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The bilaterally asymmetrical larval form of stomopneustes variolaris (Lamarck).

Introduction

Echinoderms are well known for the bilateral symmetry of their larvae and the pentamerous radial symmetry of the adults (e.g., Hyman, 1955; Ruppert et al., 2004). Each class of echinoderms has a distinctive larval form. Bilaterally symmetrical feeding larvae occur in four classes--Asteroida, Ophiuroida, Holothuroida, and Echinoida. The fifth class, Crinoida, has nonfeeding larvae with subtle bilateral symmetry (e.g., Lacalli and West, 1986; Nakano et al., 2003). Developmental regulation of the symmetry shift between larval and adult stages within this distinctive deuterostome phylum is currently receiving substantial attention at the level of patterns of gene expression (e.g., Morris and Byrne, 2005; Mooi et al., 2005; Hibino et al., 2006; Morris, 2007).

The feeding larva of a sea urchin, called an echinopluteus, is recognized by its anteriorly directed, bilaterally symmetrical pairs of arms supported by calcitic skeletal rods and lined by a ciliated band used to swim and feed. The skeleton starts as a pair of calcite spicules that form the body skeleton and rods that project into the postoral arms and anterolateral arms of the young pluteus. Eventually up to eight separate skeletal rods are present, and in fully developed larvae (except some diadematids) there are four to five named pairs of arms that project anteriorly and sometimes another pair of arms that is directed laterally or posterolaterally (Fig. 1). In spatangoids, feeding larvae possess an unpaired posterior process that resembles an arm (see, e.g., Emlet et al., 2002). The shapes of echinoplutei are characteristic of the order or family to which the species belongs (Mortensen 1921, 1931, 1937, 1938; Wray, 1992).

Ophiuroids also possess bilaterally symmetrical feeding larvae, called ophioplutei, with anteriorly directed arms, and they resemble echinoplutei. Ophioplutei possess up to four pairs of arms, but all are supported by a pair of branched calcite spicules. While the same names are used to label arms in echinoplutei and ophioplutei, the structures are not believed to be homologous. Feeding larvae of asteroids and holothuroids lack larval arms but still exhibit bilateral symmetry in lobes and folds of the ectoderm as well as in the arrangement of the ciliated band.

Departures from distinctive larval bilateral symmetry such as reductions or complete loss of arms, lobes, or ciliated bands are known in some nonfeeding larval forms of echinoderms. Such variations may be due to loss of function of traits associated with feeding larvae or because direct-developing forms have returned to the plankton (e.g., Raff, 1992; McEdward, 1992; Morris, 1995; Selvakuma-raswamy and Byrne, 2000; Emlet, 1995, 2006). Slight asymmetries in length within pairs of echinoid larval arms have also been described (e.g., Collin, 1997), but consistent bilateral biases in larval shape are not known. The origin of the adult rudiment on the left side of the larval gut in echinoids and asteroids is an obvious departure from bilateral symmetry for internal structures such as coeloms and gene expression territories. The allocation of materials to the rudiment could lead to a bias in bilateral symmetry of the larval shape overall due to redistribution of larval cells or the need to balance the swimming forces generated by ciliated structures, but this is not obvious in the literature over the past century.

[FIGURE 1 OMITTED]

The present study describes the echinopluteus and newly metamorphosed juveniles of the tropical Indo-Pacific echinoid Stomopneustes variolaris (Lamarck 1816). S. variolaris is the only living species in the family Stomopneustidae, order Phymosomatoida, within the superorder Stirodonta along with arbacioid and salenioid echinoids (e.g., Smith 2005; Smith et al., 2006). The early larval stages of this species have been described previously (Mortensen, 1931; Shetty, 1960) and were said to resemble larvae of the family Echinometridae (order Echinoida, superorder Camerodonta). I raised this species through metamorphosis and found that the fully formed larva has features also present in the stirodontoid genus Arbacia. I also found a distinctive bias in lengths and pigmentation of two of the six pairs of arms. Yanagisawa (2004) published a seven-line abstract reporting that the later stage larvae of S. variolaris were essentially arbacioid in form, but had long right and short left posterolateral arms I descirbe the bilateral asymmetry here in greater detail and examine its connection with an adult bilateral asymmetry that has not been studied in detail. The asymmetry affects the way the larvae orient while swimming, but it is not obviously related to a radial bias in the shape of the test and spine lengths that is found in some adult specimens.

