Completely direct development of Abatus cordatus, a brooding schizasterid (Echinodermata: Echinoidea) from Kerguelen, with description of perigastrulation, a hypothetical new mode of gastrulation.
A great diversity of larval forms and patterns of development occur in echinoderms. They are often classified on characters such as larval and embryonic features, habitat, and nutritional type (cf. McEdward and Janies, 1993). Raft (1987) considered four patterns of development in echinoids for the transition from the classical feeding, free-swimming larva echinopluteus to completely direct development. However, in asteroids, McEdward and Janies (1993) distinguished eight possible patterns on the basis of three characters (development, habitat, nutrition) each with two states (respectively, indirect or direct, pelagic or benthic, feeding and nonfeeding). In fact, such a multifactorial classification, which encompasses all of the possible combinations, is also valid for echinoids. From a strictly embryological point of view, development is described following the distinction between direct and indirect. A general definition of indirect development, and of larva and metamorphosis, has been proposed by Geigy and Portmann (1941). According to them, a larva goes through a metamorphosis that is characterized by three processes: (1) obliteration of the larval parts of the animal, (2) formation of certain adult parts from rudiments that remained in an embryonic state, and (3) continuation of larvo-adult formations that are not affected by metamorphosis. This refers to a pattern of development in which the gastrula is followed by intermediate stages with structural features that are not directly involved in the morphogenesis of the juvenile (McEdward and Janies, 1993). These authors defined direct development as a pattern wherein the embryonic stages are followed by the morphogenesis of the juvenile, without an intervening larval stage. Considering modified developments, the problem is, what is a larva? McEdward and Janies (1993) proposed that "a larva is produced by post-embryonic morphogenesis (postgastrula) and is eliminated by the metamorphic transition to the juvenile." Thus, considering the evolutionary diversification of development, to assume a simple dichotomy between direct and indirect development is very difficult and appears to be an oversimplification. Indeed, when nutritional and swimming larval structures are reduced or even absent, development is called abbreviated (Strathmann, 1978; Emlet et al., 1987; Raff, 1987; Pearse et al., 1991; Amemiya and Emlet, 1992). Such a decrease in morphological larval complexity led some authors to arrange larvae along a continuum reaching from typical planktotrophy to the absence of any larval stage (direct development) and to the corresponding internal reorganization of the embryo (Raff, 1987; Raff et al., 1990; Olson et al., 1993). For these authors, other changes that occur in the shift to direct development are the increase of egg size and an acceleration in the appearance of juvenile features. However, even if series of reductions in larval structures have been described in some lineages, such series are not evident in most groups of echinoderms (Emlet et al., 1987). If a continuun does exist, it has been achieved through time. Thus the "terminal" observation of present larval types may give only an approximate idea of the process.
Further detailed descriptions of the developmental patterns are needed to fully understand mechanisms underlying evolutionary changes (Raff, 1992). Information on the internal morphological events occurring during modified development is limited (see Barker, 1985; Amemiya and Emlet, 1992; Parks et al., 1989), and data on echinoid species with completely direct development are scarce. Brooding rarely proceeds by strictly direct development. Benthic brooding may involve more or less simplified larval stages (McEdward and Janies, 1993). Of the perhaps 10 to 30 species with modified development (Emlet, 1990), sometimes qualified as direct (see Raff et al, 1990; Olson et al., 1993), only Goniocidaris umbulacrum (Barker, 1985, peri-oral brooder) seems to have a completely direct development, with no larval feeding appendages, no necrosis of larval tissue, and no metamorphosis (ametamorphic development [Bonar, 1978]). Phyllacanthus parvispinus, another cidarid echinoid, may be another directly developing species (Parks et al., 1989). More recently, Holopneustes purpurescens has been described as an apluteal developing species (Morris, in press).
The present article elaborates on previous information about the events occurring from spawning to settling in Abatus cordatus, an echinoid with completely direct development (Schatt 1985a, b; 1988; Schatt and De Vos, 1990). The aims of the article are to give (1) an overview of the true direct development of this brood-protecting spatangoid that is endemic to the Kerguelen Islands (South Indian Ocean, 70 [degrees] 12 [minutes] E, 49 [degrees] 21 [minutes] S) and (2) preliminary evidence for a new mode of gastrulation. The ecology and biology of this species are already well known (Magniez, 1980, 1983; Schatt, 1985a; Poulin and Feral, 1994, 1995) and are similar to those of other Abatus spp. (Pearse and McClintock, 1990). Reproductive (Magniez, 1980) and brooding cycles are annual in Kerguelen and may depend on the locality and the depth where the animals live (Schatt and Feral, 1991); it is therefore possible to obtain and study all developmental stages.
Materials and Methods
Collection and preparation of Abatus cordatus
Female Abatus cordatus (Verrill, 1876) were collected fortnightly from December 1981 to March 1983 in the fine sand of the Anse du Halage in the Golfe du Morbihan. Samples collected in 1986, 1988, 1990, and 1992 were also used to check some details.
Females were held in the laboratory (BIOMAR, Base of Port-aux-Francais) before analysis. Brooded young were collected from each of the four pouches of the female, sorted under a binocular microscope, and reared in glass vessels containing filtered seawater (Millipore 0.45-[[micro]grams] filter) which was renewed every 2 days. They were maintained in the dark, from 7 [degrees] C (in summer) to 2 [degrees] C (in winter). No food was added, since internal nutrient stores and, possibly, dissolved organic matter [DOM] (Feral, 1985; Walker and Smith, 1985) supported development through the advanced juvenile stage. Artificial fertilization was performed in order to observe the first cleavage. Oocytes were collected directly in the gonoduct. They were fertilized by sperm ([approximately]250,000 cells [multiplied by] [ml.sup.-1]) in filtered seawater.
