Feeding in a calcareous sponge: particle uptake by pseudopodia.
Sponges are some of the predominant benthic suspension feeders globally (Gili and Coma, 1998). Detailed in situ and in vitro studies have revealed feeding efficiencies in the range of 75%-99% on plankton 0.1-70 [micro]m (Reiswig, 1974, 1975; Pile et al., 1996, 1997; Ribes et al., 1999). Sponges are capable of ingesting a great variety of types and sizes of plankton, but generally with higher grazing efficiencies on plankton smaller than 10 [micro]m (Pile et al., 1996; Turon et al., 1997; Witte et al., 1997; Ribes et al., 1999). While concentration and particle size do affect retention ability (Huysecom et al., 1988; Duckworth et al., 2003), studies indicate that sponges can select nutritionally favorable cells (Frost, 1980). Two hypotheses have been proposed to explain particle selectivity: differential uptake, in which preferred particles are phagocytosed more "efficiently," and differential retention during digestion and egestion. From measurements of clearance rates of synthetic and natural particles, Francis and Poirrier (1986) concluded that since all particles were removed from the water equally, differential retention during digestion and egestion must explain selectivity. However, whether this can also explain the ability of sponge cells to differentiate between symbiotic bacteria and those in the ambient water, as demonstrated by Wilkinson et al. (1984), is not so clear. Few studies have addressed exactly how sponges take up food at the cellular level.
The sponge choanocyte is considered one of the few examples of a true sieve filter in metazoans (Riisgard and Larsen, 2001). A similar collar sieve is well known from choanoflagellates--colonial protozoans that strongly resemble the sponge choanocyte--which are now confirmed to be the sister group to sponges (Snell et al., 2001; Cavalier-Smith and Chao, 2003; King et al., 2003). The sponge choanocyte is said to be functionally identical to the choanoflagellate cell (Riisgard and Larsen, 2000): the beat of the flagellum draws water toward and through a collar of microvilli (Van Trigt, 1919; Kilian, 1952; Riisgard and Larsen, 2001; Pettitt et al., 2002). In choanoflagellates, suspended particles are retained on the microvilli when water is drawn through the collar (Fenchel, 1982, 1986; Andersen, 1989), and particles can be seen to be phagocytosed by pseudopodia arising from the base of the collar (Leadbeater and Morton, 1974). Sponge choanocytes are more difficult to observe in vivo, but studies that have examined particle uptake suggest that although choanocytes ingest India ink, carmine, bacteria, and latex microspheres, the collar microvilli do not stain with the dyes, nor are particles shown to be retained in great numbers on the collar microvilli (Van Trigt, 1919; Pourbaix, 1933; Schmidt, 1970; Willenz, 1980; Imsiecke, 1993).
Differences between the structure of sponge choanocytes and the choanoflagellate cell, and the fact that many choanocytes are arranged within single chambers in a sponge (Fig. 1), suggest that mechanisms of particle capture may differ. Choanoflagellates tend to have a short flagellum (2-3 times the height of the collar) and short, wide, flaring collars whose microvilli are more widely spaced apically than basally (0.7-0.1 [micro]m) (Fig. 1A) (Fjerdingstad, 1961b; Leadbeater, 1983; Fenchel, 1986; Orme et al., 2003), allowing relatively fast flow through the sieve (14-30 [micro]m [s.sup.-1]) (Fenchel, 1986; Larsen and Riisgard, 1994). In many choanoflagellates, microvilli contain a pair of microtubules that penetrate deep into the cell body (Leadbeader and Morton, 1974; Fenchel, 1982). In contrast, demosponges have long flagella (up to 6 times the height of the collar) and long narrow collars (Fjerdingstad, 1961a; Simpson, 1984; Langenbruch and Scalera-Liaci, 1986; Larsen and Riisgard 1994); collar microvilli are only 0.1 [micro]m or less apart, and water is estimated to flow through the sieve at 2-3 [micro]m [s.sup.-1] (Reiswig, 1975; Riisgard et al., 1993). Larsen and Riisgard (1994) argue that the long flagellum of sponges is adapted to provide a more powerful pump than the choanoflagellate cell, and that 80-100 such pumps in a single chamber are needed to create the pressure to overcome the resistance of the extensive sponge canal system. The narrow collar is deemed appropriate to fit all the cells into a chamber, and though the flow field around the collar is thought to differ from that around free-living choanoflagellates, it is presumed that the alignment of collars and their possible touching at the tips, or the presence of a mesh "strainer" linking the tips (see Weissenfels, 1992) ensures that flow passes through the collar slits (Larsen and Riisgard, 1994).
