Development and larval feeding in the capitellid annelid Notomastus cf. tenuis.

Reference: Biol. Bull. 228: 25–38. (February 2015) © 2015 Marine Biological Laboratory

Development and Larval Feeding in the Capitellid Annelid Notomastus cf. tenuis BRUNO PERNET1*, LESLIE H. HARRIS2, AND PAUL SCHROEDER3 1

Department of Biological Sciences, California State University Long Beach, Long Beach, California 90840; 2Natural History Museum of Los Angeles County, Los Angeles, California 90007; and 3School of Biological Sciences, Washington State University, Pullman, Washington 99164

Abstract. Making inferences about the evolution of larval nutritional mode and feeding mechanisms in annelids requires data on the form and function of the larvae, but such data are lacking for many taxa. Though some capitellid annelids are known or suspected to have planktotrophic larvae, these larvae have not previously been described in sufficient detail to understand how they feed. Here we describe embryos and larvae of the capitellid Notomastus cf. tenuis from San Juan Island, Washington State. Fertilized oocytes average about 58 ␮m in equivalent spherical diameter. Early embryos undergo spiral cleavage and develop into larvae that feed for about 5 weeks before metamorphosis. Larvae of N. cf. tenuis capture food particles between prototrochal and metatrochal ciliary bands and transport them to the mouth in an intermediate food groove; this arrangement is typical of “opposed band” larval feeding systems. Surprisingly, however, larvae of N. cf. tenuis appeared to have only simple cilia in the prototrochal ciliary band; among planktotrophic larvae of annelids, simple cilia in the prototroch were previously known only from members of Oweniidae. The anteriormost tier of prototrochal cilia in N. cf. tenuis appears to be non-motile; its role in swimming or particle capture is unclear. Like some planktotrophic larvae in the closely related Echiuridae and Opheliidae, larvae of N. cf. tenuis can capture relatively large particles (up to at least 45 ␮m in diameter), suggesting that they may use an alternative particle capture mechanism in addition to opposed bands of cilia.

Introduction The planktonic larval stages of annelids are extremely diverse in form and functional biology, especially with respect to larval feeding (Pernet et al., 2002; Rouse, 2006). Some annelid larvae cannot feed while in the plankton; others can feed but do not require food to develop through metamorphosis; and still others are obligate feeders, requiring food to develop through metamorphosis (Pernet and McArthur, 2006; Rouse, 2006). Those annelid larvae that do feed in the plankton use one or several of a diverse set of feeding mechanisms, including capturing particles from suspension between opposed bands of cilia (Strathmann et al., 1972; Riisgård et al., 2000; Pernet and Strathmann, 2011), ramming into particles one by one while swimming (Phillips and Pernet, 1996), or trapping particles in various kinds of external mucus strands or filters (Akesson, 1961; Pernet, 2004). This within-phylum diversity in feeding mechanisms is unusual; in most other phyla of marine invertebrates that include species with ciliated feeding larvae, the feeding larvae all share one primary mechanism for particle capture (R. Strathmann, 1987). This suggests that there may be interesting differences between annelids and other phyla in the dynamics of evolutionary change in larval nutritional modes and larval feeding mechanisms (Strathmann, 1978, 1993). Understanding these evolutionary changes requires, at a minimum, both well-supported phylogenetic hypotheses for the clade and detailed descriptions of larval form and function to interpret in the context of those phylogenetic hypotheses. Most recent analyses of annelid phylogeny differ in their conclusions in important respects (e.g., Rouse, 2000; Rousset et al., 2007; Zrzavy et al., 2009; Struck et al., 2011; Weigert et al., 2014), but this problem will presumably eventually yield to the application of new genetic data

Received 29 August 2014; accepted 2 November 2014. * To whom correspondence should be addressed. E-mail: bruno.pernet@ csulb.edu 25

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Dasybranchus caducus Mediomastus fragilis (as Heteromastus filiformis) Notomastus latericeus Notomastus cf. tenuis

Capitella teleta (⫽C. sp I of Grassle & Grassle, 1976) Capitella spp.


All known larvae of members of the genus Capitella are “barrel-shaped” (Eckelbarger and Grassle, 1987); in contrast, all known larvae of members of the other genera of capitellids are shaped more like the “classical trochophore” described by Rouse (2006).

Lecithotrophy Blake et al., 2009 Planktotrophy? (but larval feeding Grassle & Grassle, 1976; Eckelbarger & has not been observed) Grassle, 1987 260 ⫻ 180 Maternal tube Lecithotrophy Blake et al., 2009; Meyer et al., 2010 At least 12 additional species, mostly undescribed, with egg sizes ranging from 75 to ⬎300 ␮m, and substantial variation in larval size. See Grassle & Grassle, 1976; Eckelbarger & Grassle, 1987; Mendez et al., 2000; and Blake et al., 2009. 90–95 Jelly mass Lecithotrophy Bookhout, 1957 110 Jelly mass Planktotrophy Rasmussen, 1956; 1973; Hansen, 1993 150 Unknown Lecithotrophy Wilson, 1933 58 Unknown Planktotrophy This study 312 50 Capitella capitata Capitella jonesi (⫽C. sp. III of Grassle & Grassle, 1976)

Maternal tube Maternal tube

Larval nutrition Species

Site of embryonic development Egg size (␮m) (diameter unless otherwise noted) Egg size and developmental modes of members of Capitellidae

