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Larval development of two N. E. Pacific Pilidiophoran Nemerteans (Heteronemertea; Lineidae).

Abstract. Unique to the phylum Nemertea, the pilidium is an unmistakable planktonic larva found in one group of nemerteans, the Pilidiophora. Inside the pilidium, the juvenile develops from a series of epidermal invaginations in the larval body, called imaginal discs. The discs grow and fuse around the larval gut over the course of weeks to months in the plankton. Once complete, the juvenile breaks free from the larval body in a catastrophic metamorphosis, and often devours the larva as its first meal. One third of nemertean species are expected to produce a pilidium, but the larvae are known for very few species; development from fertilization to metamorphosis has been described in only one species, Micrura alaskensis. Known pilidia include both planktotrophic and lecithotrophic forms, and otherwise exhibit great morphological diversity. Here, we describe the complete development in two lineiform species that are common to the northeast Pacific coast, Micrura wilsoni and Lineus sp. "red." Both species possess typical, cap-shaped planktotrophic pilidia, and the order of emergence of imaginal discs is similar to that which is described in M. alaskensis. The pilidium of Lineus sp. "red" resembles pilidia of several other species, such as Lineus flavescens, and potentially characterizes a pilidiophoran clade. M. wilsoni has relatively transparent oocytes and a pilidium with what appears to be a unique pattern of pigmentation. The adults of both species are more commonly observed in intertidal zones than their larvae are in the plankton.

Introduction

Nemertean worms comprise a lophotrochozoan phylum with about 1275 described species (Kajihara et al., 2008), most of which are marine. As is the case for most benthic marine invertebrates, nemerteans exhibit a biphasic life history with a pelagic larval stage. The two life-history stages can have dramatically dissimilar morphology. The most distinctive nemertean larva is called the pilidium, and was first described in 1847 by Johannes Muller. He suspected that it was some sort of marine invertebrate larva, but at the time could not make the connection to a nemertean (Muller, 1847). The pilidium is a unique larval form; its morphology is unmistakable and found in no other marine invertebrate. Furthermore, it is observed in only one clade of nemerteans, called the Pilidiophora (= Heteronemertea + Hubrechtidae; Thollesson and Norenburg, 2003; Andrade et al., 2014, but see Andrade et al., 2012; Kvist et al., 2014), which comprises about a third of all nemertean species (Kajihara et al., 2008). Unique for pilidial development is the formation of the juvenile body from seven or eight separate rudiments, called imaginal discs (Salensky, 1912; Maslakova, 2010a). Paired discs that arise as invaginations of larval epidermis include the cephalic discs, trunk discs, and cerebral organ discs, which give rise to the juvenile head, trunk, and cerebral organs, respectively. Two unpaired rudiments, which may be mesenchymal in origin--the proboscis and the dorsal disc--also contribute to the juvenile body (Maslakova, 2010a). Ultimately, these discs fuse to form a juvenile worm that erupts from its larval body in a rapid and catastrophic metamorphosis. In this transition from planktonic to benthic habitat, the juvenile pilidiophoran typically ingests its larval body (Cantell, 1966a, 1969; Lacalli, 2005; Maslakova, 2010a; von Dohren, 2011; Maslakova and von Dassow, 2012; Hiebert et al., 2013).

Development is known in any detail in only a few species of pilidiophorans, because most pilidia require food and take weeks to months to develop through metamorphosis. Maslakova (2010a) published the first description of pilidial development from fertilization to metamorphosis in a common intertidal heteronemertean from the northeast Pacific, Micrura alaskensis Coe, 1901. Prior to this study, the most detailed account of pilidial development (Salensky, 1912) was based on wild-caught, unidentified pilidia. Schmidt (1930) published a description of larval development in Cerebratulus marginatus from fertilization to the formation of imaginal discs, but did not observe late development or metamorphosis. Partial descriptions of development are available for only a handful of other pilidiophorans (Desor, 1848; Burger, 1895; Coe, 1899; Wilson, 1900; Salensky, 1912; Nusbaum and Oxner, 1913; Schmidt, 1929, 1932a, b, 1934, 1964; Dawydoff, 1940; Iwata, 1958; Cantell, 1969; Schwartz and Norenburg, 2005; Schwartz, 2009; Maslakova, 2010b; von Dohren, 2011; Maslakova and von Dassow, 2012; Hiebert et al., 2013; Maslakova and Hiebert, 2014; Hiebert and Maslakova, in press). Phylogenetically, all pilidiophorans are expected to produce a pilidium larva (Thollesson and Norenburg, 2003), but the specific larval morphology can be highly variable (e.g., see Leuckart and Pagenstecher, 1858; Fewkes, 1883; Dawydoff, 1940; Cantell, 1966a, b; Norenburg and Strieker. 2002; Lacalli. 2005; Maslakova and Hiebert, 2014). Furthermore, whether the larva is feeding or non-feeding cannot be easily anticipated phylogenetically. Lecithotrophic, or modified, pilidia, once believed to be rare within the group, likely evolved in several lineages (Schwartz, 2009; Maslakova and Hiebert, 2014). Until more studies track pilidial development from fertilization to metamorphosis, one cannot be certain how generally the findings reported for Micrura alaskensis (Maslakova, 2010a) may be applied.

