Nechtochaete larva of the polychaete Magelona

On April 5, 2010 my Comparative Embryology and Larval Biology class ventured outside the mouth of Coos Bay, OR in a small boat to do a plankton tow. A plankton tow consists of dragging a net with very small holes, in this case we used a 153 μm mesh, through the water column. The organisms big enough to get caught in the net are collected at the bottom in a small container. Once back in the lab we sorted the plankton, and I came across a polychaete nechtochaete larva with long tentacles on its head, and bundles of long chaetae, chitinous bristles found in annelids, two on each segment of the larva. I identified this polychaete as belonging to the genus Magelona (Fam. Magelonidae), because it has the characteristic pair of long tentacles, which are often coiled.

These large tentacles are thought to function as locomotory suspension organs (Wilson 1982).While observing the larva under a compound microscope I noticed that it would contract and expand the tentacles and move around under the cover slip. The chaetae found on the larva may also aid in defense against predators. The larva’s tentacles have also been hypothesized by Wilson (1982) to assist in the capture of prey. Lebour (1922) and Smidt (1951) observed bivalve veliger larvae in the guts of larval Magelona. During metamorphosis, the larval tentacles are replaced by proportionally smaller adult tentacles.

Lebour MV. 1922. The food of plankton organisms. Journal of the Marine Biological Association of the United Kingdom. 12: 644-677.

Smidt ELB. 1951. Animal production in the Danish Waddensea. Meddelelser Kommission fra Danmarks Fiskeri- og Havundersogelser. 11 (6): 151.

Wilson DP. 1982. The larval development of three species of Magelona (Polychaeta) from localities near Plymouth. Journal of the Marine Biological Association of the United Kingdom. 62: 385-401.

Trochophore larva of the polychaete Sabellaria

Sabellaria cementarium is a polychaete worm that lives in hard tubes constructed of sand held together with a glue-like secretion. The adult worm can be up to 7 cm long and lives in clusters subtidally (Kozloff, 1974). A few adult worms were collected by Richard Emlet and George von Dassow from the dredge (about 150 ft deep, a couple of miles south of Cape Arago, OR). Luckily, two of the worms spawned, when Paul Dunn, our TA, cracked their tubes open with forceps. One of them was a male, and another one — a female! So we were able to fertilize the eggs and start a culture.

These photos are of 11-day old trochophore larvae. The first one shows the ciliated band, called the prototroch, which encircles the larva just anterior to the mouth. The long bristles are called setae (or chaetae) and are characteristic of both the larvae and adults of polychaete worms. The setae serve as defense against planktonic predators (Pennington & Chia, 1984). Fanning out the setae (second picture), the larva can nearly double its diameter (140μm without the setae, and 250μm with setae spread out).

In the third photo you can also see the two reddish eyespots anterior to the prototroch. This trochophore will continue adding new segments, each segment bearing more setae. Once it has more than three setigers (segments with setae) it will find a suitable place to settle and build its sand tube. In some areas species of Sabellariaform extensive reefs, because their larvae prefer to settle on the tubes of adult worms of their species.

Kozloff, E.N. 1974. Seashore Life of the northern Pacific Coast; an illustrated guide to northern California, Oregon, Washington, and British Columbia. U of Washington P: Seattle.

Pennington, J. T., & Chia, F.-S. (1984). Morphological and Behavioral Defenses of Trochophore Larvae of Sabellaria cementarium (Polychaeta) against Four Planktonic Predators. Biological Bulletin. 167 (1), 168-175.

Juvenile brittle star in polarized light

I dissected this juvenile brittle star (Class Ophiuroidea) from a brood pouch of the adult, Amphipholis squamata. Through careful removal of the legs and the mouth plate, I was able to extract the brood pouch (also called genital bursa) containing the juvenile brittle star pictured here. This species is placental and broods its young instead of releasing gametes into the water column. This specimen is approximately 3 millimeters in diameter and is photographed under a system of polarizers. These cause the calcareous spicules present in the juvenile to glow on a dark background. See another blog post by Kristina Sawyer which pictures a similar specimen under regular transmitted light. The intricate skeleton of the juvenile forms the basis for the skeleton of the adult brittle star.

