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Polar body formation in Spisula oocytes: function of the peripheral aster.

Introduction

Polar body (PB) formation is common to all sexually reproducing animals, but many details of this critical process and its products remain to be elucidated. We do not know the extent to which the mechanisms involved in generating such extreme asymmetry resemble those of normal somatic cell division, or whether these mechanisms are the same in all animals. Moreover, demonstrations of such fundamental features as PB division (Saiki and Hamaguchi, 1997), the PB F-actin ring pattern (Pielak et al., 2003, 2004), Aurora kinase involvement in PB extrusion (Schumacher et al., 1998; Castro et al., 2003), and cytoskeletal differences between PB1 and PB2 (Alliegro and Alliegro, 2005) are relatively recent.

This paper addresses one major step in PB formation: deposition of the F-actin ring involved in PB extrusion. For this work we used Spisula solidissima, the large "surf clam" found in the eastern United States--an organism whose commercial importance (Weinberg and Helser, 1996) is more than matched by its value in biomedical research. Spisula is a prodigious gamete producer, and ripe animals retain viable eggs and sperm for several months if maintained at appropriate temperatures. Shed oocytes can be kept viable for at least 2 days (Hunt et al., 1992), remaining at the germinal vesicle stage--prophase of meiosis I--until activated by sperm, KCl, or other agents (Allen, 1953; Longo et al., 1991). Activated oocytes proceed through meiotic stages in near synchrony, including germinal vesicle breakdown (GVBD), formation of the metaphase I meiotic apparatus, translocation of the metaphase I spindle to the cortex, completion of meiosis I with extrusion of PB1, formation of the meiosis II spindle, and completion of meiosis II with PB2 extrusion. This entire sequence occurs in about 30 min under laboratory conditions (Allen, 1953) and is followed, in sperm-activated oocytes, by nuclear fusion and embryogenesis. Thus, Spisula gametes have served in studies of the meiotic cell cycle (Swenson et al., 1987; Hunt et al., 1992; Katsu et al., 1999; Yi et al., 2002; Dube and Eckberg, 1997) and in related work on the organization, function, molecular composition, and biogenesis of centrosomes (Wu and Palazzo, 1999; Schnackenberg et al., 2000), meiotic apparatus (Suprenant and Rebhun, 1984; Haimo, 1985; Kuriyama et al., 1986; Suddith et al., 2001), nuclear envelope (Maul et al., 1987; Dessev and Goldman, 1988; Dessev et al., 1989), and PBs (Longo and Anderson, 1970; Pielak et al., 2003, 2004; Alliegro and Alliegro, 2005). One deficiency recognized in recent years, a lack of genetic data for the species, is currently being addressed (Marine Biological Laboratory, 2003).

In previous work on cytoskeletal events during PB formation in Spisula (Pielak et al., 2003, 2004), we found that the metaphase I peripheral aster spread onto the cortex in a circular pattern containing relatively few central microtubules. This aster disassembled during anaphase, and a ring of thickened F-actin appeared that had essentially the same cortical location, dimensions, and pattern as did microtubules in the earlier aster. The late anaphase spindle remained docked at the F-actin-poor center of the ring (the "ring opening"), where PB extrusion then occurred. The observed spatial correspondence immediately suggested that microtubules of the peripheral aster are a causal agent in generation of the PB F-actin ring.

The present studies were designed to test this hypothesis by altering the number of peripheral asters and their cortical contact patterns prior to PB formation. Two methods were employed. First, exposure to lovastatin (LV), followed by washout, induced both metaphase arrest (Turner et al., 1995) and multiple asters under our conditions, an effect modified and enhanced by hexylene glycol (HG; Rebhun and Sawada, 1969; Saiki and Hamaguchi, 1997). Second, cytochalasin D (CD) was used to block PB1 extrusion, with extra asters being generated following washout prior to meiosis II (Longo, 1972; Hertzler, 2002). Both approaches gave strikingly consistent positive results, producing multiple peripheral asters associated with multiple, overlapping F-actin rings.