Materials And Methods

Adult individuals of Stomopneustes variolaris were collected at low tide from a rocky intertidal bench known as Duwa Reef at Negombo, Sri Lanka, in April 2007 (7[degrees]12'15.3"N, 79[degrees]49'0.3"E). Adults were transported in buckets of seawater to the University of Colombo and maintained in recirculating aquaria at ambient temperatures (28-31 [degrees]C). Adults spawned when injected with 0.3 ml of 0.1 mol [l.sup.-1] acetylcholine in filtered seawater (FSW). Eggs were removed from the adult's aboral surface by glass pipette, suspended in FSW, and allowed to settle. Sperm was also collected with pipette from the aboral surface of adults and activated by diluting it in FSW. A suspension of eggs was fertilized with dilute sperm, and larval cultures were set up following the methods described by Strathmann (1987).

Embryos and larvae were maintained in 500 to 700 ml of FSW in 1-liter glass jars. The water temperature of larval cultures was 28 to 31 [degrees]C. Initially, the densities of embryos were greater than 10/ml, but later stages were less than 1/ml. Every 2 to 3 days, larvae were concentrated by removing water through a coarse filter that did not allow larvae to pass. Individual larvae were moved with a pipette from the remaining water into new FSW. After water changes, larvae were fed a mixture of Dunaliella tertiolecta and Chaetoceros gracilis that was centrifuged to remove culture medium and resuspended in seawater. After the first week, larval cultures were stirred with a paddle system similar to that described by Strathmann (1987).

Larvae with well-developed rudiments that appeared ready to metamorphose were placed in dishes containing FSW and a glass shard or shell fragments collected from the intertidal of Duwa reef. Before these substrata were used to induce settlement, they were observed with a microscope to ensure that potential predators and naturally settled sea urchin juveniles were absent. These substrata contained biofilms but lacked coralline algae or other fouling organisms.

At regular intervals throughout development, larval or juvenile stages were photographed with a digital camera attached to the trinocular port of a dissecting or compound microscope. For scale, a stage micrometer was photographed at the same magnification as the larval/juvenile stage, and this image was used to set the scale in pictures of organisms. Occasionally, cross-polarized light was used to reveal birefringent calcareous larval spicules and developing skeletal elements of juveniles. Preserved 1- and 2-day-old juveniles were dehydrated through an ethanol series and cleared in a solution of benzyl alcohol and benzyl benzoate to observe the apical plates.

Specimens of adult S. variolaris were borrowed from the Los Angeles County Museum of Natural History (lots: LACM EC-518; AHF Cat. No. 871.1; LACM 89-81.1). Two bare tests and nine complete specimens preserved in ethanol were examined for test shape and length of spines relative to adult radial symmetry. The interambulacral and ambulacral regions are labeled following Loven's system (Hyman, 1955). This system labels ambulacra with Roman numerals and interambulacra with Arabic numerals, proceeding counterclockwise. Genital plates are located at the tops of interambulacra, and ocular plates are locted at the top of the ambulacra. Located at interambulacrum 2 (IA2), the madreporite is also a genital plate (G2). When looking down on the apical surface, the ocular plate adjacent to and clockwise from the madreporite is located at the top of ambulacrum II (All).

Results

Two females shed small, spherical, optically opaque eggs on 20 and 24 April 2007. The female that spawned on 20 April had eggs less than 70 [micro]m in diameter, but no quantitative data were collected. The female that spawned on 24 April had eggs with a mean diameter of 63 [micro]m ([+ or -]2, [micro]m SD, n = 20). Sperm, newly released each day from a single male, were used to fertilize eggs of both females. The small size and opaqueness of the eggs and early embryos prevented me from describing these stages.