or analytical techniques. Surprisingly, however, there are also relatively few primary data on the functional biology of larvae of many annelid taxa, including some taxa that are well known or ecologically important as adults (e.g, glycerids, nephtyids, and phyllodocids; Rouse, 2006). One of these taxa is Capitellidae. Adult capitellids are common in marine sediments, where they feed on deposited organic material (Fauchald and Jumars, 1979). A subset of capitellids, members of the genus Capitella, are best known as “pollution indicators” (e.g., Grassle and Grassle, 1974; Sanders et al., 1980; Tsutsumi and Kikuchi, 1984). One of these species, C. teleta, is a relatively new model system in the evolution of development and has been used to explore questions about topics such as spiralian cell lineages, germ cell specification, and the development of segments (e.g., Seaver and Kaneshige, 2006; Meyer et al., 2010; Giani et al., 2011); its genome has also been sequenced and studied (Simakov et al., 2012). Larvae of this species develop as lecithotrophs in the maternal tube and are competent to metamorphose within hours after release from it (Seaver et al., 2005). Sparse data are available on a few other species in the genus Capitella (e.g., Grassle and Grassle, 1976; Eckelbarger and Grassle, 1987; Mendez et al., 2000), and on single species in each of three other genera of capitellids as well—Dasybranchus, Mediomastus, and Notomastus (Wilson, 1933; Rasmussen, 1956; Bookhout, 1957) (Table 1). The genera Capitella, Mediomastus, and Notomastus are thought or known to include species with planktotrophic larvae (Rasmussen, 1956; Ecklebarger and Grassle, 1976; Hansen, 1993; Schroeder, 1998; Pernet and Schroeder, 1999), but no detailed descriptions of the form or feeding biology of the planktotrophic larvae of any capitellid are available (see Rouse, 2006). Such information is necessary for an eventual understanding of how larval nutritional mode and feeding mechanisms have evolved in annelids. Here we describe development and larval feeding in Notomastus cf. tenuis, adults of which are abundant in intertidal sediments at some sites in the state of Washington. In a meeting abstract, Schroeder (1998) reported that members of this or a very similar species collected in the southern part of Puget Sound, Washington, produced small eggs that developed into planktotrophic larvae. Pernet and Schroeder (1999), again in a meeting abstract, reported that larvae of Notomastus sp. (spawned from adults collected from San Juan Island, WA) captured particles using opposed bands of cilia. Because both reports are meeting abstracts, however, the data are not available for review by other workers. We present new information on development, larval form, and larval feeding in members of N. cf. tenuis. This represents the first detailed description of the planktotrophic larvae of a capitellid annelid. These data will be useful in exploring the evolution of opposed bands of cilia in annelids in general, and more specifically, within the

Reference

B. PERNET ET AL.

Table 1

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LARVAE OF NOTOMASTUS CF. TENUIS

capitellids. Further, members of this species of Notomastus provide an interesting system in which to compare development with the relatively well-known C. teleta, to test hypotheses about differences between planktotrophic and lecithotrophic development. Materials and Methods Collection and identification Adults were collected, using a shovel, from intertidal mud in North Bay, San Juan Island, Washington, near the seaward end of Argyle Creek. This is the same population from which members of “Notomastus sp.” were collected by Pernet and Schroeder (1999). Worms were identified as Notomastus cf. tenuis using the redescription by GarciaGarza et al. (2012) and by comparison to verified examples of N. tenuis and other Notomastus species in the collections of the Natural History Museum of Los Angeles County (N. tenuis specimens listed in Garcia-Garza et al., 2012). One characteristic of N. tenuis is a conspicuous enlargement of the body in the first abdominal chaetigers. The San Juan Island specimens lack this enlargement but match N. tenuis in all other characters. As the enlargement may be an artifact of preservation, we use the name “cf. tenuis” to reflect our uncertainty about the identification. Ethanolfixed and formalin-fixed voucher specimens were deposited at the Natural History Museum of Los Angeles County (lots LACM-AHF Poly 6533 and LACM-AHF Poly 6534, respectively). Collections were made in 6 years from 1998 to 2014 (Table 1). Adults were maintained in the laboratory for up to 2 weeks in glass bowls submerged in flowing seawater tanks at about 11 °C, near ambient seawater temperature. For comparison with N. cf. tenuis, we also reared larvae of the serpulid annelid Serpula columbiana Johnson, 1901. Adults of S. columbiana growing on small stones were collected from Argyle Creek, a few hundred meters from the collection site for N. cf. tenuis, and maintained in flowing seawater tanks until spawning. Spawning, fertilization, and larval culture Gametes of N. cf. tenuis were obtained by puncturing adults with forceps, allowing gametes to spill out of the coelomic cavity. Segments appear to be well-isolated from each other by septa, so each segment had to be punctured individually. Oocytes liberated from the coelom were pipetted into clean filtered seawater (FSW; mesh size ⬃5 ␮m). Collected oocytes were passed through Nitex mesh (mesh size 200 ␮m) to remove body tissue and other large debris, then collected on 50-␮m-mesh Nitex to remove smaller contaminating particles such as coelomocytes; collected oocytes were then resuspended in clean FSW. Cleaned oocytes were set aside for 10 –30 min at 11 °C while sperm were obtained. Sperm were released in morulae

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of hundreds of sperm cells, with their flagellar tails aligned. Sperm morulae from one or several segments of a male were incubated in a small volume of FSW for about 15 min at room temperature to stimulate the activation of individual sperm. This sperm suspension was then used to fertilize cleaned oocytes. Fertilized oocytes were resuspended in clean FSW 15–30 min after the addition of sperm. One day after fertilization, swimming embryos were decanted into 1-liter beakers. These were partially immersed in a flow-through sea table to keep temperatures at about 11–13 °C. Cultures were stirred continuously using a swinging paddle system like that described by M. Strathmann (1987). Larvae were first fed 3 days after fertilization, with a mixture of cells of Isochrysis galbana, Nanochloropsis sp., and Rhodomonas sp.; additional food was added every 2– 4 days. Food concentrations were not measured. Cultures were cleaned by reverse filtration through appropriatelysized Nitex mesh filters approximately once a week. Metamorphic competence was assessed by exposure of larvae to sediment collected from the field site, sieved through a 315-␮m mesh, frozen at ⫺20 °C for several days, then defrosted. Gametes of S. columbiana were obtained and fertilized as described in M. Strathmann (1987; in that book, the species is referred to as S. vermicularis), and larvae were reared as described above for N. cf. tenuis. Microscopy Routine observations of gametes, embryos, and larvae were made using a Nikon 80i compound microscope with differential interference contrast optics, and photographed using a Nikon Coolpix 4500 digital camera mounted on the microscope. All stages were mounted under coverslips supported at their corners with modeling clay; before mounting, larvae were relaxed by incubation in a 1:1 solution of seawater/0.37 mol l–1 MgCl2. Measurements were made using a calibrated ocular micrometer. The external morphology of larval stages was examined at higher magnification by scanning electron microscopy (SEM). To prepare larvae for SEM, they were first relaxed by incubation in a 1:1 solution of seawater/0.37 mol l–1 MgCl2 for about 15 min, and then fixed in 2% OsO4 (in 1.25% NaHCO3, ph 7.2) at 4 °C for about 2 h. Fixed larvae were rinsed three times in deionized water, then dehydrated in an ascending concentration series of ethanol solutions (30%, 50%, 70%, 85%, 95%, 100%, 100%). They were critical-point dried using ethanol as a transitional fluid, mounted on stubs with adhesive carbon tabs, and sputtercoated with gold-palladium before being examined with a JEOL JCM-5000 Neoscope or a JEOL JSM-35 scanning electron microscope. The beat patterns of cilia and movements of captured particles were examined by high-speed video microscopy,


