Biol. Bull. 203: 80-86. (August 2002)
© 2002 Marine Biological Laboratory
Embryonic Velar Structure and Function of Two Sibling Species of Crepidula With Different Modes of Development
O. R. Chaparro1,*,
J. L. Charpentier1 and
R. Collin2,
1 Instituto de Biología Marina Dr. J. Winter, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile
2 Committee on Evolutionary Biology, University of Chicago, 1025 E. 57th St., Chicago, Illinois 60637, and the Field Museum, 1400 S. Lake Shore Dr., Chicago, Illinois 60605
* To whom correspondence should be addressed. E-mail: ochaparr{at}uach.cl
Current address: Smithsonian Tropical Research Institute, P.O. Box 2072, Balboa, Ancon, Republic of Panama. E-mail: collinr{at}naos.si.edu
 |
Abstract
|
|---|
The structure and function of the embryonic velum of two closely related species of Crepidula with different modes of development are examined. The velum of C. dilatata, a direct developer whose embryos feed on nurse eggs, does not differ substantially from the velum of C. fecunda, a species with planktotrophic larvae. Although velar ciliation develops earlier in embryos of C. dilatata, embryos of both species were able to feed on small particles, using the opposed-band ciliary mechanism. However, the embryos of C. dilatata lose this ability as they grow. The embryos of C. dilatata were not able to swim, whereas those of C. fecunda swam consistently in vials of seawater. This difference in swimming ability is probably due to differences in velum-body size allometry between the two species.
|
Introduction
|
|---|
In marine invertebrates, the evolution of direct development from the putative ancestral state of development with planktotrophic larvae is often accompanied by modifications of morphological structures associated with swimming and feeding (Strathmann, 1978; Hadfield and Iaea, 1989; Emlet, 1994; Byrne, 1995; Byrne and Cerra, 1996; Wray, 1996; Hadfield et al., 1997). The functional modifications associated with the loss of feeding in pelagic echinoderm larvae are relatively well understood. For example, the ciliary bands that are arranged into complex loops along larval arms in feeding larvae are reorganized into transverse bands or fields of cilia around a cylindrical body in nonfeeding larvae (Emlet, 1994). Among echinoderm larvae, such modifications are believed to maximize feeding efficiency in planktotrophic larvae and maximize swimming efficiency in nonfeeding planktonic larvae (Emlet, 1994). Modifications or loss of larval feeding or swimming structures are often so extreme that re-evolution of such structures appears to be difficult. Loss of these complex structures is cited as evidence that feeding larvae, once lost, cannot be regained (Strathmann, 1978). The morphological modifications associated with the loss of feeding larvae in other groups of marine invertebrates are less well understood. In marine gastropods, for example, the evolution of nonfeeding or direct development is usually associated with encapsulated development. The functional requirements of development within a capsule may be very different from the requirements of free-living development. For example, the embryo may need structures to circulate intracapsular fluid (Strathmann and Chaffee, 1984), ingest intracapsular fluid (Fioroni, 1966; Morrill, 1982; Rivest, 1992; Moran, 1999), or ingest nurse eggs or siblings (Rivest, 1981).
The morphological structure associated with both feeding and swimming in gastropod larvae is the velum. In planktotrophic larvae, the velum is normally a pair of large flat lobes of tissue edged with two opposed bands of compound 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; Strathmann and Leise, 1979). Gastropods with planktotrophic larvae, and especially those with long-lived planktotrophic larvae, often have large elaborate vela with as many as six or eight lobes (Fretter and Pilkington, 1970; DiSalvo, 1988). In larvae of Crepidula fornicata, the velar lobes grow significantly larger, in relation to shell size, when larvae are cultured with low concentrations of food rather than with high concentrations (Klinzing and Pechenik, 2000). In swimming larvae, the velum is lost at metamorphosis. Those species with abbreviated pelagic phases or complete intracapsular development often show a reduction in the size of the velum, loss of ciliary bands, and shortening of cilia (Fioroni, 1966). Hadfield et al. (1997) showed that "archaeogastropod" larvae, which are characterized by swimming, nonfeeding larvae, have well developed preoral cilia but lack the food groove. In embryos of Crepidula adunca, a species with direct development from large yolky eggs, the velum is reduced to a small ridge with cilia only slightly longer than those on the surrounding mouth, tentacles, and head vesicle (Collin, 2000).
