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Laboratory spawning and development of the Bahama lancelet, Asymmetron lucayanum (Cephalochordata): fertilization through feeding larvae.

Introduction

The phylum Chordata comprises three extant clades--Cephalochordata, Tunicata, and Vertebrata--with the first, commonly called lancelets or amphioxus, basal to the other two (Delsuc et al., 2008; Putnam et al., 2008). Currently, 32 lancelet species are recognized: 24 in the genus Branchiostoma, 7 in the genus Asymmetron, and 1 in the genus Epigonichthys (Poss and Boschung, 1996; Nishikawa, 2004; Zhang et al., 2006; Kon et al., 2006, 2007). Lancelets have long been regarded as useful proxies for the latest invertebrate ancestor of the vertebrates and have been intensively studied at all levels of biological organization (reviewed by Gans, 1996; Garcia-Fernandez, 2006). However, almost all of this effort has been concentrated on a few species of Branchiostoma, while next to nothing has been learned about the biology of the less accessible lancelets in the genera Asymmetron and Epigonichthys. In the area of developmental biology, the discrepancy is especially marked--the development of Branchiostoma has been meticulously studied, most notably by Hatschek (1893) and Conklin (1932), whereas there have been no descriptions of the embryos and early larvae of the other two genera.

Andrews (1893) discovered the Bahama lancelet, Asymmetron lucayanum, living in the lagoon at Bimini, Bahamas, in shallow water (ankle-deep at low tide). Since then, other populations of the same species (or species complex) have been found elsewhere in the tropics (reviewed by Kon et al., 2007), but always at subtidal depths. During his visit to Bimini, Andrews obtained one spawning by the lancelets. Disappointingly, he noted that the resulting embryos "developed only as far as the gastrula stage, when they were destroyed by an accident." Andrews (1893) included no other information on development, except for describing very late, almost metamorphic larvae that he captured in the plankton. Even so, his results were tantalizing enough to induce us to go to Bimini, where we found the Bahama lancelet living in abundance where he had originally discovered it over a century before. Here we report on a laboratory spawning of Asymmetron lucayanum and describe development of the species through the early feeding larva.

Beginning in late embryology, several aspects of morphogenesis diverge between Asymmetron and Branchiostoma. One of these differences was unexpected and especially striking: in Branchiostoma, the two most anterior body cavities (the preoral pit and the rostral coelom) originate by enterocoely, whereas in Asymmetron, their likely homologs arise by schizocoely. At present, the significance of these new findings is obscured by uncertainty about whether characters in the genus Asymmetron are more likely to be primitive or derived within the cephalochordates. It will, therefore, be important to resolve the main course of cephalochordate evolution by further phylogenetic analyses with nuclear genes and by sequencing the genome from a species of Asymmetron.

Materials and Methods

Collection of animals and biometry

We collected 106 specimens of Asymmetron lucayanum on 15 November 2009 during an early afternoon low tide in the Bimini Lagoon when the water was about 10 cm deep and the water temperature was 25 [degrees]C. The habitat is a seagrass bed, previously described by Scoffin (1970), between Sand Cay (formerly called Stokes Cay or Tokas Cay) and Pine Cay (Fig. 1). The global positioning system coordinates of the collection site are 25.72297[degrees]N, 79.29388[degrees]W. The substrate was scooped to a depth of a few centimeters with a 20-cm-diameter geology sieve with a mesh opening of 710 [micro]m (British grade 22; American grade 25). The sieve retained some sediment and all of the lancelets between 10 mm and 23 mm (the maximum length). The captured animals, surrounded by a small amount of sediment, were transferred by spoon to a seawater-filled container. Back in the laboratory the same afternoon, the animals were cleaned by pipetting them from the residual sediment into a dish of clean seawater. Each lancelet was observed individually in a few milliliters of seawater in a 6-cm petri dish, and the body length was measured to the nearest millimeter. Then, using a dissecting microscope, we examined the gonads, which are visible within the relatively transparent body, to determine the animal's sex (the sperm-filled testes appear homogeneous, whereas ovaries are filled with oocytes). Finally, with a calibrated ocular micrometer in the dissecting scope, we measured (to the nearest 0.01 mm) the dorsoventral height of the animal and one of its gonads (Fig. 2, AH and GH, respectively) as viewed from its right side halfway between the head and tail. The percentage of gonad height to animal height is the gonad index.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Spawning in the laboratory and determining the schedule of development

