Laboratorio de Embriologia Animal, Departamento de Ciencias Biol6gicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. PabeII6n 11, Ciudad Universitaria, 1428, Buenos Aires, Argentina

(Accepted 24 November 1999)

Abstract

There is a vast literature on the reproductive behaviour of cichlid fishes, most of which describes spawning strategies and parental care. However, descriptive information on the early development of cichlids is scarce. In this study, embryos and larvae of laboratory-reared Cichlasoma dimerus are described. The early ontogeny is documented from oocyte activation until the beginning of the juvenile period. At a water temperature of 25 ± 0.5 'C and a 12:12 h photoperiod, cleavage is finished in 1 0 h and the first somites appear at 26 h of development. The larvae hatch during the beginning of the third day and are deposited by both parents in a pit they have dug out in advance. Yolk-sac larvae present three pairs of adhesive glands over the head, these transient larval organs being characteristic of substrate-brooding cichlids. After another 5 days, the fry swim freely and begin to feed exogenously. Since the yolk-sac is not completely reabsorbed until 2 or 3 days later, there is a period of combined endogenous and erogenous food supply. The juvenile stage is reached on the 42nd day from spawning.

Key words: development, substrate-brooder, Cichlasoma dimerus, Cichlidae, Perciformes

INTRODUCTION

Cichlidae is a family of fresh and brackish water species found in Central and South America, Africa, Madagascar, West Indies, Israel, Syria, coastal India and Sri Lanka. It is a large family that comprises about 105 genera and 1300 species, making it the second largest perciform family (Nelson, 1994).

Species of the family have highly organized breeding activities. Two general forms of parental care can be recognized: (1) mouth-brooders, which are usually polygamous and generally only the female carries the fertilized eggs and newly hatched eggs in the mouth; (2) substrate-brooders, which are usually monogamous and both sexes may care for the eggs (Baerends & Baerends van Roon, 1950; Fryer & lles, 1972; Barlow, 1991; Keenleyside, 1991). Mouth-brooding or oral incubation is common in several groups of African cichlids, while most American cichlids are substrate-spawners (Fryer & lles, 1972; Keenleyside, 1991; Nelson, 1994).

In substrate-brooding species the eggs are always spherical or elliptical and stick firmly to the substrate, with a sticky mucous layer or attachment filaments around the chorion. Egg size varies greatly, as does colour. Larvae of many species have cement glands over the head and swing their tails rapidly while attached to the substrate. Some species that build their nests in running water have moderately developed embryonic respiratory organs; others from lentic nest sites have highly developed yolk-sac and fin fold vessels (Balon, 1975).

The South American cichlid C dimerus is common in quiet shallow waters of the Paraguay and most of the ParanA river basins (Kullander, 1983) and lives in pairs which defend territories. The females lay their eggs on a cleaned substrate and one or more pits are dug on the bottom, to which the offspring are transferred by mouth after hatching. Both parents fan the eggs and the young fry, and guard their young when these, in a school, start swimming around (Staeck & Linke, 1995).

There is a vast literature on the reproductive behaviour ofcichlid fishes, most of which describes spawning strategies and parental care (Baerends & Baerends van Roon, 1950; Fryer & lles, 1972; Barlow, 1991; Keenleyside, 1991). But, considering the number of species and their wide distribution, it is surprising that there is little descriptive information on the early development of cichlids.

Several detailed studies of the early ontogeny of fishes have been made on species traditionally considered asmodels of teleostean or even vertebrate development, such as Fundulus heteroclitus (Armstrong & Child, 1965; Trinkaus, 1984, 1992), Salmo gairdneri (Ballard, 1973a,b,c), or the zebrafish Danio (Brachydanio) rerio (Warga & Kimmel, 1990; Kimmel et al., 1995). Many other studies, at various levels of detail, have been made on hundreds of species, both freshwater and marine. However, there are few studies on the family Cichlidae, which belongs to the largest order of vertebrales, namely Perciformes. Most of the descriptiva works on the early development of cichlids were carried out on African species (Fryer & lles, 1972; Balon, 1977; Kuwamura & Mihigo, 1988; Galman & Avtalion, 1989; Holden & Bruton, 1992, 1994). In comparison, there are few descriptions of Neotropical cichlid development and these are generally less detailed (Balon, 1960; Jones, 1972; Martínez & Murillo, 1987; Cabrera, Murillo & Mora, 1988; Contreras & Díaz, 1990).

