Table Of ContentReference: Bio/. Bull. 203: 80-86. (Auaust 2002)
Embryonic Velar Structure and Function of Two
Sibling Species of Crepidula With Different Modes
of Development
O. R. CHAPARRO1 *, J. L. CHARPENTIER1, AND R. COLLIN"t
llnstituto de Biologfa Marina Dr. J. Winter, Facnltad de Ciencias, UniversidadAustral de Chile,
Valdivia, Chile; and 2Committee on Erolntiona/y Biology, University ofChicago, 1025 E. 57th St.,
Chicago, Illinois 60637, and the Field Museum. 1400 S. Lake Shore Dr., Chicago, Illinois 60605
Abstract. The structure and function of the embryonic larval arms in feeding larvae are reorganized into transverse
velum of two closely related species of CrepiJnla with bands or fields of cilia around a cylindrical body in non-
different modes of development are examined. The velum feeding larvae (Emlet. 1994). Among echinoderm larvae,
of C. dilatata. a direct developer whose embryos feed on such modifications are believed to maximize feeding effi-
nurse eggs, does not differ substantially from the velum of ciency in planktotrophic larvae and maximize swimming
C. fecnnda, a species with planktotrophic larvae. Although efficiency in nonfeeding planktonic larvae (Emlet, 1994).
velar ciliation develops earlier in embryos of C. dilatata, Modifications or loss of larval feeding or swimming struc-
embryos of both species were able to feed on small parti- tures are often so extreme that re-evolution of such struc-
cles, using the opposed-band ciliary mechanism. However, tures appears to be difficult. Loss of these complex struc-
theembryosofC. dilatata lose thisability asthey grow. The tures is cited as evidence that feeding larvae, once lost,
embryos ofC. dilatata were not able to swim, whereas those cannot be regained (Strathmann, 1978). The morphological
of C.fecnnda swam consistently in vials of seawater. This modifications associated with the loss of feeding larvae in
difference in swimming ability is probably due to differ- other groups of marine invertebrates are less well under-
ences in velum-body size allometry between the two spe- stood. In marine gastropods, for example, the evolution of
cies. nonfeeding ordirect development is usually associated with
encapsulated development. The functional requirements of
Introduction development within a capsule may be very different from
In marine invertebrates, the evolution of direct develop- the requirements of free-living development. For example,
ment from the putative ancestral state ofdevelopment with the embryo may need structures to circulate intracapsular
planktotrophic larvae is often accompanied by modifica- fluid (Strathmann and Chaffee. 1984), ingest intracapsular
tions ofmorphological structures associated with swimming fluid (Fioroni, 1966; Morrill. 1982; Rivest. 1992; Moran,
and feeding (Strathmann. 1978; Hadtield and laea. 1989; 1999), or ingest nurse eggs or siblings (Rivest, 1981 ).
Emlet, 1994; Byrne, 1995; Byrne and Cerra. 1996; Wray. The morphological structure associated with both feeding
1996; Hadfield et al., 1997). The functional modifications and swimming in gastropod larvae is the velum. In plank-
associated with the loss of feeding in pelagic echinoderm totrophic larvae, the velum is normally a pair of large flat
larvae are relatively well understood. For example, the lobes oftissue edged with two opposed bands ofcompound
ciliary bands that are arranged into complex loops along cilia, extending from behind the head. The anterior band of
cilia, the preoral band, and the posterior, postoral band flank
a "food groove" lined with shorter cilia (Werner. 1955;
Received 10July 2001; accepted 23 May 2002. Strathmann and Leise, 1979). Gastropods with planktotro-
*To whom correspondence should be addressed. E-mail: ochaparr@
uach.cl phic larvae, and especially those with long-lived planktotro-
tCurrent address: Smithsonian Tropical Research Institute. P.O. Box phic larvae, often have large elaborate vela with as many as
2072, Balboa. Ancon, Republic ofPanama. E-mail: collinrCs'naos.si.edu six or eiht lobes (Fretter and Pilkinaton. 1970; DiSalvo.
80
EMBRYONIC VELUM IN CREP1DULA 81
Figure 1. Scanningelectron micrographsofdifferentdevelopmental stagesofCrepidulafecunda (left side
pictures: A-C) and C. dilalata (right side pictures: A'-C"). (A) Gastrula. (B) Intracapsular veliger; arrow in
enlarged area indicates the food groove. (C) Light micrograph of the same stage as in B, showing the
opposed-band ciliation and organized metachronal wave in both species.