The above studies, measurements, and models concern flow within the chamber of demosponges, all of which have a leuconoid body plan with incurrent canals leading to numerous ovoid chambers, as illustrated in Figure 1B. A small number of sponges in the Calcarea have vastly different chambers. Calcareous sponges make up only 5% of all Porifera and are the only group with ascon (choanocyte-lined tubes) and sycon (long choanocyte-lined chambers branching off a central cavity) as well as leucon body plans. In the calcareous sponge Sycon coactum, water enters through pores 10-20 [micro]m apart into long chambers lined by several thousand pumping choanocytes (Fig. 1C). Furthermore, unlike those in demosponge collars, microvilli in Sycon are rarely
neat filaments. As described in early studies (Haeckel, 1872; Schulze, 1875; Duboscq and Tuzet, 1939), the collar microvilli in Sycon frequently form short, lumpy projections that are often partially fused together. Although periods of anoxia can cause abnormal shapes of the choanocyte and its collar, our examination concurs with the earlier studies which suggest that collars are inherently variable in morphology regardless of fixative or handling. Early researchers suggested that the morphology of the collar microvilli reflect the state of feeding of the cell (Bidder, 1892; Duboscq and Tuzet, 1939). No recent work has examined this question, and it remains unclear how these chambers and the choanocytes that line them function in filter feeding.
To determine how choanocyte collars in large syconoid chambers filter, we fed the sponges bacteria and three sizes of latex microspheres. Sponges were fed both in situ, by divers using scuba, and in vitro. The sponges were preserved at intervals after feeding, by instant immersion in a large volume of fixative, and processed for examination by scanning electron microscopy.
Materials and Methods
Specimens of the calcareous sponge Sycon coactum were fed latex beads (Molecular Probes, OR) and heat-killed Escherichia coli bacteria both in situ and in vitro at the Bamfield Marine Sciences Centre, Bamfield, Canada. Solutions of beads in three sizes (1.0, 0.5, and 1.0 [micro]m) were diluted to 1 X [10.sup.9] in seawater collected from 20-m depth and mixed with 10% bovine serum albumin to prevent clumping, according to the manufacturer's instructions. For in situ feeding, sponges attached to dock piles at 10-m depth were fed by scuba divers. The sponges were first enclosed in 4 X 8 cm resealable zipper-type plastic bags, and 2 ml of the solution of 1-[micro]m latex beads was injected into the bag, for a final concentration of 4 X [10.sup.7] [ml.sup.-1]. Bags were removed from the sponges after 10 min; after 20 min the sponges, together with some wood substrate, were carefully cut off the piles, slipped into plastic collection bags, and taken to the surface. Two sponges were fixed immediately (i.e., 30 min post-feeding) in a cocktail fixative consisting of 1% Os[O.sub.4], 2% glutaraldehyde in 0.45 mol [l.sup.-1] sodium acetate buffer pH 6.4, with 10% sucrose in the final mixture; the vial with fixative was kept cold until it was added to the sponges, and fixation was carried out on ice. The remaining sponges were suspended from the dock in mesh cages at 10-m depth and fixed at 2 and 6 h post-feeding as above. Sponges fed in vitro were slid, without removal from seawater, into 23-ml tubes of water from 10-m depth, and solutions of the three sizes of latex beads were added, to a final concentration of 1 X [10.sup.8] [ml.sup.-1]. Stock solutions of bacteria were more dilute; the final working concentration was 1 X [10.sup.4] [ml.sup.-1] in seawater. Two sponges from each treatment were fixed at 5, 10 and 20 minutes post-feeding. Sponges were not removed from seawater at any time before or during fixation.