At 19 h post-fertilization, late prism to early 2-arraed plutei swam near the surface of culture bowls. Larvae developed into bilaterally symmetrical 2-armed plutei with postoral arms (Fig. 2A inset). By day 4, anterolateral arms began to grow (Fig. 2A); at this stage larvae readily consumed algal food, as indicated by their pigmented stomachs. By day 6, 4-armed plutei had postoral arms 400 [micro]m. long and anterolateral arms 80 [micro]m long (Fig. 2B). The postoral arms were supported by fenestrated spicules, while those of the anterolateral arms were simple rods. By day 8, postoral arms were about 600 [micro]m long; anterolateral arms were about 200 [micro]m long; and a third pair of arms, the posterodor-sal arms, were present in some larvae (Fig. 2C). The spicules of these arms were simple rods with limited fenestration at their bases.

Over subsequent days, 6-armed larvae gained two more arm pairs. Growing from the dorsal arch spicule, preoral arms developed and were supported by a simple U-shaped spicule (Fig. 2D). Soon afterward, anterodorsal rods formed as branches of the dorsal arch spicule and grew to support the anterodorsal arms (Fig. 2E).

Already by day 6 in some 4-armed larvae and by day 8 in all 4- or 6-armed larvae, the postoral arms were unequal in length. Of the 20 or more larvae closely examined at the 6-armed stage, all had left postoral arms longer than their right postoral arms (e.g., Fig. 2C). This asymmetry became more obvious as larval arms lengthened during the larval period (Fig. 2E, F). In addition, the left postoral arm had more red pigment at its tip than the right postoral arm (Fig. 2G, H). All arms increased in length (Fig. 3).

By day 15, the last arms--the posterolateral arms--began to form, and these arms also grew asymmetrically. The right posterolateral arm grew long and was oriented either perpendicular to the main body axis (Fig. 2E) or in a more posterior direction about 45[degrees] between perpendicular and parallel to the main body axis (Fig. 2F). The left posterolateral arm remained relatively short and showed a variation in angle relative to the main body axis similar to that of the right posterolateral arm. The tip of the right posterolateral arm was colored a deep red with pigment cells (Fig. 21); the left arm tip had little pigment. Like the other arms, both posterolateral arms were lined by a ciliary band. All arms increased in length. By day 28, when some larvae were competent to metamorphose, arm lengths from tip to base where each joins the larval body were as follows: left postoral, 1.4 mm; right postoral, 1.2 mm; right posterolateral, 1.6 mm; and left posterolateral, 0.19 mm (Fig. 3B). The length from left postoral arm tip to right posterolateral arm tip was 3.1 mm. Other larvae had arm lengths similar to these, but in one the length of the left postoral arm exceeded that of the right posterolateral arm. Advanced larvae also developed a pair of dorsal lobes at the base of the posterodorsal arms and a pair of ventral lobes as the base of the postoral arms (Fig, 3A).

When larvae were swimming in beakers, they were oriented so that their anterior-posterior axis was oblique to vertical, with the right side of the larva higher than the left side. The long right posterolateral arm was pointed outward and upward in the water column and complemented the longer left postoral arm. When viewed from above, the right posterolateral and the left postoral arms projected widely from the rest of the larval body (Fig. 3A). The cilia on both of the posterolateral arms were arranged in lateral bands. Cilia on the right posterolateral arm responded to algal cells by reversing the direction of beat and directing the cells toward the base of the arm. No observations were made on cilia of the shorter left posterolateral arm.

[FIGURE 2 OMITTED]

Competent larvae had 5 to 8, most commonly 7, pedicellariae. One pedicellaria was always located at the posterior end of the larval body between the posterolateral processes. The other pedicellariae were located on the dorsal and ventral surfaces of the larval body near the bases of the postoral and posterodorsal arms. When 7 pedicellariae were present, there were 3 on each of the larval dorsal and ventral surfaces. With fewer pedicellariae, one or more was missing from either the dorsal or ventral surfaces; with more than 7, an extra pedicellaria was on the dorsal surface (Fig. 3B inset).