Preparation for light microscopy and scanning electron microscopy
Developmental stages were fixed in 3% glutaraldehyde with 0.2 M sodium cacodylate, at pH 7.4 (final concentrations, 1100-1200 mOsM). Serial semi-thin sections (1 [[micro]meter] thick) were cut after embedding in araldite. They were stained with toluidine blue (pH 11.5).
For scanning electron microscopy (SEM), developmental stages were fixed in the same mixture and postfixed with 2% osmium tetroxide in the same buffer. Embryos and juveniles were dehydrated in graded ethanol and dried by the critical-point method. They were mounted on aluminum stubs, coated with gold in a sputter coater, and observed with an ISI DS 130 scanning electron microscope.
For DNA staining, eggs were fixed in buffered 10% paraformaldehyde, then rinsed in 150 mM NaCl, 50 mM TRIS, pH 7.5, containing 0.1 [[micro]grams] [multiplied by] [ml.sup.-1] Hoechst 33258.
Gametes, spawning, and fertilization
The spherical egg is 1300 [[micro]meter] in diameter and is negatively buoyant. It is deep orange, densely yolked, and opaque [ILLUSTRATION FOR FIGURE 1A OMITTED]. The sperm has a narrow, pointed head, 15 [[micro]meter] long and 1 [[micro]meter] wide, with a tail 25 [[micro]meter] long [ILLUSTRATION FOR FIGURE 1B OMITTED]. During the breeding season, just after spawning, the eggs are moved to the brood pouches [ILLUSTRATION FOR FIGURE 2A OMITTED] by the spines and pedicellaria. Fertilization probably occurs during the movement of the eggs from the gonopore to the pouches. The fertilization membrane and a fertilization cone [ILLUSTRATION FOR FIGURE 2B OMITTED] lift off from the surface of the egg after about 34 min at 6 [degrees] C, leaving a large perivitelline space of 65 [[micro]meter]. In vitro, the fertilization cone is always oriented to the bottom. The axis of polarity is approximately the axis of gravity and will be the same until the post-gastrular stage. The cleavage will affect the pole opposite the site of sperm entry, which is defined as the vegetal pole (facing downward). The fertilization membrane is present to the end of gastrulation.
Development before hatching
Cleavage and blastulation. A horizontal constriction, whose plan is perpendicular to the animal-vegetal axis, occurs 2 days after fertilization [ILLUSTRATION FOR FIGURE 3A OMITTED]. It separates the egg into two parts, with a nucleus in the animal part and a non-nucleated vegetal part filled with yolk only. The second cleavage division [ILLUSTRATION FOR FIGURE 3B OMITTED], perpendicular to the constriction and involving only the animal part, forms two blastomeres. The blastomeres are yolky, ovoid to spherical, and 100 to 200 [[micro]meter] in diameter [ILLUSTRATION FOR FIGURE 3C OMITTED]. Synchrony of division is lost as early as the third cleavage.
The vegetal yolk mass progressively decreases in size while the blastomeres increase in number [ILLUSTRATION FOR FIGURES 2D AND 4A OMITTED]. The yolk mass was not observed to be taken up by blastomeres [ILLUSTRATION FOR FIGURE 3C OMITTED]. The yolk mass disappears and segmentation finally becomes total [ILLUSTRATION FOR FIGURES 4B AND 4C OMITTED]. The space between the blastomeres communicates with the perivitelline space [ILLUSTRATION FOR FIGURE 4C OMITTED]. A blastocoele was never observed. The blastula is filled with cells that originate from the blastomeres. The cell nucleus is rarely visible because of the abundance of yolk; moreover, the large size of the mitotic apparatus (almost 100 [[micro]meter], [ILLUSTRATION FOR FIGURE 3B OMITTED]) obstructs the nuclear area in 1- to 2-[[micro]meter]-thick sections. On semi-thin sections, often only clearer areas ("ghosts" of nuclei) can be seen in some cells [ILLUSTRATION FOR FIGURE 4C OMITTED]. All the cells of the blastula are filled with lipid(?)-rich spheroids.
Eighteen days after fertilization, the blastomeres associate into lobes, giving the blastula a wrinkled appearance. The blastomeres become smaller and smaller through divisions, thus increasing the ratio of surface area to volume. The wrinkling may be due to a lack of space in the tight-fitting fertilization membrane. The embryo is convoluted by furrows that gradually spread over its lumpy surface. About 26 days after fertilization, the stereoblastula is totally wrinkled [ILLUSTRATION FOR FIGURES 2E AND 4D OMITTED] and consists of a monostratified columnar epithelium enclosing a mesenchyme mass.