Materials and Methods

Ripe individuals of Spisula solidissima (Dillwyn, 1817), maintained at 11-13[degrees]C to inhibit spawning, were obtained from the Aquatic Resources Division of the Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts. Gonadal tissue was removed by dissection and suspended in about 150-300 ml of filtered (Whatman #2) natural seawater (F-NSW) depending on gonad size. After tissue removal by filtration through a double cheesecloth layer, the egg suspension passing through was allowed to settle ([approximately equal to]20-30 min), the supernate was removed, and the eggs were washed twice more by resuspension and settling. After determining approximate total egg volume by gentle hand-centrifugation, final resuspension was in F-NSW refiltered at 0.45-[micro]m (= double-filtered, or DF-NSW) to a concentration of about 1% (v/v). Eggs were stored at this concentration in DF-NSW containing 50 [micro]g/ml gentamycin (Hunt et al., 1992), maintained in suspension at 16[degrees]C in a rocking water bath. Such eggs retained the capacity for normal PB formation and (when sperm-activated) normal cleavage, but were used for 2 days only because, beyond that, increased time to PB formation suggested some deterioration.

Spisula oocytes were activated by using excess KCl, a standard method for this species (Allen, 1953; Costello and Henley, 1971). Eggs were gently hand-centrifuged and resuspended to 10% (v/v) in DF-NSW, and one volume of suspension was mixed with 7.6 vol. DF-NSW and 1.4 vol. 0.5 M KCl (t = 0). Eggs were monitored in phase contrast for GVBD, sedimented gently, resuspended in DF-NSW or experimental media to about 1% (v/v), and maintained in suspension by gentle rocking on a nutator at 22[degrees]C. Samples were treated as described in particular experiments and then prepared for fluorescence labeling and microscopy (see below). The timing of stages became slightly extended with longer-term storage of ripe animals in cold seawater, and this was adjusted for by monitoring a range of time-point samples in each preparation.

Fluorescence labeling and microscopy

For rapid monitoring of chromosomal stage, oocytes were routinely fixed in seawater containing 2% or 4% formaldehyde (equally effective), stained with DAPI (3 or 6 [micro]M), and examined and photographed using a standard Zeiss epifluorescence microscope. For cytoskeletal labeling, oocytes were simultaneously permeabilized and fixed for 15 min in PEM (100 mM PIPES, 5 mM EGTA, 1 mM Mg[Cl.sub.2], pH 6.8, using NaOH) containing 0.6% Brij-58 and 2% formaldehyde, then washed twice in phosphate-buffered saline (PBS). Labeling was performed in PBS using the following agents: for F-actin, Alexa Fluor 568-phalloidin (Molecular Probes); for microtubules, a mixture of mouse monoclonal anti-[alpha] and anti-[beta] tubulins (Sigma T9026, T-4026) at their recommended working dilutions, pretagged with Alexa Fluor 488-labeled anti-mouse Fab fragments (Zenon; Molecular Probes); for DNA, DAPI. Labeling protocols were as described in prior work, without vitelline membrane removal (Lee et al., 2002; Pielak et al., 2003, 2004). Samples were viewed both in a standard Zeiss epifluorescence microscope and by confocal fluorescence microscopy using the Zeiss Laser Scanning System LSM 5 PASCAL, with image processing via Zeiss LSM Image Examiner (2004) and Adobe Photoshop (vers. 3.0) software. Aster or ring diameters were measured on images of optimum clarity, using Zeiss Image Examiner software. Relative pixel intensity was measured on the original 3-D reconstructed image in face view, using the Scion Image PC version of NIH-Image software (beta vers. 4.0.2).

Treatment with aster-perturbing agents

Following activation, Spisula oocytes proceed continuously from GVBD through meiosis and the extrusion of two PBs. Therefore, we tested lovastatin (LV; Calbiochem) as a possible means to provide a time window for addition of aster-perturbing agents such as hexylene glycol (HG) that require oocyte penetration. LV, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, induces reversible metaphase I arrest of KCl-activated Spisula oocytes (Turner et al., 1995). Using DF-NSW as the primary medium, as for other experimental agents and solvent controls described below, activated oocytes were incubated in LV at 3.5 [micro]M, 35 [micro]M, 52.5 [micro]M, and 70 [micro]M. Under our conditions, metaphase stability was most repeatable at 52.5 [micro]M (1.5 X conc. used by Turner et al., 1995), and this concentration was used routinely at about 12 min post-activation, when metaphase spindles were still centrally located and en route to the cortex. Solvent-only controls were exposed to 0.15% DMSO in DF-NSW. Oocyte samples were processed for fluorescence microscopy after various LV incubation periods before washout and during meiotic resumption after two washes in DF-NSW.