[FIGURE 4 OMITTED]

Two larvae were observed that differed from the pattern described above. One of these had a long left posterolateral arm and a short right posterolateral arm, and its right postoral arm was longer than its left postoral arm, a pattern opposite to that normally seen (Fig. 4A). Greater amounts of red pigment were associated with the longer arms. Finally, the rudiment was on the right side of the larva, indicating a case of situs inversus (e.g., Ohshima, 1922). The second larva with an aberrant pattern did not develop a rudiment. In this case its postoral arms were of equal length and had little red pigment at the tips. The posterolateral arms were both well developed, though the left one was longer, and both had large amounts of red pigment at their tips (Fig. 4B). This phenotype of arm pigmentation was as if the larva had two right sides. This individual also lacked pedicellariae. These two unusual larvae suggest a developmental link between the asymmetry of the larval arms and the formation of the juvenile rudiment.

Larvae metamorphosed in response to biohlms present on the glass shards or shell fragments in their culture dishes. Twenty-four hours after larvae were introduced to the bio-films, juveniles were 440 to 480 [micro]m in test diameter. They had 5 primary podia, some short juvenile spines projecting in pairs from the aboral surface near the podia, and pedicellariae retained from the larval body (Fig. 5A, B). Three adult spines were beginning to form in each interambulacrum (between the primary podia). By the second day after metamorphosis, considerable growth of juvenile and adult spines had occurred (Fig. 5C, D). The test diameter was slightly larger, ranging from 475 to 495 [micro]m, and overall diameter was 770 to 820 [micro]m. The adult spines were about 50 [micro]m long and visible from oral and aboral views (Fig. 5C, D). Juvenile spines, also up to 50 [micro]m long, were in pairs near each of the 5 primary podia, and about 12 to 15 additional juvenile spines were distributed over the aboral surface. At this time, a pair of podia buds was present on the oral surface adjacent to each primary podium (Fig. 5D). Five days after metamorphosis, one juvenile had reduced primary podia, active paired podia, and an open mouth.

[FIGURE 5 OMITTED]

Eleven adult specimens (2 dry bare tests and 9 with spines, preserved in alcohol) of Stomopneustes variolaris were examined for asymmetry of the test and spines. Both bare tests had a gently sloping aboral surface with the "high" side located arouud interambulacrum 3 (IA3) and the "low" side around ambulacrum I (AI, see Fig. 6A). Seven of the 9 individuals with spines had longer spines on one side than the other. In these individuals the side with long spines was AI, and the opposite side with short spines was TA3. Rough measurements made with calipers of the test height on several of these adults with spines indicated that the IA3 side was 1 to 2 mm higher than the AI side.

Cleared juveniles, 1 day and 2 days post-metamorphosis, had genital plates in the orientations expected for newly metamorphosed sea urchins (Fig. 6C-F). These newly metamorphosed juveniles had no obvious asymmetry of the test or spines that could be tied to larval or adult bilateral asymmetry. Remnants of the larval rods could be seen in genital plates G1 and G3 (Fig. 6C) and G2 (Fig. 6D). G1 was associated with the right anterolateral rod, G2 formed from the dorsal arch spicule, and G3 formed on the right posterodorsal rod. G4 formed at the base of the posterolateral rod, and G5 formed on the right postoral rod. Due to rapid growth of calcitic plates, resolution of apical plates on day 2 was limited.

Discussion

Stomopneustes variolaris has long been placed in the superorder Stirodonta because of the structure of its Aristotle's lantern: keeled teeth, small epiphyses, and an open foramen magnum over each tooth (Jackson, 1912). The Stirodonta includes a number of extinct stem group lineages and has living taxa in three orders: Salenioida, Arbacioida, and Phymosomatoida (Durham, 1966; Smith, 2005; Smith et al., 2006). Nothing is known of the larval forms of salenioids; larvae and juveniles of several arbacioids have been described (see below). The Phymosomatoida has two extant species--Glyptocidaris crenularis, whose larva and juvenile have been described (Fukushi, 1960), and Stomopneustes variolaris, the subject of the present study. The Stirodonta is the sister taxon to the superorder Camarodonta, which includes the orders Temnopleuroida and Echinoida, the latter of which includes families Toxopneustidae, Strongylocentrotidae, Echinometridae, Echinidae, and Parechinidae. The clade containing both Stirodonta and Camarodonta is currently considered sister taxon to the Superorder Irregularia, which includes spatangoids (heart urchins) and clypeasteroids (sand dollars) (Smith et al., 2006).