Larval morphology may be phylogenetically relevant for nemerteans, a group for which taxonomy is mostly based on characters of adult morphology, which are few and often not unique. Many traditionally established nemertean genera are non-monophyletic, according to molecular phylogenetic analyses, and are in need of revision (e.g., Thollesson and Norenburg, 2003; Andrade et al., 2012). New genera should be defined based on monophyly and, ideally, characterized by morphological (as well as molecular) synapomorphies. Where adult morphology fails to provide such synapomorphies, larval features may provide additional characters that will help to define closely related groups or genera. However, the development of few nemertean species is known. Here, we describe the development from fertilization to metamorphosis of two common, northeast Pacific nemertean species Micrura wilsoni Coe, 1904, and an undescribed lineiform species, which we call Lineus sp. "red," because it lacks a caudal cirrus, is reddish-brown in color, and appears to be related to a number of described Lineus species, such as L. bilineatus, L. flavescens, and L. torquatus.

Materials and Methods

Micrura wilsoni is a lineiform heteronemertean known from the Pacific coast of North America, and reported to occur from Oregon to Mexico (Coe, 1904; Gibson, 1995; Roe et al., 2007; T. C. Hiebert and S. A. Maslakova, unpubl. data). It resides intertidally and subtidally, in rocky habitats, where it is somewhat common (T. C. Hiebert and S. A. Maslakova, pers. obs.), meaning that on any given trip to their known habitat, we can find one or a few individuals, but sometimes none. Reproductive adults of M. wilsoni were collected under rocks and from the holes of bivalves (Fam. Pholadidae) boring the rocks intertidally from Middle Cove and North Cove at Cape Arago, near Charleston, OR (Fig. 1A, B), in June of 2013. Adults are up to 15 cm in length, dark brown to black, but with very thin and irregular transverse lines of somewhat lighter color, and a characteristic white anterior margin of the head (Roe et al., 2007; Fig. IB). Adults lack ocelli, possess typical lineiform lateral cephalic slits, a ventral mouth, and a long caudal cirrus at the posterior end (Fig. IB). The cirrus may be lost during collecting, but readily regenerates if worms are kept in the lab for a few weeks, even without food.

Lineus sp. "red" is an undescribed lineiform species from southern Oregon that groups closely with Lineus flavescens, L. bilineatus, L. torquatus, and Cerebratulus montgomeryi on molecular phylogenies (T. C. Hiebert and S. A. Maslakova, unpubl. data). Adults are fairly common intertidally in the mudflats of Coos Bay, OR, as well as among the roots of surfgrass, in silty shell hash, and under rocks in coves near Cape Arago, OR. Reproductive adults were collected in February of 2012 from mudflats along the South Slough in Coos Bay, OR (Fig. 1A, C). Adults vary in color from dark red or brownish black to ochre (Fig. 1C), with the anterior darker than the rest of the body, and are approximately 6-10 mm long. The head is rectanguloid in shape and only slightly demarcated from the body. Adults lack ocelli, and possess typical lineiform lateral cephalic slits and a ventral mouth. The body is rounded in the foregut region and flattened dorso-ventrally in the midgut region, where it is often transversely creased and coiled like a wrinkled ribbon. A caudal cirrus is absent; instead, the worm tapers to a blunt end.

