CILIATE PROTISTS FROM CABIÚNAS LAGOON ( RESTINGA DE JURUBATIBA , MACAÉ , RIO DE JANEIRO ) WITH EMPHASIS ON WATER QUALITY INDICATOR SPECIES AND DESCRIPTION OF Oxytricha marcili sp

We found 34 species of ciliate protists in the samples collected by the margins of Cabiúnas Lagoon during 2001. The ciliates were cultivated in the laboratory, where they were examined in vivo and identified through silver impregnation techniques. A new species, Oxytricha marcili (Ciliophora, Oxytrichidae), was found and characterized as follows: in vivo length about 60-80 μm x 30-40 μm wide; on average 22 adoral membranelles; 18 left marginal cirri; 18 right marginal cirri; and 3 small caudal cirri. All specimens analyzed presented 7 frontal cirri (3 anterior + 4 posterior), 1 buccal cirrus, 4 ventral cirri (3 postoral + 1 pre-transverse), and 5 transverse cirri. Among the species found, some are considered as water quality indicators ranging from alpha-mesosaprobity to polysaprobity and isosaprobity.

Among the complex of coast lakes is Cabiúnas Lagoon (also called Jurubatiba Lagoon), located in the Parque Nacional da Restinga de Jurubatiba, in the city of Macaé.Cabiúnas Lagoon presents a rich diversity of microorganisms, among among them the ciliate protists found in both benthos and plankton.In the benthos, a considerable amount of ciliate species may be encysted.These can be stimulated to excystment in the laboratory by adding crushed rice or wheat grains to the sample flasks containing the cysts.The resulting bacterial populations serve as food for the ciliates.Also, water conditions inside the flasks change over time in the laboratory.This variation and the presence of food can be associated with conditions accompanying excystment of many species present in sediments, making it possible to observe an ecological succession in the laboratory.
Endemic species are common in coastal lakes and lagoons (Dragesco & Dragesco-Kernéis, 1991).In the studied lagoon we found Oxytricha marcili, a new species of the genus Oxytricha Bory de Saint-Vincent in Lamouroux, Bory de Saint-Vincent & Deslongchamps, 1824.This genus is the one with most valid species characterized (about 50 in accordance to Berger & Foissner, 1997) within the subfamily Oxytrichinae Jankowski, 1979.Due to such a high count of known species and synonyms, characterization of this and other oxytrichid genus must be well detailed and are subject to constant revisions and redescriptions (Borror, 1972;Corliss, 1979;Jankowski, 1979;Small & Lynn, 1985;Tuffrau, 1987;Berger & Foissner, 1997;Eigner, 1997).
Coastal lakes and lagoons are water bodies of great economic and ecological importance because of their high productivity and intense utilization, including as aloctone residue deposits, for activities such as acquiculture and recreation (Spaulding, 1994).According to Gomes & Godinho (1999), efforts are urgently needed to further understand natural interactions occurring in these ecosystems, in order to find ways to preserve water bodies by either preventing them from harm or, if that has already been done, repairing the damage.
The present paper consists in a taxonomic study of the diversity of species composing the fauna of ciliates present in Cabiúnas Lagoon.These organisms, that play an important role in bacterial population control, have until now never been the object of such studies in this region.Therefore, the importance of this study lies in the fact that it identifies diverse ciliate species considered by many authors (Sládecek, 1973;Sládecek et al., 1981;Wegl, 1983;Mauch et al., 1985) as indicators of water quality.Puytorac et al. (1987) affirms that ciliates can be used as biological indicators of water quality in relation to dissolved oxygen concentration variations.