Although in situ experiments were designed to cause the least disturbance to the sponge during particle uptake, the additional time involved with doing this experiment by scuba meant that the first fixation was considerably delayed after feeding. In vitro feeding allowed more control during addition of the "food" solutions, and sponges could be fixed soon after being exposed to particles. Sponges were maintained in cold seawater collected from 10-m depth, and particles were added within 10 min of collecting animals. Nevertheless, careful and minimal handling of all specimens during collecting, feeding, and fixation meant that the first animals were fixed some 5-10 min after particles were added. Controls were carried out to assess the effect of handling alone. In these experiments, after collection, sponges were knocked repeatedly for 5 min (while still submerged in seawater) prior to fixation, and others were fixed without the handling involved in adding beads or bacteria. To assess the possibility that the method of fixation affected cell morphology, five fixation protocols were used, and two types of anesthetics were applied in separate experiments (Eerkes-Medrano and Leys, 2006). To assess the effects of anoxia on sponge morphology, sponges were fixed after 30 min of sitting in cold still seawater.
After 4 h in fixative, pieces of sponge were rinsed twice in distilled water and decalcified in 5% ethylene diamine tetra-acetic acid (EDTA) disodium salt overnight. Decalcified pieces were dehydrated through a graded ethanol series and fractured in liquid nitrogen while in a vial of 100% ethanol. Fractured pieces were critical-point dried, mounted with nail polish on aluminum stubs, coated with gold, and viewed in a field emission scanning electron microscope at 5 kV.
Pieces of fractured choanocyte chambers were surveyed for interactions between particles and choanocytes. The number and type of interactions of particles with collars and the cell surfaces at each time point (5, 10, 20, 30 min, 1, 2, and 6 h post-feeding) were counted on 312 images.
The structure of choanocyte chambers and morphology of cells and particles were identical in sponges fed in situ and in vitro. Briefly, feeding chambers are finger-shaped cavities that radiate from a central atrial tube (Fig. 2A, B). Each chamber is carpeted by a single layer of cuboidal cells called choanocytes (Fig. 2C). About 10,000 choanocytes 3.5 [micro]m in diameter were estimated to line an average-sized chamber (450 [micro]m long, 100 [micro]m in diameter) (Fig. 2B, C). Ostia (incurrent pores) were 10-20 [micro]m apart in all specimens, with about 20 cells per ostia; however, few ostia were ever visible from the inside of the chamber (Fig. 2C, D). All the water vents out of the chamber through a single excurrent opening (the apopyle) into the atrial cavity (Fig. 2B).
At the apical surface each choanocyte has a 15-[micro]m-long flagellum that is surrounded by a ring of microvilli (Fig. 2E, F). The shape of collar microvilli was highly variable in all sponges. Some collar microvilli were long and thin (2-3 [micro]m long, 0.1 [micro]m in diameter) and individual microvilli were linked by fine filaments, leaving a space between microvilli [less than or equal to] 0.1 [micro]m (Fig. 2E), but many collars were short and their microvilli were thick ([less than or equal to]0.5 [micro]m long, up to 0.15 [micro]m thick). In other collars, microvilli were fused to one another (Fig. 2F). Experimentation with a variety of fixatives and techniques has shown that fixation procedure, excess handling, and periods of anoxia can greatly affect the shape of the choanocyte cell body, but it was not clear that collar structure was equally sensitive. In our feeding experiments we took great care to reduce excess handling and processing time, but we still found that the morphology of collars was inconsistent, even among neighboring choanocytes in the same chamber. However, all choanocytes in this experiment, regardless of collar shape, were readily capable of particle uptake.
Full details of the fine structure of Sycon coactum are presented elsewhere (Eerkes-Medrano and Leys, 2006).
Time of particle uptake
Choanocytes of all sponges fixed 5-10 min after being fed were already filled with phagosomes containing either beads or bacteria, suggesting that uptake occurs within only a few minutes of particles entering the animal. Yet, in specimens fixed up to 1 h after feeding, chambers still contained beads or bacteria. Many instances of phagocytosis were found in sponges fixed 2 h after feeding, but no beads or bacteria were found in sponges fixed 6 h after feeding. While the time of uptake of particles was independent of particle size or type, the location and method of uptake varied considerably with both particle size and type.