Gastrulation. Within a few days, the wrinkled blastula transforms into a young, externally smooth gastrula [ILLUSTRATION FOR FIGURE 2F OMITTED]. It will develop to hatching in the fertilization membrane that always keeps the same overall size (1.3 mm in diameter). A large depression, corresponding to the blastopore, appears at the vegetal pole 30-35 days after fertilization. The gastrula consists of a central mesenchyme surrounded by a monolayered epithelium, whose cells are cylindrical, still yolk-rich, and 100 [+ or -] 10 [[micro]meter] in height. The vegetal part of this gastrular epithelium invaginates to form the archenteron [ILLUSTRATION FOR FIGURE 5D OMITTED], while the animal part of the epithelium becomes thinner [ILLUSTRATION FOR FIGURE 5B AND E OMITTED]. The early gastrula is filled with yolky mesenchyme cells [ILLUSTRATION FOR FIGURE 5D OMITTED] in each of which the nucleus is difficult to see because of the very abundant yolk [ILLUSTRATION FOR FIGURE 5E OMITTED]. As early as the onset of invagination, cells emerge from the thinner part of the epithelium and enter the perivitelline space at the animal pole [ILLUSTRATION FOR FIGURE 5B OMITTED]. Their microanatomy is similar to that of the mesenchyme, suggesting that they originate from it, through the ectoderm [ILLUSTRATION FOR FIGURE 5B OMITTED]. At the animal pole, the gastrular epithelium may become locally disorganized [ILLUSTRATION FOR FIGURE 5E OMITTED]. During gastrulation, these mesenchyme-like cells move around the embryo, from the animal pole to the deepened blastopore in the perivitelline space, forming a lateral cap on half of the surface of the embryo [ILLUSTRATION FOR FIGURES 2F AND 6A OMITTED]. More and more cells invade the perivitelline space as the diameter of the gastrula decreases [ILLUSTRATION FOR FIGURES 2G AND 6B OMITTED]. Finally, the perivitelline space is again completely free of cells [ILLUSTRATION FOR FIGURE 6C OMITTED]. In fact, all the migrating cells have moved to the blastopore of the embryo to form, at the end of gastrulation, a "vitelline plug" [ILLUSTRATION FOR FIGURE 6C OMITTED] that closed and thus isolated the archenteron in the mesenchyme [ILLUSTRATION FOR FIGURE 7C OMITTED]. All that remains visible of the mass of migrating cells at the surface of the embryo is the vitelline plug at the blastopore. During gastrulation, the lumen of the archenteron is always empty. The advanced gastrula is still filled with mesenchyme cells [ILLUSTRATION FOR FIGURE 7C OMITTED], and interphase and mitotic nuclei are often observed in the mesenchyme [ILLUSTRATION FOR FIGURE 7D OMITTED]. At the end of gastrulation (50-65 days), part of the animal ectoderm thickens and forms two tissue layers separated by a lumen. This cell mass will become the vestibule [ILLUSTRATION FOR FIGURE 5C OMITTED]. The external layer becomes the ceiling of the vestibule. The internal layer (floor of the vestibule) and the underlying mesenchyme will give rise to the oral surface of the juvenile. Other cell masses differentiate within the mesenchyme [ILLUSTRATION FOR FIGURES 7A AND B OMITTED]. They grow hollow and give rise to the coelomic cavities. No connection between these cell masses and the archenteron was ever observed on serial sections. Gastrulation ends with hatching (65 days after fertilization). The embryo breaks through the enclosing fertilization membrane [ILLUSTRATION FOR FIGURES 2H AND 6D OMITTED].
Post-gastrular stage and differentiation of the oral surface of juvenile
The embryo is now spherical (1.3 mm in diameter). A small aboral depression is the only mark of the blastopore [ILLUSTRATION FOR FIGURE 2I OMITTED]. During the post-gastrular stage, from 65 days to about 130 days after fertilization, a red pigmentation spreads over the surface of the postgastrula embryo and finally concentrates at the oral pole [ILLUSTRATION FOR FIGURE 2J OMITTED]. It is surrounded with an epidermis 70 to 80 [[micro]meter] thick [ILLUSTRATION FOR FIGURES 8A AND C OMITTED]. The developing vestibule, arising from the ectodermal cell mass, is now separated from the embryonic epidermis [ILLUSTRATION FOR FIGURE 8A OMITTED] and completely surrounded by mesenchyme [ILLUSTRATION FOR FIGURES 8A AND B OMITTED]. The coelomic cavities differentiated from the mesenchymal cell masses of the gastrula increase in volume around the gut [ILLUSTRATION FOR FIGURES 8C AND 9A OMITTED]. The gut wall consists of three tissue layers: a digestive epithelium (high and narrow cylindric cells filled with yolk), an intermediate layer of mesenchyme, and a thin coelomic epithelium [ILLUSTRATION FOR FIGURES 8B, C OMITTED]. At the end of the post-gastrular stage, the digestive tube is suspended in the coelomic cavity by means of mesenteries [ILLUSTRATION FOR FIGURES 8C AND 9A OMITTED]. The future oral surface of juvenile has formed from the ectodermic floor of the vestibule and the underlying mesoderm [ILLUSTRATION FOR FIGURES 8B, C, AND 9E OMITTED]. It is separated from the external environment by the epidermis of the postgastrula embryo and the epidermis of the ceiling of the vestibule [ILLUSTRATION FOR FIGURES 8C, 9A AND E OMITTED]. The epidermis of the floor of the vestibule forms five external folds that will become the five primary podia [ILLUSTRATION FOR FIGURES 8B, 9B AND C OMITTED]. Internal folds of that floor will give rise to the neural plate [ILLUSTRATION FOR FIGURES 9A AND D OMITTED] and the five radial nerves [ILLUSTRATION FOR FIGURE 9B OMITTED]. These folds are epineural folds that are growing over the juvenile oral surface to form the epineural sinus, as in the juvenile rudiment of planktotrophic larvae (von Ubisch, 1913; Hyman, 1955; Emlet, 1988). At the same time, the underlying mesoderm begins to differentiate the aquiferous ring, which is extended under each fold to form five aquiferous tubes [ILLUSTRATION FOR FIGURES 9B AND D OMITTED]. The radial nerves and the aquiferous tubes provide the primary organization of the ambulacral zone of the oral surface of the juvenile. The epidermis of the oral surface develops more buds; these will evolve into additional podia in the ambulacral zone and into spines [ILLUSTRATION FOR FIGURES 9C AND E OMITTED]. Between 80 and 85 days after fertilization, a tiny fold of the ectoderm ceiling of the vestibule forms and grows toward the embryonic epidermis [ILLUSTRATION FOR FIGURES 8D OMITTED]. Fifteen days later, the epidermal ceiling of the vestibule merges into the oral embryonic epidermis [ILLUSTRATION FOR FIGURE 8F OMITTED], forming a tiny opening to the external environment [ILLUSTRATION FOR FIGURES 2K, 8E AND F OMITTED]. At the end of the post-gastrular stage, the epidermis of the oral (animal) pole becomes thinner and thinner until it turns almost transparent [ILLUSTRATION FOR FIGURE 8E OMITTED]. Finally, this opening becomes bigger and bigger. The lips move to the aboral (vegetal) pole. The movement involves the total exteriorization of the oral surface of juvenile. At this stage (130 days), the "old" postgastrula embryo measures about 1.6 mm in diameter. That means a growth of 0.3 mm in diameter. Classical echinoid larval structures (arms, ciliary bands, larval tissue, or larval skeleton) have never been observed during the postgastrular stage.
