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F U L L P A P E R

Early ontogeny of aquarium-raisedMoenkhausia sanctaefilomenae(Characiformes: Characidae)

Brian E. Walter

Received: 18 January 2011/ Revised: 28 September 2011/ Accepted: 18 October 2011 / Published online: 16 December 2011

The Ichthyological Society of Japan 2011

Abstract The ontogeny of the characiform fish Moenk-

hausia sanctaefilomenae, from early embryogenesisthrough the early larval period, is presented. Fertilized eggs

were slightly elliptical, measured 0.6 mm in diameter, and

were surrounded by a fertilization envelope 0.8 mm in

diameter. Much of the early embryogenesis is complete

after 12 h, with cleavages complete after 2.5 h and gas-

trulation complete after 3 additional hours. The initial

formation of organs needed for predatory behaviors occurs

within 72 h. Growth of the cranial elements is quite dra-

matic and allows for the capture of relatively large prey at

the onset of exogenous feeding. Elaboration of these ele-

ments continues into the early larval period.

Keywords Development Embryo Larva

Pharyngula Tetra

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-Ferreira2010;

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;

Mirande2009; 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 15 maxillary

teeth, and, traditionally, a complete lateral line (Eigenmann

1903; Gery 1977). However, the relationship of Moenk-

hausia with other taxa within Characidae has remained

elusive. Gery (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 ofMoenkhausiaitself 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 sub-

ject of various life history studies, most of which have

focused on reproductive behaviors and larval feeding

strategies (Borges et al.2000; Lourenco 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.

B. E. Walter (&)

Department of Biology, Illinois Wesleyan University,

Bloomington, IL 61702-2900, USA

e-mail: bwalter@iwu.edu

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Ichthyol Res (2012) 59:95103

DOI 10.1007/s10228-011-0257-8

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Materials and methods

Animal husbandry. Moenkhausia sanctaefilomenae is

indigenous to the Brazilian river systems of the Sao

Francisco, Paraba do Sul, and Parana 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 thebreeding colony cannot be definitively established. Verifi-

cation of the species was performed in order to discrimi-

nate 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 (Fowler1932; 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 shrimpArtemiasp. (Aquatic Foods,

Fresco, CA, and Brine Shrimp Direct, Ogden, UT), andfrozen 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 5.66.7, average 6.1), con-

ductivity (range 235472 lS, average 378 lS), and tem-

perature (between 26 and 27C). 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 27C. 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 con-

trast 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 parafor-

maldehyde, in phosphate-buffered saline), immediately

after the data were recorded, and they were registered with

Illinois Wesleyan University [five 98 h specimens (IWU:

ICH:0000100005) and five 122 h specimens (IWU:ICH:0000600010)].

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 agreater 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.

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 (Fuiman1984).

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 fer-

tilization envelope surrounds the embryo, separated from

the embryo via the perivitelline space (Fig. 1a). The fer-

tilization envelope is slightly elliptical with an approximate

diameter of 0.8 mm. Visible on the surface of the fertil-

ization 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 (Riehl1991; Kunz2004). An adhesive pedestal

(Fuiman1984) associated with the micropyle anchors the

embryo to the substrate. Later during development, the

anteriorposterior 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.

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

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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 27C, cleavage

cycles occur every 12 min, eventually producing a blas-

toderm 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. 1ce).

Gastrulation phase.Gastrulation inM. 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. 1fg). The cells of the blasto-

derm 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.1fh). At approximately 60% epiboly,

Fig. 1 Early embryonic period through hatching. Cleavage (ae),gastrulation (fi), and segmentation (js) 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 topand

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 hi indicate the change in

body plan that occurs during gastrulation. Stages during the segmen-

tation 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, wherethe 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, blastblastodisc, fe fertilization

envelope, h developing head region, hrtheart, kv Kupffers vesicle,

mes mesencephalon,ncneural crest,notnotochord,ovotic vesicle, ps

perivitelline space, pro prosencephalon, rhom rhombencephalon, tb

tailbud,Vvegetal pole, ye yolk extension. The scale barin a is equal

to 0.5 mm and applies to ao. 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 rs

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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. Thisregion, 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. 1fg), and as gastrulation continues, the yolk

cell becomes spherical and then oblong along the animal

vegetal axis (Fig. 1hi).