Location of particle uptake
Most particles were found in association with the cell surface, lodged between neighboring choanocytes, rather than on the collar microvilli (Fig. 3, Fig. 4A, C, D). Few large particles (1-[micro]m and 0.5-[micro]m beads and bacteria) were found adjacent to collars, except where particles were very dense. For example, in an image of 180 cells, only 1-2 beads were found adjacent to a collar (Fig. 4A, B). In regions where beads or bacteria were dense, the ratio of particles to collar was 1:4 (4 particles on the collars of 16 cells; e.g., Fig. 4C, D). Only 0.1-[micro]m latex beads were found in large numbers on collar microvilli, but these beads adhered even more often to the cell surface (Fig. 5A-C). A comparison of collar interactions with 1-[micro]m beads over time is shown in Figure 6. Those beads adherent on collars were found on the outside of the collar, and in a few instances one or several adjacent microvilli were wrapped around particles.
Mechanisms of particle uptake
Most 1.0- and 0.5-[micro]m beads and some bacteria were engulfed by broad lamellipodia at the cell surface (Fig. 7, 8, 9). More particles were engulfed at the side of the cell than at its apical surface, but the number of interactions of particles with the apical surface increased with time (Fig. 8C).
Many cells also produced extensions of the cell surface that reached several micrometers out from the lateral or apical surface to contact beads or bacteria (Figs. 8, 10). The average length of extensions was twice that of the collar microvilli (membrane extension: 2.7 [+ or -] 1.6 [micro]m, range 0.5-16 [micro]m; collar length: 1.2 [+ or -] 0.5 [micro]m, 0.4-3.5 [micro]m, n = 44) (Fig. 8B). The longest extensions were found in contact with aggregates of a mucoid material, but in pieces fixed 30 min after being fed in situ, several 7-[micro]m-long extensions were found wrapped around clumps of 1-[micro]m latex beads (Fig. 10A-E). A total of 30 lateral and 12 apical extensions were counted in 44 images; 9 extensions were directed into the chamber, while 33 contacted particles that were between choanocytes. In two cases the extensions were clearly formed by fusion of collar microvilli (e.g., Fig. 10C). Extensions for 0.5-[micro]m beads typically involved more than one bead (Fig. 10F), and often two or more cells were in contact with the same particle (Fig. 10F, G). Although extensions did contact natural bacteria (Fig. 9A), fewer extensions touched heat-killed bacteria than latex beads or natural particles. Some heat-killed E. coli were engulfed with a lamellipodium (Fig. 9B), but in most instances the bacteria appeared to simply sink into the cell (Fig. 9C).
Only large particles (large beads and bacteria) were found in contact with flagella (24 instances observed). No pseudopodial extensions were found with 0.1-[micro]m latex beads.
Uptake of natural food
The validity of the varied mechanisms of particle uptake we introduced experimentally is substantiated by examples of natural uptake of intact or broken diatoms (2-3 [micro]m) by five separate choanocytes (Fig. 9D, E). Diatoms were always ingested at the apical surface of the cell, as were aggregates of mucous-bound material (2-3 [micro]m in diameter) (Fig. 9F).
Sycon coactum began to phagocytose particles immediately after they were introduced and uptake continued for at least 2 h after the initial feeding. All sizes and shapes of particles were ingested, and all choanocytes took up particles regardless of size or shape of collar microvilli. However, the mechanism of uptake varied with the size and type of particle. Only the finest particles (0.1-[micro]m beads) adhered to collar microvilli in great numbers. Most particles contacted the cell surface, and larger particles and clumps of particles were attached to extensions of the cell surface. The longest extensions were produced 30-60 min after particles were introduced to the sponges. The results suggest that although the collar of syconoid sponges may entrain flow around the surface of the choanocyte, uptake of particles greater than 0.1 [micro]m is independent of the collar.
