Morphology of Parastrongylidium estevesi comb . nov . and Deviata brasiliensis sp . nov . ( Ciliophora : Stichotrichia ) from a sewage treatment plant in Rio de Janeiro , Brazil

In samples of raw sewage collected from a sewage treatment plant in Rio de Janeiro (ETE-Penha), we found populations of two species, Deviata estevesi Paiva & Silva-Neto, 2005 and Deviata brasiliensis sp. nov. The organisms were studied in vivo under phase contrast microscopy, differential interference contrast (DIC), and after protargol-impregnation. The population of D. estevesi exhibited more extensive variation in cirral pattern than previously described. The interphasic organisms of new species D. brasiliensis sp. nov. are distinguishable from their congeners based on a series of morphometric features: cirral row R3 usually presents 1-3 cirri behind the right frontal cirrus, on average there are four macronuclear nodules, and, during morphogenesis of cell division, primordium V of the proter originates from the anterior region of cirral row R5 instead of row R6, as in the type species D. abbrevescens Eigner, 1995. In D. estevesi, the ventral cirral rows replicate by within-row primordia, which develop independently for the proter and for the opisthe, suggesting that it belongs to or is closely related to Parastrongylidium, hence the combination P. estevesi comb. nov. is established.


MATERIAL AND METHODS
Samples of raw sewage water were collected from the primary settling tanks of Penha Sewage Treatment Plant (ETE-Penha), located in the city of Rio de Janeiro, RJ, Brazil, during the period from March 2005 to July 2006.The water character-istics in the samples from which the species were found were: dissolved oxygen concentration = 2.2 mg/l; pH = 7; temperature = 27.8 °C.
Aliquots of the samples were split into Petri dishes where cultures were made with addition of mineral water and crushed rice grains to favor the growth of bacteria to serve as primary food source for the ciliates (FOISSNER et al. 2002).
The ciliates were primarily examined in vivo under phase and differential interference (DIC) contrasts to check for the presence of cortical granules, verify the body outline, flexibility, contractile vacuole, details of the cytoplasm and other taxonomic features (BERGER 1999, FOISSNER et al. 2002).Protargolimpregnation (DIECKMANN 1995) and scanning electron microscopy preparations (SILVA-NETO 1994) were made to examine the ciliature, nuclear apparatus and divisional morphogenesis.Data from tables I and II were obtained from protargol-impregnated specimens observed at 1000x magnification (bright field, oil immersion).All measurements are in micrometers (µm).The ventral and lateral cirral rows were numbered according PAIVA & SILVA- NETO (2005) and follow their morphogenetic origin.Statistical procedures were conducted using the software BioEstat 2.0 (AYRES et al. 2004).Schematic drawings of protargolimpregnated specimens were first sketched at 1000x magnification plus a 1.6x optovar device, with the aid of a camera lucida attached to a Carl Zeiss Axioskop 20 microscope, and then refined with computer image edition software.Morphology of Parastrongylidium estevesi comb.nov.and Deviata brasiliensis sp.nov.
Both species studied in this paper occurred on 10 of 27 sewage samples.Deviata brasiliensis sp.nov.was also present in fresh samples in natura, co-existing with Paramecium aurelia Ehrenberg, 1838 and testate amoebae.P. estevesi grew concomitantly with Blepharisma sinuosum Sawaya, 1940 and P. aurelia.Observations on cytoplasmic inclusions in protargol slides of P. estevesi comb.nov.indicate this organism feeds on flagellates in addition to bacteria.Encystant specimens and conjugant pairs of P. estevesi comb.nov.were observed in our cultures (Figs 3 and 6), however, detailed morphological descriptions under those conditions were not carried out in the present study.
Figs 1-20, Tab.I Redescription: the organisms measure approximately 90 x 40 µm in vivo, displaying roughly ellipsoid outline, narrowed anteriorly and broader at the posterior end, varying from 2:1 to 6:1; with the body slightly dorso-ventrally flattened, flexible and contractile.They exhibit dark coloration under low magnification, conspicuous compact crystals scattered through the cytoplasm alongside with variable sized granulation, but lacked cortical granules.A spheroid contractile vacuole is observed at mid-body, away from the lateral margins, with a dorsal opening pore (Figs 7-9).
The adoral zone of membranelles (AZM) is formed of 20-34 membranelles and occupies about 37.5% of the body length.The undulating membranes are aligned side by side, seldom intersecting each other optically, with the paroral distal end slightly ahead of the endoral (Figs 1,4,10,and 12).Frontal and buccal cirri are indistinguishable from ordinary cirri.
The ventral ciliature comprises on average eight cirral rows on the right and five cirral rows on the left of the AZM (Tab.I), with row R3 ending approximately at the equatorial region of the body (Figs 1,4,10,and 12).On the dorsal side there are two dikinetid rows, with the right one being posteriorly shortened.A small file of three to five dikinetids is located close to the posterior end of the body, behind the right dorsal kinety (Figs 2,5,11,and 13).The nuclear apparatus is composed of usually two macronuclear nodules of roughly spheroid to ellipsoid or ovoid shapes, containing variable sized argentophilic granules.On average, the anterior nodule measures 13.6 x 8.1 µm and the posterior one 12.7 x 8.3 µm.Two spheroid micronuclei are located near each macronuclear nodule (Figs 1 and 12).In some specimens, the macronuclear nodules are constricted at their equatorial region, becoming dumbbell shaped.A morphometric characterization of the population of P. estevesi comb.nov.from ETE-Penha is shown in table I.
Divisional morphogenesis (Figs 14-20): stomatogenesis of the opisthe is either parakinetal, beginning with proliferation of basal bodies originating adjacent to row R3, or from disaggregating of the posterior-most cirri to the formation of the early oral primordium (Fig. 15).The undulating membranes primordium (primordium I) develops from the right margin of the oral primordium and its distal end produces the cirral row R1.In the proter, the cirral row R1 is formed likewise from the distal end of the undulating membrane primordium.The cirral primordium II of the opisthe develops from primordium I, whereas in the proter, primordium II develops within the cirral row R2.Rows R3-R6 each develops two independent withinrow primordia for each divider, viz.primordia III-VI (Figs 16 and 18).The left marginal row (L3) of the opisthe develops from within its respective parental row (Fig. 18).Further middleto-late dividers are lacking in ours slides.Based on the configuration of the ciliature in late dividers (Figs 19 and 20) it is possible that all the ventral cirral rows of P. estevesi replicate by within-row proliferations of basal bodies, forming independent primordia on each divider.The AZM of the proter remains unchanged during the observed divisional morphogenesis stages, and the undulating membranes disaggregate and form the undulating membrane primordium in early to middle dividers.Later, this primordium splits longitudinally into the new paroral and endoral membranes (Fig. 19).
The dorsal kineties replicates by intrakinetal process.The right kinety forms two distinct separated primordia (one for each divider), whereas within the left kinety, the primordia are only narrowly separated (Figs 17 and 20).Later, the right dorsal row split and formed short posterior fragments, one per divider (Fig. 20).In the divisional morphogenesis of the nuclear apparatus the complete fusion of the macronuclear nodules occurs and further divisional events proceed as in most stichotrichs (Fig. 14).The parental somatic ciliature is likely entirely resorbed by the end of the morphogenetic process.