Arm formation in pluteus larvae

I was interested in how the sand dollar pluteus larva develops. Here are a few pictures of successive developmental stages of this species, Dendraster excentricus. The top picture shows an early four-armed pluteus larva which is three to four days old. It has formed the first two pairs of arms called the post-oral and antero-lateral arms. The post-oral arms form first and are the longest. One can also see the calcareous spicules supporting the antero-lateral arms.

The next photo shows a 14-day old larva which is more advanced. It has longer antero-lateral arms, which project outwards from the anterior end and frame the mouth of the pluteus. The formation of extra arm pairs extends the length of the continuous ciliated band which surrounds the larval mouth and is used to capture microscopic food particles. The longer the ciliated band, the more efficient pluteus can feed. The small bumps at the base of the post-oral arms are the newly developing postero-dorsal arms.

Although one can barely see them, one can already distinguish the small calcareous spicules, which support the new pair of arms, using cross-polarized light. The four spicule rods supporting the antero-lateral pair of arms (towards the midline) and the longer post-oral pair of arms are clearly visible. The shorter postero-dorsal spicule is visible on the left side.

The final pair of arms to form are the pre-oral arms, which, true to their name, form just anterior to the mouth of the pluteus larva. These arms are more or less parallel to the anterolateral arms, and can be seen as small bumps between the antero-lateral arms. On this bottom picture you can also see an unpaired rudiment of the juvenile sand dollar (a bean shaped mass to the left of the larval stomach - which is a large darkish oval occupying the majority of space inside the larval body.

Fertilization in a sea urchin and a starfish

During the first two weeks of class we focused on the early development of sea urchins (e.g. Strongylocentrotus purpuratus) and sea stars (e.g. Pisaster ochraceous), both in the phylum Echinodermata. By either physically shaking or injecting the adults with 0.53 M KCl, we encouraged the release of gametes and fertilized them to observe development. One of the first changes that can be seen following fertilization of echinoderm eggs is the formation of the fertilization envelope, a visible membrane surrounding a fertilized egg that acts as a physical barrier to prevent polyspermy (fertilization by multiple sperm). It forms from the vitelline layer as it lifts off the egg plasma membrane and is hardened by the enzymes released by cortical granules. This photo shows two eggs of a purple sea urchin, Strongylocentrotus purpuratus - one fertilized (and surrounded by the fertilization envelope), and one unfertilized (without the envelope).

One of the largest noticeable differences between sea stars and sea urchins in early development is the formation of polar bodies. A polar body is a tiny sister-cell of the primary oocyte, produced during meiosis. It contains discarded DNA, and very little of anything else. Polar bodies are not usually observed in sea urchin, because meiosis is completed within the ovary, and spawned eggs have already parted with their polar bodes.
However, we were able observe polar bodies in sea stars. This is because in sea stars, sperm entry occurs before the oocytes have completed meiosis (cell division, reducing the number of chromosomes). Polar bodies form after fertilization and are trapped within the fertilization envelope. The photos here show an immature unfertilized oocyte (with a large nucleus and a nucleolus inside) and a fertilized secondary oocyte with homogenenous cytoplasm, a tight fertilization envelope around it, and one polar body (at about 5 o'clock), in the ochre sea star Pisaster ochraceous. The fertilization envelope in starfish is much closer to the surface of the egg than in sea urchins.

DNA sequence identifies a larval nemertean

Marley Jarvis has finished her rotation project in my lab. Her project was to try to identify several planktonic larvae using DNA sequence data, while learning some basic molecular techniques (DNA extraction, PCR, gel electrophoresis etc.). Among other things,  we have sequenced portions of two mitochondrial genes (16S rDNA and Cytochrome Oxidase Subunit I) from the pilidium, which based on its morphology, I preliminary identified as belonging to the palaeonemertean Family Hubrechtidae, and likely the genus Hubrechtella (see my earlier post this year). It was a surprise to find this larva, because, no hubrechtids are currently known to occur on the Pacific Coast of North America. We have matched the 16S sequence derived from this pilidium to the sequence, I obtained earlier from the hubrechtid species from the Sea of Japan, Hubrechtella juliae Chernyshev, 2003. The uncorrected sequence divergence is 0.7% for 16S. Sequence divergence of less than 1% for this region of 16S, suggests that the larva belongs to Hubrechtella juliae, or a very closely related species (very likely morphologically indistinguishable). Because this pilidium larva is at a very early developmental stage (before formation of any of the juvenile rudiments, called imaginal discs), and because of what we know about the dominant currents in the Pacific Ocean, it is highly unlikely that this larva was carried here from the Sea of Japan. A more likely explanation is that Hubrechtella juliae occurs on the Pacific Coast of North America, but we have not found the adults yet.