The collar as a filter
Sponges are generally thought to filter with a true collar sieve (Riisgard and Larsen, 2000, 2001), yet in reality we know very little about the means by which particles are brought to, and interact with the collar microvilli, or how they are engulfed by the cell. To the best of our knowledge there are only two examples of sponge filters in which water is forced by physical barriers to pass through the collar microvilli. In the glass sponges Oopsacas minuta and Rhabdocalyptus dawsoni, particles are thought to be trapped at the collar microvilli by a thin layer of the syncytial trabecular reticulum that surrounds the distal region of each collar (Perez, 1996; Wyeth, 1999). The freshwater demosponge Spongilla lacustris has a similar feature made of a glycocalyx mesh that lies between the tips of adjacent collars and is thought to function like a strainer, forcing water through the collar microvilli (Weissenfels, 1992). In other sponges, no such barrier seems to exist, unless fixation techniques do not preserve it, and chambers could be interpreted as being leaky. In Sycon there is no membrane or protein mesh that links the tips of adjacent collar microvilli, yet all choanocytes were still capable of taking up particles. However, most particles were phagocytosed at the base of the collar; only 0.1-[micro]m beads were found adhering to collars in substantial numbers, but these were even more numerous below the collars on the cell surface. Interestingly, those 0.1-[micro]m beads that were attached to the collar were not retained by the fine mesh of the filter pores as would be expected for a true sieve filter (Rubenstein and Koehl, 1977); instead, they adhered to the outside of individual microvilli. Adhesion to the filter surface is not considered to occur in a true sieve (LaBarbera, 1984), but particles are also said to adhere to collars in choanoflagellates, which allows them to be transported down toward the surface of the cell (Lapage, 1925). The large numbers of 0.1-[micro]m beads found at the base of collars 20 mins after feeding suggests that membrane transport of adherent particles may also occur down microvilli in Sycon.
It is generally imagined that particles such as bacteria that are caught on the collar microvilli, if not transported to the base of the collar by membrane transport, are phagocytosed by pseudopodial extensions that grasp them off the collar. However, in 312 images of more than 50 specimens, we did not find one example of this. Pesudopodia were involved in feeding, but they did not phagocytose particles attached to the collar. We found that most large particles (bacteria and 0.5-1.0-[micro]m beads) were engulfed either directly by the cell surface or with pseudopodial extensions, rather than the collar.
Phagocytosis by pseudopodial extensions
Phagocytosis of larger particles by pseudopodia has been shown to occur in many demosponges and is not in itself unusual (Schmidt, 1970; Willenz, 1980; Imsiecke, 1993). Pinacocytes can phagocytose particles as large as 5.7 [micro]m on the surface of the sponge (Willenz and Van de Vyver, 1982), and it is generally assumed that the pinacocyte epithelium that lines canals can take up particles as large as 50 [micro]m (Weissenfels, 1992). Phagocytosis of particles at the apical surface of choanocytes is less common, since demosponge collars do not appear to allow large items within the collar microvilli. An exception may be the periflagellar sleeve of Suberites massa, a permanent sheath-like extension of the choanocyte surface (Connes et al., 1971) that is thought to be involved in feeding (Simpson, 1984).
What is unusual and intriguing, however, is the manner in which the choanocyte surface in Sycon coactum extends laterally and apically to phagocytose the latex microspheres or bacteria, and the observation that often several cells extend pseudopodia apparently for the same particle (e.g., Fig. 10G). Some pseudopodia are up to twice the height of the collar microvilli and contact a group of latex beads or piece of mucoid material well above the collar microvilli. The images capture a remarkable feat of the cell membranes. Not only can large amounts of membrane be generated over a relatively short time (lateral extensions 1-2 [micro]m long were present in sponges fixed 5-10 min after being fed), but in some manner the cells must sense the particles even through they are several micrometers away from the cell surface; how this is achieved is unknown. The presence of membrane extensions 5 min after feeding indicates that the sponges are relatively quick to detect food particles. However, the increase in both collar and surface interactions for 1.0-[micro]m beads over time shows that it takes time before the entire population of choanocytes in the chamber becomes involved in phagocytosis of particles.
