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Microtubule arrays during ooplasmic segregation in the medaka fish egg (Oryzias latipes).


Ooplasmic segregation in the medaka egg consists of the approximately simultaneous streaming of ooplasm toward the animal pole, the saltatory movement of parcels toward the animal pole and vegetal pole, and the movement of oil droplets toward the vegetal pole (Abraham et al., 1993a; Webb and Fluck, 1993). These movements are roughly simultaneous and are essentially completed during the first cell cycle. Streaming of ooplasm toward the animal pole is inhibited by cytochalasin D and thus presumably requires microfilaments (Webb and Fluck, 1993), whereas saltatory movements and the movement of oil droplets toward the vegetal pole are both inhibited by microtubule poisons and thus presumably require microtubules (Abraham et al., 1993a). All of these movements can be easily observed in the optically clear medaka egg, in which a thin ([approximately equal to] 15 [[micro]meter] thick) peripheral layer of ooplasm surrounds a large, central yolk vacuole.

In a number of diverse animals, including annelids (Astrow et al., 1989), ctenophores (Houliston et al., 1993), echinoderms (Harris et al., 1980), ascidians (Sawada and Schatten, 1988), and amphibians (Elinson and Rowning, 1988; Houliston and Elinson, 1991; Schroeder and Gard, 1992; Elinson and Palacek, 1993), a network of microtubules that forms during the first cell cycle is required for the movement of specific components of the ooplasm, including the pronuclei. In the present study, we describe the development of such an array of microtubules during the first cell cycle of the medaka egg. Previous studies of microtubules in telcost embryos either have provided very little information (Beams et al., 1985) or have been done on later stages of development (Strahle and Jesuthasan, 1993; Solnica-Krezel and Driever, 1994).

We also pursued the hypothesis that gradients of cytosolic free [Ca.sup.2+] organize the multimolecular assemblies involved in ooplasmic segregation in the medaka egg. Zones of elevated calcium are present at the animal and vegetal poles of the medaka egg during segregation (Fluck et al., 1992). Moreover, injection of 5,5[prime]-dibromo-BAPTA (hereafter referred to as dibromo-BAPTA), a weak calcium buffer that dissipates cytosolic calcium gradients (Pethig et al., 1989; Speksnijder et al., 1989; Fluck et al., 1992), inhibits ooplasmic segregation in this egg (Fluck et al., 1994). In the present-study we monitored the effects of this buffer on the spatiotemporal pattern of microtubules and on pronuclear movements. A preliminary account of these findings has been published (Abraham et al., 1993b).

Materials and Methods


Methods for removing gonads from breeding medaka and for the in vitro fertilization of eggs have been described previously (Abraham et al., 1993a). Some eggs were incubated in 100 [[micro]molar] colchicine (dissolved in buffered saline solution: 111 mM NaCl; 5.37 mM KCl; 1.0 mM Ca[Cl.sub.2]; 0.6 mM MgS[O.sub.4]; 5 mM HEPES, pH 7.3) for 1 h at room temperature (20 [degrees]-25 [degrees] C) before they were fertilized.

Using a high-pressure microinjection system, we injected enough 50 mM dibromo-BAPTA (tetrapotassium salt; containing 5 mM HEPES, pH 7.2, and sufficient Ca[Cl.sub.2] to set [[[Ca.sup.2+]]] at 100 nM) to raise the cytosolic concentration to 2.7 mM (Fluck et al., 1992). Injections were made within 10 [degrees] arc toward the vegetal pole from the equator within 6 min after fertilization. Control eggs received a comparable volume (ca. 1.5 nl) of 150 mM KCl, 5 mM HEPES, pH 7.2.