1988). In larvae of Crepidula fomicuta, the velar lobes "archaeogastropod" larvae, which are characterized by
grow significantly larger, in relation to shell size, when swimming, nonfeeding larvae, have well developed preoral
larvae are cultured with low concentrations of food rather cilia but lack the food groove. In embryos of Crepidula
than with high concentrations (Klinzing and Pechenik, adunca. a species with direct development from large yolky
2000). In swimming larvae, the velum is lost at metamor- eggs, the velum is reduced to a small ridge with cilia only
phosis. Those species with abbreviated pelagic phases or slightly longer than those on the surrounding mouth, tenta-
complete intracapsulardevelopment often show a reduction cles, and head vesicle (Collin, 2000).
in the size ofthevelum, lossofciliary bands, and shortening The absence of a feeding, swimming larval stage is not
ofcilia (Fioroni, 1966). Hadfield el al. (1997) showed that always associated with the loss offunction ofthe velum. In
82 O. R. CHAPARRO ET AL
some species of Littorina, the embryonic velum is used to
ingest albumin from the egg capsules (Moran, 1999). In C. fecunda
these species the velum is as large, relative to embryonic
body size, as the velum in species with planktotrophic E
larvae (Moran 1999), suggesting that this novel function is '
selecting against loss ofstructure and function ofthe velum.
In embryos of Petaloconchus nwntereyensis, the velum is k 004
JO
modified to ingest intracapsularyolk but has lost the preoral >0) 002
cilia. Other embryonic modifications associated with intru-
capsular development are reviewed in Fioroni (1966). In
species with direct, nonswimming development, the velum 200 250
is lost before hatching. Shell length (pm)
The present study was undertaken to examine structural
020
and functional modifications of the embryonic velum asso-
ciated with the evolution ofnurse-egg feeding. We compare C. dilatata
the embryonic development, especially the velar develop-
ment, of two closely related species of gastropods with
differing modes of development: Crepichda fecunda Gal- (a0> oio
lardo 1979, a species with planktotrophic development, and (8
C. tlilatata Lamarck 1822, a species with direct develop-
ment in which the embryos consume nurse eggs (Gallardo, "55 oos
1976. 1977, 1979; Chaparro and Paschke, 1990). We mea-
sured the ability, relative to velar morphology, of the em- 000
bryos to capture particles and to swim. 200 400 600 800 1000 1200 1400
Shell length (\im)
Materials and Methods Figure2. Scatterplotofvelum areaversusshell length for Crepidula
fecunda and C. dilatata.
Adult Crepidulu dilatata were collected subtidally in the
estuary ofRio Quempillen at the north end ofChiloe Island.
Chile (4T52'S, 7344'W), and C. fecunda were collected recorded using an inverted microscope connected to a Sony
from the intertidal ofBahiade Yaldad in the south ofChiloe DXC-C1 videocamera and a Sony PVM-1953MD monitor.
Island (4308'S. 7344'W). Similarly, to test for swimming ability, embryos were ex-
Prior to examination by electron microscopy, broods capsulated, and groups of 20 were placed in 20 ml of
were removed from the females, individually incubated for seawater. After 5 min, the number of embryos swimming
5 min in 0.5% solution of chloral hydrate, fixed in a 3% above the bottom ofthe vial was recorded. The shell length
solution of gluteralderhyde with cacodylate buffer (pH = and velar characteristics of embryos used in both experi-
7.4), and maintained at 4 C for 1 h. Embryos were subse- ments were measured, using Scion Image PC (Scion Cor-
quently excapsulated, rinsed for 10 min in phosphate buffer, poration, Frederick, MD), from video images.
dehydrated in a graded acetone series, and critical-point
dried. They were attached to stubs with double-sided tape, Results
gold-coated, andexamined with a Bausch & Lomb Nanolab
Enihryonic morphology
2000 scanning electron microscope.
To test for the ability of the embryos to feed on small Embryos of both Crepidula dilatata and C. fecunda de-
particles, the embryos were excapsulated and placed in velop from eggs with average diameters of250 and 212 /im
seawater at ambient temperature. Nontoxic red beads 2-10 respectively (Gallardo, 1977). However, the embryos of C.
/am in diameter (Chaparro et ai, 1993) were added to the dilatata consume uncleaved nurse eggs (Gallardo, 1977)
solution. After 2 h the embryos were removed from the and hatch as crawling juveniles of about 1100-1200 /Ltm
solution and fixed in formalin until they were analyzed. The shell length (Chaparroand Paschke, 1990), whereasthoseof
presence ofred particles in the larval orembryonic stomach C. fecunda do not consume nurse eggs, and they hatch as
was determined afterfixation. One hundred embryos at each planktotrophic larvae at a size of400-500 p.m. Despite this
stage of development were examined under a compound difference in mode of development, the embryonic mor-
microscope, and the percentage of embryos that had in- phologies of both species are similar.
gested particles (as indicated by red particles in the gut) was In both species, spiral cleavage and gastrulation by epi-
recorded. The method ofparticle capture was observed and boly result in a very slightly flattened gastrula with acentral
EMBRYONIC VELUM IN CREPIDULA 83
100 and the foot begins to differentiate and becomes ciliated. In
embryos of both species of 200-300 jam, the small head
C. fecunda
80 vesicle is distinct and covered with cilia, the velum clearly
shows opposed band filiation, and the foot has an opercu-
.C 60 lum and a medial band ofventral cilia (Fig. IB. B', 1C. C').