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B. PERNET ET AL. Table 2

Months and years in which adults of Notomastus cf. tenuis were sampled from North Bay, San Juan Island, Washington

suggesting that there is among-year variation in the timing of the onset of the reproductive period. Gametes

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 1998 1999 2001 2004 2011 2014

— —

—

—

**

** **

**

**

**

—

** **

Dashes (—) indicate months in which sampling occurred but no gametebearing adults were found, and asterisks (**) indicate months in which sampling occurred and gamete-bearing adults were present.

using the methods of Pernet (2003). Briefly, video sequences of larvae restrained under a coverslip supported by modeling clay were recorded using a Motionscope 1000-S high-speed camera (Redlake Imaging, Inc.) mounted on a compound microscope with DIC optics. In some cases larvae were relaxed in a 1:1 solution of seawater/0.37 mol l–1 MgCl2 to prevent muscle contraction during observations, but in others unrelaxed larvae were observed to rule out the possibility that MgCl2 affected ciliary structure (i.e., simple vs. compound) or movements. Images were collected at 250 or 500 frames per second. Video sequences replayed at 30 frames per second were routed from the camera through an analog-digital converter to a Macbook Pro computer and saved as iMovie files (Apple Computer, Inc.). The range of particle sizes that could be captured and ingested was examined by offering larvae of various ages spherical particles of a range of diameters. In some experiments, we offered larvae polystyrene divinylbenzene spheres ranging in diameter from 5 to 45 ␮m (one size at a time); in others, we used Sephadex particles ranging in size from 10 to 130 ␮m (in a suspension consisting of a mixture of particle sizes across that range). Particle concentrations were unmeasured. Larvae were incubated in particle suspensions in 20-ml glass scintillation vials, with periodic gentle inversion of the vials, for periods of 10 –20 min. After incubation, larvae were killed with dilute formalin, and the sizes of ingested beads were determined. Results Reproductive periodicity Mature, gamete-bearing adults of Notomastus cf. tenuis were typically present in the intertidal zone of North Bay, San Juan Island, during the months of June, July, and August (Table 2). In 1999, the year in which the most frequent sampling was done, mature adults first appeared in the population in mid-June and disappeared from the population in midAugust. In 2004, mature adults were observed on 10 May,

No natural spawnings of N. cf. tenuis were observed, so descriptions are based on gametes dissected from the coelomic cavity. Sperm from the coelomic cavities of males were invariably found in morulae of hundreds of spermatids bound together at the head end, with their flagella aligned in parallel (Fig. 1A). After a few minutes of incubation in seawater, these morulae began to break down, releasing active single sperm cells. Individual sperm cells had roughly spherical heads about 2 ␮m in diameter and were similar in shape to sperm of annelids in which fertilization is external (e.g., Franzen, 1956; Rouse and Jamieson, 1987). Unfertilized primary oocytes were oblate spheroids about 76 ␮m in major diameter (n ⫽ 10 oocytes measured from each of two females; means for oocytes from each female were 75.7 ⫾ 2.2 [SD] ␮m and 76.3 ⫾ 1.8 ␮m). Oocytes placed on a glass slide always settled with their minor diameter perpendicular to the slide, so the minor diameter was difficult to measure. Oocytes were surrounded by a distinct egg envelope that was tightly appressed to the cell membrane; observations of oocytes in a suspension of sumi ink particles revealed that there was also a thin (⬃3–5 ␮m thick) jelly coat outside of this egg envelope. The cytoplasm was pale straw brown in color in transmitted light, and each unfertilized oocyte had a large, well-defined nucleus. Unfertilized oocytes incubated in seawater were observed periodically for up to 2 h; these did not undergo any obvious changes in form (e.g., rounding up or germinal vesicle breakdown) during this time. Embryogenesis A schedule of developmental events at 10 –13 °C is provided in Table 3. Within 30 min of fertilization, many oocytes had undergone two obvious morphological changes— germinal vesicle breakdown and a change in overall shape (Fig. 1B). Though maturing oocytes were still oblate spheroids, they became more spherical in shape. The grand mean of the major diameter of fertilized oocytes of eight females was 65.5 ⫾ 1.8 ␮m (n ⫽ 10 oocytes measured per female). Embryos rolled onto their sides had minor diameters of about 45 ␮m (n ⫽ 2). Thus, we estimate the volume of an average fertilized oocyte of N. cf. tenuis to be roughly 101,087 ␮m3; the diameter of a sphere with this volume would be about 57.8 ␮m. The first polar body was produced about 30 min after fertilization (Fig. 1C). Polar bodies were produced at one end of the minor axis of the maturing oocyte and thus were not easily visible unless oocytes were rolled into a lateral orientation. Embryonic cleavage was typically spiral. The first two cleavages were meridional and equal (Fig. 1D,



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Figure 1. Gametes and embryos of Notomastus cf. tenuis. (A) Sperm morula. (B) Fertilized oocyte. (C) Zygote with first polar body. (D) Two-cell stage. (E) Four-cell stage (animal pole view). (F) Eight-cell stage (animal pole view). (G) Eight-cell stage, in side view; animal pole is up. (H) Blastula, in side view. (I) Gastrula, in side view. Scalebar shown in (A) applies to all panels. Ar, archenteron; bl, blastocoel; pb, polar body.

E). Third cleavage was equatorial, dexiotropic, and also roughly equal (Fig. 1F, G). Ciliated, swimming blastulae with a small blastocoel were formed about 16 h after ferTable 3

tilization (Fig. 1H); blastulae had sparse equatorial cilia. The vegetal-most cells of blastulae were slightly darker in color than the rest of the embryo and formed a thick plate prior to gastrulation. Gastrulation, which appeared to be by invagination, commenced about 24 h after fertilization (Fig. 1I).