The procedures for indirect immunofluorescence were those developed by Gard (1991) for the study of microtubules in Xenopus laevis eggs. At regular intervals after fertilization, eggs were transferred to fixative solution at room temperature (3.7% formaldehyde, 0.25% glutaraldehyde, 0.2% Triton X-100, 5 mM EGTA, 1 mM Mg[Cl.sub.2], 80 mM potassium PIPES, pH 6.8). After 4 h, the eggs were dechorionated with fine forceps and post-fixed in absolute methanol (-20 [degrees] C) overnight. The eggs were then washed with phosphate-buffered saline (PBS: 128 mM NaCl; 2 mM KCl; 8 mM Na[H.sub.2]P[O.sub.4]; 2 mM K[H.sub.2]P[O.sub.4], pH 7.2) and incubated in 100 mM sodium borohydride (in PBS) for 6 h. The eggs were then washed with cold Tris-buffered saline (TBS: 155 mM NaCl; 10 mM Tris-Cl, pH 7.4; 0.1% Nonidet P-40); transferred to Lab-Tek chamber slides (Thomas Scientific, Swedesboro, New Jersey); incubated with a monoclonal mouse anti-[Alpha]-tubulin antibody (ICN, DM1A; diluted 1:250 with TBS containing 2% bovine serum albumin); washed with TBS for 24 h; incubated with the secondary antibody (rhodamine-conjugated goat anti-mouse IgG, diluted 1:25 with TBS containing 2% bovine serum albumin); and washed for 24 h with TBS. We performed two kinds of antibody controls: omission of the primary antibody and omission of both the primary and the secondary antibodies. To stain the nuclei, we incubated the eggs for 30 min in a solution of Hoechst 33258 (10 [[micro]gram] [ml.sup.-1]) in TBS.

The eggs were mounted between a coverglass and a microscope slide as described previously (Abraham et al., 1993a) and examined by one of three methods. Conventional epifluorescence microscopy was performed with a Nikon Optiphot microscope coupled to a Dage-MTI SIT camera and video monitor; in later replicates, the SIT camera was coupled to a Dage-MTI DSP-2000 image processor. In either case, the image on the monitor was photographed through a Ronchi grating (Rolyn Optics Co., Covina, California; Inoue, 1981). Some eggs were examined with a laser scanning confocal microscope (Zeiss LSCM 410) at the Marine BiologiCal Laboratory.

We also acquired and processed images at the Science and Technology Center at Carnegie-Mellon University. Images at each focal plane were acquired on a multimode microscope (Biological Detection Systems, Inc., Pittsburgh, Pennsylvania) equipped with a 576 x 384 Thompson chip, cooled CCD camera (Photometrics Ltd., Tucson, Arizona). Three-dimensional fluorescence microscopy used the nearest neighbor algorithm to correct for out-of-focus fluorescence (Biological Detection Systems, Inc.) The final three-dimensional representation was generated with the program ANALYZE (Biomedical Imaging Resource, Mayo Foundation, Rochester, Minnesota) on the SGI, Inc., ONYX workstation.

The results summarized herein represent 11 replicate experiments in which we fixed eggs at various intervals after fertilization and three replicate experiments in which we examined the effects of dibromo-BAPTA on microtubules. In all, we have examined a total of 117 eggs from 17 females, including 20 eggs into which we injected dibromo-BAPTA and 8 eggs into which we injected KCl.


Formaldehyde and glutaraldehyde were obtained from Electron Microscopy Sciences (Fort Washington, Pennsylvania); Triton X-100, Nonidet P-40, sodium borohydride, bovine serum albumin, Hoechst 33258, and colchicine from Sigma (St. Louis, Missouri); 5,5[prime]-dibromo-BAPTA from Molecular Probes (Eugene, Oregon); anti-[Alpha]-tubulin antibody from ICN (Costa Mesa, California); and rhodamine-conjugated goat anti-mouse IgG from Organon Teknika (Malvern, Pennsylvania).


The various events that constitute ooplasmic segregation in the fertilized medaka egg have already been described in detail (Abraham et al., 1993a) and are only summarized below. To indicate the relative temporal position of these events, we have used a normalized time ([T.sub.n]) scale in which the time between fertilization and the beginning of cytokinesis is 1 unit. In the range of room temperatures in which the experiments were done (20 [degrees] - 25 [degrees] C), 1 unit of normalized time corresponds to 75-110 min. Fertilization in the medaka egg is followed by a cortical granule reaction and a contraction, during which the ooplasm and its contents appear to be pulled toward the animal pole. The second meiotic division is completed by [T.sub.n] [approximately equal to] 0.12 and is followed closely by a second animal-pole-directed contraction at [T.sub.n] [approximately equal to] 0.15. This second contraction heralds the beginning of ooplasmic segregation in the form of streaming of ooplasm toward the animal pole, saltatory movements of parcels toward both polar regions, and movement of oil droplets (a class of ooplasmic inclusions) of various sizes toward the vegetal pole. At [T.sub.n] = 1.0, the blastodisc divides into two blastomeres. By this time the oil droplets have formed a crude ring around the vegetal pole.