Studies on the early ontogeny of fishes are important not only to increase the knowledge about the developmental features of the different species but also to have a model for comparison when normal patterns of development are altered. Also they have a practicar application in aquaculture and fisheries biology. Moreover, larval fishes often display characteristic patterns of pigmentation which can be used to identify species at an early point in their lives. Early life-history characters may be useful also in assessing phylogenetic relationships (Richards & Leis, 1984; Stiassny & Mezey, 1993; Britz, 1997).

The aim of the present study was to describe the early life-history stages of C dimerus, from oocyte activation until the beginning of the juvenile period, under laboratory conditions. This work will then contribute to increase knowledge about the early development of cichlids and might result in an aid to systematic and phylogenetie studies.

MATERIALS AND METHODS

Adult specimens used in this study were captured in Esteros del Riachuelo, Corrientes, Argentina (27'25'S, 58'1 5'W). They were kept in large aquaria at 25 ± 0.5 'C and 12:12 h photoperiod that were well aerated and provided with externas filtration. Fishes were normally fed with pelleted commercial food and Tubifex worms.

The adaptation of adult individuals to aquarium conditions took c. 1 month. From that moment, pair formation and establishment of territorios within the aquaria took place. Single pairs were transferred to smaller aquaria of 45 1, with a layer of gravel and smooth stones on the bottom for egg deposition.

Eggs and larvae describes in this work were obtained from 26 spawnings of 3 pairs. A part of the brood guarded by parents was sampled periodically. On the 14th day from spawning, each offspring was isolated in a 15-1 aquarium, in which their development was followed until they reached the juvenile stage. Temperature and photoperiod conditions were the same as those of the adults. The fry were fed with freshly hatched nauplii of Artemia salina during the first stages, and then with powdered dry food.

Specimens alive or recently fixed in 4% formalin were observed and measured microscopically. The main morphological and functional features of each developmental stage were describes and the hours or days of development were determined, considering the moment of insemination as 0 h. Embryos and larvae were photographed under a Nikon microphot FX microscope and a Nikon SMZ-U stereomicroscope, respectively.

To study the structure of cement glands, larvae were fixed in Bouin's liquid, embedded in paraffin and sections, 8 lim thick, were stained with haematoxylincosin. The slides were examined and photographed under the Nikon microscope. Other larvae were stained in toto for mucopolysaccharides in accordance with the PAS (periodic acid Schiff reaction) technique of Peters & Berns (1982).

The progress of ossification was studied using a modification of the clearing and staining procedures proposed by Potthoff (1984) and Taylor & Van Dyke (1985). Alcian blue (20 mg in a solution of 70 ml absolute ethanol, 30 ml glacial acetic acid, for 24 h) and alizarin red S (75 mg in 100 ml 1% KOH for 28 h) were used to stain cartilage and bone, respectively.

For scanning electron microscopy (SEM), freshly spawned, fertilized eggs and larvae were fixed in 3% glutaraldehyde in 0.1 m phosphate buffer, pH 7.8, for 24 h. Specimens were subsequently washed with phosphate buffer and gradually dehydrated to ethanol 100%. Samples were then critical-point dried, coated with gold, and examined and photographed under a Jeol JSM 11.