Approximate developmental schedule for Notomastus cf. tenuis at 10 –13 °C

Larval development Event Fertilization First polar body First cleavage Second cleavage Third cleavage Ciliogenesis Swimming blastulae Gastrulation Episphere eyespots Feeding Telotroch First accessory eyespot Chaetae Metamorphosis

Time 0 30 min 2 h 15 min 3h 3 h 45 min 15 h 16 h ⬃24 h ⬃42–48 h ⬃70–75 h ⬃16 d ⬃24–30 d ⬃24–30 d ⬃35 d

After the onset of feeding at 70 –75 h, development rate varies substantially among cultures; times recorded are for rapidly developing cultures.

By 3 days after fertilization, larvae had developed a nearly complete set of external ciliary structures: an apical tuft composed of a group of long cilia surrounded by shorter cilia, three equatorial ciliary bands (a preoral prototroch, circumoral food groove, and postoral metatroch), and a ventral neurotroch (Fig. 2A, B). In all observations of living and fixed larvae, the prototroch and metatroch appeared to consist of simple, not compound, cilia. The prototroch was broad and included five distinct tiers of cilia. There was no dorsal gap in the prototroch, but there was a dorsal gap in the metatroch, as well as a mid-ventral gap in the metatroch (for the neurotroch). At this stage the digestive system was also complete, consisting of a wide mouth leading into a spacious oral cavity, stomach, intestine, and anus (illustrated in a 12-day old larva in Fig. 2C, D). Larvae began to


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Figure 2. Early larval stages of Notomastus cf. tenuis. (A) Scanning electron micrograph of a 72-h-old larva, in side view. (B) Scanning electron micrograph of a 72-h-old larva, in posterior view. The mouth (mo) is obscured by a cluster of captured particles. (C) Light micrograph of a 12-d-old larva, in side view. (D) Light micrograph of a 12-d-old larva, in side view. At, apical tuft; ey, eye; int, intestine; mo, mouth; mt, metatroch; pi0, episphere pigment band; pi1 and 2, first and second hyposphere pigment bands; pt, prototroch; st, stomach.

capture and ingest particles about 70 –75 h post-fertilization at 10 –13 °C. By one week after fertilization, some larval epidermal cells contained a pale green pigment. At this age, these pigment cells were mostly concentrated into three equatorial bands: one just anterior to the prototroch, one just posterior to the metatroch, and one at the posterior end of the larva (Fig. 2C). Growth involved the addition of tissue to the posterior end, as well as the addition of new structures. Larval length, diameter at the level of the prototroch, and length of protrochal cilia are plotted in Figure 3. Larval length increased from about 140 ␮m in 1-week-old larvae to about 300 ␮m in 5– 6-week-old larvae. Diameter at the level of the prototroch was relatively constant, ranging from about 140 to 160 ␮m. The longest prototrochal cilia were about 40 ␮m long; prototrochal cilia did not increase in length over the course of development.

Figure 3. Body length, diameter at the level of the prototroch, and length of prototrochal cilia of larvae of Notomastus cf. tenuis from 1– 6 weeks of age. Each point represents the mean of measurements of five larvae from 1 to 3 families of larvae.

At about 16 d post-fertilization, a new circumferential ciliary band, the telotroch, appeared just anterior to the anus (Fig. 4A, B). The telotroch was incomplete ventrally where the neurotroch passed through it. As development continued, additional bands of green pigment cells were added to the epidermis posterior to the prototroch (Fig. 4C–E). Staging of these larvae could be conveniently carried out by counting these post-prototrochal pigment bands. For staging purposes, we refer to the pigment band immediately anterior to the prototroch as pi0, and the pigment band immediately posterior to the metatroch as pi1; subsequent pigment bands are assigned numbers that increase with increasing distance posteriorly. The maximum number of post-prototrochal pigment bands ever seen in cultures was eight, in larvae that were 4 –5 weeks old (Fig. 4D, E). By the time a total of 4 or 5 post-prototrochal pigment bands was present (⬃3– 4 wk), pairs of red accessory eyespots began to appear laterally. The first pair of accessory eyespots always appeared just behind pi1 (Fig. 4C). The second pair typically appeared between pi3 and pi4, with additional pairs appearing gradually between all posterior pigment bands except for the last pair (Fig. 4D). In a few individuals, accessory eyes also appeared between pi2 and pi3, but this was uncommon. The greatest number of pairs of accessory eyes observed was six, in larvae that were 4 –5 weeks old (Fig. 4E). Three-week-old larvae were observed to be strongly photopositive, but the ontogeny of phototaxis was not observed over the full course of larval development, so we are not sure if its onset was associated with the development of accessory eyes. When larvae had six pigment bands posterior to the prototroch, chaetae appeared in three segments: between pi2 and pi3, pi3 and pi4, and pi4 and pi5. The first three chaetigers each contained a pair of capillary chaetae on each side, one in a dorsal and one in a ventral follicle (presumably notopodial and neuropodial). As development



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Figure 4. Late larval stages of Notomastus cf. tenuis. (A) Light micrograph of a 23-day-old larva, in side view. (B) Scanning electron micrograph of a 28-day-old larva, in side view. Most external larval ciliary structures are clearly visible; note in particular the prototroch and metatroch. This larva was derived from a slow-growing culture, and is unusually small for its age. (C) Light micrograph of a 28-day-old larva, in side view. Hyposphere pigment bands are labeled pi1–pi7. The first pair of accessory eyes, between pi1 and pi2, is indicated by a white arrow. (D) Light micrograph of a 32-day-old larva, in side view. This larva has eight hyposphere pigment bands. Four pairs of accessory eyes are present, betweein pi1 and pi2, pi3 and pi4, pi4 and pi5, and pi5 and pi6. (E) Light micrograph of another 32-day-old larva, in side view. This larva has five pairs of accessory eyes (the same four pairs as in the larva in (D), plus a pair between pi6 and pi7). At, apical tuft; fg, food groove; mo, mouth; mt, metatroch; ne, neurotroch; pi1–pi8, hyposphere pigment bands; pt, prototroch; st, stomach; te, telotroch.

continued, additional chaetigers containing 1 or 2 hooded hooks on each side were added posteriorly. The greatest number of chaetigers observed was six, in larvae that were 5– 6 weeks old. Larvae were metamorphically competent at the five- or six-chaetiger stages. Metamorphosis Spontaneous metamorphosis of larvae in culture jars was never observed. However, five- or six-chaetiger larvae held in seawater with small amounts of sediment from the adult habitat typically metamorphosed within a few hours.