Embryological cultures

Primary oocytes and sperm were obtained by dissection. Oocytes had conspicuous germinal vesicles (e.g., Fig. 2A) when dissected. After approximately 30 min in filtered seawater (FWS) (0.45 jam), oocytes "rounded up" underwent germinal vesicle breakdown (GVBD), and were fertilized with dilute sperm suspension. Oocytes were fertilized in 150-ml glass culture dishes, in concentrations such that a monolayer formed on the bottom of the culture dish. Dishes were surrounded by flowing seawater at ambient sea temperature (9-12 [degrees]C). After 24 h, larvae were suspended at approximately 1 larva per ml in a 1-gallon glass jar. Swimming larvae were maintained at this concentration with constant stirring using plexiglass paddles (Strathmann, 1987). Larvae were fed Rhodomonas lens Pascher & Ruttner (CCMP 739) at concentrations of approximately [10.sup.4] cells/ml, and water was changed every 3 d by reverse filtration. After about 2 wk, larval concentration was further reduced to approximately 1 larva per 5 ml. Throughout development, individual larvae were removed from culture and photographed using a Leica DFC400 digital camera (Leica, Wetzlar, Germany) mounted to an Olympus BX51 compound microscope (Olympus America, Inc., Melville, NY) equipped with differential interference contrast (DIC) optics. Larvae were imaged live in a small drop of FWS on a glass slide, covered, and gently trapped with a glass coverslip supported by small clay feet at the corners (Strathmann, 1987).

Confocal microscopy

Ten to fifteen Micrura wilsoni larvae were relaxed in a 1:1 mixture of FSW and 0.34 mol [1.sup.-1] Mg[Cl.sub.2] for approximately 30 min, and subsequently fixed in 4% paraformaldehyde for 1 h at room temperature. After fixation, larvae were rinsed in several short (< 1 min) washes in phosphate-buffered saline (PBS) (pH 7.4; Thermo Fisher Scientific, Waltham, MA), and permeabilized in PBS with 0.1 % Triton X-100 (PBT) in three 10-min washes. To visualize cell nuclei and musculature, larvae were labeled with Hoechst 33342 (2 [micro]mol [1.sup.-1]; Thermo Fisher Scientific) and Rhodamine Phalloidin (165 nmol [1.sup.-1]; Sigma-Aldrich Corp., St. Louis, MO), respectively. The fluorescently labeled larvae were washed in PBS (three 10-min washes), emersed in 50% glycerol, and mounted and imaged in 90% glycerol. Confocal stacks of 0.5-0.75-[micro]m sections were gathered using an Olympus FluoView 1000 confocal system mounted on an Olympus IX81 inverted microscope equipped with a UPlanSApo 20X 0.85 NA oil lens (Olympus America, Inc.). Z-projections were reconstructed using ImageJ version 1.46 (Wayne Rasband, National Institutes of Health, Bethesda, MD). Lineus sp. "red" larvae were not preserved for or imaged using confocal microscopy.

Results

Micrura wilsoni development

Recently dissected oocytes are approximately 100-110 [micro]m in diameter, relatively transparent, possess a distinct germinal vesicle, and are surrounded by a chorion tightly apposed to the surface (Fig. 2A). Sperm is of "primitive" (Strieker and Folsom, 1998) type, with a head piece 4-5 [micro]m in length (Cluster 4-5 in fig. 11 of von Dohren et al., 2010). The developmental timeline described here is based on observations of a single larval cohort, with seawater temperature at 12 [degrees]C (Table 1). Following GVBD in seawater, the egg envelope lifts slightly off the oocyte surface and becomes even more conspicuous following fertilization (Fig. 2B). First and second polar bodies are apparent at 1 and 2 h post-fertilization (PF), respectively (Fig. 2B, C). The first cleavage occurs at 2.5 h PF. Due to transparency of the eggs, cell nuclei, cleavage spindles, and asters are clearly visible with DIC optics (Fig. 2B-D). Cleavage is spiral, equal, and holoblastic, as is typical of nemerteans. Spherical blastulae, rather than the "blastosquare" described for Micrura alaskensis (Maslakova, 2010a), form by 17 h PF (Fig. 2E). Embryos become ciliated by 24 h, at which point the blastulae begin to rotate within their chorions (Fig. 2F). Invagination of the archenteron is apparent by 46 h PF (Fig. 2G). Gastrulae hatch from the egg chorion by 46 h, and swim with a small apical tuft pointing forward, and a densely ciliated region, which represents the nascent ciliated band (arrowheads, Fig. 2G) encircling the vegetal (posterior) pole. At 51 h PF, mesenchymal cells can be seen inside the blastocoel, near the base of the archenteron (Fig. 2H). By 66 h, the young pilidia have a blind gut differentiated into a funnel-shaped esophagus and a round stomach, and stubby lateral lappets, as well as the anterior and posterior (with respect to the axis of the future juvenile) lobes (Fig. 21). The larval lobes and lappets are spanned by a primary ciliated band, which is used in larval feeding (von Dassow et al., 2013) and swimming.