Characteristics of the freshly spawned egg

The eggs of C. dimerus have an ovoid shape, with the horizontal axis longer (1.65 ± 0.05 mm) than the vertical axis (1.25 ± 0.05 mm) (Fig. la). They have a smooth translucent chorion surrounded by a layer of mucous secretion which makes them stick to the substrate and to one another at the moment of deposition (Fig. 2a, b). The micropyle, situated at the animal pole of the egg, is cone-shaped. The diameters of its outer and inner openings are 90 um and 1um, respectively (Fig. la). The mucous layer is less evident around the animal pole; instead, a set of numerous thin filaments is present in this region (Fig. 2c). This filament tuft is not permanent, and can only be observed until the first stages of segmentation (up to the four or eight blastomere stage). The cytoplasm is uniform in appearance and the yolk is bright, with numerous tightly packed globules (Fig. la).

Fig. 1. Embryonic development of Cichlasoma dimerus. Light microscope photographs. (a) Oocyte stage (0 h); (b) zygote stage (10 min); (c) zygote stage (1 h 25 min); (d) two-blastomere stage (1 h 45 min); (e) four-blastomere stage (2 h 05 min); (f) eightblastomere stage (2 h 45 min); (g) 64-blastomere stage (4 h 55 min); (h) early-blastula stage (5 h 30 min); (i) late-blastula stage (7 h 10 min). b, blastomeres; bd, blastodise; bl, blastoderm; ch, chorion; mi, micropyle; ps, perivitelline space; y, yolk. All photographs are at the same magnification. Scale bar = 500 gm

Under the SEM, the funnel-shaped configuration of the micropyle is clearly observed (Fig. 2d). The external  chorionic surface shows slight prominences separated by furrows, which may correspond to imprints of  follicular cells (Fig. 2e).

Embryonic development

Zygote stage (10 min-1 h 25 min). Fertilization activates cytoplasmatic movements, easily evident within 10 min.

Fig. 2. Freshly spawned eggs of Cichlasoma dimerus. (a) (c) Light-microscope photographs; (d), (e) SEM photographs. (a) A pair of eggs attached by the mucous secretion; (b) adhesive mucous layer at a higher magnification; (c) filament tuft surrounding the micropylar area; (d) micropylar region; (c) external chorionic surface (mucous coat has been removed for clarity). ch, chorion; ft, filament tuft; ml, mucous layer. Scale bars: (a) = 200 um; (b), (c) = 100 um; (d) = 25 um; (c) = 5 um.

Perivitelline space is formed and cortical cytoplasm begins to stream toward the animal pole (Fig. lb), where the nucleus is situated, to form a prominent domed layer of cytoplasm known as blastodisc (Fig. le). The cytoplasm of the blastodisc is continuous with a thin layer of cytoplasm which surrounds the yolk, called the yolk cytoplasmatic layer.

The cleavage is typically meroblastic discoidal, creating a cellular region above the yolk mass. The first divisions are synchronous and result in fairly stereotyped arrays of blastomeres (Fig. Id-g).

Two-blastomere stage (1 h 45 min). The first cleavage furrow is meridional. It arises near the animal pole and progresses rapidly toward the vegetal pole, dividing the blastodisc into two blastomeres of equal size (Fig. Id).

During the first cleavage, second polar body extrusion takes place. The polar body appears on the surface of the segmented blastodisc, close to the micropyle (Fig. 3).

Four-blastomere stage (2 h 05 min). The second cleavage occurs in a single meridional plane at a right angle to the first, giving rise to four blastomeres in a 2 x 2 array (Fig. le).

Eight-blastomere stage (2 h 45 min). Cleavages occur in two separate planes, parallel to the first one, and on either side of it, producing a 2 x 4 array of cells when viewed from the animal pole. In a lateral view, only four blastomeres are clearly observed (Fig. If).

The fourth and fifth cleavages define 16 and 32 blastomeres, respectively. These blastomeres lie in a single layer that may be partially curved on the yolk surface.

64-blastomere stage (4 h 55 min). The sixth cleavage is the first to occur along a latitudinal plane and so, for the first time, some of the blastomeres completely cover the other ones (Fig. 1 g).