Metamorphosed juveniles attached to sediment or to the container wall by a thin, sticky mucus tube. Juveniles removed from tubes 1–2 days after metamorphosis were about 350 ␮m in length, with six chaetigers (the anterior three bearing capillary chaetae and the posterior three hooded hooks: Fig. 5A). These early juveniles had lost all larval ciliary bands, as well as the apical tuft. The mouth was present only as a small mid-ventral hole just posterior to the former site of the prototroch in juveniles 2 days after metamorphosis; these juveniles never contained sediment in their guts. In addition, 2-day-old juveniles each bore a pair of laterodorsal eyes on the posterior edge of the prostomium


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Figure 6. Frames from high-speed video of larvae of (A) Notomastus cf. tenuis and (B) Serpula columbiana. In both cases, anterior is up. Arrows indicate the anteriormost tier of prototrochal cilia, which appear to be non-motile. These frames were exported from Supplemental Videos S2 and S3 (http://www.biolbull.org/content/supplemental).

sediment particles. By 2 weeks after metamorphosis, juveniles had added a seventh chaetiger posteriorly, containing hooded hooks, and had reached about 500 –590 ␮m in length. Juvenile development and growth were not followed beyond this point. Larval feeding

Figure 5. Juveniles of of Notomastus cf. tenuis. (A) Lateral (top) and ventral (bottom) views of juveniles 2 days after metamorphosis. Anterior is to the right. Numbers indicate chaetigers. The first three chaetigers contain capillary chaetae; posterior chaetigers contain hooded hooks. The inset shows a higher magnification view of the nuchal organ (inset scale 2.5 ␮m). (B) Lateral (top) and ventral (bottom) views of juveniles 7 days after metamorphosis. Anterior is to the right. Chaetigers are similar to those in (A). The inset shows a higher magnification view of the nuchal organ (inset scale 2.5 ␮m). Nu, nuchal organ; mo, mouth; pr, prototroch.

and a pair of lateral nuchal organs on the posterior edge of the prostomium. Each nuchal organ consisted of a pit from which numerous cilia emerged (Fig. 5A, inset). Two-dayold juveniles also retained the accessory eyes present in the larval stage. Seven days after metamorphosis, juveniles still had six chaetigers and had not increased in length substantially. The most obvious developmental change in these specimens was the appearance of the juvenile mouth, a ventral, laterally oriented slit between the prostomium and the peristomium. At this stage, some juveniles had small particles in their guts, suggesting that they had made the transition from larval feeding on suspended particles to juvenile feeding on

Video recordings at 250 and 500 frames per second showed that the active strokes of motile prototrochal cilia were from anterior to posterior, and that the active strokes of metatrochal cilia were from posterior to anterior (Video Supplement 1; http://www.biolbull.org/content/supplemental). The anteriormost tier of prototrochal cilia consisted of cilia that were considerably shorter (⬃20 ␮m) than those of the four more posterior tiers (⬃40 ␮m; Fig. 3), and apparently non-motile; in all observations of unrelaxed and relaxed larvae, these cilia were held straight and oriented at an angle of about 40° above the equatorial plane (Fig. 6A; Video S2). The remaining prototrochal cilia passed through this tier of non-motile cilia on both active and recovery strokes. The length and orientation of the non-motile cilia were such that they encompassed the entire recovery stroke height of the posterior tiers of motile prototrochal cilia. The anteriormost of the three tiers of prototrochal cilia of larvae of Serpula columbiana also consisted of short, apparently non-motile cilia, oriented about 40° above the equatorial plane (Fig. 6B; Video S3). In larvae of N. cf. tenuis, the active strokes of the food groove cilia (located between the prototroch and metatroch) were from dorsal to ventral, toward the mouth; these movements were inferred from the movements of particles overlying them (e.g., Fig. 7). When larvae of N. cf. tenuis were immobilized by gentle pressure under a coverslip and viewed in a suspension of potential food particles (e.g., cells of Rhodomonas sp. or



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Figure 7. Series of frames from a high-speed (125 frames per second) video sequence showing capture and transport of a 4.5 ␮m diameter bead by a 20-day-old larva of Notomastus cf. tenuis. The bead was captured in the left dorsal quadrant of the opposed bands, moved to the food groove, and carried ventrally toward the mouth, where it was swallowed. Frames are labeled by their times (ms) relative to the first frame. Mo, mouth.

polystyrene beads), particles could be seen being accelerated by the prototrochal cilia and trapped between prototroch and metatroch. Once in the food groove, particles were transported to the mouth, where they were swallowed (Fig. 7; Video S4). Twenty-seven of these transport events were captured on high-speed video. In a few instances, when larvae were observed in ventral view, particles were seen being delivered to the mouth from both the left and the right sides of the opposed band system simultaneously. We did not observe rejection of particles at the mouth by larvae of N. cf. tenuis. Larvae of N. cf. tenuis consumed polystyrene divinylbenzene spheres of all sizes offered (5– 45 ␮m in diameter). Larvae consumed Sephadex particles from 10 to 30 ␮m in diameter. Discussion Embryogenesis There is substantial variation in egg size among species of Capitellidae (Table 1). To our knowledge, early development has been observed in only three species in the clade: Capitella capitata and C. teleta, both of which have large eggs and lecithotrophic larvae (Eisig, 1898; Meyer et al., 2010), and Notomastus cf. tenuis, which has small eggs (⬃1/40 the volume of those of C. teleta) and planktotrophic larvae (this study). Early development in N. cf. tenuis differs substantially from that of the two Capitella spp., which are very similar to each other in embryogenesis (Meyer et al., 2010). For example, the first two cleavages in embryos of N.