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.1hi). This likely has resulted from con-

vergent 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.1ij), most likely driven by

the continued convergent extension forces (Warga and

Kimmel1990; Concha and Adams1998). By 90% epiboly,

the broad expanse of cells that will form the notochord can

be discriminated from surrounding tissues. Early organo-

genesis 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 1013 min.

When generated, each somitic myotome rapidly broadens

along the dorsal ventral axis and produces a myomere with

a characteristic chevron morphology (Fig.1jo). 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.1lo). 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 Kupffers vesicle (Fig. 1l), a structure that, according torecent evidence, has a role in leftright asymmetry (Essner

et al. 2005). Kupffers 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.1mo).

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, thecentral nervous system continues to grow and elaborate.

The three primary vesicles of the brain (the prosencepha-

lon, mesencephalon, and rhombencephalon) can be distin-

guished 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 23 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 rhomb-

encephalon (Fig. 1s). This gland (called the casquette),

in addition to its role in adhesion, has been shown to reg-

ulate 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

Fig. 2 Micropyle on the fertilization membrane, dissected from the

embryo. The scale barequals 0.125 mm

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with the adhesive gland (Willemse and Denuce1973). The

fertilization envelope ofM. 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. Like-

wise, the growth from the tailbud has ceased. Thus,

hatching serves as a convenient point in which to distin-

guish between the segmentation phase and the pharyngulaphase in M. sanctaefilomenae.

Pharyngula phase. The term pharyngula was origi-

nally 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 pha-

ryngeal arches. The pharyngeal arches specifically define

the pharyngula (Ballard 1981) and become prominentduring this time. Other phenomena include the straighten-

ing of the embryo along the anteriorposterior axis,

development of the gas bladder, elaboration of the circu-

latory 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 1415 hpf), and

specimens were recorded to be 1.9 mm BL. The straight-

ening 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). Fol-

lowing 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.

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 headtail 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. 3bc, 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 thisspace 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 2428 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. Thepectoral 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, con-

sisting 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 dem-

onstrate flight responses as well as simple lunging behav-

iors 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 exog-

enous sources. Although fish were allowed access to vari-

ous 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 Artemialarvae.

During the early phase of the larval period, growth along

the anteriorposterior axis is not dramatic. In contrast,

organogenesis continues within the head and trunk. This is

most apparent in the head, where the cranial skeletal ele-

ments 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

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elongate, tapered shape (Fig.4). Much of this morpho-

genesis occurs via the allometric growth of the Meckels

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. 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 27C. 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. fg Close-up photographs

of the specimens in c and d,

respectively.ad Adhesive

gland, arch pharyngeal arches

producing the lower jaw and gill

elements,finfoldmedian finfold,

gasgas bladder, hrtheart, not

notochord, pecfin pectoral fin

anlage,ov otic vesicle. The

scale barin a equals 0.5 mm

and applies to ae. The scale

barin f equals 0.25 mm and

applies to fand g

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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) andProchilodus

lineatus (Ninhaus-Silveira et al. 2006), have been previ-

ously 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 fertiliza-

tion 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 28C), whileB. orbignyanus hatches

by 18.5 hpf (at 25C; Reynalte-Tataje et al. 2004). Simi-

larly, Moenkhausia sanctaefilomenae hatches by 12 hpf at

27C. 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. lineatusby 3 hpf (at 28C; Ninhaus-Silveira et al.2006),

which is quite similar to what is seen in M. sanctaefilom-

enae. Interestingly, these events occur similarly despite the

size differences apparent between the species. Moenkhau-

sia 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. orbigny-

anus reaches 79.5 cm TL (Godoy1975), while M. sancta-

efilomenae 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 Cyprinifor-

mes, 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 evi-

dence suggests that the superficial-most layer of cells, the

yolk cell itself, or both, might play a role (Lyman Ginge-

rich et al.2006). Studies using Danio reriohave led to the

hypothesis that the yolk extension forms in order to facil-

itate the redistribution of yolk throughout the body of the

Fig. 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

27C. 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

barin a is equal to 1.0 mm and

applies to all panels

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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

Cooper2009). Compared toD. rerio, the yolk extension of

M. sanctaefilomenaeis 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).

Acknowledgments This work was funded by an Artistic and

Scholarly Development Grant provided by Illinois Wesleyan Uni-

versity. All specimens were maintained and utilized in compliance

with Illinois Wesleyan University IACUC protocol 07-002.

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