All laboratory procedures were carried out at 27 [+ or -] 1 [degrees]C. In our collection, about a quarter of the lancelets had gonad indices of 30% or greater. These animals (11 males and 16 females) were segregated from the others and placed together in a dish 20 cm in diameter containing 500 ml of seawater. The dish was continuously exposed to the room light during the late afternoon and early evening of collection and then placed in the dark at 2100 h. When the dish was observed 90 min later, its bottom was covered with thousands of one- and two-cell embryos. The adult lancelets, whether spawned or not, were removed by pipette and their post-spawning gonad indices were measured.

The embryos were transferred from the spawning container to clean seawater in petri dishes (9-cm diameter) and fixed periodically in 4% paraformaldehyde buffered with 3-(N-morpholino)propanesulfonic acid with added NaCl to elevate the osmolarity (Holland et al., 1996). Fixation did not appreciably change the dimensions of the embryos. Starting on the third day of development, when the mouth first opened, the larvae were fed algae (80% Isochrysis + 20% Tetraselmis) that had been centrifuged free of their medium before addition to the culture at a concentration of approximately 105 algal cells per ml. The ciliary tuft of living larvae was moving too rapidly to photograph, so we stopped it by running PFA under the coverslip. Whole mounts of fixed and living developmental stages were photographed under differential interference contrast optics. About 10 specimens at each developmental stage were prepared for histology. They were treated whole for 1 h in 0.05% Ponceau S in 0.1% aqueous acetic acid, which stained the cytoplasm red. The specimens were than embedded in Spurr's resin and sectioned at 3-[micro]m with a glass knife. After sectioning, the nuclei in the specimens were stained dark blue with 0.1 % aqueous azure A, and the slides were mounted in immersion oil.

Results

Dark induction of spawning

The collection of 15 Nov. 2009 comprised 106 specimens of Asymmetron lucayanum: 44 males, 56 females, and 6 unsexable because the gonads were minute or invisible under the dissecting microscope. After the ripest animals had been placed together in the dark for 90 min on the evening of collection, 11 of 11 males and 7 of 16 females spawned (Fig. 3). All gonads of each spawned animal emitted virtually all of their gametes, except for two of the several dozen ovaries of one female, which remained full of large oocytes with germinal vesicles (these unspawned ovaries were not taken into account for calculating the gonad index). The seven spawned females cumulatively produced several thousand eggs, about 10% of which were not fertilized, as judged by their lack of an elevated fertilization envelope.

[FIGURE 3 OMITTED]

Schedule of development; morphology of embryos and early larvae

The later embryos and larvae of cephalochordates have some unusual anatomical characters with names unfamiliar to most biologists. To make the present work more accessible, we have included a diagram (Fig. 4) illustrating these specialized features. The figure also compares aspects of the developmental anatomy of Branchiostoma and Asymmetron and emphasizes differences between the two.

[FIGURE 4 OMITTED]

Table 1 summarizes the time course of development at 27 [degrees]C and includes some behavioral observations on the living embryos and larvae. The development times in the table are based on the assumption that spawning and fertilization took place after 30 min in the dark: thus the first fixed sample is 1 h after fertilization. In this sample about half the fertilized embryos were at the one-cell stage (Fig. 5A, B) and the other half were at the two-cell stage (Fig. 5C). The mean diameter of the one-cell stages is 130 [+ or -] 8.7 [micro]m (SD, n = 10), and the diameter of the surrounding fertilization envelope is 345 [+ or -] 54.4 [micro]m (SD, n = 10). Thus, the perivitelline space of Asymmetron, like that of Branchiostoma, is exceptionally wide. A few of the embryos, otherwise developing normally, are enclosed by two concentric fertilization envelopes (Fig. 5B).
Table 1
Schedule for development of Asymmetron lucayanum at 27[degrees]C, with
notes on embryonic and larval behavior

             Stage                   Time after      Figure
                                   fertilization