"c5) Subsequentdevelopment reflectsthe growth andelaboration
<" 40 ofthese structures. The shell grows to contain the yolk and
O developing viscera. The lobes ofthe velum grow wider, and
20 the preoral cilia grow to a length of 80 /am. Larvae of C.
fecunda hatch at a shell length of 400-500 /am. after the
reabsorption ofthe embryonic kidneys and the head vesicle.
200 280 320 360 400
In C. di/atatii. further morphogenesis occurs in the cap-
Shell length (pm) sule. The tentacles begin to form at a shell length of about
500 /am. Both tentacles are large and the apical ciliarly tufts
are well developed, as is the foot, in embryos with a shell
C. dilatata length of 700 /am. At this stage the velar cilia reach their
maximal length of 80 /am. Embryos between 800 and 1 100
.C 60 P 5 /am in shell length undergo intracapsular metamorphosis.
0c) The pedal glands become visible, and the velum gradually
. 40 shrinks. By the time the embryo is about 1000 /am in shell
m
length, the velum and embryonic kidneys are no longer
20 - visible. This is also near the length at which the embryos
usually exhaust the supply of nurse eggs (Chaparro and
Paschke. 1990). Among advanced embryos, the shell length
600 800 1000 1200
Shell length (pm)
(A
10
Figure 3. A comparison ofcilia length versus shell length for Crep- 15
idii/u fecunda and C. dilatata. The vertical bars represent the standard 'E
80
deviation. S.
60
blastopore. In C. dilatata (Fig. 1A'), the gastrula is some- 40
what more elongate than the round gastrula of C. fecunda JD
(Fig. 1A). The initial gastrula is not ciliated, but patches of E 20
cilia in the areaofthe presumptive velumandhead vesiclecan in C. fecunda
be seen in scanning electron micrographs of later gastrulas. 150 200 250 300 350 400
In both species, an intracapsular trochophore stage fol-
lows the gastrula. This stage is characterized by elongation Shell length (pm)
of the embryo and flattening of the posterior dorsal area
(A
where the shell begins to form. In C. dilatata, clearly 0>
defined patches of cilia around the mouth, at the ventral
anterior (presumptive head vesicle) and ventral posterior HQJ. 80 -
(telotroch) areas of the embryo, and lateral to the mouth
(presumptive velum) begin to develop. In C.fecunda, these
cilia are less obvious, and their elaboration occurs later, (O/)
relative to other morphological events, than in the embryos
of C. dilatata. E
As the edge ofthe velum extends laterally from the body LU C. dilatata
wall, the distinct bands of cilia can be observed along its
edge in C. dilatata. It is at this stage that the preoral cilia
first appear in C. fecunda', the postoral and oral cilia appear Shell length (\\m)
slightly later. At this stage, in both species, the embryonic Figure 4. Scatter plot of the proportion of embryos that ingested
kidney is clearly visible on each side of the embryo, the particles versus shell length for Crepiilulufecunda and C. dilatata. Each
shell begins togrow aroundthe posteriorend ofthe embryo. point represents atrial with 100 embryos.
84 O. R. CHAPARRO ET AL
Figure 5. Video sequence of a captured particle (encircled) moving along the food groove towards the
mouth. Left side (A-C): Crepidiilnfecunda embryo. Right side (A'-C'): C. dilatata embryo.
at any given developmental stage shows variation due to to shell length (Fig. 2), and it reaches its maximal size,
differences in the number of nurse eggs consumed by each about 0.06 mm2, near hatching. The size of the velum
embryo. relative to shell length in C. dilatata is initially the same as
in C. fecunda. but the velum grows more slowly relative to
Allometry shell length. Comparisons of larvae at a comparable shell
In C.fecunda, the velum grows throughout intracapsular length (<600 ju,m) showed a significant difference in velum
= =
development. The velar area increases linearly with respect size relative to shell length (ANCOVA. n 374: /
EMBRYONIC VELUM IN CKEPIDULA 85
I/)
86 O. R. CHAPARRO ET AL
The energetic contributions ofthese different feeding mech- DiSalvo. L. H. 1988. Observations on the larval and post-metamorphic
anisms should be examined in greater detail. lifeofConcholepasconcholepas(Brugiere. 1789)inlaboratoryculture.
A previous study that addressed the possibility of the EmleVte,ligRe.rB3.0:1939548.-36B8o.dy form and patterns ofciliation in non-feeding
re-evolution of feeding gastropod larvae concluded that lanaeofechinoderms functionalsolutionstoswimmingintheplank-
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