Microtubule network dynamics during ooplasmic segregation

We identified three regions of the medaka egg on the basis of the spatiotemporal pattern of microtubules in them [ILLUSTRATION FOR FIGURES 1-3 OMITTED]: (1) a region within 30 [degrees] ([approximately equal to] 300 [[micro]meter]) arc of the animal pole; (2) a region within 60 [degrees] ([approximately equal to] 600 [[micro]meter]) arc on both sides of the equator; and (3) a region within 30 [degrees] ([approximately equal to] 300 [[micro]meter]) arc of the vegetal pole. In all regions of the egg except the animal pole, all the microtubules were within 2-3 [[micro]meter] of the surface of the egg and were visible in a single optical section.

[T.sub.n] [approximately equal to] 0.02, 0.08, and 0.16. The earliest time at which we fixed eggs was [T.sub.n] = 0.02; that is, immediately after the contraction that follows the cortical granule reaction. Though we searched the ooplasm everywhere on these eggs (and all of the ooplasm is peripheral, see Introduction), we saw no microtubules parallel to the surface of the egg but saw instead a punctate pattern of fluorescence [ILLUSTRATION FOR FIGURE 1A OMITTED]. Analysis of these eggs by three-dimensional fluorescence microscopy suggests that the sources of fluorescence were microtubules oriented perpendicular to the surface of the egg [ILLUSTRATION FOR FIGURE 1B OMITTED]. This punctate pattern was a prominent feature of eggs fixed at [T.sub.n] = 0.02 and 0.08 and was less prominent at later stages.

In eggs fixed at [T.sub.n] [approximately equal to] 0.08, we saw a very sparse network of microtubules oriented parallel to the surface of the egg but having no apparent preferred orientation with respect to the animal-vegetal axis of the egg. This network was roughly confined to the animal hemisphere, and we saw no microtubules in most of the vegetal hemisphere [ILLUSTRATION FOR FIGURE 1C, D OMITTED].

In eggs fixed at [T.sub.n] [approximately equal to] 0.16 - by which time ooplasmic segregation has begun in the form of streaming, saltatory movements, and oil droplet movement - a network of microtubules was present throughout the ooplasm, and the network in the equatorial region was denser than at [T.sub.n] [approximately equal to] 0.08 [ILLUSTRATION FOR FIGURE 1E OMITTED]. The network did not yet appear to have a preferred orientation.

To test for autofluorescence, we examined fixed eggs that were incubated with no antibodies and that were processed for viewing up to the first wash with TBS. We saw no fluorescence at all in these eggs (data not shown). We also examined eggs that were incubated with the secondary antibody but not with the primary antibody. In these eggs, we saw diffuse fluorescence but no linear elements or punctate sources of fluorescence [ILLUSTRATION FOR FIGURE 1F OMITTED]. In eggs incubated with 100 [[micro]molar] colchicine before fixation and incubated with both the primary and secondary antibodies, we saw diffuse fluorescence but no microtubules or any punctate sources of fluorescence (data not shown).

The pattern of microtubules in eggs fixed at [T.sub.n] [approximately equal to] 0.16 is summarized in Fig. 3A.

[T.sub.n] = 0.24, 0.3, 0.4, 0.76. In ooplasm near the equator, the network of microtubules was denser than at [T.sub.n] = 0.16 ([ILLUSTRATION FOR FIGURE 2A OMITTED]; compare with [ILLUSTRATION FOR FIGURE 1E OMITTED]) but still showed no preferred orientation with respect to the animal-vegetal axis. In contrast, oriented microtubule networks were present near the vegetal pole and animal pole by [T.sub.n] = 0.24 and [T.sub.n] = 0.3, respectively. Near the vegetal pole, an array of (mostly) parallel microtubules was present [ILLUSTRATION FOR FIGURE 2B OMITTED]. This "vegetal mat" extended about 30 [degrees] arc (300 [[micro]meter]) in all directions from the vegetal pole and covered at least 30% of the area of the vegetal hemisphere. The orientation of microtubules was uniform throughout the vegetal mat. Beyond the edges of the mat, the network of microtubules abruptly lost its orientation and became the network characteristic of interpolar ooplasm. Using the z-sectioning function of the LSCM 410, we determined that the thickness of the microtubule network in the vegetal mat ([approximately equal to] 2.7 [[micro]meter]) was the same as the thickness of the unoriented microtubule network just outside the mat. The two networks appeared to be continuous with each other and were in the same optical section, suggesting that the two networks are located at the same depth in the ooplasm.