Hexylene glycol (HG, = 2-methyl-2,4-pentanediol; Sigma) reversibly increases aster size and stability (Spisula: Rebhun and Sawada, 1969; starfish: Saiki and Hamaguchi, 1997). During LV-induced metaphase time windows, HG (1% v/v) was added either "early" at t = 20 min post-activation, with t = 25 min washout (5 min HG incubation), or "late" at t = 25 or 30 min, with washout at 35 or 42 min, respectively (10 or 12 min HG incubation). Both late addition times gave the same results, so that HG use is described below as either early or late. For all experiments with LV plus HG, maximum oocyte exposure to LV was thus 30 min.

As in previous work (Pielak et al., 2004), cytochalasin D (CD; Calbiochem) was used to inhibit F-actin ring assembly prior to PB1 formation. Eggs were sedimented gently and resuspended in 1.0 [micro]g/ml CD or 0.1% DMSO (solvent control) at about 15 min post-activation, when metaphase spindles were still in transit to the cortex. Various times for initiating CD washout were then tested to determine which would block extrusion of PB1 while permitting that of PB2. Washout when solvent-only controls were still in metaphase ([approximately equal to]20 min) was too early, allowing sequential formation of both PBs. Washout when controls were at late anaphase ([approximately equal to]27-30 min) was too late; no PBs formed, and oocytes contained four pronuclei. However, washout with controls at anaphase onset ([approximately equal to]25 min post-activation) permitted subsequent PB2 formation and was used in the experiments.

Results

F-actin ring formation in untreated oocytes

In the normal cytoskeletal sequence that precedes PB formation, the metaphase meiotic spindle moved toward the egg surface, the peripheral aster spread fountain-like onto the oocyte cortex, and subsequently in anaphase, most astral microtubules disassembled (Fig. 1A-C). The F-actin ring appeared in the cortical location of the previously spread aster, with similar outer diameter ([approximately equal to]20-22 [micro]m), and with the spindle docked at the center of the F-actin-deficient ring opening (Fig. 1C, D). Rotated 3-D reconstructions of cortical F-actin from optical section stacks revealed the circular pattern of the ring, with chromosomes positioned in the ring opening (Fig. 1E).

The normal pattern of F-actin deposition was also observed in solvent-only controls (Fig. 1F, G), again with an outer ring diameter of about 21-22 [micro]m, as measured in optical sections of three rings. Thus, for comparison with the normal pattern, altered cortical F-actin deposition is described throughout this paper in terms of the "ring" and "ring opening." As examined in scans of pixel intensity across the ring, cortical F-actin was most dense within the ring, and the level in the ring opening resembled that of surrounding cortex (Fig. 1H).

Lovastatin-induced metaphase arrest, followed by washout

Lovastatin (LV) was initially tested to determine its utility in providing a time window for administration of hexylene glycol (HG) by means of metaphase arrest. First, we confirmed LV-induced metaphase arrest (Fig. 2A, B), as well as PB production in about 90% of oocytes after 30 min arrest and washout (Fig. 2C, D). However, we were surprised to discover that, under our conditions, LV-induced metaphase arrest lasted only about 30 min, after which anaphase began even without washout. More importantly for our objectives, most of the LV-treated oocytes displayed multiple asters with interconnected spindles (Fig. 2E). In a majority of oocytes, only one of these asters was in contact with the cortex; but in about 15%-20%, the meiotic apparatus was oriented such that two or three asters touched the cortex. In anaphase, such oocytes contained adjacent docked spindles (Fig. 2F) that were associated either with a single enlarged, circular or avoid F-actin ring (>30 [micro]m; Fig. 2G) or with double F-actin rings (Fig. 2H-J). Each ring opening was occupied by a spindle centrosomal region (e.g., Fig. 2H, J), and dual ring openings shared a strip of thickened F-actin between them that varied in width in different oocytes (e.g., Fig. 2H-J).