Chernyshev AV. 2003. Novy vid roda Hubrechtella (Nemertea, Anopla) i obosnovanie semeistva Hubrechtellidae. [A new species of the genus Hubrechtella (Nemertea, Anopla) from the Sea of Japan, and establishement of the family Hubrechtellidae]. In Russian. Biologiya Morya. 29(5): 368-370. 

Marine gastropod escaping its chorion

On Aprl 26^th, 2010 at 11:25 AM, I started a culture of a marine gastropod (snail) Calliostoma ligatum. When I looked at it 3 days later, the veliger larvae were still in their chorions (egg envelopes). While in the chorions, veligers beat long cilia on their velum really fast, then stop for a moment to take a breather, and then continue moving. By beating the cilia on their velum really quickly against the chorion, they were able to deform the chorion. This eventually ruptured the chorion and the larvae hatched! The first picture shows a veliger larva (complete with a shell and a foot, like a miniature snail) in the process of deforming its chorion with its velum. As you can see, the chorion is flattened on the side where it contacts the velum, while the rest of the envelope is still more or less rounded.

Eight days after fertilization, I looked at my Calliostoma culture again. At this point, most of the embryos were dead or abnormal. Larvae can be so temperamental! There were some larvae resting at the bottom of the dish that looked normal and moved their cilia. While under a cover slip, they used cilia on their velum to move around, and they moved FAST! Those I could observe had two eyespots and two tentacles forming in the apical area. At metamorphosis, the velum will degenerate, and the miniature snail will start crawling using its foot. This picture shows the veliger on its side, with one of the eyespots facing us. The velum is out on this picture (top). You can also see the foot (left) and the operculum (trap door) attached to it. At this time, I witnessed one of the other juveniles moving on its foot, — all I could see was the shell waddling around the slide.

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Laboratory culture of Strongylocentrotus franciscanus (red urchin)

On 17 March 2010, I collected 4 adult red urchins, Strongylocentrotus franciscanus, from the Lighthouse Island channel, near Charleston, OR, during low tide. They were found in burrows, holes in the rocky outcroppings created by repetitive scraping by their spines and teeth, alongside some purple urchins, S. purpuratus. This is a relatively rare find, because S. franciscanus is mostly subtidal. Red urchins have longer spines and tube feet, as well as a larger test diameter compared to purple urchins, and often they are more reddish than purple. In my Comparative Embryology Class, we were looking at the development of S. purpuratus and I was interested to follow the development of a closely related species. I induced spawning in the adult urchins in the lab by injecting them with 5 ml of 0.5 M potassium chloride. I then collected eggs and sperm, and started a culture that afternoon. According to Strathmann (1987), S. purpuratus eggs range from 78 to 80 µm, while eggs of S. franciscanus are between 130 and 140 µm. The eggs I fertilized averaged 125 µm in diameter (n=10) and the fertilization envelope expanded in about a minute after addition of sperm to eggs. It raised about 18 µm (n=10) from the surface of the eggs. After 16 hours at 13°C, the embryos reached the blastula stage shown here. The fertilization envelope is still seen around the blastula, which rotates within the envelope. Eggs of S. purpuratus would take over 20 hours to reach the blastula stage at the same temperature.

Strathmann, M. 1987. Phylum Echinodermata, Class Echinoidea. In Reproduction and Development of Marine Invertebrates of the Northern Pacific Coast. P. 512. University of Washington Press, Seattle.