cf. tenuis are equal (Fig. 1), but the first two cleavages in C. teleta and C. capitata are unequal, leading to the formation of recognizably larger D quadrant blastomeres (Eisig, 1898; Meyer et al., 2010). These differences in the symmetry of early cleavages may be indicative of major differences in the mode of D-quadrant specification (Freeman and Lundelius, 2002). Gastrulation in N. cf. tenuis appears to occur by invagination (Fig. 1), but gastrulation is by epiboly in the two species of Capitella. These differences are typical of comparisons between annelid species with small eggs and those with large eggs (Anderson, 1973; Schroeder and Hermans, 1975). What is unusual about this particular comparison, however, is the substantial quantity of data available on the genome and the embryonic and larval development of C. teleta (e.g., Seaver and Kaneshige, 2006; Meyer et al., 2010; Giani et al., 2011; Simakov et al., 2012; Amiel et al., 2013). For those interested in exploring evolutionary changes in development associated with changes in per-offspring maternal investment then, capitellids may thus be a particularly valuable group. Viable gametes of at least three species of capitellids with relatively small eggs (Dasybranchus caducus, N. latericeus, and N. cf. tenuis: Wilson, 1933; Bookhout, 1957; this study) can be obtained by dissection, greatly facilitating study of their embryos or larvae. (Note that N. cf. tenuis is the only one of these three species with planktotrophic larval development; Table 1.) In addition, capitellids and echiurids are consistently recovered as sister taxa in molecular phylogenetic analyses (e.g., Bleidorn et al., 2003; Rousset et al., 2007; Struck et al., 2007, 2011; Zrzavy et al.,


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2009; Weigert et al., 2014), suggesting that embryos of echiurids might also be interesting to compare to those of capitellids. Viable gametes of several species of echiurids with planktotrophic development are also easily obtained (Conn, 1886; Torrey, 1903; Newby, 1940). Larval form Larvae of capitellids seem to fall into two major groups in terms of morphology. Larvae of members of the genus Capitella are very distinctively barrel-shaped and bear three main ciliary bands: prototroch, telotroch, and neurotroch (Eckelbarger and Grassle, 1987). They do not bear an apical tuft of cilia (Eckelbarger and Grassle, 1987). Most of these species are known to have lecithotrophic development, although several (specifically, C. sp. 1a and C. sp. III [⫽C. jonesi] of Grassle and Grassle, 1976) have been suggested to have planktotrophic development (Eckelbarger and Grassle, 1987). It should be noted that feeding has never been observed in these “planktotrophic” larvae of Capitella spp.; planktotrophy was apparently inferred from their small egg sizes (75 and 50 ␮m, respectively) and relatively long planktonic periods (Grassle and Grassle, 1976). In contrast, larvae of members of the capitellid genera Dasybranchus, Mediomastus, and Notomastus are shaped more like the “classical opposed band trochophore” larvae of serpulid annelids (e.g., fig. 9A of Nielsen, 1987, and fig. 6.2A of Rouse, 2006), with a roughly conical episphere (the region anterior to the prototroch), and a roughly conical hyposphere (the region posterior to the prototroch) (Fig. 2). These larvae all bear prototrochal, telotrochal, and neurotrochal ciliary bands, as well an apical tuft of cilia at the anterior end. Some of the capitellid larvae in this group also bear metatrochal cilia (described further below). Another similarity among most of the larvae of this second group is the presence of striking green epidermal “chromatophores,” found primarily in equatorial bands in the hyposphere. These were remarked on by Bookhout (1957) in Dasybranchus caducus, Rasmussen (1956) in Mediomastus fragile, and in this study in N. cf. tenuis. Though Wilson (1933) does not describe these green structures in N. latericeus, he does describe “bands of colourless but refringent ectodermal globules” in similar positions as the green epidermal cells in these other species. Epidermal cells containing green pigment are also found in the larvae of numerous echiurids, including both planktotrophs and lecithotrophs (e.g., Bonellia viridis: Agius and Jaccarini, 1981; Urechis uncinatus: Kazama et al., 2005; Thalassema mellita: Conn, 1886; Torrey, 1903). A similar pigment has been isolated from adults of Bonellia viridis. This pigment—a chlorin called bonellin—is toxic to many eukaryote cells in very low concentrations, and thus has been inferred to serve a defensive role in adults of B. viridis (de Nicola Giuduce, 1984). It is not clear if the green pigment

in the larvae of capitellids or echiurids is bonellin or a related compound; if it is, it could serve as a chemical defense in larval stages. As larvae of N. cf. tenuis near metamorphosis, pairs of red spots very similar in appearance to the episphere eyes began to appear in specific post-prototrochal segments (Fig. 4C–E). Such “accessory eyespots” are not known from the larvae of any other capitellids (e.g., Wilson, 1933; Rasmussen, 1956; Bookhout, 1957), or from the larvae of echiurids (e.g., Conn, 1886). Though accessory eyes were still present in recently metamorphosed juveniles of N. cf. tenuis, adults do not seem to have segmental eyes, so presumably they are lost during subsequent growth. The monoclonal antibody (22C10) labels the sensory cell of both larval and juvenile eyes of C. teleta (Yamaguchi and Seaver, 2013). Labeling larvae of N. cf. tenuis with this antibody might provide evidence that “accessory eyespots” actually are photoreceptors. Ultrastructural and functional data are also needed to definitively make this determination.

Larval feeding Larvae of N. cf. tenuis bear prototrochal and metatrochal bands of cilia, with an intermediate food groove (Fig. 4B). High-speed video sequences indicated that prototrochal cilia have their active strokes from anterior to posterior, and metatrochal cilia have their active strokes from posterior to anterior (Video S1, http://www.biolbull.org/content/ supplemental). Particles trapped between the “opposed” prototrochal and metatrochal ciliary bands are carried ventrally to the mouth, presumably by the food-groove cilia (Fig. 7; Video S4). This morphology, ciliary kinematics, and behavior of captured particles is characteristic of opposed band larval feeding. Though some larvae that feed with opposed bands feed primarily on small particles (⬎⬃20 ␮m: e.g., Hansen, 1991, 1993), larvae of N. cf. tenuis were able to consume spherical particles up to 45 ␮m in diameter. This ability to capture relatively large particles might indicate that these larvae are capable of using both their opposed bands of cilia and an alternative mechanism to capture particles, as described by Miner et al. (1999) for an echiurid and an opheliid. As noted above, molecular phylogenetic analyses typically link capitellids and echiurids as sister taxa; some of these analyses also suggest that opheliids are close relatives of the capitellid/echiurid clade (e.g., Rousset et al., 2007; Zrzavy et al., 2009; Struck et al., 2011; Weigert et al., 2014). The presence of opposed bands of cilia is probably not limited to N. cf. tenuis among the capitellids. For example, Rasmussen (1956) noted that the planktotrophic larvae of Mediomastus fragilis (as Heteromastus filiformis) bear a prototroch with two rows of cilia, “an anterior row consisting of long and strong cilia and another posterior row of



