytes during low tide. The subtidal population was sam- display apical microvillae and a basal membrane pled through scuba diving off Galheta I., Pontal do associated with numerous elongated mitochondria Paraná (25 30'S, 48 15'W), Paraná State, Brazil, (Warnau et al. 1998, Warnau and Jangoux 1999). from a depth° of 3-4 m.° After being collected, adult There is also evidence of an excretory role of male and female urchins of both populations were phagocytic coelomocytes associated with rectum placed inside Styrofoam boxes with some water enterocytes, the axial complex, ambulacral feet, from the collection site and were transported to the and peristomial gills (Cobb and Sneddon 1977, laboratory. In the laboratory in Curitiba, they were Bachmann et al. 1980; Cavey and Märkel 1994). maintained in a stock tank (160 L) containing sea In addition to the evidence from enterocytes and water (SW) with a salinity of 35 ppt, a temperature the extrusion of phagocytic coelomocytes in sever- of 20-24 C, constant aeration, and biological filtra- al tissues, the axial gland has been demonstrated tion. The° average test diameter of the urchins of to display podocytes, a structure typically involved both populations used in this study was 77 mm. in ultrafiltration, which strengthens the idea that the excretory function is carried out not by a single Experiments excretory organ, but through a division of labor between several organs and tissues (Boolootian The control group of the intertidal population 1966, Cavey and Märkel 1994). (4 urchins) was kept for ~16 hours in 35 ppt SW, This study was conducted in order to: 1) eval- being dissected the day after being collected in the uate the structural effects of hypo- and hypersaline field. All other urchins were kept for 3 d in the stresses on echinoderm epithelial tissues, for the stock tank containing 35 ppt SW to allow them to 1st time ever, looking at 2 ecologically distinct pop- acclimate to laboratory conditions. After that peri- ulations of urchins, 1 intertidal population, naturally od, 2 animals were transferred and maintained in subjected to daily tidal fluctuations in salinity, and 1 30 L experimental aquaria, with constant aeration, population inhabiting a stable subtidal environ- pH control (7.5-8), a temperature of 22-24 C, bio- ment; and 2) gather additional morphological evi- logical filtration, and SW adjusted to a salinity° of 35 dence on the putative ion-transporting or excretory ppt (controls for the subtidal population), 25 ppt roles attributed to certain echinoderm tissues, by (hyposaline SW), or 45 ppt (hypersaline SW). In looking for abundant mitochondria and/or the pres- order to increase the salinities, adequate amounts ence of excretory cells. Two external tissues that of marine salt were added. Evaporated marine are directly bathed by seawater, and 2 internal tis- salt was purchased from a local aquarium shop. sues were examined. The external tissues were Diluted SW was produced by appropriate dilution the peristomial gills and adoral ambulacral feet, of full-strength SW with filtered (using activated and the internal tissues were the coelomic wall of charcoal and cellulose filters) tap water. Each the ambulacral system and the intestinal rectum. experiment, for each of the 3 salinities and both The ultrastructures of echinoid tissues were previ- populations, was performed in duplicate, always ously described: the peristomial gills by Cobb and with 2 urchins per aquarium, yielding a total of 4 Sneddon (1977); the ambulacral feet and the urchins for each salinity and each population. The channels and structures of the ambulacral system only exception was for the subtidal population by Cavey and Märkel (1994); and the intestinal exposed to 25 ppt: only 3 urchins were used. Both rectum by Warnau et al. (1998). The echinoderm intertidal and subtidal urchins in 25 ppt, and subti- model used here was the sea urchin, Echinometra dal controls in 35 ppt were maintained for 5 d in lucunter Linnaeus 1758. the experimental aquaria. However, exposure of urchins from both populations to 45 ppt was for Santos-Gouvea and Freire -- Salinity Effects on Echinoid Tissues 205 only 40 h, due to morbidity and eventual mortality buffer with the same NaCl concentrations as in the after 48-72 h in this concentrated, hypersaline SW. primary fixative, plus 1% osmium tetroxide for 1.5 h A 29% decrease (10 ppt, 35 to 25 ppt), and a 29% on ice. Fragments were dehydrated in an ethanol increase (10 ppt, 35 to 45 ppt) in salinity was the series, infiltrated, and embedded in Araldite 502 protocol employed, to allow a direct comparison resin, using propylene oxide as the vehicle. All tis- between the responses of the urchins. The time of sue fragments were oriented to allow transverse exposure to dilute SW (120 h, 5 d) was 3 times the sections through the material. Semi-thin (500 nm) time of exposure to hypersaline SW (40 h), evi- and thin (50 nm) sections were prepared with a dencing the much-higher tolerance of the urchins Leica Ultracut UCT Ultramicrotome (Wetzlar, to SW dilution compared to SW concentration. Germany). Semi-thin sections were stained with toluidine blue (1%), and photographed using a Tissue dissection Zeiss Axiophot photomicroscope (Oberkochen, Germany). Thin sections were contrasted with 5% After exposure to either the control or experi- uranyl acetate and lead citrate (Reynolds 1963), mental conditions, urchins were anesthetized in and examined with a JEOL JEM 1200 EXII trans- magnesium chloride kept isosmotic to the control mission electron microscope (Tokyo, Japan), at 80 or experimental SW: 0.36 M MgCl2 for 35 ppt, kV of accelerating voltage.