Sticholonche zanclea ( Protozoa , actinoPoda ) in fecal Pellets of coPePods and Euphausia sp . in Brazilian coastal waters

fecal pellets produced by mesozooplanktonic copepods (Centropages velificatus and Paracalanus parvus) and macrozooplanktonic euphausiacea (Euphausia sp.) were examined using scanning electron microscopy. fragments of the protozoan Sticholonche zanclea were found in both copepod and in Euphausia sp. fecal pellets, even when the abundance of the protozoan in the water was low. the results suggest that S. zanclea is an important food resource for different trophic levels, including mesoand macrozooplankton, in Brazilian coastal waters.

there is little published information on predation on protozoans under natural conditions.evidence of the ingestion of protozoans comes, mostly, from predation experiments done in situ or in the laboratory (Paffenhöfer et al., 1995), through the use of biomarkers (kivi & setala, 1995; li et al., 1996), filming (Verity & Paffenhöfer, 1996), or through identification of hard parts such as loricae and skeletal material in gut contents or in fecal pellets (conover, 1982).
a previous investigation (eskinazi-sant'anna, 2000) showed that the protozoan S. zanclea forms Braz.J. Biol., 66(3): [839][840][841][842][843][844][845][846][847]2006 part of the natural diet of mesozooplanktonic copepods such as Parvocalanus crassirostris, Paracalanus quasimodo, Temora stylifera, and T. turbinata in the são sebastião channel (ssc).the aim of the present study was to verify whether S. zanclea is also utilized as a food resource by other zooplankters in the ssc. in order to achieve this, the contents of fecal pellets of the copepods Paracalanus parvus and Centropages velificatus and of the euphausiacean Euphausia sp. were examined under scanning electron Microscopy (seM).these organisms have distinct feeding habitats and body sizes, representing different trophic niches in the ssc.
stUdy ArEA the são sebastião channel is located on the northeast coast of são Paulo state (from 23° 41' s -45° 10' w to 23° 53' s -45° 30' w). the channel is bounded on the west by the slopes of the serra do Mar, and on the east by são sebastião island (fig.1). the funnel-shaped channel is curved along its 25 km length, with the narrowest part (2 km wide) in the center, widening toward both ends to 7.2 km at the southern entrance and 5.6 km at the northern entrance.depths along the channel range from approximately 20 m at the entrances down to 50 m in the middle part (Gianesella et al., 1999).water temperature and salinity vary annually from 18 to °c and 35 to 37‰, respectively.total chlorophyll (chl a + pheopigments) averages 4.3 mg chl-a m -3 and can vary annually from 0.16 to 6.42 mg chl-a m -3 (Gaeta et al., 1995).the temperature in the region is warm year-round, with typical monthly means from 19.5 °c in winter to 25.5 °c in summer.annual precipitation oscillates between 1500 and 2000 mm, with a dry period from June through august and a rainy period from January through March.

MAtErIAl And MEthOds
sampling campaigns were carried out in January and July 1997, July 1998, and January 1999, at two stations (P1 and P2) in the ssc, Brazil.surface-water samples (250 ml) for phytoplankton and protozooplankton analyses were taken with a van dorn bottle (5 l).all samples were fixed with lugol's solution.
Phytoplankton and protozooplankton were enumerated using the inverted microscope method (lund et al., 1958) under magnifications of 40x, 60x and 100x.Meso and macrozooplankton were collected during 2 min surface tows with a 150 µm mesh net.adult females of the copepods Centropages velificatus and Paracalanus parvus, and individuals of Euphausia sp. were sorted under a wild M8 stereomicroscope, isolated within 20 min of collection, and transferred to Petri dishes containing collected surface sea water, and then left for 30 to 120 min to produce fecal pellets.the expelled fecal pellets (15 from C. velificatus, 22 from P. parvus, and 25 from Euphausia sp.) were individually removed by pipette and placed in petri dishes containing a mixture of filtered sea water (nucleopore cellulose acetate membrane filters 20 µm) and 10 µm screened surface sea water.the pellets were then left for 24 h at 24 °c (± 2 °c) for microbial stripping of their peritrophic membranes (turner, 1984b,c).all pellets were subsequently preserved in 4% formalin-seawaterglutaraldehyde solution.ashore, preserved pellets were individually removed by pipette, transferred to nucleopore filters, and then left to adhere.filters with attached pellets were washed in distilled water for salt elimination, dehydrated in a graded ethanol series (50, 60, 70, 80, 90, 95, and 100%), left to dry at room temperature, and then coated with gold.the fecal pellets were examined with a zeiss dsM-950 scanning electron Microscope (seM).