Study area
Cabiúnas Lagoon is perpendicular to the coast, having originated from a barrage of rivers, whose valleys were sculptured in the crystalline shield that composes the ground of the region, flooding the ridges formed by sand cords of restinga.It presents water of medium dark coloration (light penetration = 1.3 m from the bottom), aquatic surface of 0.34 km², average depth of 3.2 m, temperature around 24.8ºC, pH = 6.6, and average salinity of 1.1 ppt (Petrucio, 1998).
We collected samples of water and sediments from 4 sampling stations (Fig. 1) during 2001.Station 1 is about 1m distant from the margin, which has abundant vegetation (Typha domingensis).Samples were collected from about a 20 cm depth.Water is clean transparent and salinity = 1.3 ppt.Station 2 is about 3 m distant from the margin, from which abundant tufts of algae grow.Samples were collected from about a 70 cm depth.Water is clean transparent and salinity = 1.4 ppt.Station 3 is right by the margin, and samples were collected from about a 15 cm depth.Water is medium dark, of yellowish coloration.Salinity = 0.7 ppt.Station 4 is located far from the coast, right after the second sand cord.Samples were collected right by the margin.Water in this station is medium dark, of yellowish coloration, darker than that at Station 3. Salinity = 0.6 ppt.The description of the sampling stations is based on both field observations and data obtained in laboratory from recently collected samples.
Dissolved oxygen concentration measured during 2001 in an area close to where the sampling stations were established is according to Projeto ECOLagoas of Laboratório de Limnologia, Departamento de Ecologia, Instituto de Biologia, CCS, Universidade Federal do Rio de Janeiro.

Methodology
Samples were brought to the laboratory, where they were first examined under a stereoscopic micros-^ cope.After isolating the ciliates, done using micropipettes, the specimens were preliminarily observed in vivo, through bright field, phase contrast, and differential interferential contrast (DIC) microscopy.Cultures were made using both water from the location in which the ciliates were isolated and that had been filtered, and mineral water from Petrópolis, Rio de Janeiro.To all cultures we added crushed rice or wheat grains to induce bacterial growth, thus making possible ciliate population growth.
For scanning electron microscopy, ciliates were fixed with a mixture of 2 ml of glutaraldheyde 10% + 1 ml of osmium tetroxyde 2% + 1 ml of buffer sodium cacodylate 0.2 M, as proposed by Silva-Neto (1994).The cells were then washed successively with distilled water and some drops of sodium hypochlorite 0.3%, in order to remove fixative excess and attached particles.We used a JEOL: JSM-5310 scanning electron microscope to make observations and acquire electromicrographic images of the specimens.
The photomicrographic images were obtained through digital and analogical cameras attached to Zeiss and Olympus optical microscopes.
Living specimen morphological data were obtained using coverless slides (except for DIC observations) and at 200x magnification.All countings and measurements in the morphometric table were made using protargol slides and at 1.000x magnification.All measurements were in µm.Schematic drawings of protargol stained specimens were made using a modular camera lucida, and are based on the most representative specimens; in vivo drawings are based on freehand sketches and micrographs of specimens observed using phase contrast.All drawings are oriented with the anterior end of the organism directed to the top of the page.