Bryozoan coronate larvae

I study the cheilostomate bryozoan Schizoporella unicornis (Johnston), which encrusts hard substrates and looks like a bright orange patch, as large as a quarter coin or bigger. A recent paper by Tompsett et al. (2009) has suggested that S. unicornis on the west coast is in fact S. japonica. I have found colonies of this bryozoan on the California mussels (Mytilus californianus) growing on the floating docks of the inner boat basin in Charleston, OR. Bryozoans are colonial animals; each individual within a colony is called a zooid. S. unicornis broods its embryos in modified zooids called ovicells. When exposed to bright light for several hours, S. unicornis releases lecithotrophic (non-feeding) coronate larvae, which are approximately 300 μm in length.

The larvae swim towards the light, actively changing their shape with muscular contractions. The larva appears bright orange in reflected light (although it looks brownish in transmitted light, as you can see) with two dark reddish pigment spots one on each side of the apical organ. The entire body surface is covered with cilia (the outer ciliated epithelium of a coronate larva is called corona ciliata). The cilia of the corona ciliata beat in a clockwise direction when viewed from the apical pole. The internal sac is well defined and visible through the body wall at the broader posterior end of the larva. The thin tri-radial dark line (top picture) is the lumen of this thick-walled epidermal invagination. This invagination is everted during metamorphosis, helps the larva attach to the substratum, and makes up a significant portion of the epidermis of the founding zooid of the colony.

Between the two dark red pigment spots lies the sensory region called apical organ (it looks like a finely outlined oval). Ventral to the apical organ is a smaller lighter red pigment spot (middle and bottom pictures) marking the location of the ciliated cleft, which contains a bundle of longer stronger beating cilia, called the vibratile plume (see bottom picture, at about seven o-clock). The vibratile plume is a sensory structure which plays a role in selecting the appropriate substratum for larval settlement.

These larvae are fascinating, but ephemeral. If a suitable substrate is available, they will settle within hours of being released, and metamorphose (transform) into the founding zooid (ancestrula) of a new bryozoan colony!

Tompsett S, Porter JS, Taylor PD. 2009. Taxonomy of the fouling cheilostome bryozoans Schizoporella unicornis (Johnston) and Schizoporella errata (Waters). J Nat Hist. 43:2227-2243.

Asteroid Bipinnaria Larva

Last week in my embryology class, I took some pictures of the starfish bipinnaria larvae (Evasterias troschelii). Like other planktotrophic echinoderm larvae, bipinnaria has a complete tri-partite gut. The mouth is a clear rounded triangular shape in the anterior third of the animal (upper left on this photo). The mouth leads to the esophagus (a wide tube below the mouth), which connects to the stomach. I found it interesting that I was able to see some color in the larval gut, which comes from the unicellular algae that we feed to the larvae. The large pinkish egg-like shape in the posterior third of the animal (bottom right) is the larval stomach. The elongated shapes on either side of the esophagus and stomach are the larval coeloms. The left coelom is going to make up the majority of the water-vascular system in the adult starfish. The dark ribbon-like shape is the larval ciliated band (out of focus on this picture).

This picture shows the same larva in the same orientation, only with different parts in focus. It is easy to see the intestine (narrow tube in the posterior third of the larva) with the same red pigment as in the stomach, only darker! The intestine curves up and leads to the anus, located on the ventral side (in focus here). These larvae are able to swim and feed using a ciliated band composed of many tightly opposed epithelial cells, each with a cilium that looks like a little hair. The cilia (plural of cilium) beat continuously to create a water current away from the mouth, and locally redirect the flow toward the mouth, when they encounter food particles. One cannot distinguish individual cilia on this photo, however the portion of the ciliated band that flanks the mouth above and below is distinguishable and sharply in focus. The portion of the band that is in focus above the anus is called "postoral", and the portion of the band above the mouth is called "preoral". During development of the bipinnaria larva the preoral and postrodal portions of the ciliated band separate to form two separate loops (preoral and postoral).

Actinotroch of Phoronis vancouverensis

These pictures are stacks of confocal images of two different actinotroch larvae of the horseshoe worm Phoronis vancouverensis (Phylum Phoronida). P. vancouverensis is a rather inconspicuous phoronid which lives in small (a few centimeters long) muddy tubes in clumps, attached to some sort of hard substratum (a rock, a floating dock) often in somewhat muddy surroundings. This species broods its larvae in the crown of tentacles, called the lophophore. I gently shook the larvae out of the lophophore of an adult and prepared them for confocal microscopy with my students while teaching the Comparative Embryology course at the Friday Harbor Labs in the Summer 2007.