short cilia stopping on each side of the mouth.” This posterior row is in the position of the metatroch in N. cf. tenuis. We suggest that Rasmussen simply did not identify it as a ciliary band separate from the prototroch. This is not uncommon among studies of larval form carried out before the functional significance of metatrochal ciliation was known (e.g., see Pernet and Strathmann, 2011). This interpretation is consistent with Hansen’s (1993) statement that larvae of M. fragilis (as M. fragile) capture particles using opposed bands of cilia. Of course, additional study is needed to verify the post-oral position of the putative metatroch, as well as the beat pattern of its cilia. Observation of transport of captured particles in the food groove would also serve as useful evidence of opposed band feeding in this species. Another interesting example is provided by the lecithotrophic larvae of N. latericeus. Wilson (1933) indicated that these larvae bear “a narrow metatroch of short posteriorly directed cilia immediately behind the prototroch.” Again, verification of metatrochal ciliary beat pattern and particle transport is needed for this species. If verified, the possession of metatrochal cilia by these lecithotrophic larvae is an interesting parallel to the situation in sabellid annelids, where numerous species with lecithotrophic larvae possess functional opposed bands of cilia (Pernet, 2003). However, some lecithotrophic larvae of capitellids do not seem to bear metatrochal cilia. For example, there is no indication that the larvae of Dasybranchus caducus have metatrochs (Bookhout, 1957). Likewise, metatrochal cilia have not been described from any species in the genus Capitella. Several species in this genus are putatively planktotrophic, though this inference was not based on observations of particle capture or ingestion, but instead apparently on egg size and on the length of the planktonic period in the laboratory (Eckelbarger and Grassle, 1987). Verification that any of the putatively planktotrophic species of Capitella can actually capture and ingest particles would be very useful in understanding how larval feeding and feeding mechanisms have evolved in Capitellidae. Generating and testing hypotheses on how larval nutritional mode and larval feeding mechanisms have evolved in these diverse capitellids will also require robust phylogenetic hypotheses for the group; these are currently lacking. Pernet and Strathmann (2011) listed 10 taxa of annelids that included at least one species known to feed using opposed bands of cilia (their table 2). This included Capitellidae, though Pernet and Strathmann (2011) suggested that additional published evidence would be necessary to make this case definitively. The data reported here effectively make this case. Such improvement of our knowledge of the distribution of opposed band and other larval feeding mechanisms among the annelids is one of the basic requirements for future analyses aimed at understanding the evolution of larval feeding and larval feeding mechanisms among the annelids.

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As noted above, molecular phylogenetic analyses typically link capitellids and echiurids as sister taxa; opheliids are also often recovered as close relatives of the capitellid/ echiurid clade. Several echiurids are known to have metatrochal cilia. Conn (1886) clearly described and illustrated a post-oral band of cilia in his study of the life history of Thalassema mellita, a species with planktotrophic larvae. The larvae of Urechis caupo also have metatrochal cilia and use them to capture particles in an opposed band system (Miner et al., 1999), and Korn (1960) showed metatrochal cilia as present in his illustration of larvae of Echiurus abyssalis. Larvae of the opheliid Armandia brevis also possess opposed bands of cilia (Miner et al., 1999). In addition, the feeding larvae of U. caupo, A. brevis, and N. cf. tenuis are all able to capture relatively large particles, an unusual ability among larvae that can capture particles using opposed bands of cilia (Miner et al., 1999; this study). As capitellids, echiurids, and opheliids are apparently closely related to each other (along with some taxa that do not include any species with feeding larvae, e.g., Arenicolidae, Maldanidae, and Terebellidae) and share the unusual ability to capture large particles, it seems plausible that the most recent common ancestor of all three taxa had planktotrophic larvae that fed using the same mechanism(s) used by U. caupo, A. brevis, and N. cf. tenuis. Robust tests of this hypothesis will require better taxon sampling in phylogenies of the three families and their close relatives, as well as studies of larval form and function in a broader diversity of capitellid, echiurid, and opheliid species. Our examination of larval form and feeding in N. cf. tenuis resulted in two surprising observations. The first is that in this species, prototrochal cilia appeared to be simple, not compound. Prototrochs of most annelid and mollusc larvae are composed of compound cilia arising from multiciliated cells (Nielsen, 1987). The only exceptions we are aware of are some members of Oweniidae and possibly Magelonidae; larvae of these species bear at least some simple cilia arising from monociliated epidermal cells (Emlet and Strathmann, 1994; Bartolomaeus, 1995; Smart and von Dassow, 2009). Though we have no direct evidence on whether or not prototrochal cells of N. cf. tenuis are monoor multiciliated, the high density of cilia in that band (Figs. 2A, B; 4B) suggests that they arise from multiciliated cells. Two lines of evidence are consistent with the hypothesis that larvae of N. cf. tenuis have simple prototrochal cilia. First, we never observed typical “sword-shaped” (wider at the base than the tip: see fig. 3 of Nielsen, 1987) compound cilia in fixed (viewed via scanning electron microscopy) specimens of N. cf. tenuis; instead, cilia always appeared to be unconnected to each other. Using the same methods, we were easily able to identify compound prototrochal cilia in fixed larvae of numerous sabellid annelids (Pernet, 2003). As noted by Nielsen (1987), “some species are exceedingly difficult to fix with the compound cilia intact”; it is possible


