Introduction

Characiform fishes are a group of tropical fishes indigenous to North America, South America, and Africa. This is a large group, comprised of at least 1,680 members, with new species being described on a regular basis (Nelson 2006; Zanata et al. 2009; Ferreira and Netto-Ferreira 2010; Sousa et al. 2010). Current efforts are being made to ascertain the evolutionary history of this group, based upon anatomical and molecular data (Calcagnotto et al. 2005; Mirande 2009; Javonillo et al. 2010). However, despite the abundance of characiform fishes, very little has been documented in regard to their life histories. Fuiman (1984) provides an overview of what is known from past records. More recently, ontogenetic examinations of Characidae have been made on Astyanax mexicanus (Yamamoto et al. 2003; Jeffery 2009), as well as various Brycon species (Reynalte-Tataje et al. 2004; Vandewalle et al. 2005).

The characid genus Moenkhausia, consisting of over 65 species, has been defined by the presence of caudal fin scales, a diagnostic tooth arrangement that includes two rows of premaxillary teeth and between 1–5 maxillary teeth, and, traditionally, a complete lateral line (Eigenmann 1903; Géry 1977). However, the relationship of Moenkhausia with other taxa within Characidae has remained elusive. Géry (1977) originally placed Moenkhausia into the Tetragonopterinae subfamily. However, based upon sequence data from a variety of mitochondrial and nuclear genes, Calcagnotto et al. (2005) suggest that members of Tetragonopterinae can instead be recategorized into a number of smaller, potentially monophyletic groups. More recent work by Mirande (2009), using a wide range of anatomical and morphological characters, substantiated the division of Tetragonopterinae, placing Moenkhausia into a “Hemigrammus clade,” a diverse group classified with only limited resolution. In addition, the monophyletic organization of Moenkhausia itself was also cast into doubt. Continued refinement of these groups may necessitate the inclusion of additional data, such as those regarding life history. Moenkhausia sanctaefilomenae has been the subject of various life history studies, most of which have focused on reproductive behaviors and larval feeding strategies (Borges et al. 2000; Lourenço et al. 2008; Alanis et al. 2009; Tondato et al. 2010). This article presents the early ontogeny of M. sanctaefilomenae. Embryogenesis is detailed, and an examination is given through the early larval period.

Materials and methods

Animal husbandry. Moenkhausia sanctaefilomenae is indigenous to the Brazilian river systems of the São Francisco, Paraíba do Sul, and Paraná Rivers as well as various river systems of Uruguay and Paraguay (Benine 2002; Nion et al. 2002). As the adults were acquired from various sources, the exact origins of the fish within the breeding colony cannot be definitively established. Verification of the species was performed in order to discriminate M. sanctaefilomenae from its most similar cogeners, based upon numbers of scale rows relative to the lateral line, scale numbers along the lateral line, and the number of scales bearing lateral line canals (Fowler 1932; Benine et al. 2009). Males and females were housed in separate 80-l aquaria with water of low hardness (under 2.0 GH) and acidic pH (approximately pH 6.0). The fish were fed a diverse array of foods, including manufactured flake foods, frozen and reared brine shrimp Artemia sp. (Aquatic Foods, Fresco, CA, and Brine Shrimp Direct, Ogden, UT), and frozen and live bloodworms Chironomidae gen. sp.

Spawning of adults and rearing of progeny. Over the course of 10 months, 13 pairings were performed. Individual females and males were chosen at random from their respective community tanks and placed in a dimly lit, 40-l aquaria with 20 l of water. Pairs spawned under a range of conditions, including pH (range 5.6–6.7, average 6.1), conductivity (range 235–472 μS, average 378 μS), and temperature (between 26 and 27°C). Spawning was successful for 10 of the 13 pairings to yield an average of 1,334 (±548 SD) embryos produced. In all successful cases, spawning occurred during the morning within 2 days of the pairings.