We preserved the larvae and stained them with fluorescent phallodin (a toxin, derived from the deathcap mushroom Amanita phalloides), which binds to filamentous actin. Muscles are highlighted because they are full of actin, a protein which enables cellular contractility. So, most of what you see on these pictures are muscle fibers. There is also quite a bit of actin in the cell cortex (the region of the cytoplasm adjacent to the plasma membrane). So, the outlines of epidermal cells are often also labeled with phalloidin.

The anterior end of the larva has a large preoral hood (upper right). The mouth opens under the hood. The first picture is a side view. The second picture is a ventral view. The hood is lifted, and we are looking straight into the larval mouth. Posterior to the mouth is a set of tentacles, which bear a ciliated band used in capturing microscopic food particles. At the posterior end (bottom left) is another ring of ciliated cells, which propels the larva through the water. You cannot see the cilia in this preparation (because they are not fluorescent), but you can see the outlines of the small cells which compose the larval ciliated bands.

Actinotroch larva of Phoronopsis harmeri

This is a dark field microphotograph of an actinotroch larva that, according to Dr. Elena Temereva, a Russian specialist on phoronid development who examined the larva, belongs to the horseshoe worm  Phoronopsis harmeri (Temereva 2009). This species, also known as Phoronopsis viridis, is a common intertidal species on the sandflats in the Pacific Northwest. This particular larva was caught in a plankton tow I took in August 2006 in a channel separating the San Juan Island and the Shaw Island in Puget Sound, WA. This larva had 16 tentacles and was about 0.9 mm long. Its broad preoral hood is up. The semi-transparent tube inside is the stomach. A ring of cilia at the posterior end (down) is the telotroch, which propels the larva through the water. This larva had numerous pigment granules (which appear golden in reflected light and black in transmitted light) along the tentacles, the margin of the hood, the telotroch, and even the protocoel (a small coelomic sack in the hood).

Temereva EN. 2009. New data on distribution, morphology and taxonomy of phoronid larvae (Lophophorata: Phoronida). Invertebrate Zoology 6(1): 47-64.

Pteropod Limacina sp.

This is a picture of a pteropod mollusc Limacina sp. This pelagic snail swims with its modified foot (which looks like two wings).  Although pelagic and tiny (only a few millimiters in diameter), this is not a larva, but an adult. This beautiful semi-transparent specimen is photographed though a dissecting microscope on a dark background. It came out of a plankton tow taken by Alan Shanks on October 7, 2009 just outside the mouth of Coos Bay, OR (near buoy K). George von Dassow made a time-lapse movie of early embryonic develpment in Limacina and made it available along with many other time-lapse videos of development on the website of the Center for Cell Dynamics (Friday Harbor Labs, University of Washington). Scroll down to the movie called: "Early development in the pteropod Limacina".

Blastomere Separation: Part Two

Development continues! On April 9, 2010, three days after fertilization, the embryos I surgically cut in half at the four-cell stage (see my previous post) reached the early prism stage (top). You can see the two calcium carbonate spicules which provide skeletal support for the larval body. In addition to spicules, the tripartite gut is starting to form. The control embryo (second from top), shown for comparison, is at approximately the same developmental stage, as indicated by the pair of spicules and the tripartite gut. The control is larger than the experimental embryo (they are photographed at the same magnification).

On April 16, 2010, ten days after fertilization, the experimental embryos have proceeded to the pluteus stage. The larvae appear to develop normally, if slow. The spicules are shaped like a chair (viewed from the side), as they are supposed to be at this stage. This larva also shows a small hydrocoel (a coelomic sack) which will later develop into the water-vascular system , characteristic of echinoderms. The hydrocoel is connected to the outside via a hydropore canal visible as a small strand of tissue reaching to the surface of the larva in the upper left quarter of the picture. Also, if you look closely, you will see some of the cilia at the tips of the larval arms which surround the larval mouth (to the left in this picture). These cilia make up the circumoral ciliated band, used in feeding.