The brooding cycle continues with two juvenile stages. Externally, these two stages differ markedly in spine length (Magniez, 1979): stage 1 (130-197 days) has short spines [ILLUSTRATION FOR FIGURE 2M OMITTED]; stage 2 (197-254 days) has much longer spines [ILLUSTRATION FOR FIGURE 2N OMITTED]. Both are orange with much red pigment in the epidermis. Except for the gut, all organs of the juveniles essentially originate from the differentiation of the floor of the oral surface of the juvenile.
Short-spined juvenile (juvenile stage 1). The early juvenile stage [ILLUSTRATION FOR FIGURE 2L OMITTED] is characterized by a complete exteriorization of the stomodeal area and of the five ambulacral areas that end in primary podia. They will extend to the aboral pole during this phase. Juvenile stage 1 is oblong in outline but becomes spherical (1.8 mm in diameter) as development proceeds.
At the oral pole, the coelomic cavity increases in volume and progresses to the aboral pole. The gut is suspended in the coelomic cavity by means of mesenteries. The digestive tube convolutes on itself, forming two loops as revealed by serial sections [ILLUSTRATION FOR FIGURE 10B OMITTED]. The upper loop is the most developed and goes counterclockwise; the smaller, second one goes clockwise. The ends of the gut are still not differentiated. Although the oral end is virtually in its final position, near the stomodeal area [ILLUSTRATION FOR FIGURE 10B OMITTED], it is not yet connected to the ectoderm [ILLUSTRATION FOR FIGURE 10C OMITTED]. The latter begins to differentiate the stomodeum at the oral pole [ILLUSTRATION FOR FIGURE 10C OMITTED]. The other end of the gut is in contact with the remaining mesenchyme [ILLUSTRATION FOR FIGURE 10B OMITTED], where the aboral mesentery differentiates. The mesenchyme is still filling the vegetal half of the juvenile [ILLUSTRATION FOR FIGURE 10A, B OMITTED]. The three tissue layers forming the gut wall are very similar to that of the postgastrula embryo. The nervous system is made of the neural plate [ILLUSTRATION FOR FIGURE 10C OMITTED] and the five radial nerves (internal folds), which reach to the proximity of the primary podia (external folds). The aquiferous system begins to differentiate podial anlagen. Its gross anatomy is similar to that of the postgastrula embryo.
The advanced stage differentiates many small, non-mobile spines of ca. 0.1-0.3 mm in height [ILLUSTRATION FOR FIGURE 2M OMITTED] with nonfunctional tubercles and podia buds. The proctodeal area is now differentiated. It is surrounded by the five primary podia and is still closed by the ectoderm (as is the stomodeal area).
The very first skeletal stereom elements are now visible in the oral mesoderm, mainly in the interambulacral areas at the oral pole.
Long-spined juvenile (juvenile stage 2). The body of juvenile stage 2 is covered with many long, movable spines. It is spherical, about 1.95 mm in diameter without the spines. The epidermis includes many pigment cells, especially in the podia and at the bases of the spines [ILLUSTRATION FOR FIGURES 2N, 11A OMITTED]. Both are pigmented deep red. During this phase the remaining mesenchyme cells have completely disappeared [ILLUSTRATION FOR FIGURES 11B AND C, 12A OMITTED]. Ectodermic invaginations of the stomodeal and the proctodeal areas join the digestive tube, but it remains closed [ILLUSTRATION FOR FIGURE 12B OMITTED]. The mouth and the anus [ILLUSTRATION FOR FIGURE 12E OMITTED] will open only 1 week before the release of the juvenile (245-250 days after fertilization).
The digestive tube is now complete [ILLUSTRATION FOR FIGURES 11B AND C, 12A OMITTED]. Differentiation of the endoderm into digestive epithelium begins from the ends toward the middle part of the gut. This epithelium includes secretory cells and other cell types with well-developed microvilli [ILLUSTRATION FOR FIGURE 12E OMITTED]. The coelomic epithelium consists of flagellated choanocyte-like cells. The hemal system and the siphon differentiate in the connective tissue (Fig. 11C). Digestive annexes (expansions of the stomach and the intestinal caecum) are also formed [ILLUSTRATION FOR FIGURE 11C OMITTED].