Beyond the sixth cleavage, it becomes more difficult to discern any stereotypical array of blastomeres. Cleavage planes are no longer regularly patterned and cell cycles begin to lose synchrony.

Fig. 3. Cichlasoma dimerus. Light-microscope   photograph of second polar body extrusion, which takes place during the first cleavage. f, first cleavage furrow; spb, second polar body; Scale bar 25 um

Fig. 4. Blastoderm of Cichlasoma dimerus. Cross-section at blastula stage. be, blastocoel; db, deep blastomeres; el, enveloping layer; p, periblast; y, yolk; ycl, yolk cytoplasmatic layer; ysn, yolk syncytial nuclei. Scale bar = 1 00 um.

Blastulation extends from the 128-blastomere stage to the onset of gastrulation. The blastula consists of a multicellular blastoderm in which three cell types can be distinguished: a superficial layer of cells, firmly attached to each other, known as enveloping layer; a yolksyncytial layer or periblast that lies over the yolk; between these two layers, a group of inner loosely arranged cells called deep blastomeres. The small intercellular spaces that exist between the deep cells constitute the blastocoel or segmentation cavity (Fig. 4). Early-blastula stage (5 h 30 min). The blastoderm looks like a roundish mound perched on the top of the yolk. A constriction can be observed where the marginal cells of the blastoderm meet the yolk mass (Fig. lh).

Late-blastula stage (7 h 10 min). The animal-vegetal axis of the blastula shortens, with the blastoderm compressing down upon the yolk. The constriction at the blastoderm margin diminishes and finally disappears (Fig. li).

The subsequent gastrulation process is characterized by different morphogenetic movements. The major cell movement to occur is epiboly, which results in a rearrangement of the blastoderm relative to the yolk. During epiboly, the blastoderm, with the periblast as its leading edge (Trinkaus, 1984, 1992), spreads gradually ver the yolk mass, toward the vegetal pole. As the

blastoderm flattens around the yolk, inner cells intercalate outward among those of more superficial layers (Warga & Kimmel, 1990; Helde et al., 1994), and this is termed radial intercalations. The yolk mass prominently changes its shape at the same time that epiboly and radial intercalations occur. It bulges toward the animal pole, occupying territory simultaneously vacated by the deep blastomeres during their outward movements. As a consequence, the blastoderm thins considerably, changing from a piled cell mound (Fig. li) to an inverted cup-shaped cell multilayer of nearly uniform thickness (Fig. 5a).

Germ-ring stage (15 h 30 min). The thickened marginal region that circumscribes the entire rim of the blastoerm is terme t e germ ring and, although passively pulled along by the periblast, it visually represents the advancing edge during the epiboly movement (Fig. 5a). The germ ring consists of two cell layers. The more superficial layer is called the epiblast, and the inner is termed the hypoblast. The cells remaining in the epiblast when gastrulation ends correspond to the definitive ectoderm 1 while thb hypoblast gives rise to derivatives classically ascribed to both the mesoderm and endoderm (Kimmel et al., 1995).

Embryonic-shield stage (16 h 20 min). Convergence movements produce a local accumulation of deep cells at the position of the germ ring that corresponds to the future dorsal side of the embryo, the so-called embryonic shield (Fig. 5b).

As more cells converge upon the embryonic shield, mediolateral intercalations cause the shield to narrow and elongate toward the animal pole. This process is termed extension (Langeland & Kimmel, 1997) and morphologically defines the anterior-posterior axis of the embryo (Fig. 5e-f).

Evacuation-zone stage (19 h). As epiboly continues and the shield extends toward the animal pole, the blastoderm clearly becomes thinner than elsewhere on the antero-ventral side, above the margin. This region is the evacuation zone; cells leave it by both epiboly and convergence toward the embryonic axis (Fig. 5c). 75%-epiboly stage (21 h). When 75% of the yolk mass has been covered by the blastoderm, the shape of the embryo itself increases along the animal-vegetal (anterior-posterior) axis (Fig. 5d).