This is another picture of the same half-size pluteus larva as above, just at a different focal plane. Unlike some larvae, plutei (plural of pluteus) are planktotrhopic, meaning that they feed on plankton. Therefore, if a larva is to be considered normal it must be able to feed. In this picture (side view) you can clearly see the complete tripartite gut which consists of the esophagus (on the left), the stomach (a clear shape roughly in the center of the larva), and the intestine (an oval shape under the stomach). Note some green particles in the stomach. These are cells of the unicellular green alga Dunaliella tercioleta that I have been feeding to the larvae. See also my next post.

Hydrocoel in 8-Armed Ophiopluteus

On April 12, 2010, my fellow students and I went on a boat trip in and outside the mouth of Coos Bay, Oregon. We did a plankton tow with a 153-micron plankton net in the bay near Buoy #10, and in the open ocean (about a mile off shore) near Buoy #1. I wasn’t able to help collect the sample in the open ocean because the boat was rocking a lot, even though it was a very nice day for Oregon. Don’t worry; I didn’t throw up.

While using a dissecting microscope to sort through the sample from the ocean later that day, I stumbled on this larva nestled within many diatoms. I was surprised that I was able to see the larva at all, because it was nearly translucent, though it was a relatively big larva, with a “wingspan” over 2 mm. The larva had 8 long, slender arms, which were set at a wide angle. I determined that it was an 8-armed ophiopluteus (a larval brittle star). My Invertebrate Zoology professor Richard Emlet, who happens to know a lot about echinoderm larval development, suggested that it might belong to one of three local species of brittle stars, Ophiopholis aculeata, Ophiura luetkeni, or Ophiura sarsii.

The coolest part about this larva was the 5-lobed hydrocoel, which is the large coelomic sack on the left side of the esophagus, well visible on this photo. This stage suggests that the larva was around a month old (R. Emlet, personal communication). Each lobe of the hydrocoel will become an arm of the water vascular system in the adult brittle star. The hydrocoel will migrate around and surround the esophagus before metamorphosis.

Brooded Juvenile Brittle Star

I found this juvenile by dissecting the central disk of an adult brittle star Amphipholis squamata. It was a little less than 1 mm in diameter. In this picture you can discern the developing ossicles, or small calcareous plates, that will cover the central disk and the rays, and form the skeleton. The central disk is visible as the dark pentagon in the center.

These viviparous brittle stars give birth to live young that are brooded internally. The parent can nourish the juveniles until they reach 2 mm and are large enough to crawl away. In the second picture you can see 10 tube feet on the juvenile brittle star, these will be used for locomotion as it crawls away.

Due to the small size of the adults (only 3-5 mm), they utilize a different reproductive strategy than larger sea stars. Instead of investing energy in producing large numbers of small eggs to free-spawn into the water column (and allow them to develop into feeding ophiopluteus larvae - as pictured in the next post), these small animals produce only a few small eggs (about 100 μm) in each gonad, and invest energy in brooding them internally until they are large enough to crawl away. Although it is a larger investment per egg, this direct development strategy ensures that the young develop to the juvenile stage. Amphipholis squamata can brood multiple cohorts simultaneously, and in our dissections we found brooded young in multiple stages of development within the same adult brittle star.

Amphipholis squamata is also interesting because it is a simultaneous hermaphrodite and appears to be capable of self-fertilization. The grey colored adult brittle stars feed on diatoms and detritus, and can be found under small rocks on sand or gravel in intertidal zones worldwide (Kozloff 1974).

Kozloff, E.N. 1974. Seashore Life of the Northern Pacific Coast; an illustrated guide to northern California, Oregon, Washington and British Columbia. U of Washington P: Seattle.

Blastomere Separation in Purple Urchins

In the late eighteen hundreds Driesch and Fiedler independently conducted a similar experiment - they successfully separated blastomeres of a sea urchin embryo at the 2-cell stage, and showed that each half gives rise to a complete half-size pluteus larva. This demonstrates that sea urchins can regulate their development. I am attempting to repeat such an experiment using purple sea urchin, Strongylocentrotus purpuratus.