The oral area now includes a nerve ring, an aquiferous ring, and a hemal ring, which surround the future mouth [ILLUSTRATION FOR FIGURE 12B OMITTED]. Five ambulacral canals, which originate from the stomodeal rings, consist of a nerve tube and an aquiferous tube and extend to the proctodeal area [ILLUSTRATION FOR FIGURE 12C OMITTED]. The nerve ring and tube consist of an epineural sinus and three tissue layers: the epineural, ectoneural, and hypo-neural systems [ILLUSTRATION FOR FIGURE 12C OMITTED]. One podium (external expansion) and its ampulla (internal expansion) protude from the aquiferous canal alternately on each side along the radial ambulacra [ILLUSTRATION FOR FIGURE 12C AND D OMITTED]. They form the ambulacral area [ILLUSTRATION FOR FIGURES 2N, 11A OMITTED]. Spines and buds of pedicellaria develop in the interambulacral area. Spines of the advanced juvenile stage 2 have a functional tubercle (muscles, catch apparatus, and skeleton) and are 0.5 to 1.2 mm in height. Juvenile stage 2 individuals are able to move, even in the pouches. The skeleton differentiates from the oral area, in the connective tissue toward the aboral area. When the broods are released from the pouches, a complete test is built by anastomosing perforated plates [ILLUSTRATION FOR FIGURE 12D OMITTED].
Compared to other echinoids that produce a feeding larva or develop from a large egg through an abbreviated development, the most obvious peculiarity of Abatus cordatus is the lack of any larval stage and the tremendous increase in the length of its direct development. The juveniles become free-living 8.5 to 9 months after fertilization. The main characteristics of this development are (1) incomplete cleavage that becomes holoblastic; (2) external migration of mesenchyme cells, in the perivitelline space, from the animal to the vegetal pole during gastrulation; (3) hatching that occurs at the end of the gastrulation; (4) differentiation of the vestibule as soon as the end of gastrulation is attained; and (5) production of a juvenile directly from the gastrula without any larval stage. We will discuss some features of this completely direct development.
Large gametes and lecithotrophy
Chia et al. (1975) described the possible implications of sperm morphology for mode of fertilization and phylogeny. Raff et al. (1990) proposed a correlation between sperm morphology and developmental mode and phylogeny. Among nonfeeding developers, Abatus cordatus, with a 15-[[micro]meter]-long spermatozoid head and an egg 1300 [[micro]meter] in diameter, has the longest echinoid sperm after Phyllacanthus parvispinus (20 [[micro]meter]; Raff et al., 1990), whose egg is not as large (700 [[micro]meter] in diameter; Mortensen, 1921). Moreover, the egg of Araeosoma fenestratum (1290 [[micro]meter] in diameter; Eckelbarger et al., 1989a) corresponds to a sperm-head length of 8.5 [[micro]meter]. A. cordatus is near the upper limit of the range of sperm-head length for non-feeding developers (6.3 to 20 [[micro]meter], Raff et al., 1990). These data show that spermatozoid length is only roughly correlated with mode of development. The mean length ([+ or -] 95% confidence interval), calculated from the compilation of Raft et al. (1990) and including A. cordatus, is 10.6 [+ or -] 7.2 [[micro]meter]. This indicates that an elongate-headed spermatozoid is consistent with a greater ability to penetrate a very large egg, with or without a thick jelly coat, but does not give any idea about the more-or-less transformed or eliminated larval phase. Furthermore, the longest sperm reported in echinoids (26 [[micro]meter]) was described from Aspidodiadema jacobyi, a species with planktotrophic development (Eckelbarger et al., 1989b).
Contrary to what is considered usual for the so-called direct developers (Raff et al., 1990; Wray and Raff, 1991a; Byrne, 1992), the yolky eggs of A. cordatus are not buoyant, in spite of their high lipid content (Magniez, 1983; Lawrence et al., 1984). They sink, as do the eggs of Patiriella spp. (asterinid seastars with pseudo-direct development; Byrne, 1992). Lawrence et al. (1984) estimated the energetic density of A. cordatus egg to be 12.31 J [center dot] [mm.sup.-3], a relatively high value, close to that of brooding asteroid species (Lawrence et al., 1984; McClary and Mladenov, 1990). Because the eggs of A. cordatus are spawned through dorsal gonopores, negative buoyancy assists the eggs in reaching the dorsal brood chambers.
The large, nutrient-rich egg of A. cordatus should provide the energy source required for its 9-month-long development. However, epidermal uptake of DOM is a possible enhancement of nutrient supply in the brood chambers of A. cordatus, as demonstrated for adult, larvae, and broods in different echinoderm classes (Bamford, 1982; Feral, 1985; McClary and Mladenov, 1990).
An incomplete and superficial segmentation becomes holoblastic and generates a wrinkled stereoblastula
The first horizontal constriction, which occurs 2 days after fertilization, delimits a non-nucleated vegetal structure (as revealed by Hoechst staining). This phenomenon has no equivalent among echinoderms. It is analogous to the very beginning of the formation of the polar lobe of some mollusc species in which the first cleavage is accompanied by the formation of a protusion at the vegetal pole of the egg (Verdonk and van den Biggelaar, 1983). However, experiments must be performed to determine what role (other than nutrient storage) is played by this structure, which totally regresses during cleavage, in Abatus cordatus.