The shape of Micrura wilsoni pilidia resembles that of other, so-called typical pilidia (e.g., unassigned Lineus and Micrura larvae, Lacalli, 2005; M. alaskensis, Maslakova, 2010a), and is not unlike a deer hunter's cap with the earflaps pulled down. Young pilidia begin to feed on Rhodomonas lens at 3 d after fertilization (Fig. 3A, B). They have a prominent apical organ, from which arises the apical tuft, marking the larval apical pole, which is also the larval anterior, based on the direction of swimming (Fig. 3A). The two lateral lappets, and the anterior and posterior larval lobes-so named for the future antero-posterior (AP) axis of the juvenile worm inside-are rounded. As in a typical pilidium, the primary larval ciliated band spans the lobes and lappets (Fig. 3C); and a stiff larval cirrus is present on the posterior lobe (not shown). Leading into the gut is a funnel-shaped, ciliated esophagus with two distinct, ciliated ridges along its posterior (again, with respect to the future juvenile AP axis) wall (arrowhead. Fig. 3A). Lateral lappets are relatively small (e.g., 225 [micro]m and 280 [micro]m at 24 d and 63 d, respectively) compared to the larval episphere (e.g., 320 [micro]m and 450 [micro]m at 24 d and 63 d, respectively) (Fig. 3D, G, and Table 1). After approximately 9 d, the first pair of juvenile rudiments begins to invaginate from the larval epidermis (Fig. 3C). These invaginations become the cephalic discs (Fig. 3C, D), which will give rise to the head of the juvenile worm. Two black pigment patches, or chromatophores (Cantell, 1969), one on either side of the anterior larval lobe, develop by 13 d (arrowhead, Fig. 3C). After 22 d, all three pairs of imaginal discs are present (Fig. 3D). In addition to the cephalic discs, the trunk discs, which give rise to the juvenile trunk, and the cerebral organ discs, which give rise to cerebral organs, can be seen surrounding the larval esophagus. The unpaired proboscis rudiment, described in an unidentified pilidium (Burger, 1894), in Cerebratulus marginalus (Schmidt, 1930, 1937), and in M. alaskensis (Maslakova, 2010a), is also present at this time (Fig. 3D, inset). By 40 d, all discs fuse around the larval esophagus (Fig. 3E). At this time, the unpaired dorsal disc is observed posterior to the larval gut. The juvenile is complete by 63 d after fertilization, possesses a caudal cirrus (Fig. 3G), and, surprisingly, two small patches of pigment, which we assume to be eyes (Fig. 3F, G, I), that are lacking in the adult.

The black pigment patches on the anterior lobe remain prominent until metamorphosis. These pigment patches appear to be groups of pigment-containing cells that extend into the episphere from the ciliated band, are irregular or stellate in shape (Fig. 3C, H), and are observed only on the anterior lobe. However, without additional investigation (e.g., transmission electron microscopy), we cannot be certain whether this pigment is cellular or extracellular. There was variation in the size of this patch among individuals; however, the location and presence were consistent within the entire cohort.

The larval musculature of Micrura wilsoni is similar to what has been described for pilidia of M. alaskensis (Maslakova, 2010a; von Dassow et al., 2013), with prominent radial muscles in the lobes and lappets, a thick muscle strand along the primary ciliated band, and muscle strands that cross the lappets at their base, allowing the pilidium to constrict the lappets (Fig. 4A). Another prominent muscular strand originates at the apical organ, and divides into two strands, each attached to one side of the larval esophagus (arrowheads, Fig. 4A). Nuclear (Hoechst) staining (Fig. 4B) reveals that cells are arranged most densely in the apical organ, the stomach, the esophageal ciliary ridges, the imaginal discs, and within the primary ciliated band near the pilidial axils, the recesses between the larval lobes and lappets, described by Bird et al. (2014) (asterisks, Fig. 4B).