rEsUlts
analysis of the fecal pellets of C. velificatus, P. parvus, and Euphausia sp.indicated the presence of S. zanclea fragments and remains of diatoms, mainly Chaetoceros sp., Thalassiotrix frauenfeldii, and Thalassionema nitzschioides.Unidentified centric and pennate nanoplanktonic cells (< 20 µm) were also observed.fecal pellets of Euphausia sp.contained dinoflagellate remains (fig.2) and amorphous material, which was also present in the fecal pellets of C. velificatus (fig.3).overall, the diatom remains observed in the fecal pellets of copepods and Euphausia sp. did not reflect the composition of the available phytoplankton.Phytoplankton abundance in ssc ranged from 25.4 to 717 x 10 3 cells.l - .the assemblages were dominated mainly by flagellates and diatoms (mainly Cerataulina pelagica, Leptocylindrus danicus, Skeletonema costatum, and Cylindrotheca closterium).in January 1997, phytoplankton assemblages at station P1 were dominated mainly by flagellates and large diatoms (> 50 µm), represented by L. danicus.at station P2, the same pattern of phytoplankton assemblage composition was observed, together with the occurrence of C. closterium and Asterionella japonica (fig.4).
in fecal pellets of C. velificatus and P. parvus, fragments of diatoms (Thalassionema nitzschioides and Thalassiotrix frauenfeldii) and a protozoan (S. zanclea) were observed.remains of T. nitzschioides and S. zanclea were detected in the fecal pellets of Euphausia sp. in July 1997, flagellates comprised almost 90% of the phytoplankton community at P1, although in the fecal pellets of all three zooplankters, the main fragments found were of S. zanclea and diatoms (mainly T. frauenfeldii).
identification of S. zanclea fragments was only possible under seM, which revealed their characteristic ornamentation, consisting of a series of fringes along the spine (fig.5).these spines were the only item identified in some pellets (fig.6).fragments of S. zanclea were found in more than 80% of the pellets of the species studied, during both summer and winter.these were present even when the relative abundance of this protozoan corresponded to less than 10% of the total microzooplankton in the ssc (fig.7), comprising a minimal fraction of the microzooplankton density which ranged from 5 to 101 org.l -1 (fig.8).dIscUssIOn all the zooplankters examined fed in situ upon S. zanclea. in the fecal pellets of the copepods, the proportion of S. zanclea spines was in some cases higher than the proportion of diatom frustules.the same composition pattern was observed in the fecal pellets of Euphausia sp. S. zanclea is apparently an important food resource for different trophic levels in the ssc.Grassé (1953) described the species S. zanclea as a peculiar organism.it was considered as an intermediary form between heliozoarians and radiolarians, which did not have a defined taxonomic Braz.J. Biol., 66( 3 position at that time.S. zanclea is common in the plankton of the Mediterranean sea (tregouboff & rose, 1957).However, in Brazilian waters its distribution seems to be limited to the southeast coast, since there are no published records of its occurrence in other areas of Brazil.that these species are present during all seasonals in the ssc.also, they seem to be a preferential food source for the meso-and macrozooplankton, even when they are available in low abundance.they may be preferred because of the small size of their cells (< 50 µm).remains of flagellates were not identified in the fecal-pellet contents of either copepods or Euphausia sp. this is surprising, because flagellates may represent a reasonable food source, in view of their small size and ease of digestion because of the lack of a shell or rigid cell walls.rapid processing of these cells within the digestive tract probably accounts for the difficulty in identifying their fragments in the fecal pellets, even with seM.although fecal-pellet analysis is considered a reliable method to determine food preference, there are some limitations to the technique, such as the difficulty of appropriately preserving for identification, particles lacking hard parts or composed only of soft tissue, e.g., flagellates.the amorphous material observed in the fecalpellet contents of C. velificatus and P. parvus and Euphausia sp. may indicate that these soft-celled organisms were indeed ingested.
another important observation made through analysis of fecal pellets of copepods and Euphausia sp. was the presence, within a single fecal pellet, of remains of phytoplankton cells and protozoan fragments ranging over two orders of magnitude in their longest dimension.according to turner (1986), the presence of food fragments of different sizes could be indicative of selective grazing on large particles.therefore, ingestion of S. zanclea by the meso-and macrozooplankton in ssc seems to indicate that these zooplankters are actively selecting this protozoan, because of its size (> 300 µm) and its low abundance in the environment.However, corroboration of this conclusion requires more conclusive experimental treatments.
Predator-prey interactions are important for identifying the links of the planktonic food chains, as well as for interpreting seasonal changes in the structure and species composition of the aquatic community.Protozoans are likely to be an important alternative food resource for zooplankton, when phytoplankton abundance is low or when phytoplankton quality is reduced (i.e., during periods of nutrient limitation) (schmidt et al., 1998).the phytoplankton standing-stock in the ssc is similar to levels observed in other tropical coastal areas (eppley et al., 1978; webber et al., 1996) and to those reported by turner & tester (1989) and kleppel et al. (1996) in previous studies on the natural diet of copepods.
Because phytoplankton is available in the ssc, questions that arise are: why is S. zanclea predated on by such different zooplanktonic organisms, with different food habits and at high frequency?also, what would be the competitive advantage for the active capture of a species that is relatively large (> 300 µm) and rare in the environment?the search for additional food sources can occur when phytoplankton biomass falls to very low levels, or when drastic changes in phytoplankton composition occur (smetacek, 1980).the ssc is dominated by coastal water (cw: t > 20 °c; s > 36‰), but during spring and summer a distinct flow of nutrient-rich, high-salinity, low-temperature water (central water from the south atlantic, cwsa: t < 20 °c; s > 35‰) can be detected in the deepest layers.the cw is superficial, with oligotrophic characteristics, marked by the supremacy of flagellates, which comprise around 70% of the total number of organisms, and enriched seasonally by intrusion of the cwsa (Gianesella-Galvão et al., 1997).diatoms need high nutrient concentrations, and for this reason are scarce in oligotrophic waters.Because diatoms are the main food item ingested by copepods (turner, 1991), the quantitative dominance of flagellates in the ssc waters, also confirmed in the present investigation, may be the determining factor for incorporation of heterotrophic food sources, such as the protozoan S. zanclea, into the algal diet by the local zooplankton.

Fig. 1 -
Fig. 1são sebastião channel, at the northern coast of são Paulo state, and the position of the sampling points.

Fig. 5 -
Fig. 4abundance (upper) and composition (lower) of the phytoplankton in the são sebastião channel where fecal pellets of zooplankton were collected.