After the incomplete and superficial cleavage, the segmentation of A. cordatus becomes holoblastic, as in most echinoderms that produce yolky eggs (Mortensen, 1921; Raff, 1987; Olson et al., 1993). This cleavage gives a wrinkled blastula, a phenomenon usually observed in lecithotrophic echinoderms (Mortensen, 1921; Fell, 1941, 1946; Okazaki and Dan, 1954; Amemiya and Tsuchiya, 1979; Byrne and Barker, 1991; Henry et al., 1991; Komatsu et al., 1991; Amemiya and Emlet, 1992; Byrne, 1992; McEdward, 1992; Olson et al., 1993) and some planktotrophic asteroids (Henry et al., 1991). In A. cordatus, the blastula - which is filled with mesenchyme cells - is in fact a stereoblastula. Syncytial cleavages, also resulting in stereoblastulae, were described for the holothuroid Cucumaria glacialis (Mortensen, 1894) and the asteroid Fromia ghardaqana (Mortensen, 1938) and are posited for some other echinoids (Mortensen, 1903, 1905).
Wrinkled blastulae have been described in four of the five classes of extant echinoderms (asteroids, holothuroids, ophiuroids, and echinoids) with no reference to the systematic position of the species (Fell, 1946; Patent, 1970; Komatsu et al., 1990, 1991; Henry et al., 1991; McEdward and Janies, 1993; Cerra and Byrne, 1995). Wrinkled blastulae have been seen most frequently in starfish (Henry et al., 1991). In asteroids, the smallest ovum developing through a wrinkled blastula is 125 [[micro]meter] in diameter (Luidia quinaria) and the largest is 1200 [[micro]meter] in diameter (Mediaster aequalis) as noted by Komatsu et al., 1991. These authors concluded that the size of the ovum has no bearing on the occurrence of the wrinkled blastula. In echinoids, a wrinkled blastula was described for species having large ova with more or less abbreviated development (Peronella japonica, Phyllacanthus imperialis, P. parvispinus, Asthenosoma ijimai, Heliocidaris erythrogramma; see Komatsu et al., 1991; Wray and Raff, 1991a; Raff, 1992). As emphasized by Cerra and Byrne (1995), there is no relationship between the mode of development and the presence of wrinkled blastulae. The wrinkling may be caused by the lack of space in the fertilization membrane (Chia, 1968; Byrne and Barker, 1991; Henry et al., 1991; Cerra and Byrne, 1995). Our observations confirm that wrinkle formation is the result of packing an enlarged surface area in a confined space. Cell-marking experiments would be required to test whether the wrinkles result also from differences in the physical properties and construction of the blastular epithelium (Henry et al., 1991).
In Abatus cordatus, wrinkles are not lost prior to gastrulation. Gastrulation may be initiated within one of the wrinkles, as observed in Heliocidaris erythrogramma (Williams and Anderson, 1975).
The gastrula is made of a monostratified epidermis surrounding the mesenchyme, which consists of nucleated yolky cells. The nuclei (interphase and mitotic) are quite difficult to see because the cells are big and the yolk is still very abundant. The observed lipid(?)-rich spheroids are similar in shape to the lipid droplets reported by Henry et al. (1991) in Heliocidaris erythrogramma, but they are not secreted into a cavity, which does not exist in Abatus. Lipid(?) droplets are inclusions of the mesenchyme cells. Epithelial cells do keep their own spheroids until the juvenile stages. No apocrine secretion is taking place at any time ([ILLUSTRATION FOR FIGURE 7 OMITTED] in Henry et al.).
Generally, echinoid embryos hatch from the fertilization membrane just before the beginning of gastrulation, even in cidaroids with abbreviated development (Parks et al., 1989; 18 h after fertilization in Phyllacanthus parvispinus.) In other species, hatching occurs at the beginning of gastrulation (10 h after fertilization in Peronella lesueuri - Mortensen, 1921; 15-16 hours after fertilization in Heliocidaris erythrogramma - Williams and Anderson, 1975). In Abatus cordatus, hatching occurs at the end of gastrulation, ca. 65-70 days after fertilization. Species that hatch at the end of gastrulation or even later are found among the asteroids. In Patiriella regularis (indirect development), hatching by rupture of the fertilization membrane occurs 25 h after fertilization, and gastrulae become free-swimming feeding larvae (Byrne and Barker, 1991). In Asterina gibbosa (abbreviated development), hatching occurs 85 to 96 h after fertilization and produces a thoroughly transformed brachiolaria that rapidly adheres to the substratum (Marthy, 1980). In both cases, blastula and gastrula are hollow (Byrne and Barker, 1991; Ludwig, 1882).
A hypothetical new mode of gastrulation produces a pre-adult: perigastrulation
Gastrulation in echinoderms involves two simultaneous but relatively independent processes, invagination to form the archenteron and migration of the primary mesenchyme cells. As the archenteron approaches the wall of the animal region, pseudopodia-forming cells become disengaged from the archenteron and form a mass of secondary mesenchyme cells that spread over the inner surface of the adjacent ectoderm (see Burke, 1990; Burke et al., 1991, for the mechanism). Modifications in gastrulation have been an important component of evolution in echinoderms and other animals (Wray and Raff, 1991b; Raff, 1992). Cell movements that occur during gastrulation establish the topological relationships between cells of the primary germ layers, setting up all the future inductions between tissues.