1- and 2-cell                          1 h        5A-C
4- and 8-cell                          2 h        5D
16- and 32-cell                        3 h        not shown
64- and 128-cell                       4 h        not shown
Blastula                               5 h        5E
Mid gastrula                           9 h        5F
Late gastrula (a)                      12 h       5G-I (b)
Just-hatched neurula                   15 h       5J-L
Early neurula                          19 h       5M, N
Mid neurula (c)                        24 h       5O
Late neurula (d)                       32 h       5P, Q
Hatchet-shaped embryo (d), (c)         50 h       5R-Y
Early larva (mouth just open) (f)      3 days     5Z-Z"; 6A-F
Feeding larva                          5 days     6G-Q

(a) Newly produced ectodermal cilia cause embryo to rotate in
fertilization envelope.
(b) Late gastrulae shown here were removed from fertilization envelope
to facilitate their orientation for photography.
(c) Mid neurulae aggregate at meniscus all around container
circumference regardless of the direction of light.
(d) Ciliating embryos hover throughout top few centimeters of water; no
light influence.
(e) Occasional, sporadic muscular undulation begins.
(f) Embryos throughout water column or crawling on bottom of container.


From cleavage through mid gastrula (Fig. 5C-F), the embryos of Asymmetron closely resemble those of Bran-chiostoma species (Holland and Yu, 2004). However, at the late gastrula stage (Fig. 5G-I), Asymmetron begins to differ from its congener in having a gut with a markedly smaller average diameter. Around the time of hatching, as in Bran-chiostoma, the ectoderm overgrows the neural plate (Fig. 5J-L). Then, during the early neurula stage (Fig. 5M), the lateral sides of the neural plate curl dorsally and fuse to form the neural tube, and the endoderm gives rise to the notochord and somites (Fig. 5N). By mid neurula (Fig. 50), the body is elongating and the epidermal cells of the tail fill with black pigment granules. Strikingly, at the mid neurula (and subsequent) stages of Asymmetron, Hatschek's left and right diverticula do not form; in mid neurulae of Branchio-stoma, by contrast, these diverticula arise by evagination from the anterior end of the pharynx (Fig. 4).

By the late neurula stage (Fig. 5P) the first pigment spot is detectable in the neural tube. Moreover, a small cluster of cells (Fig. 5Q, single arrow) of unproven (but probably endodermal) origin has accumulated just anterior to the pharynx. Subsequently, in the hatchet-shaped embryo, the rostral coelom opens up, evidently by schizocoely, in the cell cluster anterior to the pharynx. The rostral coelom, when it first appears (Fig. 5R-T, single arrows), is located in the midline of the body--this coelom subsequently shifts somewhat to the right of center (Fig. 6J, asterisk) by the early larval stage. In addition, near the anterior end of the pharynx of the hatchet-shaped embryo, a diverticulum evaginates from the endoderm midventrally and extends anteriorly (5V, arrow); this evagination is the initial part of the duct of the club-shaped gland (a peculiar structure reviewed by Holland et al, 2009). At the level of the mid pharynx, there is a slight thickening of the endoderm on the right side (Fig. 5W, arrow), presaging the formation of the endostyle and glandular portion of the club-shaped gland. Near the posterior end of the pharynx, the endoderm is closely apposed to the ectoderm (Fig. 5X, arrow) where the first gill slit will open, just to the left of the ventral midline. Posterior to the pharynx, the gut diameter narrows markedly (Fig. 5Y).

[FIGURE 6 OMITTED]

At the early larval stage (Fig. 5Z-Z", Fig. 6A-F), reached after 3 days of development, the mouth opens on the left side of the head. At about the same time, the gut also establishes two other openings to the exterior--the first gill slit (Fig. 6E) and the anus (Fig. 5Z, inset), respectively just to the left and just to the right of the ventral midline. This contrasts with Branchiostoma, in which the first gill slit and anus first appear, respectively, just to the right and far to the right of the midline (Stokes and Holland, 1995). On the left side of the larval head, a plaque of ectoderm thickens into the preoral pad (Fig. 5Z', single arrow; Fig. 6A, arrowhead) and the club-shaped gland's duct opens to the exterior (Fig. 5Z', arrowhead; Fig. 6B, arrow). The pharyngeal endoderm on the right side differentiates into the endostyle (Fig. 6C, tandem arrows) and, just posterior to that, into the glandular portion of the club-shaped gland (Fig. 5Z"; Fig. 6D, tandem arrows). The inner opening of the duct of the club-shaped gland still opens ventrally (Fig. 6D, single arrow) into the pharyngeal lumen. At the level of the endostyle and club-shaped gland, the epidermis along the ventral, right side of the head is differentiated into a mucus gland (Fig. 6D, arrowhead) in virtually the same position as a similar gland in Branchiostoma larvae (Lacalli, 2008). More posteriorly, a subenteric blood vessel runs along the ventral side of the post-pharyngeal gut (Fig. 6F); Holland et al. (2003) suggested that this vessel represents the initial stage of heart development in amphioxus.