The absence of a feeding, swimming larval stage is not always associated with the loss of function of the velum. In some species of Littorina, the embryonic velum is used to ingest albumin from the egg capsules (Moran, 1999). In these species the velum is as large, relative to embryonic body size, as the velum in species with planktotrophic larvae (Moran 1999), suggesting that this novel function is selecting against loss of structure and function of the velum. In embryos of Petaloconchus montereyensis, the velum is modified to ingest intracapsular yolk but has lost the preoral cilia. Other embryonic modifications associated with intracapsular development are reviewed in Fioroni (1966). In species with direct, nonswimming development, the velum is lost before hatching.
The present study was undertaken to examine structural and functional modifications of the embryonic velum associated with the evolution of nurse-egg feeding. We compare the embryonic development, especially the velar development, of two closely related species of gastropods with differing modes of development: Crepidula fecunda Gallardo 1979, a species with planktotrophic development, and C. dilatata Lamarck 1822, a species with direct development in which the embryos consume nurse eggs (Gallardo, 1976, 1977, 1979; Chaparro and Paschke, 1990). We measured the ability, relative to velar morphology, of the embryos to capture particles and to swim.
|
Materials and Methods
|
|---|
Adult Crepidula dilatata were collected subtidally in the estuary of Rio Quempillen at the north end of Chiloé Island, Chile (41°52'S, 73°44'W), and C. fecunda were collected from the intertidal of Bahía de Yaldad in the south of Chiloé Island (43°08'S, 73°44'W).
Prior to examination by electron microscopy, broods were removed from the females, individually incubated for 5 min in 0.5% solution of chloral hydrate, fixed in a 3% solution of gluteralderhyde with cacodylate buffer (pH = 7.4), and maintained at 4 °C for 1 h. Embryos were subsequently excapsulated, rinsed for 10 min in phosphate buffer, dehydrated in a graded acetone series, and critical-point dried. They were attached to stubs with double-sided tape, gold-coated, and examined with a Bausch & Lomb Nanolab 2000 scanning electron microscope.
To test for the ability of the embryos to feed on small particles, the embryos were excapsulated and placed in seawater at ambient temperature. Nontoxic red beads 2–10 µm in diameter (Chaparro et al., 1993) were added to the solution. After 2 h the embryos were removed from the solution and fixed in formalin until they were analyzed. The presence of red particles in the larval or embryonic stomach was determined after fixation. One hundred embryos at each stage of development were examined under a compound microscope, and the percentage of embryos that had ingested particles (as indicated by red particles in the gut) was recorded. The method of particle capture was observed and recorded using an inverted microscope connected to a Sony DXC-C1 video camera and a Sony PVM-1953MD monitor. Similarly, to test for swimming ability, embryos were excapsulated, and groups of 20 were placed in 20 ml of seawater. After 5 min, the number of embryos swimming above the bottom of the vial was recorded. The shell length and velar characteristics of embryos used in both experiments were measured, using Scion Image PC (Scion Corporation, Frederick, MD), from video images.
|
Results
|
|---|
Embryonic morphology
Embryos of both Crepidula dilatata and C. fecunda develop from eggs with average diameters of 250 and 212 µm respectively (Gallardo, 1977). However, the embryos of C. dilatata consume uncleaved nurse eggs (Gallardo, 1977) and hatch as crawling juveniles of about 1100–1200 µm shell length (Chaparro and Paschke, 1990), whereas those of C. fecunda do not consume nurse eggs, and they hatch as planktotrophic larvae at a size of 400–500 µm. Despite this difference in mode of development, the embryonic morphologies of both species are similar.
In both species, spiral cleavage and gastrulation by epiboly result in a very slightly flattened gastrula with a central blastopore. In C. dilatata (Fig. 1A'), the gastrula is somewhat more elongate than the round gastrula of C. fecunda (Fig. 1A). The initial gastrula is not ciliated, but patches of cilia in the area of the presumptive velum and head vesicle can be seen in scanning electron micrographs of later gastrulas.
View larger version (176K):
[in this window]
[in a new window]
|
Figure 1. Scanning electron micrographs of different developmental stages of Crepidula fecunda (left side pictures: A–C) and C. dilatata (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.
|
|
In both species, an intracapsular trochophore stage follows the gastrula. This stage is characterized by elongation of the embryo and flattening of the posterior dorsal area where the shell begins to form. In C. dilatata, clearly defined patches of cilia around the mouth, at the ventral anterior (presumptive head vesicle) and ventral posterior (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, relative to other morphological events, than in the embryos of C. dilatata.