Fertilized eggs from successful spawnings were scattered throughout the tank, and they were collected with a siphon. Healthy embryos were separated from dead embryos or unfertilized eggs, and they were reared in 20 l of water in 40-l aquaria (at approximately 30 fish/l density). The water was prepared as 0.3 g Instant Ocean in 10.0 l deionized water (recipe modified from Westerfield 1995). Water temperature was maintained at 27°C. Young fish from 3 to 4 days of age were fed Paramecia multimicronucleatum (Carolina Biological, Burlington, NC), No BS Fry Food (Mike Reed Enterprises, Sutter Creek, CA), and reared Artemia larvae. Those from 5 days and beyond were fed live brine shrimp larvae.

Analysis. Specimens were routinely examined using a Nikon SMZ1000 stereoscope with oblique coherent contrast optics and a Leitz Ortholux II compound microscope. Motile specimens were briefly relaxed in 0.02% tricaine prior to observation. Measurements such as body length (BL) (Leis and Trnski 1989) were performed using an ocular micrometer. Photos were taken using a Nikon D200 camera, with image processing performed using Adobe Lightroom and Photoshop. Embryonic specimens were allowed to develop after the data were recorded, so no representatives of these stages were preserved. Older samples were fixed in 4.0% formaldehyde (from paraformaldehyde, in phosphate-buffered saline), immediately after the data were recorded, and they were registered with Illinois Wesleyan University [five 98 h specimens (IWU:ICH:00001–00005) and five 122 h specimens (IWU:ICH:00006–00010)].

Results

Examples of Moenkhausia sanctaefilomenae specimens at various stages during the embryonic period are shown in Fig. 1. Balon (1975, 1999) divides the embryonic period into three discrete phases: the cleavage egg phase, the embryo phase, and the eleutheroembryo (“free embryo”) phase. However, from an embryological perspective, it is more meaningful to divide the embryonic period into a greater number of distinct phases as follows: cleavage, gastrulation, segmentation, and pharyngula. Each phase can be definitively characterized via morphological criteria and embryological phenomena, and it is highly probable that each phase occurs consistently from taxa to taxa.

Fig. 1
figure 1

Early embryonic period through hatching. Cleavage (a–e), gastrulation (f–i), and segmentation (j–s) phases are shown. Stages during the cleavage phase include the single-cell zygote (a), 2-blastomere stage (b), 16-blastomere stage (c), 1,000-blastomere stage (d), and discoblastula (e). The animal pole is toward the top and the vegetal pole toward the bottom. Stages during the gastrulation phase include dome (f), 50% epiboly (g), 80% epiboly (h), and bud (i). The developing head and tail bud in h–i indicate the change in body plan that occurs during gastrulation. Stages during the segmentation phase include 2-somite (j), 5-somite (k), 12-somite (l), 17-somite (m), 20-somite (n), and 21-somite (o, removed from the fertilization envelope). p Dorsal view of a 2-somite specimen, showing the notochord. q Dorsal view of an 8-somite embryo, where the neural crest cells can be seen in the head region. r Lateral view of the tailbud of 29-somite embryo. s Lateral view of the head region of a newly-hatched embryo. The three major brain regions can be seen. A animal pole, ad adhesive gland, blast blastodisc, fe fertilization envelope, h developing head region, hrt heart, kv Kupffer’s vesicle, mes mesencephalon, nc neural crest, not notochord, ov otic vesicle, ps perivitelline space, pro prosencephalon, rhom rhombencephalon, tb tailbud, V vegetal pole, ye yolk extension. The scale bar in a is equal to 0.5 mm and applies to a–o. Scale bar in p is equal to 0.4 mm for p and 0.36 mm for q. The scale bar in r is equal to 0.15 mm and applies to r–s

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The fertilized eggs are demersal, and, in the aquarium environment, are dispersed along the bottom of the tank. The eggs are slightly adherent; they stick to the substrate on the bottom of the tank but are easily dislodged with minimal force. Overall, the eggs have a yellow cast. No lipid droplets can be seen. They are relatively small, as characteristic for characid fishes (Fuiman 1984).