After 5 days of development, the larval mouth is much enlarged, and most specimens have microalgae in the gut (Fig. 6G). On the left side of the head, the preoral pad (Fig. 6H, twin arrows) appears in some specimens to be underlain by a small vesicle (Fig. 6I and J, single arrow), which, unlike the preoral pit of Branchiostoma, does not open to the exterior (as diagrammed in Glardon et al., 1998); even so, the vesicle could well be comparable to Hatschek's left diverticulum. A little farther posteriorly on the same side of the head is a second epidermal thickening, the oral papilla (Fig. 6K), which bears the long cilia that cumulatively form the ciliated tuft (Fig. 6G, asterisk). The endostyle is well differentiated (Fig. 6I and J, twin arrows) and includes a dorsoventrally oriented tract of conspicuously ciliated cells. The inner opening of the club-shaped gland has moved up the right side of the pharynx (by evagination of the latter) to open into the gut lumen dorsolaterally (Fig. 6M, single arrow). The mucus gland is still present along the right ventral side of the head (Fig. 6L, single arrow), and the single gill slit still opens from the pharyngeal lumen just to the left of the ventral midline (Fig. 6H and I, arrowhead). Posterior to the pharynx, the peritoneal wall of the subenteric blood vessel can be seen contracting rhythmically several times a minute, although amoebocytes in the blood plasma remain stationary in spite of the contractions (Fig. 6N-P). In the tail region of the 5-day larva, a conspicuous neurenteric canal (Fig. 6Q, single arrow) connects the neurocoel (Fig. 6Q, twin arrows) with the lumen of the hindgut (Fig. 6Q, tandem arrows).

None of our larvae survived beyond the 10th day in culture, although they were cleaned and fed daily. The fed larvae had algae in their guts, although it is possible that we were feeding them indigestible algal species. An additional problem, which we had not previously encountered in raising Branchiostoma, was that Asymmetron larvae have a strong tendency to get stuck at the air-water interface and die. Thus, by maintaining the larvae in petri dishes, we exacerbated trapping in the surface film. At 10 days, the larvae had not advanced morphologically over the 5-day stage described above.

Discussion

Spawning periodicity of the Bahama lancelet

Andrews (1893) reported that specimens of Asymmetron lucayanum collected from the Bimini Lagoon in 1892 were sexually mature in June and that several of them spawned that month on a single (unspecified) date. He also mentioned that the animals seemed to become progressively less ripe as the summer progressed. In the present work, our animals spawned on 15 November 2008. Therefore, at a minimum, the Bimini amphioxus spawns in late spring/early summer and in the fall. We are currently gathering gonad index data for the Bimini population of Asymmetron at roughly monthly intervals to establish whether spawnings can also be obtained during the cool of the winter and the heat of the summer. Andrews (1893) makes no mention of the time of day when his animals spawned, but ours spawned when put in the dark at 2100 h. Thus it is likely that ripe males and females of Asymmetron emit their gametes shortly after sundown, as has been shown experimentally and under natural conditions for lancelets in the genus Branchiostoma (Wiley, 1891; Stokes and Holland, 1996; Watanabe et al., 1999).

Concentric fertilization envelopes

In our spawning of Asymmetron lucayanum, a small percentage of the embryos raised two concentric fertilization envelopes (e.g., Fig. 5B); moreover, at least some of the doubly enclosed embryos developed normally and hatched. The production of concentric fertilization envelopes has not been reported for any other cephalochordate, or indeed for any kind of animal. In a transmission electron microscopy (TEM) study of the formation of the fertilization envelope of Branchiostoma floridae, Holland and Holland (1989) demonstrated that this structure is always single and evidently forms by the organization of exocytosed cortical granule material on the inside of a vitelline layer. This has proven to be the commonest mode of fertilization envelope formation in invertebrate deuterostomes since first demonstrated by Endo (1961). For the doubly enclosed embryos of A. lucayanum, nothing is known about how the inner fertilization envelope forms. Even if there were two temporally separate waves of cortical granule exocytosis, the material released would not be expected to be constrained by a second vitelline layer--unless such a layer could form in just a few seconds following the first wave of cortical granule exocytosis. Alternatively, the released cortical granule contents of Asymmetron might self-organize into a membrane-like structure. A TEM study would be needed to test these alternatives.