As the edge of the velum extends laterally from the body 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 slightly later. At this stage, in both species, the embryonic kidney is clearly visible on each side of the embryo, the shell begins to grow around the posterior end of the embryo, and the foot begins to differentiate and becomes ciliated. In embryos of both species of 200–300 µm, the small head vesicle is distinct and covered with cilia, the velum clearly shows opposed band ciliation, and the foot has an operculum and a medial band of ventral cilia (Fig. 1B, B', 1C, C'). Subsequent development reflects the growth and elaboration of these structures. The shell grows to contain the yolk and developing viscera. The lobes of the velum grow wider, and the preoral cilia grow to a length of 80 µm. Larvae of C. fecunda hatch at a shell length of 400–500 µm, after the reabsorption of the embryonic kidneys and the head vesicle.
In C. dilatata, further morphogenesis occurs in the capsule. The tentacles begin to form at a shell length of about 500 µm. Both tentacles are large and the apical ciliarly tufts are well developed, as is the foot, in embryos with a shell length of 700 µm. At this stage the velar cilia reach their maximal length of 80 µm. Embryos between 800 and 1100 µm in shell length undergo intracapsular metamorphosis. The pedal glands become visible, and the velum gradually shrinks. By the time the embryo is about 1000 µm in shell length, the velum and embryonic kidneys are no longer 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 at any given developmental stage shows variation due to differences in the number of nurse eggs consumed by each embryo.
Allometry
In C. fecunda, the velum grows throughout intracapsular development. The velar area increases linearly with respect to shell length (Fig. 2), and it reaches its maximal size, about 0.06 mm2, near hatching. The size of the velum relative to shell length in C. dilatata is initially the same as in C. fecunda, but the velum grows more slowly relative to shell length. Comparisons of larvae at a comparable shell length (<600 µm) showed a significant difference in velum size relative to shell length (ANCOVA, n = 374; f = 227.987, P < 0.001). The velum in C. dilatata continues to grow until it reaches an area of about 0.12 mm2. At large shell sizes the velar area decreases as the velum is absorbed prior to hatching.
The preoral cilia grow to a length of about 80–90 µm in embryos of both species (Fig. 3), but the relationship between the lengths of cilia and shell differs between the two species. In C. dilatata, the direct developer, the cilia reach their maximal length at a shell length of approximately 700 µm (near the size of maximal velar area); the same maximal length is attained in C. fecunda at a shell length of about 320 µm. The velar cilia of C. fecunda continue growing until hatching, as does the velum.
View larger version (19K):
[in this window]
[in a new window]
|
Figure 3. A comparison of cilia length versus shell length for Crepidula fecunda and C. dilatata. The vertical bars represent the standard deviation.
|
|
Feeding and swimming
The embryos of both C. dilatata and C. fecunda were able to capture and ingest small particles from suspension in seawater (Fig. 4). A high percentage of C. fecunda embryos from a shell length of 175 µm to the hatching size of 350–450 µm showed the ability to capture and ingest beads. In C. dilatata, the small embryos (shell length less than 400 µm) ingested particles with high frequency. Observation of both species showed that the embryos use the opposed-band ciliary mechanism to capture particles and to move them along the food groove to the mouth (Fig. 5A, A', 5B, B', 5C, C').
View larger version (19K):
[in this window]
[in a new window]
|
Figure 4. Scatter plot of the proportion of embryos that ingested particles versus shell length for Crepidula fecunda and C. dilatata. Each point represents a trial with 100 embryos.
|
|
View larger version (164K):
[in this window]
[in a new window]
|
Figure 5. Video sequence of a captured particle (encircled) moving along the food groove towards the mouth. Left side (A–C): Crepidula fecunda embryo, Right side (A'–C'): C. dilatata embryo.
|
|
There was a striking difference between the species in swimming ability: embryos of C. dilatata never swam, whereas embryos of C. fecunda larger than 270 µm swam consistently in our experiment (Fig. 6).