Near the animal pole, microtubules were oriented more or less along meridian lines and appeared to radiate from a microtubule-organizing center near the male and female pronuclei, which were 38.6 [+ or -] 6.7 [[micro]meter] ([Mathematical Expression Omitted], n = 5) below the surface of the blastodisc at [T.sub.n] [approximately equal to] 0.76 [ILLUSTRATION FOR FIGURE 2D-I OMITTED]. This network of oriented microtubules extended approximately 30 [degrees] arc (300 [[micro]meter]) in all directions from the animal pole.

The pattern of microtubule distribution in eggs fixed at [T.sub.n] = 0.30-0.76 is summarized in Fig. 3B.

[T.sub.n] = 1.0. In these eggs, the blastodisc has begun to divide into two blastomeres, and essentially all oil droplets have aggregated in a crude ring near the vegetal pole. Each of these blastomeres had a microtubule-organizing center (data not shown) similar to the one present in the blastodisc from [T.sub.n] = 0.30 to at least [T.sub.n] = 0.76. The parallel organization of the microtubule network near the vegetal pole was lost by this stage [ILLUSTRATION FOR FIGURE 2C OMITTED]. The pattern of microtubules in eggs fixed at [T.sub.n] [approximately equal to] 1.0 is summarized in Fig. 3C.

Microtubule networks in eggs injected with 5,5[prime]-dibromo-BAPTA

The effects of injected KCI and dibromo-BAPTA on microtubule networks are summarized in Figures 4 and 5. The microtubule networks in eggs into which we injected KCI were indistinguishable from those found in uninjected eggs fixed at the same time ([T.sub.n] = 0.76 [from 57 min to 84 min after fertilization, depending on the temperature]; Fig. 4A [compare with Fig. 2B], 5A [compare with Fig. 2A]). The parallel organization of microtubules in the vegetal mat was not disrupted by dibromo-BAPTA [ILLUSTRATION FOR FIGURE 4B OMITTED]. However, the injection of dibromo-BAPTA had profound effects on the microtubule networks near the animal pole, where the radiating pattern of microtubules near the animal pole was disrupted [ILLUSTRATION FOR FIGURE 4C, D OMITTED]. The only apparent effect of dibromo-BAPTA on the microtubule arrays in the equatorial region was a decrease in the density of microtubules near the injection site [ILLUSTRATION FOR FIGURES 5B, C OMITTED].

Dibromo-BAPTA also inhibited the movement of the pronuclei. At [T.sub.n] [approximately equal to] 0.5, the distance between the male and female pronuclei was as follows: uninjected control eggs, 1.8 [+ or -] 3.0 [[micro]meter] ([Mathematical Expression Omitted], n = 10 eggs); eggs into which we injected KCl, 1.7 [+ or -] 3.4 [[micro]meter] (n = 4 eggs); eggs into which we injected dibromo-BAPTA, 40.4 [+ or -] 28.6 [[micro]meter] (n = 5 eggs). A one-way analysis of variance and post-hoc Student's t-tests revealed that the BAPTA-treated eggs differed significantly (P = 0.038) from the other two treatments.


The pattern of microtubules in the fertilized medaka egg varied both temporally and spatially during the first cell cycle. Temporal changes included an increase in the density of microtubules throughout the ooplasm and changes in the degree of orientation of microtubules in the polar regions of the egg. The striking feature of the region near the animal pole was the development of an array of microtubules that radiated from near the pronuclei to approximately 30 [degrees] arc from the animal pole. Though microtubules were initially ([T.sub.n] = 0.08) present near the animal pole as a sparse unoriented network, a dense network of radially oriented microtubules had formed by [T.sub.n] = 0.3. Such radial arrays of microtubules are present in fertilized eggs of a number of animals, including annelids (Astrow et al., 1989), ctenophores (Houliston et al., 1993), echinoderms (Harris et al., 1980), ascidians (Sawada and Schatten, 1988), and amphibians (Houliston and Elinson, 1991; Schroeder and Gard, 1992; Elinson and Palacek, 1993), and are associated with the sperm aster and sometimes with the female pronucleus as well.