From the exterior, gastrulation of Abatus cordatus begins with smoothing of the surface. As soon as this phenomenon begins, invagination for gastrulation begins at the vegetal pole. The overall diameter does not change up to the time of hatching. Two findings suggest that this is a novel form of gastrulation involving mesenchyme cells moving out of the gastrula and reentering it during gastrulation: (1) the original diameter of the gastrula decreases while the number of migrating cells - so-called elements vitellins (Schatt, 1985a; Shatt and De Vos, 1990) - increases in the perivitelline space; and (2) the diameter of the gastrula increases again at the end of gastrulation and the perivitelline space is clear before hatching. The perivitelline space is the only possible means for such movements to occur, the gastrula being continuously filled with cells (see diagrammatic [ILLUSTRATION FOR FIGURE 13 OMITTED]). The fate of these migrating cells is obscure. It is possible that some reenter the embryo (ingression) at the base of the archenteron. The last ones are actually included in the vitelline plug. If two populations of mesenchyme cell (one autochthonous, the other migrating) actually exist, they may form the skeleton, the connective tissue, the muscle, or the epithelium of cavities (water vascular system, mesenteries). However, this remains speculative. Fate-mapping studies will be necessary to establish the existence and the establishment of different mesenchyme cell lineages. Experimental confirmation of perigastrulation is still needed.
Because the gastrulation of A. cordatus is very different from other known gastrulations, we propose to use the term perigastrulation, tentatively until more definitive studies are available, to describe the peculiar movement of cells within the perivitelline space during gastrulation.
Animal pole ciliary tuft and ciliated epidermis of echinoderm gastrulae (Horstadius, 1973) have never been observed during the gastrulation of A. cordatus, which does not feed; these structures are also absent, or reduced, in lecithotrophic larvae (Mortensen, 1921). In contrast to the usual differentiation of the vestibule during metamorphosis (Czihak, 1971), in A. cordatus this process occurs at this end of gastrulation before any organogenesis. This phenomenon, unique in echinoids, allows differentiation of the juvenile to take place directly, without producing a larva.
A long organogenesis produces the miniature functional juvenile directly from the gastrula
The differentiation of the rudiment, which occurs during the post-gastrular stage of Abatus cordatus, is the only instance in which there is a similarity between the metamorphosis of A. cordatus and the metamorphosis of pluteus echinoid larva (Czihak, 1971). In A. cordatus, the juvenile is directly differentiated from the gastrula. The adult rudiment of Abatus arises from a thickening of the ectoderm and of the underlying mesenchyme that is already visible in the gastrula (65 days after fertilization) in place of the ectodermal invagination and of the left coelomic pouch in indirect developing echinoids (Hyman, 1955). In A. cordatus, the thickening (future vestibule) becomes isolated in the mesoderm and extends under the ectoderm of the oral pole of the embryo. The oral surface forms the neural plate, the water ring, and the five radial canals. Five primary podia arise from folds in the ectodermal floor of the cavity, just as they do in the rudiment of planktotrophic larvae (von Ubisch, 1913; Hyman, 1955; Emlet, 1988). In A. cordatus, the oral surface is thrust to the exterior at the beginning of the short-spined juvenile stage, when an opening develops in the ceiling of the cavity (130 days after fertilization). This process is similar to the opening of the outer wall of the vestibule in larvae of indirect developers such as Paracentrotus or Psammechinus (von Ubisch, 1913; Hyman, 1955). As a result, the morphogenesis of the rudiment of A. cordatus is similar to that of the other echinoids except for the formation of the cell mass that gives rise to the vestibule. Though probably independently evolved from Abatus, the pattern in temnopleuroids seems to be similar. The vestibule is also an ectodermally derived cell mass that is isolated from the ectoderm on the late gastrula (Fukushi, 1959, 1960). The details to substantiate this are missing from the Abatus series, due to the nature and timing of the material collection. In Abatus, the differentiation of the vestibule takes much longer than in any known case. Differences from the general case have been described in the abbreviated development of some euechinoids. In Peronella lesueuri, Asthenosoma ijimai, and Heliocidaris erythrogramma, the vestibule is more or less open to the exterior during the morphogenesis of the rudiment (Mortensen, 1921; Williams and Anderson, 1975; Amemiya and Tsuchiya, 1979; Amemyia and Emlet, 1992; Wray and Bely, 1994). In cidaroids, the absence of a vestibule appears to be typical (Emlet, 1988), and the morphogenesis of the rudiment occurs in contact with the exterior.
The characteristics of direct development in Abatus cordatus are not totally consistent with the hypothesis, recently advanced by several authors (Raff, 1987, 1992; Wray and Raff, 1991a; Wray and Bely, 1994), of a general acceleration, in relative time and in absolute time, in the development of adult features for the facultative feeding pluteus or the nonfeeding abbreviated pluteus. The development of A. cordatus is condensed (no larval morphogenesis) and presents a shift in the relative timing (heterochrony) of the appearance of the major features (archenteron, coelom + vestibule, hydrocoel), if compared to the typical echinoid development (archenteron, larval skeleton, larval gut, coelom, hydrocoel, vestibule). However, in this case the development does not accelerate in absolute time. The 250-day-long development of Abatus is probably a consequence of developing in cold water.
We did not observe that coeloms develop through the typical enterocoelic phenomena characteristic of most echinoderms (Czihak, 1971). The coelomic cavity of A. cordatus is apparently formed by schizocoely, similar to the schizocoely described in Heliocidaris erythrogramma by Williams and Anderson (1975). These authors described the splitting of left and right coelomic pouches immediately after gastrulation. However, the process differs from that in A. cordatus because the first pouches in H. erythrogramma develop by enterocoely. The interpretation given by Williams and Anderson was criticized by Wray and Raff (1989, 1990). Schizocoely was also hypothesized for Gorgonocephalus caryi (Patent, 1970) and Amphipholis squamata (Fell, 1946), species in which the coelomic cavity arises by splitting in the mesenchyme.