Yolk-plug stage (22 h 20 min). The portion of uncovered yolk protruding from the neighbourhood of the vegetal pole when epiboly is about to conclude is considered a yolk plug (Fig. 5e).

The anterior region of the axial hypoblast gives rise to the prechordal plate while the posterior one forms the primordium of the notochord. The dorsal epiblast thickens anteriorly to form the neural plate, the anlagen of the central nervous system.

Tail-bud stage (23 h). Epiboly finishes as the blastoderm completely covers the yolk plug. A few minutes before, just dorsal to the site of yolk plug closure, the posterior or caudal end of the embryo develops a distinct swelling, the tail bud, cells of which will contribute progeny to the tail (Fig. 5f).

Fig. 5. Embryonic development of Cichlasoma dimerus. Light-microscope photographs. (a) Germ-ring stage (15 h 30 min); (b) embryonic-shield stage (16 h 20 min); (c) evacuation-zone stage (19 h); (d) 75%-epiboly stage (21 h); (e) yolk-plug stage (22 h 20 min); (f) tail-bud stage (23 h); (g) six-somite stage (28 h); (h) 22-somite stage (36 h); (i) pre-hatching stage (53 h). agp, adhesive glands primordia; e, eye; ea, embryonic axis; es, embryonic shield; ez, evacuation zone; fb, forebrain; gr, germ ring; h,' heart; hb, hindbrain; 1, lens; m, melanophores; mb, midbrain; op, otic placode; opp, optic primordium; ov, otic vesicle; pc, presumptive pericardial cavity; s, somites; tb, tail bud; yp, yolk plug. All photographs are at the same magnification. Scale bar = 500 um.

Fig. 6. Larval development of Cichlasoma dimerus. Stereomicroscope photographs. (a) Post-hatching stage (55 h); (b) yolk-sac stage (82 h); (c) opened-mouth stage (108 h). ag, adhesive glands; ffv, fin fold vessels; h, heart; m, melanophores; no, notochord; pfb, pectoral fin bud; rp, rays primordia. Scale bars = 500 um.

Along the dorsal side, the neural plate thickens throughout the entire embryonic axis; its more anterior cells will form the brain while the more posterior ones contribute to the spinal cord.

During the metamerism phase, paired somites (derivatives of the paraxial hypoblast) develop sequentially in an anterior-posterior direction, on either side of the notochord. In addition, primordia of the primary organs become visible. The tail bud becomes more prominent and appears at the caudal end of the embryo. The body length of the embryo does not change considerably during the development of the first somites (up to the 15-somite stage approximately). Then, it increases very rapidly, as the tail curls around the original vegetal pole and grows toward the head (Fig. 5i).

Neurulation, formation of the neural tube, occurs by a process of cavitation. A transient primordium with no lumen forms first from the neural plate. Afterwards this neural rod hollows into the neural tube (Papan & Campos-Ortega, 1994).

Six-somite stage (28 h). When the embryo has approximately six somites, optic primordia become recognizable from a side view. The presumptive pericardial cavity is visible as a thin, transparent chamber extending along the surface of the yolk, anterior to the head (Fig. 5g).

Fig. 7. Adhesive glands of larval Cichlasoma dimerus. (a), (c) Light-microscope photographs; (b) stereomicroscope photograph (d) SEM photograph. (a) Anterior part of a recently hatched larva (56 h) showing the three left cement organs (focus has beeni placed on the parietal glands); (h) anterior part of an opened-mouth larva (140 h) after staining in toto with PAS; (c) longitudinal, section through one of the parietal cement glands after staining with haematoxylin-eosin; (d) dorsal view of the larva's head. ag,, adhesive glands; c, cavity of the gland. Scale bars: (a) = 200 um; (h) = 500 um; (c) = 25 um; (d) = 100 um.