Hexylene glycol addition during lovastatin metaphase arrest

Given the LV results, we tested HG addition during LV-induced time windows to determine whether it would increase astral cortical contact, enhancing or modifying the formation of multiple rings (Fig. 3). Astral spreading occurred during metaphase, as previously (Fig. 3A), and multiple spreading asters were observed (Fig. 3B). Two centrosomes were clearly present at the docking pole of some metaphase or early anaphase spindles (Fig. 3C). Single enlarged rings were most evident when HG was added "late" (Fig. 3D, E), whereas "early" HG addition produced two or more peripheral asters and multiple rings (Fig. 3F-K). Multiple asters and rings were observed more frequently when HG was used in combination with LV than when LV was used alone.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURES 3 & 4 OMITTED]

In some of these multiple-ring oocytes, F-actin stained far more intensely in the region between ring openings than in the surrounding area (Fig. 3G, H; confirmed by pixel intensity scanning). When two cortex-associated asters were adjacent and a third more distant, the result was one larger ring opening and one smaller one (Fig. 3F, G). However, if centrosomes of three peripherally spread asters were sufficiently separated, three ring openings appeared within a very large region of cortical F-actin deposition (Fig. 3I-K). Despite the unusual ring patterns, about 80%-90% of the oocytes treated with LV plus HG in different preparations ultimately produced PBs after washout, many of which appeared to be larger than control PBs (not shown).

[FIGURE 5 OMITTED]

In a minor sub-population of LV-treated oocytes, HG produced single anaphase spindles that were parallel to the cortex, a highly unusual orientation for Spisula (Fig. 4). Both centrosomal regions were docked in these oocytes, and two ring openings appeared within a common area of F-actin deposition (Fig. 4A). F-actin deposition was typically intensified between the ring openings (Fig. 4A, C, D), as in some of the multi-aster contact patterns (e.g., Fig. 3G).

Cytochalasin D inhibition of polar body formation, followed by washout

In previous work on shrimp oocytes, cytochalasin blockage of PB1 formation, followed by washout, led to co-production of two meiotic spindles and two PBs at the time of PB2 formation in controls (Hertzler, 2002). Since this was a potential means to generate two asters in simultaneous cortical contact, and perhaps unusual contact patterns, we tested the approach on Spisula oocytes. Cytochalasin effects on Spisula PB formation had been investigated earlier, but without such washout (Longo, 1972; Pielak et al., 2004).

Washout of cytochalasin D (CD) (1.0 [micro]g/ml), with controls in early anaphase, was effective in permitting the treated oocytes to proceed into meiosis II. Subsequently, when most controls exhibited a typical metaphase II spindle docked at the cortex, nearly all experimental oocytes contained two or more cortically spreading asters and interconnected metaphase spindles (Fig. 5A, B). Nevertheless, two PBs were ultimately produced at about the same time in these oocytes (Fig. 5C, D). At intermediate anaphase stages, double F-actin rings usually formed, with the two centrosomal regions of the spindles and asters occupying the ring openings (Fig. 5E-K). The dual spindles were sometimes connected at an internal centrosome and docked cortically at an angle to each other (Fig. 5H-K). In several cases, however, two docked anaphase spindles were associated with a single, very large, ovoid F-actin ring (Fig. 5L-N), with both centrosomes occupying the ring center.

Discussion

In the normal cytoskeletal sequence that precedes formation of the polar body (PB) in Spisula oocytes, cortical spreading of the peripheral aster at metaphase and cortical docking of the spindle are followed, during anaphase, by disassembly of astral microtubules and formation of the F-actin ring (Fig. 1). The diameters of both aster and ring are typically about 20-22 [micro]m, and the actin-poor center of the ring is similar in dimensions to the microtubule-deficient central region of the earlier spreading aster (Pielak et al., 2003, 2004). These observations suggest that spatial features of PB ring formation are determined by the peripheral aster.