When juveniles are ready to feed (with the opening of the stomodeum 254 days after fertilization), they leave the brood pouches. This is equivalent to the settlement of the young juvenile that develops from a planktotrophic larva; settlement occurs weeks to months after fertilization (Emlet et al., 1987).
The development of A. cordatus is strictly direct (ametamorphic). This is congruent with the fact that a histoautoradiographic study showed that there is only a unique phase of cell proliferation responsible for the first prospective organs (Schatt, 1988). In the case of the existence of larval organogenesis, before the adult organogenesis, two cell proliferations would have occurred. The adult digestive tube and the adult rudiment directly differentiate from the tissues of the gastrula. This change from a feeding larval stage to a direct development is rare. However, it occurs in ophiuroids (Fell, 1945, 1948) and asteroids (McEdward, 1992; McEdward and Janies, 1993). In order to avoid any misunderstanding, we propose to reserve the term direct development for species that exhibit completely direct development and the term abbreviated development for species that have more or less entirely eliminated the pluteus stage. The term mesogen was proposed for directly developing asteroids to refer to the developmental stages that occur between the end of the gastrula stage and the beginning of the juvenile stage (McEdward and Janies, 1993). However, we kept the term post-gastrular stage in A. cordatus because it seems to us more easily understandable and more general.
To feed or not to feed?
As stated by Strathmann (1978) and Raff (1987), loss of the feeding-larval stage is irreversible and strongly correlated with egg size. Raff (1987) hypothesized that constraints existing in the development of larval stages are eliminated by abandoning the need for a feeding stage. Increasing the amount of yolk available to the embryo seems to make this possible. The archenteron of Abatus cordatus, first isolated within the mesenchyme, elongates and differentiates into the adult gut starting with the beginning of juvenile stage 1. This gut becomes functional shortly before the stage 2 juveniles are released from the brood chambers, about 250 days after fertilization (Schatt, 1985a). Schatt (1988) showed that the dry weight is constant (ca. 0.7 mg) from spawning to 165 days after fertilization (half of the juvenile stage 1). Then, it increases to reach ca. 2.5 mg for the juvenile stage 2, at the moment where they are released before the opening of the mouth. The increase of weight of A. cordatus is essentially due to the differentiation of the adult skeleton (Schatt, 1988) and is congruent with our microscopic observations. These findings may indicate that yolk provides enough nutrients to maintain the embryos and juveniles (Lawrence et al., 1984) or that very little metabolic activity is occuring for most of the development. However, McClary and Mladenov (1900) reported evidence for nutrient translocation to brooded juveniles of the sea star Pteraster militaris as well as evidence of DOM uptake. This needs to be demonstrated experimentally for A. cordatus (see Fenaux, 1982, for a review).
Do other completely direct developing echinoids exist?
Emlet (1990) counted 14 lineages of echinoids with nonfeeding development. Of these, eight involve brood-protection (Cidaroida, Temnopleuroida, Echinoida, Spatangoida [including Schizasteridae and Brissidae], Holasteroida, Cassiduloida, Clypeasteroida). This represents 7% (15 spp.) of the regular echinoids and 35% (24 spp) of the irregular echinoids. Only a few of them are completely direct developers.
It is easy to predict that other subantarctic and Antarctic schizasterids will be found to be true direct developers. Different stages have already been described, particularly the two short- and long-spined juvenile stages (Abatus spp, Amphipneustes spp, Parapneustes abatoides, Agassiz, 1874; Verrill, 1876; Mortensen, 1910, 1951; Bernasconi, 1969; Larrain, 1973; Magniez, 1980; Schatt, 1985a; Pearse and McClintock, 1990; Schinner and McClintock, 1993; see also Schatt and Feral, 1991, for brooding schizasterid references and Poulin and Feral, in press, for other Antarctic echinoids). One can also add the abyssal Antarctic species Delopatagus brucei, formerly classified as a member of the Asterostomatidae but now considered to be in the Schizasteridae, for which De Ridder et al. (1993) mentioned the presence of juveniles in dorsal brood pouches. Feral et al. (1994) showed by means of the phylogenetic analysis of partial sequences of 28S rRNA that Delopatagus fell into the brooding schizasterid group, clustering with Amphipneustes spp., a genus closely related to Abatus.
This work has been supported by the "Institut Francais pour la Recherche et al Technologie Polaires" (program n [degrees] 195 "MacroBenthos"). We thank L. De Vos (Universite Libre de Bruxelles) for his help with SEM. Thanks are also due to C. Bernard and P. Levi (Laboratoire de Biologie des Invertebres marins, Museum national d'Histoire naturelle, Paris) for technical assistance and to M.-J. Bodiou (Observatoire Oceanologique de Banyuls) for drawings. We are grateful to S. v. Boletzky, A. Collenot, C. Houillon, H.-J. Marthy, A. Picard and E. Poulin for discussions and advice and to J. M. Lawrence and S. v. Boletzky for reading the manuscript and improving the English text. We are also grateful to the anonymous reviewers for their helpful comments.
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|Author:||Schatt, Philippe; Feral, Jean-Pierre|
|Publication:||The Biological Bulletin|
|Date:||Feb 1, 1996|
|Previous Article:||Intersex red claw crayfish, Cherax quadricarinatus (von Martens): functional males with pre-vitellogenic ovaries.|
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