22-somite stage (36 h). Primary brain differentiation (forebrain, midbrain and hindbrain) has taken place. The heart starts to beat and otic placodes become visible at both sides of the hindbrain, which is partitioned into several units called rhombomeres. Lenses are evident in the eyes and the first dendritic melanophores appear over the surface of the yolk-sac (Fig. 5h). Pre-hatching stage (53 h). Before hatching the embryo shows conspicuous muscular contractions. The tail is well extended and free from the yolk-sac. Otoliths are formed within the otic vesicles. The beating heart is placed beneath the head, slightly to the left side, and the flow of blood can be observed. Rudiments of the adhesive glands are visible at the midbrain-hindbrain boundary region (Fig. 5i).

At the moment of eclosion, i.e. 54 h, the chorion is softened and slimed by the enzymes secreted by the hatching gland. Muscular twitches of the enclosed embryo finally result in the tearing of the chorion and the release of the fry; thus the larval period begins.

Larval development

Post-hatching stage (55 h; 3.32 -± 0. 1 mm TLI). After hatching, yolk-sac larvae are transferred to a pit dug ins the gravel by the parents. These larvae present three pairs of adhesive glands over the head, this feature being characteristic of cichlids, especially of substrate. brooders (Fryer & lles, 1972; A. J. Jones, 1972; Peters & Berns, 1982, 1983). These glands are found in a specific arrangement: two pairs are situated on the parietal area, just above the eyes, and the third lies on the frontal region, close to the olfactory system (Figs 6a & 7a). Th mouth is not formed, and the gut is narrow and almosg straight. No fins are differentiated but a primordial fid fold is well developed in the sagittal plane. Melano phores are scattered over the oblong yolk-sac an( extend along the junction of the body with the ventra fin fold. Retinal pigment may be present, but the eye are not heavily pigmented and are still transluceini (Fig. 6a).

Fig. 8. Larval development of Cichlasoma dimerus. Stereomicroscope photographs. (a) Free-swimming stage (190 h); (b) unpaired-fins stage (14 days); (c) pre-juvenile stage (25 days); (d) juvenile stage (42 days). g, gills; n, nostril; rp, rays primordia; sb, swim bladder; vf, ventral fin. Scale bars: (a), (b) = 500 um; (c) = 1 mm; (d) = 2 mm.

Fig. 9. Time scale of some of the most important events during the development of Cichlasoma dimerus.

Yolk-sac stage (82 h; 4.04 ± 0.15 mm TL). The mouth has not opened. A pair of small, disc-shaped pectoral fin buds are present but have no functional value. The heart and the ventral fin fold vessels, the embryonic respiratory plexus, are clearly visible from the side. Primordia of pharyngeal arches can be distinguished and cement glands are more conspicuous. The notochord end is slightly flexed. Pigmentation has increased over the yolk-sac and the ventral trunk. Eye pigment is concentrated and opaque (Fig. 6b).

Opened-mouth stage (108 h; 4.65 ± 0.2 mm TL). The mouth and jaws are formed and mobile. The yolk-sac has become smaller. Branchial arches are more differentiated and soon afterwards gill filaments and opercular laminae start to develop. Rays primordia appear in the caudal fin and notochord flexion is more obvious. Stellate melanophores develop on the head and anterior part of the trunk. (Fig. 6c). These larvae do not swim freely; they swing their tails rapidly while still attached to the substrate by their cement glands.

In larvae stained in toto using the PAS technique, adhesive glands seem to be particularly PAS-positive (Fig. 7b). This indicates that the secretion produced by these glands mainly comprises mucopolysaccharides. A longitudinal section shows that they are cup-shaped and their cells seem full of mucous secretion (Fig. 7c). Under the SEM the distinctive crater-like structure and specific symmetrical arrangement of these transient larval organs are revealed (Fig. 7d).