Cleavage phase. The cleavage phase entails the initial cell divisions leading toward the blastula. This phase, as well as the gastrulation and segmentation phases, occurs within the confines of the fertilization envelope. The fertilization envelope surrounds the embryo, separated from the embryo via the perivitelline space (Fig. 1a). The fertilization envelope is slightly elliptical with an approximate diameter of 0.8 mm. Visible on the surface of the fertilization envelope is the micropyle (Fig. 2). The micropyle has a starburst appearance and a shallow, conical shape in the center that is most likely categorized as a type I micropyle (Riehl 1991; Kunz 2004). An adhesive pedestal (Fuiman 1984) associated with the micropyle anchors the embryo to the substrate. Later during development, the anterior–posterior axis of the embryo appears to develop orthogonal to the micropyle; it therefore seems that the location of the micropyle denotes the future left or right side of the embryo.

Fig. 2
figure 2

Micropyle on the fertilization membrane, dissected from the embryo. The scale bar equals 0.125 mm

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Just prior to cleavage, the zygote exists as a cytoplasm-rich blastodisc situated upon a large yolk mass, the “yolk cell” (Fig. 1a). At this point, the zygote has an elliptical appearance, measuring 0.7 mm along the animal-vegetal pole and 0.6 mm in diameter. The first cleavage divides the blastodisc in half (Fig. 1a). This cleavage, as well as the subsequent cleavages, are meroblastic and do not divide the yolk cell, as is consistent for teleosts (Collazo et al. 1994). The pattern produced by the cleavages is regular and occurs similarly between siblings. At 27°C, cleavage cycles occur every 12 min, eventually producing a blastoderm as a cap of cells resting atop the yolk cell (together, a discoblastula; Gilbert and Raunio 1997) within 2.5 h of the first cleavage (Fig. 1c–e).

Gastrulation phase. Gastrulation in M. sanctaefilomenae closely resembles what could be considered as classical teleost gastrulation (Collazo et al. 1994), appearing to consist of epiboly, involution, and convergent extension morphogenetic movements. Gastrulation begins with changes in the blastoderm with resulting changes in the shape of the yolk cell (Fig. 1f–g). The cells of the blastoderm merge to form a cell mass with less thickness but greater surface area, as expected during epiboly. The cell front can be observed migrating vegetally toward the vegetal pole (Fig. 1f–h). At approximately 60% epiboly, the migrating cell front thickens and produces the germ ring, suggesting that cells are involuting at the margins and redirecting themselves back toward the animal pole. This involution occurs more substantially at one site. This region, the embryonic shield, consists of a mass of cells of greater thickness than elsewhere along the germ ring. The yolk cell itself appears to alter its shape as a consequence of the cell motions. Initially, a dome forms as gastrulation begins (Fig. 1f–g), and as gastrulation continues, the yolk cell becomes spherical and then oblong along the animal–vegetal axis (Fig. 1h–i).

As gastrulation continues, a polarity can be observed within the gastrula, with a majority of cells accumulating on the one side of the embryo where the shield was observed (Fig. 1h–i). This likely has resulted from convergent extension movements, and this consistently occurs 90° from the area directly underneath the micropyle. The mass of accumulated cells represents the dorsum of the embryo. Within the mass, the forming anterior end can be seen at the animal pole, while the posterior can be observed toward the vegetal pole as the developing tail bud (Fig. 1i). As gastrulation continues, the anterior and posterior ends are spread further apart (Fig. 1i–j), most likely driven by the continued convergent extension forces (Warga and Kimmel 1990; Concha and Adams 1998). By 90% epiboly, the broad expanse of cells that will form the notochord can be discriminated from surrounding tissues. Early organogenesis of the central nervous system has begun by the end of gastrulation (5.5 h post-fertilization, hpf).

Segmentation phase. Organogenesis becomes readily apparent during the segmentation phase, and it is primarily defined by the formation of somites. Somites form in a sequential fashion from the anterior to the posterior of the embryo. A new somite is produced every 10–13 min. When generated, each somitic myotome rapidly broadens along the dorsal ventral axis and produces a myomere with a characteristic chevron morphology (Fig. 1j–o). By the time 17 somites are produced, muscular contractions have begun in the anterior-most myomeres.