Spatiotemporal relationship between spreading peripheral asters and cortical F-actin rings

In the present work we asked whether altering the number and distribution of peripheral asters would produce corresponding changes in the number and pattern of cortical F-actin rings. The answer was clear: treatment with lovastatin (LV) alone, with LV plus hexylene glycol (HG), or with cytochalasin D (CD), followed by washout, all produced oocytes with multiple cortically spreading peripheral asters (e.g., Figs. 2E; 3B, I; 5E). Subsequently, many of these oocytes produced either a single, abnormally enlarged circular or ovoid ring (>30 [micro]m diameter; e.g., Figs. 2G; 3D, E; 5 N), or multiple overlapping rings (Figs. 2H-J; 3F-K; 5E-K). Single enlarged rings appeared to be generated when centrosomes were in close proximity (e.g., Fig. 2F, G), and this was correlated with considerable increase in astral spreading diameter (long axis of [approximately equal to]40 [micro]m; Fig. 2E). However, it remains to be determined whether both increased microtubule density and astral spreading diameter are critical for cortical F-actin patterning. F-actin thickening between ring openings could be an initial stage of ring biogenesis occurring where overlap of astral microtubules provides the greatest density (e.g., Fig. 3G, H; Fig. 4C, D). Alternatively, thickening could be the result of incomplete cortical contact and spreading by the experimentally altered asters, or a symptom of an additive or even synergistic effect of astral overlap.

With respect to the temporal relationship, F-actin rings were observed in normal oocytes only in anaphase (Fig. 1). Oocytes exposed to LV, or LV plus HG, completed astral spreading and spindle docking, but were arrested at metaphase due to blockage of the degradation of Spisula oocyte cyclin A and B (Hunt et al., 1992; Turner et al., 1995). F-actin rings were not generated unless the drugs were washed out, permitting entry into anaphase (Fig. 2F; 4A). In addition, after CD-washout, multiple F-actin rings were observed only in anaphase cells (Fig. 5A, B vs. 5E, F). Therefore, both astral contact and anaphase-triggered events must determine biogenesis of the F-actin ring for Spisula PB formation. This is consistent with the importance of anaphase entry for somatic furrowing, as evident in early work on echinoderm zygotes whose asters had been removed or disrupted (Cornman and Comman, 1951; Swann and Mitchison, 1953; Mitchison, 1953; Scott, 1960), and in later studies on the appearance of actin filament bundles in the cortex (Mabuchi, 1994).

Function of asters versus centrosomal regions

The experimentally generated rings exhibited various patterns, most of which can be accounted for by varying degrees of ring overlap following a stimulus from the astral microtubules (Fig. 6A-E). However, we observed no ring openings "filled in" by F-actin from an adjacent ring (Fig. 6F, G). This cannot be explained simply by stimulus reduction caused by low microtubule density at adjacent astral centers, and therefore we suggest that, in contrast to astral microtubules, docked centrosomes may inhibit F-actin deposition. Such a mechanism would ensure reduction of mechanical resistance in ring openings, the cortical site of PB extrusion. Adjacent centrosomes would then produce one enlarged ring opening (e.g., Figs. 2G; 3G; 5N); if the centrosomes were further apart, multiple ring openings would be surrounded by F-actin from overlapping rings (e.g., Fig. 2H-J; 3F-K; 5G). The centrosomes of spindles abnormally docked parallel to the cortex would be separated by the spindle itself, generating separate ring openings (Fig. 4A-C). While we find no published evidence for or against such speculation, it is worth noting that centrosomes isolated from Spisula oocytes, though too small ([approximately equal to]2 [micro]m diameter) for simple obstruction of ring openings, contain a great many proteins of unknown function (Palazzo and Vogel, 1999).

In the androgenetic bivalve Corbicula, two PBs are produced naturally after simultaneous spreading of two metaphase I asters. Here, the metaphase I spindle always aligns parallel to the oocyte surface, and both asters spread cortically prior to dual PB extrusion (Komaru et al., 2000). This differs from CD-washout, in which dual anaphase II spindles appear nearly end-on to the cortex (Fig. 5H), but it resembles the parallel spindle-cortex alignment in oocytes treated with LV plus HG, generating dual overlapping rings (Fig. 4). The actual F-actin ring pattern in Corbicula has not yet been determined, however.

[FIGURE 6 OMITTED]

Mechanisms involved in multiple aster and ring generation

Multiple and enlarged asters and rings occurred after treatment and washout using LV, LV plus HG, or CD (Figs. 2-5). Since different means produced the same general result, multiple ring formation is aster-related, not drug-related. Multiple asters were expected for treatment with CD but not with LV; the latter may reflect use at a somewhat higher concentration and temperature than in earlier work (Turner et al., 1995). Although knowledge of the specific mechanisms that produce multiple asters is not critical to present conclusions, we believe centrosome replication to be responsible in both cases. Typical meiotic events continue during CD inhibition of ring and PB1 formation in Spisula (Pielak et al., 2004), and, in the case of LV, centrosome replication and separation apparently proceed during the metaphase block or shortly after washout (Fig. 3C).