View larger version (19K):
[in this window]
[in a new window]
|
Figure 6. Scatter plot of the number of embryos that showed swimming ability versus shell length for Crepidula fecunda and C. dilatata.
|
|
|
Discussion
|
|---|
Our observations of Crepidula embryos show that the embryonic morphology of the direct-developing C. dilatata is little modified from that of the planktotrophic C. fecunda. After consuming many nurse eggs, the embryos of C. dilatata are larger than those of C. fecunda, and the relative size of the velum is smaller; however, the operculum is not lost, and there is no elaboration of the embryonic kidneys or massive enlargement of the head vesicle as has been reported for embryos of another direct-developing Crepidula species (Moritz, 1939; Collin, 2000). The velum of C. dilatata is not lost or greatly reduced in absolute size as it is in many gastropods with direct development, and it appears to be structurally similar to the velum of the closely related planktotrophic C. fecunda. It retains the bands of both preoral and postoral cilia and has a ciliated food groove. In addition, C. dilatata retains the ability to use the opposed-band ciliation to capture and transport particles along the food groove.
The lack of change in the embryonic development in the direct-developing C. dilatata could be the result of several factors. It is possible that there are few overall modifications of development because direct development has evolved recently in this species. Gallardo (1979) suggested that C. fecunda and C. dilatata were recently diverged sibling species. The retention of a well-developed velum with a functional opposed-band ciliary mechanism in direct developing species is unusual in North American Crepidula that do not consume nurse eggs (RC, pers. obs.) It is probable that the continued use of this structure to feed on material within the capsule has maintained the velum as a functional structure. Small particles of yolk from abnormal or damaged embryos pass along the velum of young embryos of C. dilatata in the same manner as the artificial particles described here. Nurse eggs are ingested in a different manner. In C. dilatata, the nurse eggs are generally consumed after the embryos have reached a shell length of 500 µm (Chaparro and Paschke, 1990) and have developed a large ciliated velum (see above). The embryo rotates the nurse egg on the velum. The egg deforms as the cilia of the buccal region slowly draw the egg into the esophagus (OC and RC, pers. obs.). The ability of C. dilatata embryos to capture small particles before the stage at which they actively feed on nurse eggs suggests that small particles from damaged embryos may be an important source of early nutrition in young embryos. The energetic contributions of these different feeding mechanisms should be examined in greater detail.
A previous study that addressed the possibility of the re-evolution of feeding gastropod larvae concluded that Petaloconchus montereyensis has lost the velar structures necessary for swimming and feeding, making the re-evolution of feeding larvae that use the opposed-band ciliary mechanism on the velum unlikely (Hadfield and Iaea, 1989). Because, as demonstrated here, C. dilatata embryos retain the ability to capture particles by using the opposed-band mechanism, the re-evolution of feeding larvae from this species is not impossible. However, changes in the velum-body size allometry and hatching size would probably be necessary to produce a larva capable of swimming. Because the hatching size for C. dilatata is determined by the number of nurse eggs per embryo in each capsule, it is possible that velum-body size allometry is determined by the number of nurse eggs consumed. If this is the case, a drastic reduction in the number of nurse eggs per capsule could change this allometry enough to produce embryos capable of swimming.
|
Acknowledgments
|
|---|
We thank the staff of the Quempillen field station for help with field work, and R. Silva for help with electron microscopy. O. Chaparro’s research was funded by grants from Fondecyt—Chile1980984 and Dirección de Investigación—Universidad Austral de Chile. R. Collin’s travel to Chile was supported by a grant from the National Geographic Society. B. Pernet made helpful comments on the manuscript.
|
Footnotes
|
|---|
Received 10 July 2001; accepted 23 May 2002.
|
Literature cited
|
|---|
Byrne, M. 1995. Changes in larval morphology in the evolution of benthic development by Patiriella exigua (Asteroidea: Asterinidae), a comparison with the larvae of Patiriella species with planktonic development. Biol. Bull. 188:293–305.[Abstract]
Byrne, M., and A. Cerra. 1996. Evolution of intragonadal development in the diminutive asterinid sea stars Patiriella vivipara and P. parvivipara with an overview of development in the Asterinidae. Biol. Bull. 191:17–26.[Abstract]
Chaparro, O. R., and K. A. Paschke. 1990. Nurse egg feeding and energy balance in embryos of Crepidula dilatata Lamarck during intracapsular development. Mar. Ecol. Prog. Ser. 65:183–191.
Chaparro, O. R., R. J. Thompson, and J. E. Ward. 1993. In vivo observations of larval brooding in the Chilean oyster, Ostrea chilensis Philippi, 1845. Biol. Bull. 185:365–372.
Collin, R. 2000. Sex change, reproduction, and development of C. adunca and C. lingulata. Veliger 43:24–33.
DiSalvo, L. H. 1988. Observations on the larval and post-metamorphic life of Concholepas concholepas (Brugiere, 1789) in laboratory culture. Veliger 30:358–368.