Deviata brasiliensis sp. nov.
Figs 21-27, Tab.II Diagnosis: Deviata measuring about 110 x 45 mm in vivo (n = 5).With three frontal cirri and one buccal cirrus distinctly isolated from the remaining ventral ciliature; with three to six long cirral rows left and 4-5 right of AZM.Row R4 usually ends at about 4/5 of body length.On average with four macronuclear nodules of variable shape (viz.roughly ellipsoid, ovoid, fusiform or dumbbell-like).Ventral primordium V of the proter originates from anterior end of row R5.
Type locality: primary settling tanks of the Estação de Tratamento de Esgotos da Penha, ETE-Penha, a sewage treatment plant from Companhia Estadual de Águas e Esgotos (CEDAE/RJ), located in the district of Penha, Rio de Janeiro, state of Rio de Janeiro, Brazil.

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Description: specimens of D. brasiliensis sp.nov.have an elongate elliptical outline with pointed anterior end, are more or less dorso-ventrally flattened (almost cylindrical in cross-section), flexible and slightly contractile.They exhibit dark color under low magnification, cytoplasm moderately filled with compact crystals, less conspicuous than those from P. estevesi comb.nov.(Fig. 23).Cortical granules are absent.A contractile vacuole is present at equatorial level, adjacent to the left margin of the body (Fig. 23).
The oral apparatus has a short AZM that occupied about 19% of the body length and is formed by 18-31 membranelles (Figs 21 and 24).The undulating membranes are arranged as described above for P. estevesi comb.nov.The organisms exhibit conspicuous cytopharyngeal fibers measuring on average 24 µm long (Figs 21 and 26).
The frontal ciliature of D. brasiliensis sp.nov.presents three inconspicuously differentiated anterior frontal cirri, roughly aligned horizontally behind the distal membranelles (Figs 21and 24).Behind the right-most frontal cirrus there are one to three extra cirri.A buccal cirrus is located behind the middle frontal cirrus.The ventro-lateral ciliature is formed by 3-6 long cirral rows on the left and 4-5 on the right of the AZM.The first long row from the right (R4) is formed of 10-34 cirri and is shortened posteriorly, ending behind mid-body, at about 4/5 of the body length in most specimens, seldom ending at the equatorial region or at the posterior end of the body.
One specimen, however, had this row distinctly shortened, slight surpassing the AZM level (Fig. 26).In 60% of the studied specimens, the anterior cirri of R4 are misaligned in relation to the remaining cirri, sometimes resembling an extra row running parallel to its anterior end (Fig. 21).Rarely, two isolated barren kinetids (dikinetids?) are present in the space between rows R4 and L1 (Fig. 27).On the dorsal side there are two dikinetid rows, of which the right one had the anterior dikinetids densely packed but widely spaced from the equatorial region to the posterior end of the body (Figs 22 and 25).
The nuclear apparatus (Figs 22 and 24) is formed on average by four macronuclear nodules and 2-4 micronuclei.The shape of the macronuclear nodules is highly variable, ranging from roughly ovoid or ellipsoid to fusiform, being sometimes equatorially constricted, thus becoming dumb-bell shaped.The micronuclei are spheroid.The whole nuclear figure is disposed longitudinally left of the AZM, adjacent to the left margin of the body and the nodules usually are linked by thin isthmuses that stain faintly with protargol.A morphometric characterization of D. brasiliensis sp.nov. is shown in table II.