The mat of parallel microtubules at the vegetal pole of the medaka egg is reminiscent of the one found at the vegetal pole of amphibian eggs, where it is involved in the cortical rotation that forms the gray crescent and thus determines the dorsal-ventral axis of the embryo (Manes et al., 1978; Scharf and Gerhart, 1983; Vincent et al., 1987; Gerhart et al., 1989; Elinson and Rowning, 1988; Elinson and Palacek, 1993). A similar array of parallel microtubules is also present at the vegetal pole of the zebrafish (Danio rerio) egg (see Fig. 9C in Strahle and Jesuthasan, 1993). Although there is no evidence for the occurrence of a conical rotation in the medaka egg, embryos of some primitive fishes that undergo holoblastic cleavage (species in the Superorder Chondrostei within the Actinopterygii, for example the sturgeon, Acipenser guldenstadti) do form a gray crescent and thus perhaps do undergo a cortical rotation (reviewed by Bolker, 1993; see also Clavert, 1962; Ginsburg and Dettlaff, 1991). In amphibian eggs, the plane of the first cleavage is parallel to the microtubules near the vegetal pole; and in both amphibians and primitive fishes, the plane of the first cleavage bisects the gray crescent along a meridian (Bolker, 1993). We are currently pursuing the question of the relationship between the orientation of microtubules in the vegetal mat and that of the embryonic axes in the medaka egg. In both Xenopus laevis and the medaka, the parallel orientation of the microtubules in the vegetal pole region is transient. In X. laevis, this mat begins to form at [T.sub.n] [approximately equal to] 0.55 and disappears after [T.sub.n] [approximately equal to] 0.8 (Elinson and Palacek, 1993). In medaka eggs, it is present by [T.sub.n] [approximately equal to] 0.24 and is still present at [T.sub.n] [approximately equal to] 0.76; but by the beginning of the first cleavage, the network has lost its parallel organization.

In sharp contrast to the strongly oriented networks of microtubules in polar ooplasm, interpolar ooplasm contained a network of crisscrossed microtubules having no apparent preferred orientation. Microtubules were present here by [T.sub.n] = 0.08 and subsequently increased in density through [T.sub.n] = 0.24. The lack of orientation of microtubules in this region of the medaka eggs contrasts with the situation in the zebrafish egg, in which microtubules are oriented along the animal-vegetal axis during both the first cell cycle and epiboly (Strahle and Jesuthasan, 1993; Solnica-Krezel and Driever, 1994).

Because microtubule poisons inhibit the movement of oil droplets toward the vegetal pole of the medaka egg, it has been suggested that these droplets are transported along microtubules by microtubule-based motors (Abraham et al., 1993a). Further, because oil droplets move toward the vegetal pole roughly along meridians, we predicted that a network of microtubules, also oriented more or less along meridians, would extend from the animal pole to near the vegetal pole. Although such a pattern did develop near the animal pole by [T.sub.n] = 0.3, a time when oil droplets have begun to move toward the vegetal pole, we saw no such preferred orientation of microtubules in most of the interpolar ooplasm, suggesting that oil droplets move toward the vegetal pole along a biochemically distinct (Gard, 1991; Wolff et al., 1992) subset of interpolar microtubules that are oriented along meridian lines.

The movement of most oil droplets toward the vegetal pole of the medaka egg begins at [T.sub.n] [approximately equal to] 0.15-0.30 (Catalone and Fluck, 1994). The results of the present study, which show that microtubules are present before oil droplets begin to move toward the vegetal pole, suggest that this movement is triggered by some factor other than the appearance of microtubules. We have also shown that even though the movement of oil droplets is "frozen" at the antipode of the site at which dibromo-BAPTA is injected into the egg (Fluck et al., 1994), many microtubules are present in this region of the ooplasm. Factors that could initiate the movement of oil droplets toward the vegetal pole include the formation of an appropriate subtype of microtubules, the activation of a microtubule-based motor such as kinesin (Brady et al., 1990; Schnapp et al., 1992), fulfillment of a requirement that a minimum number of microtubules be associated with an oil droplet before it begins to move, or a gel-sol transition of the ooplasm (Janson and Taylor, 1993) that frees the droplets and enables them to move toward the vegetal pole.