Emlet, R. B. 1994. Body form and patterns of ciliation in non-feeding larvae of echinoderms—functional solutions to swimming in the plankton. Am. Zool. 34:570–585.
Fioroni, P. 1966. Zur Morphologie und Embryogeneses des Darmtraktes und der transitorischen Organe bei Prosobranchiern (Mollusca, Gastropoda). Rev. Suisse Zool. 73:621–876.[Medline]
Fretter, V., and M. C. Pilkington. 1970. Prosobranchia: veliger larvae of the Taenioglossa and Stenoglossa. Cons. Int. Explor. Mer. Zooplankton Sheets 129–132.
Gallardo, C. S. 1976. Historia natural y reproducción de Crepidula dilatata Lamarck en una población de Bahía Mehuín (Prov. Valdivia, Chile). Medio Ambiente 2:44–50.
Gallardo, C. S. 1977. Two modes of development in the morphospecies Crepidula dilatata (Gastropoda: Calyptraeidae) from Southern Chile. Mar. Biol. 39:241–251.
Gallardo, C. S. 1979. Especies gemelas del genero Crepidula (Gastropoda, Calyptraeidae) en la costa de Chile: una redescripción de C. dilatata Lamarck y descripción de C. fecunda n. sp. Stud. Neotrop. Fauna Environ. 14:215–226.
Hadfield, M. G., and D. K. Iaea. 1989. Velum of encapsulated veligers of Petaloconchus (Gastropoda), and the problem of re-evolution of planktotrophic larvae. Bull. Mar. Sci. 45:377–386.
Hadfield, M. G., M. F. Strathmann, and R. R. Strathmann. 1997. Ciliary currents of non-feeding veligers in putative basal clades of gastropods. Invertebr. Biol. 116:313–321.
Klinzing, M. S. E., and J. A. Pechenik. 2000. Evaluating whether velar lobe size indicates food limitation among larvae of the marine gastropod Crepidula fornicata. J. Exp. Mar. Biol. Ecol. 252:255–279.[Medline]
Moran, A. 1999. Intracapsular feeding by embryos of the gastropod genus Littorina. Biol. Bull. 196:229–244.[Abstract]
Moritz, C. E. 1939. Organogenesis in Crepidula adunca Sowerby. Univ. Calif. Publ. Zool. 43:217–248.
Morrill, J. B. 1982. Development of the pulmonate gastropod, Lymnaea. Pp. 399–483 in Developmental Biology of Freshwater Invertebrates, G. W. Harrison and R. W. Cowden, eds. Alan R. Liss, New York.
Rivest, B. R. 1981. Nurse egg consumption and the uptake of albumen in the embryonic nutrition of marine snails. Ph.D. dissertation. University of Washington, 185 pp.
Rivest, B. R. 1992. Studies on the structure and function of the larval kidney complex of prosobranch gastropods. Biol. Bull. 182:305–325.[Abstract]
Strathmann, R. R. 1978. The evolution and loss of feeding larval stages of marine invertebrates. Evolution 32:894–906.
Strathmann, R. R., and C. Chaffee. 1984. Constraints on egg masses. II. Effect of spacing, size, and number of eggs on ventilation of masses of embryos in jelly, adherent groups, or thin-walled capsules. J. Exp. Mar. Biol. Ecol. 84:85–93.
Strathmann, R. R., and E. Leise. 1979. On feeding mechanism and clearance rates of molluscan veligers. Biol. Bull. 157:524–535.[Abstract/Free Full Text]
Werner, B. 1955. Über die anatomie, die Entwicklung und Biologie des Veligers und der Veliconcha von Crepidula fornicata L. (Gastropoda, Prosobranchia). Helgol. Wiss. Meeresunters. 5:169–217.
Wray, G. 1996. Parallel evolution of nonfeeding larvae in echinoids. Syst. Biol. 45:308–322.
This article has been cited by other articles:
|
|
|
|
 
R. Collin, O. R. Chaparro, F. Winkler, and D. Veliz
Molecular Phylogenetic and Embryological Evidence That Feeding Larvae Have Been Reacquired in a Marine Gastropod
Biol. Bull.,
April 1, 2007;
212(2):
83 - 92.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Pernet
Persistent Ancestral Feeding Structures in Nonfeeding Annelid Larvae
Biol. Bull.,
December 1, 2003;
205(3):
295 - 307.
[Abstract]
[Full Text]
[PDF]
|
|
|
TOP
Abstract
Introduction