Free-swimming stage (174 h; 5.38±0.27 mm TL). Larvae start swimming freely during the eighth day of development. The oval-shaped swim bladder is visible because of its high refractiveness. The switch-over from the temporary embryonic respiratory system to the adult bronchial system has occurred. Head glands have been regressing and losing functionality, and will completely disappear during the following days. Rays primordia appear in the pectoral fins and caudal fin rays are more developed. Dorsal and anal fins show early differentiation but no fin rays are visible; they consist of thin, transparent membranes. Pigments are more thickly distributed over the beginning, middle and end of the trunk. The head is coated with round, thick melanophores (Fig. 8a). A few hours after the onset of freeswimming, these larvae start to feed exogenously; however the yolk is not totally consumed. Therefore there is a period of combined endogenous and erogenous food supply.

Unpaired-fins stage (14 days,. 5.98 ± 0.42 mm TL) Dorsal and anal fin rays differentiate simultaneousl and the first knuckles become visible in the caudal ray Gills are well formed and the gut, initially straigh starts to coil. Yolk has been completely consumed. pigment pattern of blotches along the body sides i visible (Fig. 8b).

Pre-juvenile stage (25 days; 8.22 ± 0.7 mm TL). Th pelvic fins, which are the last to form, have differen tiated. Dorsal and anal fins are more developed. Mor melanophores have formed on the head and on th lateral and dorsal parts of the body. General pigment tion gradually resembles that of the adult (Fig. 8c).

Juvenile stage (42 days,- 14.70 ± 1.2 mm TL). The fin become fully developed and the adult complement o rays is completed. Dorsal and anal fins are well pi mented and only slightly transparent. Scales hav appeared previously, around the 32nd day of develo ment, and now cover the greatest part of the bod Nostrils are clearly observed. The body is almost co pletely covered with melanophores and the skeleto presents an advanced degree of ossification, as reveale by predominant red coloration in the cleared an stained specimens. Body shape and pigmentatio pattern are similar to those of the adult (Fig. 8d).

Figure 9 shows the time scale of some of the mo important events during the early development C. dimerus.

DISCUSSION

Among substrate-spawning cichlids, two quite distin systems for egg attachment are recognizable (Wickle 1956a,b). In the primitive Asian and Madagase cichlids the eggs are non-adhesive and are attached t the substrate, or to one another, by a tuft of filamen arising from the pole opposite the micropyle. A differe system seems to characterize most African and Neotr pical species; in these fishes the eggs are attached alon their longitudinal axes by an encasing mucous layer an numerous surface filaments. In coincidence with th classification, the eggs of C. dimerus are extremely stic and adhere to the substrate and to each other by distinctive mucous layer with fine filament threa which are almost invisible with a normal microsco (Fig. 2a, b). The eggs of C. dimerus are unusual i possessing a particular filament tuft around the micropylar area (Fig. 2c). It has generally been assumed that the function of the chorionic fibrils is to attach the oviposited egg to the substrate (Brummett & Dumont, 1981). However, since the oocytes of C dirnerus adhere along their longitudinal axes, there is no contact between these filaments and the substrate. In addition, there is some evidence that fertilization is aided by the presence of sperm attractants in the micropylar region of the chorion (Suzuki, 1958). Then, the micropyle and its adjacent structures may not only serve as an access route to the oocyte but may also facilitate fertilization by attracting sperm to the appropriate region of the chorion (Dumont & Brummett, 1985). Further analysis is required to elucidate the functional significance of t hese threads.

As in most of the fish embryos with meroblastic cleavage, the first divisions occur at regular orientations so that one can see how many blastomeres are present by their arrangement (Fig. Id-g). However, certain variation in the cleavage pattern exists within teleosts. For example, the first horizontal cleavage occurs between 32-cell and 64-cell stages during the early embryogenesis of C dimerus (present study) and Danio Brachydanio) rerio (Kimmel et al., 1995). Also, it can occur between 16-cell and 32-cell stages, as in the medaka Oryzias latipes (Iwamatsu, 1994); or even earlier, between four-cell and eight-cell stages, as in the perciform ice goby Leucopsarion petersii (Nakatsuji et al., 1997).