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that N. cf. tenuis is one of these species, so our observation of only apparently simple cilia in fixed specimens is suspect. However, we also could not identify compound cilia in high-speed video footage of prototrochal cilia beating in living larvae of N. cf. tenuis. Again, using the same methods (indeed, the same microscope and camera), we were able to identify compound prototrochal cilia in living larvae of numerous sabellid annelids (Pernet, 2003) and the serpulid Serpula columbiana (Video S3). Second, compound prototrochal cilia typically increase in length over the course of larval development (reviewed in Emlet and Strathmann, 1994); however, the prototrochal cilia of N. cf. tenuis did not increase in length at all over 6 weeks of development (Fig. 3). Our current observations are thus consistent with the hypothesis that larvae of N. cf. tenuis have simple prototrochal cilia. However, our inference could of course be incorrect; additional independent observations of these larvae (as well as those of other capitellids) would be helpful in clarifying this situation. The presence of only simple cilia in the prototroch has important consequences for the function of opposed band particle capture systems: simple cilia are limited in their maximum length, which Emlet and Strathmann (1994) note imposes limits on the rate at which water can be moved per length of ciliary band. Those authors interpret the greatly expanded and sinuous prototroch of the mitraria larvae of oweniids as an evolutionary solution to the problem of maximizing clearance rates of particulate food in the face of a constraint on maximum length of simple prototrochal cilia. In this respect, it is interesting to note that the prototrochal ciliary band in larvae of N. cf. tenuis is not unusually long or sinuous. Simple prototrochal cilia and a relatively short prototrochal ciliary band seem like a poor combination of traits for a larva that can capture particles using opposed bands of cilia. It is possible that larvae of N. cf. tenuis evade these constraints on larval feeding performance by using another feeding mechanism in addition to opposed bands. The larvae of an echiurid and an opheliid (both closely related to capitellids) use both opposed bands of cilia and another feeding mechanism; the latter is used to capture large particles. This is consistent with our observations of the ingestion of relatively large particles (up to 45 ␮m diameter) by larvae of N. cf. tenuis. The second surprising observation was that the anteriormost tier of prototrochal cilia in larvae of N. cf. tenuis appeared to be non-motile. In high-speed video observations, these cilia were always held straight and at an angle of about 40° above the equatorial plane (Fig. 6A; Video S2). The remaining tiers of prototrochal cilia passed through this tier of non-motile cilia on both active and passive strokes. For comparison, we reared larvae of the serpulid S. columbiana and observed them using the same high-speed video method; these larvae also had a very distinct anteriormost tier of non-motile prototrochal cilia through which the re-

maning prototrochal cilia beat (Fig. 6B; Video S3). These “pretrochal” cilia are also clearly shown in larvae of S. columbiana (as S. vermicularis) by scanning electron microscopy in figure 9A of Nielsen (1987). Waller (1981) identified an anterior tier of short (⬃20 ␮m, like those of N. cf. tenuis) prototrochal cilia in veligers of the oyster Ostrea edulis (“inner preoral cilia,” his fig. 6); it is not known if these cilia are non-motile. However, these observations suggest that a tier of short, non-motile cilia at the anterior of the prototroch might be common among annelids and molluscs with opposed bands. That they have not been reported previously in the literature might be because their behavior can only be assessed using analysis of high-speed film or video, which has been applied to larval ciliary bands relatively rarely. Such non-motile cilia might be functionally important in opposed band larval feeding—for example, serving as a “comb” against which prototrochal cilia could wipe off adhered captured food particles on the recovery stroke. This would leave particles on the posterior side of the non-motile tier of cilia, in position to be pushed into the food groove by the next active stroke of the prototrochal cilia. This might explain how particles can be captured by direct interception by adhesion to prototrochal cilia, but subsequently released from prototrochal cilia and moved to the food groove (Romero et al., 2010). Acknowledgments We thank the director of the Friday Harbor Laboratories for providing space and resources, R. Strathmann for access to a high-speed video camera (purchased under National Science Foundation grant OCE-9633193 to RRS), and M. Sullivan for his expertise in video editing. This report is based upon work supported by NSF grant OCE-1060801 (to BP) and the Edward Meyer Bequest to the Washington State University Foundation (to PCS). Literature Cited Agius, L., and V. Jaccarini. 1981. Development of pigment in the echiuran worm Bonellia viridis. J. Zool. Lond. 193: 25–31. Akesson, B. 1961. On the histological differentiation of the larvae of Pisione remota (Pisionidae, Polychaeta). Acta Zool. 42: 177–225. Amiel, A. R., J. Q. Henry, and E. C. Seaver. 2013. An organizing activity is required for head patterning and cell fate specification in the polychaete annelid Capitella teleta: new insights into cell-cell signalling in Lophotrochozoa. Dev. Biol. 379: 107–122. Anderson, D. T. 1973. Embryology and Phylogeny in Annelids and Arthropods, International Series of Monographs in Pure and Applied Biology, Vol. 50. Pergamon Press, Oxford. Bartolomaeus, T. 1995. Secondary monociliarity in the Annelida: monociliated epidermal cells in larvae of Magelona mirabilis. Microfauna Mar. 10: 327–332. Blake, J. A., J. P. Grassle, and K. J. Eckelbarger. 2009. Capitella teleta, a new species designation for the opportunistic and experimental


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Functional analysis of the larval feeding circuit in Drosophila.

Development of the annelid axochord: insights into notochord evolution.

Owenia fusiformis - a basally branching annelid suitable for studying ancestral features of annelid neural development.

Studies on the feeding of larval Argas persicus Oken.

Feeding behavior, natural food, and nutritional relationships of larval mosquitoes.

The effect of silencing 20E biosynthesis relative genes by feeding bacterially expressed dsRNA on the larval development of Chilo suppressalis.

Spatiotemporal regulation of nervous system development in the annelid Capitella teleta.

Feeding and dentofacial development.

Hydrodynamic Constraints of Suction Feeding in Low Reynolds Numbers, and the Critical Period of Larval Fishes.

Food selection in larval fruit flies: dynamics and effects on larval development.

The development of normal feeding and swallowing.

Inhaled antibiotics in Cystic Fibrosis (CF) and non-CF bronchiectasis.

Effect of three larval diets on larval development and male sexual performance of Anopheles gambiae s.s.

Microbial shifts of the silkworm larval gut in response to lettuce leaf feeding.

ISPE and hybrid meta-DFT methods.

Protein synthesis, growth and energetics in larval herring (Clupea harengus) at different feeding regimes.

Chemical suppression of feeding in larval weakfish (Cynoscion regalis) by trochophores of the serpulid polychaeteHydroides dianthus.

Genetic and genomic tools for the marine annelid Platynereis dumerilii.

The function of the cellular haemoglobins in Capitella capitata (Fabricius) and Notomastus latericeus sars (Capitellidae: Polychaeta).

Gonad establishment during asexual reproduction in the annelid Pristina leidyi.

Exploration of the "larval pool": development and ground-truthing of a larval transport model off leeward Hawai'i.

Resource Limitation, Controphic Ostracod Density and Larval Mosquito Development.

Plasticity and regeneration of gonads in the annelid Pristina leidyi.

The efficiency of new leukocyte removal filters. CF-1 and CF-2.