Feeding larval forms of echinoids generally show clear morphological similarity within a family (e.g., Mortensen, 1921; Wray, 1992). Two-armed and four-armed larvae of S. variolaris and their skeletons were described by Mortensen (1931) and Shetty (1960). Both authors noted the resemblance of the body skeleton of S. variolaris to those of echinometrid larvae of similar stages. This resemblance was based on the presence of double recurrent rods that lead to the formation of a compound basket structure for the body skeleton. In young larvae of Arbacia, the recurrent rods are single and do not grow to the posterior to make a basket structure (Muller, 1854). Fukushi (1960) mentioned a single recurrent rod in the early larval skeleton of G. crenularis. Thus the early larvae of Stomopneustes do not resemble other stirodontoids.

Once past the 4-arm stage, larvae of S. variolaris acquire features that show their affinity with the arbacioids but not with the phymosomatoid, G. crenularis (Fukushi, 1960). Yanagisawa's (2004) success in raising S. variolaris through metamorphosis allowed him to see that later stage larvae clearly resemble those of Arbacia spp. Except for the unusual asymmetry of the postoral and posterolateral arms, the late stage larvae of S. variolaris resemble those of Arbacia spp. (Harvey, 1949, 1956; Emlet, pers. obs.) and another arbacioid, Tetrapygus niger (Fuentes and Barros, 2000), both in form and large size. The late stage larvae of Arbacia and Tetrapygus are bilaterally symmetrical and have 5 pairs of larval arms in addition to posterolateral arms. The posterolateral arms are long and, like the right posterolateral arm of S. variolaris, have deep red pigmented tips. Larvae of 5. variolaris also have dorsal and ventral paired lobes, lined by the ciliated band (Fig. 3A), and these are also found in Arbacia punctulata (Harvey, 1949, plate II, fig. 9), A. stellata (Emlet, pers. obs.), and Eucidaris thouarsi (Emlet, 1988). The maximum length of larvae of 5. variolaris exceeded 3 mm, and those of larvae of Arbacia and Tetrapygus exceeded 4 mm (Emlet, pers. obs.; Fuentes and Barros, 2000). In contrast, larvae of Glyptocidaris do not develop long posterolateral arms; their posterior skeletal element forms shor rods that do not extend the ciliated band out into arm-like processes (Fukushi, I960). Judging from the drawings and photographs in Fukushi (1960), larvae of G. crenularis lack the paired dorsal and ventral ciliated lobes; their postoral arms are less than 0.8 mm long; and the maximum length of the larval body is less than 1.2 mm. A. stellata and S. variolaris each also have 5 or more pedicellariae on the larval body, whereas larvae of G. crenularis have a single dorsal pedicellaria (Fukushi, 1960).

Spicule morphology in larvae of S. variolaris differs from that found in Arbacia and Glyptocidaris. Both postoral and posterodorsal arm rods are fenestrated along their length in Glyptocidaris (Fukushi, 1960), but fenestration in Arbacia is usually found in the more distal parts of both postoral and posterodorsal arm rods (Wray, 1992). In S. variolaris the postoral arm rods are fenestrated along their entire length, but the posterodorsal arms rods are fenestrated only at their bases and simple for most of their length. Wray (1992) hypothesized that fenestration of both postoral and posterodorsal arm rods is ancestral and noted that the loss of fenestration in the posterodorsal arm rods has occurred at least twice: (i) in several species of clypeasteroids in the genus Clypeaster and (ii) in some toxopneustids (e.g., Tripneusies spp. and Nudechinus). This condition in larvae of S. variolaris is a third instance of loss of fenestration in the posterodorsal arm rods.