The extent to which the blastoderm has spread over the yolk at the moment of the shield formation is variable within teleosts (Matkovic et al., 1985). In the eggs of C dimerus about 25% of the yolk mass is covered by the blastoderm when the shield forms (Fig. 5b). At 40% of the epiboly process, deep cells from the antero-ventral side of the blastoderm, in the vicinity of the original animal polar region, migrate rapidly by epiboly and converge toward the lengthening embryonic axis, leaving an empty space referred to as the evacuation zone (Ballard, 198 1) (Fig. 5c).

Observations on the early development of other substrate-brooding cichlids (Balon, 1959, 1960; Jones, 1972; Martinez & Murillo, 1987; Cabrera, Murillo & Mora, 1988; Kuwamura & Mihigo, 1988; Contreras & Diaz, 1990) agree with the pattern described in the present study, thus confirming the basic uniformity of cichlid development (Jones, 1972). However, variations exist among species as to the relative timing of ontogenetic events and structure formation in response to different environmental conditions, e.g. temperature and food availability. Heterochrony might also be noted among populations of the same species that occupy different ecological niches (Contreras & Diaz, 1990).

Within the first 5 days from hatching, the larvae of C. dimerus attach themselves to the substrate with the sticky secretions from six cephalic adhesive glands (Figs 6a-c & 7a-d). During this period the larvae lie in a hiding-place, guarded by the parents. In this way, in the natural environment, the adhesive glands help prevent dispersion by currents and facilitate parental care. The glands then go through a gradual regression until they completely atrophy a few days after the onset of free-swimming (Fig. 8a). Three pairs of cement glands with a similar position upon the larva's head have been described in all the substrate-brooding cichlids investigated so far (e.g. Jones, 1937; Brinley & Eulberg, 1953; Balon, 1960; Fryer & lles, 1972; Jones, 1972; Bennemann & Pietzsch-Rohrschneider, 1978; Grier, 1981; Peters & Berns, 1982, 1983; Kuwamura & Mihigo, 1988; Contreras & Diaz, 1990; Hamlett, 1990). Vestiges of these glands have also been found in mouth-brooding species, which clearly indicate that mouth-brooders were derived from substrate-spawners (Peters & Berns, 1982). The size of these transient organs seems to correspond to the size of the larva and changes with its age. According to the species, the greatest size is reached at 3-4 (e.g. C. dimerus) or 4-5 days from spawning. Then, they gradually flatten out until they completely degenerate (Fryer & lles, 1972; Jones, 1972; Peters & Berns, 1982, 1983).

Respiratory plexuses present on the yolk-sac and ventral fin fold of the non-swimming larvae (Fig. 6b, c) facilitate oxygen uptake until the bronchial system is developed. These are also temporary structures, characteristic of species that build their nests in lentic water bodies (Fryer & lles, 1972; Balon, 1960, 1975), and have been described in mouth-brooding species as well (Balon, 1975, 1977; Holden & Bruton, 1992, 1994).

By the time that larvae start swimming freely, i.e. 8th day from spawning (Fig. 8a), the skeletal and sensory systems are sufficiently developed to allow food-seeking and active avoidance behaviours. The yolk-sac serves as a supplemental source of nutrition as the larvae develop their external food-gathering abilities (Balon, 1977; Holden & Bruton, 1992,1994).

During the following month, fins become completely developed, the skeletal structures gradually ossify and the scales begin to cover the body (Fig. 8b-d). By the 42nd day, body shape and pigmentation pattern are similar to those of the adult; thus the juvenile stage is reached (Fig. 8d).

Acknowledgements

The authors thank J. P. Vittori for photographic work; P. Galeazzi and P. Comelatto for technical assistance and F. Lo Nostro, L. Strittmatter and M. Ravaglia for their valuable suggestions. This work was supported by a grant from the University of Buenos Aires, EX 095.

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