Concurrent with the formation of somites within the trunk is the growth of the tail bud to produce the post-anal tail (Fig. 1l–o). As the tail is produced, the notochord lengthens and somites continue to form within the tail. Associated with this posterior outgrowth is the appearance of Kupffer’s vesicle (Fig. 1l), a structure that, according to recent evidence, has a role in left–right asymmetry (Essner et al. 2005). Kupffer’s vesicle appears at a similar time as the formation of the second somite (6 hpf) and can no longer be seen by the 20-somite stage (9 hpf). Initially, the tail grows around the periphery of the yolk, and as the tail lengthens, a small amount of yolk extends along with it (Fig. 1m–o).

Neurulation and regionalization of the central nervous system in fishes occur during the segmentation phase (Kimmel et al. 1995). The formation of the neural tube and initial regionalization is associated with the formation and migration of neural crest cells (Fig. 1q). Over this time, the central nervous system continues to grow and elaborate. The three primary vesicles of the brain (the prosencephalon, mesencephalon, and rhombencephalon) can be distinguished by the four-somite stage. Optic vesicles are also evident by the four-somite stage, and the lenses form by the 25-somite stage. The otic vesicles form by the 14-somite stage, and, by the 25 somite stage, otoliths can be seen within the vesicles.

Somite formation from the tailbud continues to lengthen the tail (Fig. 1r) until 32 somites are formed. Toward the end of the segmentation phase (around 11 hpf, 31 somites), a number of significant phenomena occur. The heart, located just posterior to the eye between the brain and the yolk, can be observed to beat at this time. Initially, the heartbeat is slow, but increases in both speed and intensity as more of the blood-vascular system develops. Over the next 2–3 h, a large supply of blood cells can be seen within the large vessels lying superficial to the yolk on the ventral surface. A prominent adhesive gland has developed dorsal to the boundary between the mesencephalon and rhombencephalon (Fig. 1s). This gland (called the “casquette”), in addition to its role in adhesion, has been shown to regulate swimming behaviors in young fishes (Pottin et al. 2010). Pigment cells can be observed associated with the yolk as well as the dorsum, primarily in the anterior regions.

Hatching in M. sanctaefilomenae occurs around 12 h after fertilization. Hatching is typically facilitated by the secretion of enzymes from unicellular hatching glands, which are often distributed over the body of the embryo (Kunz 2004). The location of the hatching glands on M. sanctaefilomenae could not be readily identified, although it is suggested that they reside in close association with the adhesive gland (Willemse and Denucé 1973). The fertilization envelope of M. sanctaefilomenae is not robust, and it readily degrades upon exposure to proteases (e.g., 0.25% pronase E). By the time that hatching occurs, somite formation has ended, having produced 32 somites. Likewise, the growth from the tailbud has ceased. Thus, hatching serves as a convenient point in which to distinguish between the segmentation phase and the pharyngula phase in M. sanctaefilomenae.

Pharyngula phase. The term “pharyngula” was originally used to describe a vertebrate that had undergone early organogenesis (Ballard 1981). In the analysis of Danio rerio development, the term pharyngula was used to describe the period of late embryonic development prior to the transition to the larval form (Kimmel et al. 1995). It is at this time that the notable features of the chordates are apparent, including the notochord, the dorsal nerve tube, metameric muscle blocks, a post-anal tail, and the pharyngeal arches. The pharyngeal arches specifically define the pharyngula (Ballard 1981) and become prominent during this time. Other phenomena include the straightening of the embryo along the anterior–posterior axis, development of the gas bladder, elaboration of the circulatory system, and increased pigmentation. The pharyngula phase also marks the beginning of the development of the cartilaginous and osseous skeleton (Walter, in press).

An initial feature of the pharyngula phase is the straightening of the embryo in respect to the yolk. The first notable occurrence of this phenomenon is the extension of both the trunk and tail. Extension of the trunk and tail occur quite rapidly after hatching (around 14–15 hpf), and specimens were recorded to be 1.9 mm BL. The straightening of the trunk and tail is thought to be driven by the concurrent stiffening and extension of the notochord (Adams et al. 1990). However, there is evidence that the notochord may not be entirely integral for this process (Solnica-Krezel et al. 1996; Virta and Cooper 2009). Following extension, growth of the trunk and tail continues (Fig. 3) from 1.9 mm BL to 2.4 mm BL over a 16-h time frame before slowing. By this time, the median fin fold can be clearly seen along the trunk and tail.