The remarkable bilateral asymmetry in the larval arms of S. variolaris is unexpected, and its function is not clear. Yanagisawa (2004) reported the same asymmetry in posterolateral arms described here in larvae of S. variolaris he raised in the Ogasawara Islands, 1000 km south of Toyko, Japan, in the Pacific Ocean. Though his abstract does not mention the asymmetry of the postoral arms, populations in both the Indian and Pacific Oceans share this oddity of asymmetrical posterolateral arms. Sri Lankan larvae of S. variolaris showed some variation in the angle that the right posterolateral arm made with the main axis of the body. It is unclear whether this is natural variation or an artifact caused by larvae colliding with culture chamber walls during gentle stirring. At one extreme the posterolateral arm comes out perpendicular to the main body axis; this orientation is found in some spatangoids that have a pair of symmetrical posterolateral arms and a posterior projection that all grow from the posterior skeletal element--for example, in Echinocardium corrdatum (MacBride, 1914; Rees, 1953). At the other extreme the right posterolateral arm projected posteriorly and obliquely, as do the paired symmetrical posterolateral arms of Arbacia spp. (Harvey, 1949; Emlet, pers. obs.) An arrangement with a long posterolateral arm on the left side and perpendicular- to the main body axis might interfere with attachment of everted podia to the substratum during settlement. This might be more problematic for larvae settling on hard substrata and in high-energy environments such as the wave-swept intertidal habitat of Stomopneustes and less problematic for spatangoid larvae settling in soft-sediment habitats. The hypothesis that the left posterolateral arm is short to facilitate settlement by larvae of S. variolaris was not tested.

The consistent bilateral asymmetry of larvae of S. variolaris is developmentally linked to rudiment formation. Normal larvae consistently showed very short left and long right posterolateral arms and a right postoral arm that was somewhat shorter than the left postoral arm. This situation was reversed in the one larva whose rudiment developed on the right side, a case of situs inversus (Ohshima, 1922; Swan, 1966). Not only were arm lengths a mirror image of those of normal larvae whose rudiment developed on the left, but the pigmentation patterns found on the longer arms of normal larvae were also present on the longer arms of the larva with situs inversus (Fig. 3A). In the one larva that did not develop a rudiment, the posterolateral arms were not equal in length (the left one was longer), but both were long and showed the pigmentation of the long right arm in normal larvae. In addition, the postoral arms of this larva were approximately equal in length and had reduced pigment at their tips, and thus resembled the right postoral arms of normal larvae with rudiments (Fig. 3B). This larva appeared to have two right sides.

I observed several hundred larvae from the two cultures of S. variolaris that 1 raised. All except the two just described had rudiments develop on the left side and had long right posterolateral arms and short left posterolateral arms. They also had left postoral arms longer than right postoral arms and patterns of pigmentation that were consistent with what I have described above for normal larvae. The two larvae that departed from the normal pattern did so in ways that have been described previously for other sea urchin larvae. Ohshima (1922) described situs inversus in larvae of Psamtnechinus miliaris and reviewed earlier literature on plutei with double rudiments and no rudiments. He reported instances of situs inversus of from 3% to 16% and instances of no rudiment of around 5% in some of his cultures. MacBride (1911, 1918) and Czihak (1960, 1996) also described pluteus larvae with double rudiments and ones with no rudiments. All of these authors attribute these unusual larvae to development of the hydrocoel. For larvae with situs inversus, the right hydrocoel enlarged instead of the left to produce the rudiment that becomes the juvenile oral surface. For larvae with double rudiments, both left and right hydrocoel grew large and each formed a juvenile oral surface. Finally, if the hydrocoel failed to develop, then no juvenile oral surface formed. In their descriptions of aberrant larvae, the authors noted that the anatomies of the plutei, including arms and ciliated band, were otherwise similar to those of normal larvae. My interpretation of the two aberrant larvae of S. variolaris also considered the larva! arms and ciliated band to resemble normal larvae and used asymmetry of arms and pigmentation of normal larvae to understand the anatomy of the aberrant larvae.