Species succession in laboratory
Loxodes sp. was found in recently collected samples from Station 3. A very small number of specimens were observed, thus, making it very difficult to keep in cultures.This species was found in the same samples as was P. bursaria, which was successfully cultivated.
In recently collected samples from Station 1, living among populations of Coleps sp., L. lucens, U. nigricans, and P. pusilus, Coleps spetai was found.A couple of days after the cultures were made, we observed that both Coleps and L. lucens populations had disappeared.The other ciliate populations suffered no perceptible alteration.
Colpoda ecaudata was present in old cultures from Station 3 samples.This species population grew alone and was substituted after a few days by oligotrichid ciliate H. grandinella.Populations of C. inflata and Colpoda sp.grew simultaneously in cultures made from samples from stations 2 and 3, where the presence of Gastrostyla mystacea, Vorticela infusionum (Station 2 only), and T. pyriformis was detected.Small swarms of flagellate protozoan (Cryptomonas) were observed surrounding specimens of V. infusionum, suggesting that these flagellates feed on bacteria brought by the water current generated by the peritrichid's ciliature movements.Huge populations of these flagellates were also observed living among vast populations of U. nigricans.
Medium size populations of U. turbo were found in cultures made from Station 4 samples, living among small populations of P. aurelia (which was also present in cultures from Station 2 samples), Blepharisma sinuosum, and P. caudatum.After a few days, both Paramecium population sizes decreased, and a new population of S. pustulata started to grow, followed by G. scintillans about 2 weeks later.
Frontonia sp. was found in recent samples from Station 2. After cultures were made, we observed that they were soon substituted by populations of E. eurystomus, which also occurred in some cultures from Station 4, as well as in cultures from Station 3, which were replaced by C. inflata and Colpoda sp.
Huge populations of V. campanula were found growing in cultures made from Station 3 samples.In these cultures, we also observed the growth of S. ambiguum populations.V. campanula almost disappeared after 2 weeks.
Stentor sp. was found in recent collected samples from Station 2. Only a few specimens of this ciliate were observed.
Metopus striatus excysted in very old cultures (about 2 months after collection) from Station 3 samples.We observed among this species the growth of Pelagohalteria viridis (which also grew in some cultures from stations 1 and 2), O. marcili, Tachysoma sp, and Urosomoida agilis.The populations of both Tachysoma sp. and U. agilis started to increase in size as the populations of M. striatus and O. marcili decreased.In about 3 weeks, M. striatus disappeared and a somewhat constant growth was noted in these 3 species populations, plus that of another, hypotrichid ciliate Notohymena sp.We also found Vorticella chlorellata living among populations of O. marcili, in cultures where the algae Chlorella were also growing in abundance.
In most of the oldest (5 to 6 weeks) cultures obtained from stations 1, 2, and 3 samples, populations of Euplotes octocarinatus were found growing along with rotifers.
Dedication: named in honor of Marcil da Silva, grandfather of this paper's first author.
Type specimens location: found in sediments from sampling Station 3 of Cabiúnas Lagoon.
Description: body shape pyriform and elongated, with posterior region broader than anterior; dorsal side convex.Flexible, but constant in shape, tending to get rounder when squeezed under a cover slip.Two macronuclear nodules elipsoidal and very close together (x = 1.8 µm; n = 20) in the middle of body.Usually 2 micronuclear nodules attached to each macronuclear nodule; in most specimens, both are oriented in opposite directions.When in 4, 2 micronuclear nodules are attached per macronucleus.Contractile vacuole close to dorsal side, in the middle of the body, at left margin.Cytopyge found in the posterior region of cell, above pre-transverse cirrus.Colorless pellicle and greenish cytoplasm, containing small round-shaped algae (Chlorella) that stain with protargol.Some specimens were observed ingesting these algae, which they probably feed on, as suggested by the green pigment in the cytoplasm.The two marginal cirri rows tend to converge at the posterior end of the cell, but not actually join, as in O. similis Engelmann, 1862.Dorsal kineties are 4 in number, of which kineties 1, 2, and 3 each end on on a small caudal cirrus, located just above the posterior border of the cell.Kinety 4 is shortened and ends right after the equatorial region of the body, bearing no caudal cirrus (Figs.2-8).Dorsal morphogenesis in Oxytricha pattern.
Oral apparatus and conspicuous adoral zone of membranelles occupy about 37% of body length.Paroral and endoral membranes as in most species of this genus.