Metamorphosis was observed between 60 and 70 d post-fertilization (63-d-old larva, Fig. 31). Juvenile Micrura wilsoni ruptured and emerged from the larval body caudal cirrus first. Individuals proceeded to back out of their larval body while at the same time ingesting it (Fig. 31). We observed metamorphosis several times when individuals were trapped between a microscope slide and a coverslip; juvenile worms were often found at the bottom of the culturing jar with evidence of pilidial body within their guts. Thus, we believe that ingestion of the larval body is the rule in this species, as in most pilidiophorans for which metamorphosis has been documented.

Lineus sp. "red" development

Dissected oocytes are approximately 90-100 [micro]m in diameter, relatively opaque (Fig. 5B), and are surrounded by a jelly layer (approximately 130 [micro]m in diameter), but lack a chorion (a conspicuous, extracellular envelope surrounding the cleavage stages, e.g., see Micrura wilsoni, this study). Sperm heads are elongated and slightly curved, exhibiting "modified" morphology (Strieker and Folsom, 1998); and are approximately 10 [micro]m long (Cluster 3 in fig. 11 of von Dohren et al., 2010) (Fig. 5A, B). At 10 [degrees]C, the development of Lineus sp. "red" proceeds in a manner similar to that described for Micrura alaskensis (Maslakova, 2010a) and Micrura wilsoni (Table 1). We observed development in three cohorts. The first and second polar body formation occurred at 1 and 2 h post-fertilization, respectively (Fig. 5C), and the first cleavage occurred at 3.5 h PF. After 24 h, ciliated blastulae had thickened vegetal plates (Fig. 5D), and were rotating slowly within their egg jelly. Swimming blastulae compressed along the animal-vegetal axis (resembling a bean in a sagittal section) were observed as early as 48 h PF (Fig. 5E). Young pilidia with an apical tuft, a gut differentiated into an esophagus and a round stomach, and rudimentary lateral lappets developed by 4 d PF (Fig. 5F).

Pilidia were first offered food, and observed to feed, after about one week in culture (Fig. 6A). The order of emergence of imaginal discs corresponded to that observed in other pilidia (cephalic discs first, trunk discs next, and cerebral organ discs last). As in Micrura wilsoni, the shape of Lineus sp. "red" larvae also resembles that of a typical pilidium. The first two pairs of juvenile rudiments, the cephalic discs and trunk discs, are present by 15 d PF (Fig. 6B). The third pair of discs (cerebral organ discs) were observed to fuse with the trunk discs by approximately 25-27 d (arrowhead, Fig. 6D). The proboscis rudiment also appears at this time (Fig. 6C), and the unpaired dorsal disc was observed at 36 d PF (Fig. 6D). Finally, all three pairs of discs fuse around the larval esophagus, forming a toroid of juvenile tissue (torus stage, Maslakova, 2010a, table 1) at 36-44 d. Larval lappets and the episphere are similar in size (e.g., approximately 160-190 [micro]m each, Fig. 6B, D) in most individuals and developmental stages (but see Fig. 6E), unlike the body proportions reported for other Lineus species (e.g., Lacalli, 2005), where the larval episphere is relatively small. The anterior lobes of Lineus sp. "red" larvae lack pigment spots, and the complete juvenile, with a pair of anterior pigment patches, which we assume are ocelli, was observed by 53 d PF (Fig. 6E). Metamorphosis occurred between 65 and 90 d PF in three cohorts under study. Metamorphosis was observed on several occasions, during which the juveniles were seen ingesting the larval body. Often, individuals were seen at the bottom of culture dishes with a partially ingested larval body hanging out of the mouth.

In addition to laboratory-reared larvae, we have collected three wild-caught larvae (plankton samples from January 2013), which were confirmed to belong to Lineus sp. "red" using DNA sequence data (larvae collected 11 January 2013; Fig. 6F). Often, we were able to raise wild-caught larvae, collected at early developmental stages, to metamorphosis, and their morphology was similar to what was seen in laboratory-reared specimens. Lineus sp. "red" juveniles are short and stout, and lack a caudal cirrus; they crawl about the bottom of culture dishes surrounded by thin mucus. Interestingly, a row of two to three presumed ocelli is visible on the left and right sides, along the anterior margin of the juvenile head (Fig. 6G). This is noteworthy, because, as is the case in Micrura wilsoni, ocelli appear to be lacking in adults.