Developmental studies have begun to show how left-right asymmetry that leads to differential coelomic development and rudiment formation on the left side is organized in sea urchin larvae. Signaling from the rnicromeres is involved (Kitazawa and Amemiya, 2007), and the gene nodal is expressed asymmetrically in the right side archenteron and right ectoderm of the gastrula (Duboc et al., 2005). This signaling pathway apparently inhibits rudiment formation on the right side but does not obviously influence larval arm symmetry in typical (symmetrical) echinoplutei. The unusual bilateral asymmetry of larvae of 5. variolaris and its link to rudiment formation as observed indicates that genes involved in larval arm symmetry and length may be linked to genes that control rudiment formation.

The developmental control of larval arm length and symmetry is not well known. Recently, Love et al. (2007) examined gene expression patterns associated with arm growth of echinoplutei of the echinometrid Heliocidaris tuberculata. They interpreted the co-expression at the arm tips of tetraspanin, carbonic anhydrase, and advillin as unique patterning for larval arm tips. It would be important to know if these genes are still expressed in arm tips that have reached their full lengths as well as if the expression patterns of these genes differ in asymmetrical arms of S. variolaris.

Although the larvae of S. variolaris resemble those of Arbacia and Tetrapygus, the juveniles of S. variolaris are very different from these arbacioids. The main difference is that the adult spines of S. variolaris look like those of other urchin juveniles, whereas those of the arbacioids are spatulate to paddle-shaped (Harvey, 1956; Fuentes and Barros, 2000; Emlet, pers. obs.). The juveniles of S. variolaris more closely resemble those of Gixpiocidaris crenularis (Fukushi, 1960).

Many, but not all, adults of S. variolaris have longer spines on one side of their bodies. Mortensen (1935, p. 5 11) speculated that the spine asymmetry was due to adult behavior of seeking refuge in burrows or pits. He implied that the longer spines would be directed out of the pits. To my knowledge this has been neither supported nor rejected with field observations. My observations suggest that this asymmetry is fixed with respect to adult body axes. Bare tests show a more or less pronounced asymmetry with a somewhat flattened aboral surface that slants to one side when viewed laterally. The ''high side" is centered on interambulacrum 3 (e.g., contains genital plate 3). The "low side" is opposite this and is centered near ambulacrum I (e.g., ocular plate I, between interambulacra I and 5). The longer primary spines are associated with the "low side." Examination of newly metamorphosed juveniles did not show any obvious asymmetry or unusual orientation of apical plates that might indicate a morphological link between the bilaterally asymmetrical larvae and the fixed asymmetry found in adults. The adult asymmetry has not been widely studied, and additional published information appears to be lacking. It is unknown when the asymmetrical test and unequal spine lengths are first seen in small sea urchins of S. variolaris.

The bilateral asymmetry of larvae of S. variolaris is unique among sea urchin larvae described to date. Despite this asymmetry, fully formed larvae resemble arbacioid larvae in the size, number, and orientation of larval arms. The causes of this asymmetry in arm length and pigmentation warrant further mechanistic study of how they develop, and whether they are linked developmentally to rudiment formation. It is also intriguing that both larvae and adults show a fixed asymmetry. While my morphological studies of early juveniles do not show any connections between larval and adult asymmetries, these asymmetries may or may not share a common developmental basis.

Acknowledgments

This study was conducted while the author was supported by a W. J. Fulbright Senior Fellowship in Sri Lanka. I thank Professor S.U.K. Ekaratne and the University of Colombo for logistical help and for providing laboratory space and resources for this research. Tracey Smart and Katie Bennett kindly provided comments and edited the manuscript. I am grateful to reviewers for helpful comments.

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RICHARD B. EMLET

Oregon Institute of Marine Biology and the Department of Biology, University of Oregon, P.O. Box 5389, Charleston, Oregon 97420

Received 26 September 2008; accepted 9 January 2009.

E-mail: remlet@uoregon.edu
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