DISCUSSION
The new species, Oxytricha marcili, differs from other small-sized species like O. hymenostoma Stokes, 1887, O. ludibunda Stokes, 1891, O. minor Dragesco, 1966, and O. setigera Stokes, 1891 in both cell shape and ventral cirri pattern.It may also be confused with O. chlorelligera Kahl, 1932, in which cytoplasmatic inclusions of Chlorella algae were also observed, but from which it differs in characters such as average body size, ventral cirri pattern, macronuclear nodule size, and average micronuclei number.O. granulifera Foissner & Adam, 1983 (holotype of genus Oxytricha) and the subspecies O. granulifera quadricirrata Blatterer & Foissner, 1988, are other two small-sized congeners that may cause confusion, but from which O. marcili differs mainly in the organizational pattern of frontal cirri, as well as ventral cirri pattern and number of dorsal kineties.An important distinguishing detail of O. marcili is the reduced number of cirri in the posterior region of the ventral side, a reduction which has also been observed in some species such as O. granulifera quadricirrata, O. auripuncta Foissner, 1988 andO. rubripuncta Berger &Foissner, 1987.This makes them variants of the genus with the usual fronto-vental-transverse cirri number (18 FVT), but in these species, this reduction is associated with the number of transverse cirri.In O. marcili, we observe the presence of a single, instead of 2, pre-transverse cirrus in all analyzed specimens.That makes this species another kind of variation of the usual Oxytricha FVT pattern, i.e., a variation in the number of pre-transverse cirri, since no reduction is shown in the transverse cirri count.As usually characterized in most oxytrichids, the pre-transverse cirrus is shorter and less thick than transverse cirri, and has the same size asf postoral cirri .
O. marcili was included in genus Oxytricha because of usual shape of paraoral and endoral membranelles, shape of adoral zone of membranelles, number of dorsal kineties, presence of caudal cirri, and usual set of 5 transverse cirri, found in most species.It was not included in genus Urosomoida Hemberger in Foissner, 1982, a genus of oxytrichids characterized as having a single pretransverse cirrus (Berger & Foissner, 1997) because of characters such as the fragmentation of one dorsal kinety during morphogenesis, number of transverse cirri, which is constant in the studied population, and number of caudal cirri usual in most Oxytricha species.See detailed morphometric characterization of O. marcili in Table 2.
The saprobity associated with Coleps sp. and Frontonia sp. may be imprecise, since the species of these two genera present in Cabiúnas Lagoon can be different than those Sládecek used to develop his list.As for Loxodes sp., it is listed in Foissner et al., 1995, as "Loxodes spp.".As this is genus having very few species characterized, we consider that this data may be useful to our study area.
Among dissolved gases in water, oxygen is one of the most important in the dynamics and characterization of aquatic ecosystems (Esteves, 1988).It is also a useful element in determining water quality.The dissolved oxygen concentration was measured in the samples from the bottom of Cabiúnas Lagoon during every month in 2001, as shown in Fig. 16.According to Friedrich (1990), concentrations ranging from 5 to 8 mg/L, like those found during the first half of the year, may be indicative of oligosaprobic to beta-mesosaprobic, beta-mesosaprobic, and beta-to alpha mesosaprobic waters.Concentrations ranging from 7.5 to 8 mg/ L and higher, like those found in the second half of year may be indicative of oligosaprobic to betamesosaprobic waters.
Based on our results, we can conclude that the composition of ciliates present in the areas where the samples from Cabiúnas Lagoon were collected is more concentrated in the classes Oligohymenophorea and Hypotrichea, as clearly seen in the diagram in Fig. 17.We also have to consider that the growth in abundance of the most of the identified species populations was observed in laboratory cultures.The conditions present in these can be somewhat different from those observed in the field, in such a way as to allow the excystment and growth of ciliate populations like M. striatus and others that are indicators of conditions such as polisaprobity and abundant in polluted habitats.Taylor (1981), emphasizes the importance of mechanisms such as encystment which allow many species of ciliates to resist predation, physical and chemical stress, and exhaustion of food supply.The presence of these species can be useful in understanding possible modifications in the physical and chemical conditions of ecosystems caused by anthropic impacts.Classification according to Puytorac (1994).Legend: s = saprobity, according to Sládecek (1981), Foissner (1988), andFoissner et al. (1995); S(n) = cultures made with water from the sampling stations (n = sample station number); M = cultures made with mineral water from Petrópolis, Rio de Janeiro; a = alpha-mesosaprobity; b = betamesosaprobity; p = polisaprobity; i = isosaprobity.Legend: x = arithmetic mean; M = median; SD = standard deviation; SE = standard error of the arithmetic mean; CV = coefficient of variation (in %), Max = maximum value; Min = minimum value; n = sample size.All statistical procedures according to Sokal & Rohlf (1981).

Fig. 1 -
Fig. 1 -Map of the Cabiúnas Lagoon location on the Rio de Janeiro coast (from Branco, 1998).The small square marks indicate the sampling stations.

Fig. 14 -Fig. 13 -
Fig. 14 -Diagram showing distribution of classes that compose the fauna of ciliates observed in the area where samples were obtained.