Given the high volumetric pumping rates recorded from demosponges (Reiswig, 1971; Vogel, 1974), it is unknown how particles remain stationary long enough in the choanocyte chamber of Sycon to be phagocytosed by pseudopodial extensions. Several possibilities must be considered. First, could sponges have arrested their feeding current or become clogged shortly after the first beads entered the chambers? Only glass sponges (class Hexactinellida) are known to be able to arrest flagella in response to mechanical stimuli (see review by Leys and Meech, 2006). Although in Sycon the ostia close in response to excessive handling (as was seen in the controls used to assess the effects of handling), in sponges fixed during feeding experiments no ostia were closed or constricted or blocked by excess particles; thus it is unlikely that flow ceased at any point during feeding. Second, was the concentration of particles abnormally high, causing an aberrant mechanism of uptake? In fact, the concentration of bacteria fed to the sponges was 2-fold lower than in the sponge's natural environment (in July 2004, 1 X [10.sup.6] [ml.sup.-1] bacteria were counted in the Bamfield Marine Science Centre seawater system in water drawn from 25 m; Yahel et al., 2006). We used a concentration of latex beads similar to that used in previous in vitro feeding studies of sponges (Willenz, 1980; Willenz and Van de Vyver, 1982; Willenz et al., 1986), but despite attempts to disperse the particles evenly, the distribution of both beads and bacteria was very patchy in any one sponge and even within a single chamber. While one region of a chamber was filled with particles, an adjacent region had only a few particles. Although we cannot rule out the possibility that exposure to high concentrations of particles could cause abnormal feeding behavior, choanocytes took up particles in a similar manner whether particles were sparse or dense. Third, could the particles (beads and bacteria) have fallen to their present locations after flow stopped in the sponge at the moment of fixation? Although this would be difficult to disprove, the presence of lamellipodia wrapped around portions of the beads (in the same way that the lamellipodia appeared to engulf diatoms, e.g., Fig. 9D) suggests the contact was not one of chance. Furthermore, most 0.1 -[micro]m beads did not drop off the collar microvilli at fixation, nor did some 1-[micro]m beads that were found on collars (e.g., Fig. 4A,B).
The possibility that pseudopodia are used to reject or eject unwanted particles has also been considered. However, we found clear examples of egestion in samples fixed 6 h after being fed. During egestion, choanocytes lose normal morphology, become ameboid-shaped, and move up into the chamber with waste beads and broken diatom pieces. Exactly how this debris is ejected from the cell once in the chamber is not known.
The inference of living processes from static images is definitely problematic, and these difficulties will remain until a mechanism is developed to view live chambers in the process of feeding. However, consistent results with a variety of fixatives (see Eerkes-Medrano and Leys, 2006), and controls for handling suggest that phagocytosis by pseudopodia is not an artifact. Furthermore, excellent images of phagocytosis by pseudopodia in the choanoflagellate Codosiga (Leadbeater and Morton, 1974) show that choanflagellates can generate equally long membrane extensions; similar pseudopodial extensions were described by de Saedeleer (1929), who also first suggested that calcareous sponges fed much like choanoflagellates. In Codosiga it is suggested that the pseudopodia use the collar microvilli as a guide to locate the bacteria caught on the collar. In Sycon very few particles seemed to be caught on the collar, and some pseudopodia extended up from the apical surface of the cell, so it is not clear exactly how the collar is used in particle capture and uptake. One possibility is that the collar, rather than strictly sieving, may entrain the flow, drawing particles into an area of sluggish water at its base. Nevertheless, if the images are a correct representation of events frozen in time, then the fact that the largest extensions were observed in contact with natural food items suggests that the sponge may use pseudopodia to select the larger particles. If these particles (such as diatoms) are more nutritious, this mechanism could explain the particle selectivity found by Frost (1980).
Although feeding by long pseudopodia may be more common in demosponges than is generally thought, the images presented here that show uptake of whole diatoms and mucoid-coated material by choanocytes indicate that in syconoid chambers the technique has been perfected. This type of sponge may rely more on active capture of large food items than on passive filtering using the collar, to select nutritionally favorable items when food is abundant (e.g., during a summer diatom bloom). This mechanism would not be effective when food is scarce in winter, which may explain the ephemeral summer existence of this sponge.
We thank A.R. Palmer for comments on an earlier version of this manuscript, G. Braybrook for technical assistance with SEM, and the director and staff of the Bamfield Marine Sciences Centre where portions of this work were conducted. Funding was provided by grants from NSERC and Alberta Ingenuity to SPL.
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Yahel, G., F. Whitney, H. M. Reiswig, D. I. Eerkes-Medrano, and S. P. Leys. 2006. In situ feeding and metabolism of glass sponges (Hexactinellida, Porifera) studied in a deep temperate fjord with a remotely operated submersible. Limnol. Oceanogr. (In press).
SALLY P. LEYS (1) AND DAFNE I. EERKES-MEDRANO (2)
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9
Received 16 September 2005; accepted 12 June 2006.
(1) To whom correspondence should be addressed. E-mail: email@example.com
(2) Present address: Department of Zoology, Oregon State University, Corvallis, OR 97331.
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|Author:||Leys, Sally P.; Eerkes-Medrano, Dafne I.|
|Publication:||The Biological Bulletin|
|Date:||Oct 1, 2006|
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