Fig. 3
figure 3

Examples of pharyngula phase Moenkhausia sanctaefilomenae embryos are shown, including specimens at 24 (a), 30 (b), 32.5 (c), 39 (d), and 48.5 h (e) post-fertilization (hpf) at 27°C. During this time, the head extends from the yolk, while the pharyngeal arches expand to produce the jaw and gill elements. An increased level of pigmentation can be seen, especially in the developing eye. The gas bladder is clearly visible in the specimen in e. f–g Close-up photographs of the specimens in c and d, respectively. ad Adhesive gland, arch pharyngeal arches producing the lower jaw and gill elements, finfold median finfold, gas gas bladder, hrt heart, not notochord, pecfin pectoral fin anlage, ov otic vesicle. The scale bar in a equals 0.5 mm and applies to a–e. The scale bar in f equals 0.25 mm and applies to f and g

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The pharyngeal arches are present by 24 hpf, and over the next 24 h they undergo a great amount of expansion and elaboration (Fig. 3). As a consequence of this growth, the head of the embryo is extended approximately 70° away from its original position against the yolk mass [following the “head–tail angle” measurement method of Kimmel et al. (1995)]. The pharyngeal region can be observed around 30 hpf (2.45 mm BL) as a mass posterior to the eye between the head and the heart (Fig. 3b–c, f). This mass continues to grow and projects further ventro-anteriorly to lie ventral to the eye. During this time, the formation of the aortic arches can be seen, beginning with a single aortic arch at 30 hpf to four at 33 hpf and six at 39 hpf. Gill primordia are apparent by 35 hpf. By 39 hpf (2.67 mm BL), individual primordia of the pharyngeal cartilages can be seen and muscular activity occurs in the lower jaw.

The entire yolk mass is lost by the end of the pharyngula phase, and as the yolk shrinks, the digestive tract develops in its place. A lumen in the gut can be observed by 35 hpf. Yellow pigment, presumably from the yolk mass, fills this space initially, but it is lost as the lumen expands. By 39 hpf, the developing liver can be seen residing upon a cleft in the yolk.

During a large portion of the pharyngula phase, the embryo spends much of its time on its side while on the bottom of the tank. The median fin fold is present at (or immediately after) the time of hatching, and by 24–28 hpf, the embryo can perform sustained, yet sporadic swimming behaviors in the water column. Some pharyngulae attach themselves to the side of the tank via their adhesive glands. Over time, the development of the pectoral fins and the air bladder facilitates more effective swimming behaviors. The pectoral fin can be seen by 24 hpf (2.3 mm BL; Fig. 3a). It forms initially as a narrow ridge protruding from the body, just dorsal to the yolk. It continues to grow dorsally, consisting of a limb bud (containing the endoskeletal disk) and a distal finfold. The pectoral fin displays a good deal of growth, and by 48 hpf, it has reoriented itself to point posteriorly. The gas bladder begins to form at 30 hpf. It continues to increase in size, and by 48 hpf, it acquires pigmentation and begins to inflate (Fig. 3e). Using the gas bladder and pectoral fins, the embryo can now orient itself upright as well as achieve neutral buoyancy in the water column with minimal effort. The adhesive gland begins to regress by 72 hpf and is completely gone by 96 hpf. At the transition to the larval period (72 hpf), pharyngulae demonstrate flight responses as well as simple lunging behaviors associated with prey capture.

Transition to the larval period. The fish larval period is distinguished from the embryonic period via its ability to swim and remain buoyant in the water column (with minimal effort) while actively acquiring food from exogenous sources. Although fish were allowed access to various foods at 2 days post-fertilization (dpf), they were not witnessed to be feeding until 3 dpf. Therefore, 72 hpf (2.4 mm BL) was determined to be the threshold for the larval period. Even at this early time point, the larvae are able to engulf relatively large prey such as Artemia larvae.