Discussion

We describe for the first time the development of Micrura wilsoni and Lineus sp. "red," two intertidal nemertean species commonly found in southern Oregon. Both species possess typical, hat-shaped planktotrophic pilidia, and can be reared in the laboratory from fertilized, dissected gametes to metamorphosis on a diet of cryptophyte algae (Rhodomonas lens), as previously described for Micrura alaskensis (Maslakova, 2010a). The general sequence of developmental events (e.g., the order of appearance of various juvenile rudiments) corresponds to that described for M. alaskensis (Table 1) and Cerebratulus marginatus (Schmidt, 1930). However, the time to metamorphosis for both M. wilsoni and Lineus sp. "red" was nearly double that reported for M. alaskensis (Table 1). Developmental rate can vary widely, even within a single larval cohort, likely depending on individual feeding rate and possibly other factors. Thus, the most developmentally advanced larvae often metamorphose and settle to the bottom of culture jars before this stage is observed in the majority of individuals.

The larvae of Lineus sp. "red" are characterized by a juvenile that possesses presumed ocelli and lacks a caudal cirrus, and a larval episphere that is not dramatically larger or smaller than the lappets throughout development. Similar larvae have been observed by Muller (1847), Dawydoff (1940), Thorson (1946), Chemyshev (2001), and Lacalli (2005), but the species-level identities of these larvae are unknown. The larvae of Lineus sp. "red" exhibit a similar morphology (e.g., presence of juvenile (assumed) ocelli, episphere height smaller than or equal to lappets) to larvae collected by us from plankton in southern Oregon and identified by "DNA barcoding," including those of Lineus flavescens (Maslakova, 2010b; fig. 1A) and several other closely related species for which we have yet to find the adults (T. C. Hiebert and Maslakova, unpubl. data). These species form a monophyletic clade with each other, as well as with Lineus torquatus and Cerebratulus montgomeryi on molecular phylogenies (T. C. Hiebert and S. A. Maslakova, unpubl. data). Thus, it appears that this particular larval form, in which developing juveniles possess two presumed eyes and the larval episphere and lappets are of similar size throughout development, characterizes a clade of closely related species. At present, the larvae of these species cannot be differentiated using morphology alone.

Conversely, for Micrura wilsoni, we reveal a previously unknown, species-specific larval form characterized by the presence of two black pigment spots on the pilidial anterior lobe. To our knowledge, this particular larval morphotype has not been observed in any other nemertean species thus far (Fig. 7). Although the larval body is shaped like a typical (hat-like) pilidium, the larvae of M. wilsoni have additional characteristics that combine to make a unique morphotype. First, the juvenile nemertean has what are assumed to be ocelli, and a caudal cirrus. The presence of juvenile eyes and/or a caudal cirrus are not uncommon in other pilidium larvae (e.g., Cerebratulus californiensis (Fig. 8A, B); L. flavescens (Fig. 8C); Lineus sp. "red," this study), but the larval body in M. wilsoni also has pigment spots. Epidermal pigment spots, or chromatophores, occur in other pilidia, but they are usually more circular in shape and found on both larval lobes and lappets (Dawydoff, 1940; Cantell, 1969; Lacalli, 2005; Schwartz, 2009; T. C. Hiebert and S. A. Maslakova, unpubl. data). Typically, there are also more than two spots. In fact, Cantell (1969) found that the number of chromatophores increased with age in Tenuilineus albocinctus, Lineus bilineatus, and Micrura purpurea. In Micrura wilsoni, the size of the pigment spots increased over developmental time (compare Fig. 3C to Fig. 3H) and varied among individuals, but the number of spots did not increase with age; that is, there were always two spots, one on either side of the anterior lobe. The only other locally collected pilidium with pigment spots is the larva of Cerebratulus californiensis (Maslakova and Hiebert, 2014; T. C. Hiebert and S. A. Maslakova, unpubl. data; Fig. 8A, B), but the pigment spots in larvae of this species are present on both the lobes and lappets (Fig. 8A, B). These larvae are also characterized by a large, pyramidal episphere and larval lobes that are often scalloped into two to three sub-lobes (Fig. 8A, B), characters not observed in M. wilsoni larvae. Thus, the combination of juvenile eyes, caudal cirrus, and the two pigment spots on the anterior lobe appears to be unique to M. wilsoni. However, the shape of M. wilsoni larvae is not necessarily unique, and the larval dimensions and body proportions most closely resemble other described larvae in this genus. Larvae of the genus Cerebratulus tend to have relatively large epispheres. Conversely, Lineus larvae often have larger lappets and relatively small epispheres (e.g., Cantell, 1969; Lacalli, 2005; Hiebert and Maslakova, 2014). The larval body of Micrura species falls somewhere between these two genera. However, concrete comparison of larval dimensions among pilidiophorans is currently lacking, and the significance of assigning larval features to these non-monophyletic taxa based on body size and proportion alone requires further investigation.