Since astral perturbation produced marked pattern alterations, the biogenetic signal or mechanism for F-actin deposition must be recognized indiscriminately by the Spisula oocyte cortex (Fig. 7). In other species, such ring induction would account for PB formation at different cortical sites after experimental translocation of the meiotic apparatus (Hamaguchi et al., 2001), and for an indiscriminate furrowing response in somatic cytokinesis (Kawamura, 1960; Rappaport, 1985). In normal Spisula oocytes, the circular cortical F-actin ring and ring opening occupy nearly 40% of oocyte diameter before PB extrusion (Fig. 1E), but we do not yet know whether other invertebrates exhibit this ring pattern and size (Shimizu, 1990; Hamaguchi et al., 2001).

Do astral microtubules simply provide signals marking cortical regions, or do they deliver F-actin and other ring components? This question remains open both for Spisula oocytes and for PB formation. In several other systems there is evidence that microtubules determine F-actin distribution and furrow position (grasshopper spermatocytes; Alsop and Zhang, 2004), addition of new furrow membrane (sea urchin zygotes: Shuster and Burgess, 2002), and furrow induction via transported proteins (mammalian cells: Canman et al., 2003); in addition, direct actin-microtubule interaction has been reported in Drosophila embryos and in vitro (Sider et al., 1999; Foe et al., 2000; Waterman-Storer et al., 2000). With respect to PB ring signaling in Spisula oocytes, Rho is a good candidate for study. Rho family GTPases are believed to determine properties of F-actin rings involved in somatic cytokinesis and oocyte wound-healing (Mabuchi et al., 1993; Nishimura et al., 1998; Saint and Somers, 2003; Somers and Saint, 2003; Yonemura et al., 2004; Benink and Bement, 2005).

[FIGURE 7 OMITTED]

Regardless of molecular mechanism, peripheral asters clearly function to induce F-actin rings associated with PB extrusion in Spisula. Thus, at the cellular level at least, PB biogenesis in Spisula differs from that in mammals. The mammalian meiosis I spindle has truncated asters (Szollosi et al., 1972), and cortical spindle positioning involves actin filaments and formin-2 rather than astral microtubules (Leader et al., 2002; Maro and Verlhac, 2002). Comparison of the pathways to PB formation taken by mammalian, non-mammalian vertebrate, and invertebrate oocytes would be of considerable interest, and Spisula provides an excellent model system for such work.

Note added in proof: Evidence for RhoA involvement in establishing the polar body F-actin ring in Xenopus, as well as somatic contractile rings in echinoderms, is provided in a new paper by Bement et al. (2005).

Acknowledgments

We are indebted to Louie Kerr of the MBL and Rudi Rottenfusser of Carl Zeiss Inc. for the excellent confocal microscopy facilities, and to the Howard Hughes Medical Institute Undergraduate Science Education Program in Biology (HHMI 5200267), the Hunter College Avon-Tukman Fund, NSF 9808368, and PSC-CUNY 65218, for support.

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RAFAL M. PIELAK, CHRISTOPHER HAWKINS, AUNG PYIE, JENNIFER BAUTISTA, KYENG-GEA LEE, AND WILLIAM D. COHEN*

Department of Biological Sciences, Hunter College, New York, New York, and the Marine Biological Laboratory, Woods Hole, Massachusetts

Received 18 March 2005; accepted 16 May 2005.

* To whom correspondence should be addressed. E-mail: cohen@genectr.hunter.cuny.edu or bcohen@mbl.edu

Abbreviations: CD, cytochalasin-D; GVBD, germinal vesicle breakdown; HG, hexylene glycol; LV, lovastatin; PB, polar body; PB1 and PB2, first and second polar bodies.
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Author:Pielak, Rafal M.; Hawkins, Christopher; Pyie, Aung; Bautista, Jennifer; Lee, Kyeng-Gea; Cohen, Willi
Publication:The Biological Bulletin
Geographic Code:1USA
Date:Aug 1, 2005
Words:5804
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