During the early phase of the larval period, growth along the anterior–posterior axis is not dramatic. In contrast, organogenesis continues within the head and trunk. This is most apparent in the head, where the cranial skeletal elements are continuing to develop. Beginning around 72 hpf (2.4 mm BL) and continuing through 8 dpf (3.3 mm BL), the head alters its appearance from a blunt shape to a more elongate, tapered shape (Fig. 4). Much of this morphogenesis occurs via the allometric growth of the Meckel’s cartilage of the lower jaw and the ethmoid plate of the anterior cranium, but the continued growth of the bones associated with these cartilages (the maxillary and the dentary) also contribute (Walter, in press). Within the trunk, both the gas bladder and digestive tract continue to enlarge. The digestive tract takes up an increasingly greater proportion of the trunk as the phase continues, reflecting the emphasis on feeding during this phase.

Fig. 4
figure 4

Transition from the embryonic period to the larval period. Examples of a late pharyngula phase specimen at 72 hpf (a) and finfold larval phase specimens at 98 hpf (b), 122 hpf (c), and 146 hpf (d) at 27°C. Note the dramatic growth in the head, protruding jaw elements sufficient for prey capture, and the expansion of the digestive tract. The gas bladder also increases in size over this timeframe. The scale bar in a is equal to 1.0 mm and applies to all panels

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Discussion

As a group, characiform fishes have not been the subject of many embryological examinations. Although particular aspects of development have been examined for a handful of characiform species (Vandewalle et al. 2005; Jeffery 2009), the ontogenies of only a few, notably Brycon orbignyanus (Reynalte-Tataje et al. 2004) and Prochilodus lineatus (Ninhaus-Silveira et al. 2006), have been previously reported.

Based upon what is known regarding the ontogeny of characiform fishes, a number of characters seem to be consistent within this group. These fishes appear to hatch quite early, while still only part way through their embryonic period. The embryo escapes from the fertilization envelope well before the acquisition of productive locomotory function or exogenous feeding ability (Fuiman 1984). Ninhaus-Silveira et al. (2006) report that P. lineatus hatches by 14 hpf (at 28°C), while B. orbignyanus hatches by 18.5 hpf (at 25°C; Reynalte-Tataje et al. 2004). Similarly, Moenkhausia sanctaefilomenae hatches by 12 hpf at 27°C. In all cases, the hatched embryos appear to be at the pharyngula stage when hatched. Accordingly, the earlier phases of the embryonic period occur quite rapidly following fertilization. For example, gastrulation begins in P. lineatus by 3 hpf (at 28°C; Ninhaus-Silveira et al. 2006), which is quite similar to what is seen in M. sanctaefilomenae. Interestingly, these events occur similarly despite the size differences apparent between the species. Moenkhausia sanctaefilomenae, with an oocyte diameter of 0.7 mm, measures 2.3 mm TL at hatching, while B. orbignyanus, with an oocyte diameter well above 1.0 mm (exact dimensions not reported by Reynalte-Tataje et al. 2004) measures 4.46 mm TL at hatching. As adults, B. orbignyanus reaches 79.5 cm TL (Godoy 1975), while M. sanctaefilomenae reaches 7.0 cm SL (Reis et al. 2003).

The yolk extension is known to occur in only a few teleost taxa, including the Characiformes, the Cypriniformes, and the Anguilliformes (Virta and Cooper 2009). Even in these taxa, the formation of the yolk extension (and shape of the yolk sac itself) appears to be an evolutionarily labile phenomenon. The forces directing the formation of the yolk extension are currently unknown, although evidence suggests that the superficial-most layer of cells, the yolk cell itself, or both, might play a role (Lyman Gingerich et al. 2006). Studies using Danio rerio have led to the hypothesis that the yolk extension forms in order to facilitate the redistribution of yolk throughout the body of the embryo. The reshaping of the yolk would allow for the effective rhythmic contractions of the trunk musculature necessary for the hatched fish to avoid predation (Virta and Cooper 2009). Compared to D. rerio, the yolk extension of M. sanctaefilomenae is unremarkable and relatively short-lived; however, its appearance does coincide with a change in the shape of the yolk (e.g., compare Fig. 1k, o).