Finally, it is noteworthy that we observed what we believe to be juvenile ocelli in both Micrura wilsoni and Lineus sp. "red," which lack eyes as adults. This is important, because it means that the presence of juvenile eyes in a wild-caught pilidium larva does not necessarily indicate that the species possesses eyes as an adult.

Acknowledgments

We acknowledge support from the faculty and staff of the Oregon Institute of Marine Biology. We thank Brittney Dlouhy-Massengale and Summer 2013 COSEE Prime intern Leeah Whittier for occasionally caring for Micrura wilsoni culture in June 2013. This work was partially supported by NSF grants OCE-1030453 to Craig Young and SAM, and IOS-1120537 to SAM.

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TERRA C. H1EBERT (*) AND SVETLANA A. MASLAKOVA

Oregon Institute of Marine Biology, University of Oregon, 63466 Boat Basin Road, Charleston, Oregon 97420

Received 14 May 2015; accepted 21 October 2015.

(*) To whom correspondence should he addressed. E-mail: terrah@uoregon.edu

Table 1
Reproductive timelines for the Pilidiophoran species Micrura alaskensis
from Stricker (1987) and Maslakova (2010a), and M. wilsoni and Lineus
sp. "red" (this study)
                        Micrura alaskensis (1)  Micrura alaskensis (2)

Reproductive season             May-July            July-August
Temperature               10-12 [degrees]C        11 [degrees]C
Oocyte diameter              75 [micro]m           75 [micro]m
First cleavage                    4h                     2h, 15 m
Blastula                         17 h                   16 h
Gastrula                         26 h                   24 h
Young pilidium                   40 h                   40 h
Feeding pilidium                 62 h                   66 h
Cephalic discs                     -                     7 d
Trunk discs                        -                     9 d
Cerebral organ discs               -                    14 d
Head and trunk
stage (2)                          -                    24 d
Torus stage (2)                    -                    28 d
Metamorphosis                      -                    35 d
Larval height at
3-disc stage                       -                     -
Larval height prior to
metamorphosis                      -                     -

                           Micrura wilsoni (3)

Reproductive season                June
Temperature                     12[degrees]C
Oocyte diameter                100-110 [micro]m
First cleavage                   2 h, 30 m
Blastula                        17 h
Gastrula                        46 h
Young pilidium                  66 h
Feeding pilidium                72 h
Cephalic discs                   9 d
Trunk discs                     17 d
Cerebral organ discs            22 d
Head and trunk
stage (2)                        -
Torus stage (2)                 40 d
Metamorphosis                   60 d
Larval height at
3-disc stage                   540-545 [micro]m (at 24 d)
Larval height prior to
metamorphosis                  730-735 [micro]m (at 63 d)

                             Lineus sp. "red" (4)

Reproductive season               Jan-March
Temperature                          10 [degrees]C
Oocyte diameter                      90-100 [micro]m
First cleavage                        3 h, 30 m
Blastula                             24 h
Gastrula                             48 h
Young pilidium                       96 h
Feeding pilidium                    120 h
Cephalic discs                       10 d
Trunk discs                          15 d
Cerebral organ discs                 25 d
Head and trunk
stage (2)                            27 d
Torus stage (2)                      36 d
Metamorphosis                        65 d
Larval height at
3-disc stage                        350-380 [micro]m (at 25-27 d)
Larval height prior to
metamorphosis                       415-175 [micro]m (at 65 d)

(1) Strieker, 1987.
(2) Maslakova, 2010a.
(3) See Figures 2 and 3 of this study.
(4) See Figures 5 and 6 of this study.
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Author:Hiebert, Terra C.; Maslakova, Svetlana A.
Publication:The Biological Bulletin
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Date:Dec 1, 2015
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