We have also demonstrated in the present study that injection of dibromo-BAPTA into medaka eggs affects the pattern of microtubules in the egg. This relatively weak calcium buffer ([K.sub.D] = 1.5 [[micro]molar]; Pethig et al., 1989) is believed to act as a shuttle buffer, one that binds [Ca.sup.2+] at the high end of a [[Ca.sup.2+]] gradient and releases it at the low end; in other words, the buffer facilitates the diffusion of [Ca.sup.2+] within the cell (Speksnijder et al., 1989). The time between injection of dibromo-BAPTA and fixation of the eggs in the present study was sufficient for the buffer to diffuse to both the animal pole and vegetal pole, to dissipate cytosolic [Ca.sup.2+] gradients near the poles, and to inhibit the formation of the blastodisc and the movement of oil droplets toward the vegetal pole (Fluck et al., 1992, 1994).

Our results suggest that at least some of the effects of dibromo-BAPTA on ooplasmic segregation in the medaka egg are caused by its disruption of microtubule formation and organization, which are controlled by a number of calcium-binding regulatory proteins (Weisenberg, 1972; Schliwa et al., 1981; Keith et al., 1983; Lieuvin et al., 1994). Facilitated diffusion theory predicts that calcium buffer concentrations similar to those found effective in the present study act by dissipating zones of cytosolic [[Ca.sup.2+]] in the micromolar range (Speksnijder et al., 1989). An example of a protein that is responsive to changes in [[Ca.sup.2+]] in this range is calmodulin (Cheung, 1980), which regulates the activity of proteins that interact with microtubules (Ishikawa et al., 1992).

The inhibition of pronuclear movements by dibromo-BAPTA is consistent with the explanation that the buffer disrupts the organization of microtubules near the animal pole. In a number of species, including the medaka, the movement of the pronuclei is inhibited by microtubule poisons (Hiramoto et al., 1984; Sawada and Schatten, 1989; Abraham et al., 1993a), suggesting that microtubules are necessary for these movements.

The parallel array of microtubules near the vegetal pole was apparently resistant to the effects of the buffer, suggesting that microtubules in this region of the egg differ in some way from those in other regions of the egg. This possible difference could be probed by microinjecting a microtubule poison, for example demecolcine, directly into the vegetal ooplasm.

The suggestion that the medaka egg has two independent microtubule networks is consistent with the situation in X, laevis, in which two independent networks of microtubules are present during the first cell cycle: one is near the animal pole and is associated with the pronuclei, while the other is the parallel array of microtubules near the vegetal pole (Elinson and Rowning, 1988; Houliston and Elinson, 1991; Elinson and Palacek, 1993; Sardet et al., 1994). Moreover, in both amphibian eggs (Houliston and Elinson, 1991; Elinson and Palacek, 1993) and medaka eggs (Webb and Fluck, unpub. obs.), vegetal cortical arrays of microtubules form in parthenogenetically activated eggs. Using the "Colcemid-UV" method (Hiramoto et al., 1984; Webb and Fluck, 1993), we are currently pursuing the question of the existence of independent arrays of microtubules in the animal pole region, vegetal pole region, and interpolar ooplasm. Our data at this time do not permit us to say whether microtubules are continuous from one region of the egg to another.


Supported by NSF DCB-9017210 and NSF MCB-9316125 to RAF, NSF BIR 9211855 to Lionel F. Jaffe and ALM, and grants from Franklin & Marshall College's Hackman Scholar Program and Harry W. and Mary B. Huffnagle Fund to VCA. Louis Kerr provided technical assistance in the use of the laser scanning confocal microscope in the Central Microscope Facility at the Marine Biological Laboratory, Tamika Webb assisted in the preparation and examination of one batch of eggs, and Bob Golder assisted in the preparation of Figure 3. The research at Carnegie-Mellon University was supported by National Science Foundation and Technology Center grant DIR-8920118. We thank Robbin L. DeBiasio for image acquisition, data processing, and 3-D representation; and Gregory M. LaRocca for technical assistance and image acquisition.

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Author:Abraham, V.C.; Miller, A.L.; Fluck, R.A.
Publication:The Biological Bulletin
Date:Apr 1, 1995
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