Developmental features of Asymmetron: fundamental traits or peculiarities?

Kon et al. (2006) interpreted their mitochondrial genome data as indicating that Asymmetron diverged deeply--during the mid Mesozoic--from the clade including Branchio-stoma plus Epigonichthys. Unfortunately, there is as yet no consensus about what such data imply for reconstructing the ancestor of the cephalochordales. Kon et al. (2007) and Yasui and Kaji (2008) proposed, for example, that the single row of gonads limited to the right side of the adult body of Asymmetron is primitive, whereas the presence of two rows of gonads, one on either side of the midline of Branchio-stoma, is derived.

In contrast, mitochondrial genome data suggested just the opposite to Zhong et al. (2009) and led them to agree with Andrews (1893), who believed that the peculiarities of Asymmetron are "secondary departures from a type more like the common European form" (i.e., Branchiostoma). At present, this fundamental disagreement impedes speculation about cephalochordate (and even chordate) evolution--even if one ignores the added difficulty that a representative of an anciently diverged lineage may have lost some original characters and evolved some highly derived ones.

The consequences of favoring one of these viewpoints over the other can be illustrated by focusing on a Hatschek's left diverticulum during Branchiostoma development. There have been numerous and varied speculations about possible homologs of this transient evagination in other deuterostomes. It has been considered to correspond, at least in part, to the protocoel of hemichordates (MacBride, 1909), the original foregut of chordates (van Wijhe, 1913), the pre-premandibular head cavities of craniates (Neal, 1915), or the premandibular head cavities plus part of the adeno-hypophysis of craniates (Goodrich, 1917). One of these speculations might have a chance of being correct if the basal cephalochordate was Branchiostoma-like in having Hatschek's left diverticulum; in that case the absence of the structure in Asymmetron would represent a loss in that evolutionary line. However, if basal cephalochordates were Asymmetron-like in lacking Hatschek's left diverticulum, the evolution of that structure would be a derived peculiarity of the genus Branchiostoma and could not be homologized with any feature of other chordates. A parallel situation exists for the phylum Hemichordata, wherein enteropneusts form their coeloms largely by enterocoely, but pterobranchs do so by schizocoely (Lester, 1988); however, an evolutionary explanation of the difference is hampered by current uncertainty as to whether pterobranchs are basal or derived within the phylum (Sato et al., 2008).

The present discussion calls attention to the importance of settling the current disagreement about the course of evolution within the cephalochordates. The controversy could probably be resolved by adding nuclear genes to the phylogenetic analysis of the group and by sequencing the genome of a species of Asymmetron for comparison with that of Branchiostoma floridae (Putnam et al., 2008). Additional work on the developmental genetics of Asymmetron should not only provide a robust cephalochordate phylogeny, but also permit interesting comparisons with Branchiostoma. For example, divergences in regulatory elements and other aspects of gene networks between the two genera might be clearly related to the marked differences in left-right asymmetry between Branchiostoma and Asymmetron.

Acknowledgments

We are grateful to Greg Rouse, who photographed our living adult lancelets, and to the staff of the Bimini Biological Field Station (Sharklab) for all their help. Above all, we are indebted to Sharklab's director, Samuel H. "Doc" Gruber, whose commitment to marine biological studies in Bimini so wonderfully prepared the ground for our research there. This work was supported in part by NSF research grant IOS 07-43485.

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NICHOLAS D. HOLLAND* AND LINDA Z. HOLLAND

Marine Biology Research Division, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, California 93093.

Received 3 July 2010; accepted 26 July 2010.

* To whom correspondence should he addressed. E-mail: nholland@ucsd.edu
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Author:Holland, Nicholas D.; Holland, Linda Z.
Publication:The Biological Bulletin
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Date:Oct 1, 2010
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