Divisional morphogenesis (Figs 28-39): the stomatogenesis of D. brasiliensis sp.nov. is parakinetal alongside to the median region of the shortened ventral row (R4) (Fig. 29).The oral primordium grows posteriad and incorporates posterior-most cirri of row R4 (Figs 30 and 31).It is worth to mention that one specimen exhibit the oral primordium located between rows R4 and  L1.In this specimen the stomatogenesis seems to have occurred de novo, because the parental ciliature seems to remain intact (Fig. 32), but it could also be related to the posterior-most cirrus of R4, since the posterior end of the oral primordium is located near the posterior cirri of this row.In the opisthe, primordia I and II develop from the oral primordium, and primordium III from row R4.The origin of primordia IV and V could not be unambiguously determined.Either both primordia develop from the posterior region of row R5, or primordium IV develops from R5 and primordium V originates from row R6, further deviating to R5, as in D. abbrevescens.Primordium VI of the opisthe is a long primary primordium that originates from row R6.In the proter, primordium I originates from the anterior region of the undulating membranes; primordium II develops from the buccal cirrus, and primordium III from the cirri behind the right frontal cirrus.Primordia IV and V originate from the anterior region of rows R4 and R5 respectively; the long primary primordium VI splits and forms two secondary primordia, one for each divider (Figs 33,35,36,and 38).Row R7 produces two withinrow independent primordia, one for each divider.The cirral rows located left of the AZM replicate likewise, by forming two independent within-row primordia, one for each divider (Fig. 36).Noteworthy, one specimen (Figs 28 and 36) exhibited seemingly reorganizing adoral membranelles in the proximal half of the proter's AZM.The paroral and endoral membranes disaggregate and become the undulating membranes primordium only in middle to late dividers.The two dorsal dikinetid rows replicate by intrakinetal process, forming one independent primordium for each divider (Figs 34,37,and 39).The nuclear apparatus divides as usual, with replication bands developing on each nod-ule and complete fusion of the macronuclear nodules (Fig. 38), thus does not require further explanation.The parental somatic ciliature is very likely entirely resorbed by the end of the morphogenetic process.Etymology: this species is named "brasiliensis" after the country where it was discovered.

Remarks
The morphology of P. estevesi comb.nov.from ETE-Penha fits in the description of the type population provided by PAIVA & SILVA-NETO (2005) (Tab.II).Anyhow, we observed a greater variability in the cirral pattern of the presented population, more noticeably concerning the number of cirral rows.We found organisms with eight (n = 18), nine (n = 6), and ten (n =

Figures
Figures 15-20.Schematic drawings of dividing specimens of P. estevesi comb.nov.after protargol-impregnation: (15-16) early morphogenesis of the ventral ciliature, arrows in figure 16 show the early frontal row R1 primordium detaching from the undulating membranes primordium in the opisthe and proter; (17) dorsal side of specimen in 16, showing dorsal kineties primordia (arrows); (18) ventral side of middle divider, arrow points row L3 primordium of the opisthe; (19) ventral side of late divider, arrows show undulating membrane primordium splitting longitudinally into endoral and paroral; (20) dorsal side of same specimen, arrows show fragments of the right kinety primordia, which became the short file of dorsal dikinetids at the posterior end of interphasic specimens.(OP) oral primordium of the opisthe.Ventral cirral primordia are numbered in romans.Scale bar: 10µm.

Figures 35 -
Figures 35-39.Schematic drawings of dividing specimens of D. brasiliensis sp.nov.after protargol-impregnation:(35-37) middle dividers; (35) middle divider showing split of primary primordium VI (arrow); (36) ventral side of middle divider, arrows indicate R7 primordia (right side of the body) and primordia of cirral rows left of AZM (left side of the body), asterisk marks supposedly reorganizing membranelles in the proter; (37) dorsal side of same specimen; (38-39) late divider; (38) arrows point to a row which is likely a fragment of primordium IV and may explain the misaligned pattern found in some specimens; (39) dorsal side of same specimen.Ventral cirral primordia are numbered in romans.Scale bar: 10 µm.

Table I .
Continued.
* ** The largest values were observed in specimens with two macronuclear nodules.