Within about a minute of sperm contacting the egg plasma membrane, a fertilization envelope forms around the egg to prevent polyspermy (penetration by additional sperm). A hyaline layer forms directly on the surface of the egg plasma membrane to help hold the blastomeres in the dividing egg together. I had to remove the fertilization envelope and the hyaline layer in order to separate the blastomeres. To accomplish this I fertilized the eggs in filtered sea water (FSW). Within thirty seconds of fertilization I replaced the FSW with calcium-magnesium-free sea water. This prevents the fertilization envelope and hyaline layer from hardening, and allows one to remove them by sheering the eggs with a pipette. Once the eggs are denuded (their coats removed) they become very sticky, so I coated the culture dishes and glass knives used for surgery with BSA (Bovine Serum Albumin) to prevent the embryos from sticking to things and being damaged. The top two pictures show two 8-cell stage embryos of S. purpuratus - one with the fertilization envelope, and one without (denuded).

Without the fertilization envelope and hyaline layer, I was able to cut the eggs in half using a fine glass capillary, that I pulled over a flame. I cut some eggs in half at the 2-cell, and some at the 4-cell stage. The half-embryos proceeded to develop. This picture shows two embryos - a control (top left) and a half-embryo (bottom right) as they undergo gastrulation, by invagination, to form the gut. The experimental embryo is about half the size of the control.

I am attempting to rear these half-embryos all the way through to metamorphosis, so stay tuned to see the outcome and (hopefully) miniature juvenile urchins! See part two.

Bryozoan cyphonautes larva

This cyphonautes larva belongs, I believe, to a bryozoan species from the genus Conopeum (Rafferty 2002). I fished it out from an otherwise remarkably uninteresting plankton tow off the F dock in the Charleston marina (Charleston, OR) on April 30, 2010. It is distinguished from other kinds of cyphonautes larvae by the relatively opaque triangular shell encrusted with small particles. Each valve has a characteristic curved lateral ridge. The darker mass inside is the gut. Cyphonautes is a planktotrophic larva. At the apex of the shell (top) is the apical organ. The long cilia of corona ciliata are visible at the base of the triangle. These are used to propell the larva through the water, and to collect microscopic food particles.

Rafferty, K. 20o2. Bryozoa. In: An identification guide to the larval marine invertebrates of the Pacific Northwest. Edited by Alan Shanks. Oregon State University Press.

Actinotroch larva of Phoronis pallida

This actinotroch larva belongs to the horseshoe worm Phoronis pallida. George and I found quite a few of these in a plankton tow we took from the floating dock in Port Orford, OR in October 2009. This actinotroch is distingushed by a rather small size (about 600 micron long) compared to larvae of other local phoronid species. Like the actinotroch of Phoronis vancouverensis, it is opaque (Johnson 2002). One can tell it apart from the larva of P. vancouverensis by a pigment band located posterior to the tentacles on the ventral side (ventral is to the left and anterior is up on this picture), and the bow-tie shaped blood corpuscular mass in the hood (you can see the reddish mass in the hood on this picture taken from the side, but the bow-tie shape is only visible in frontal aspect). The telotroch - a prominent ring of long cilia at the posterior end is used to propell the larva through the water.

In that same plankton tow we found LOTS and LOTS of bryozoan cyphonautes larvae. We were able to distinguish at least three kinds: the transparent and triangular ones which I think might belong to Membranipora, the oval-shaped non-trasparent ones, which might belong to Electra, and the encrusted triangular ones which might belong to a species of Conopeum (see next post).

Johnson, K. 2002. Phoronida. In: An identification guide to the larval marine invertebrates of the pacific northwest. Edited by Alan Shanks. Oregon State University Press.

Calliostoma veliger larva

On 04/26/2010 I started a culture of a marine snail species, Calliostoma ligatum. After observing the early cleavage stages in the first ten hours after fertilization, I didn’t look at the culture for a week. By then, the embryos had turned into veliger larvae. Through the dissecting microscope, the veliger’s velum looked irridescent. The effect was generated by the rapid beating of the long cilia along the rim of the velum. I put a veliger under the compound microscope and found that I could not see the rainbow effect under transmitted light.

However, it was really cool to watch the larva retracting its foot and velum and closing its operculum (trap door) to seal itself into its shell. Then after several seconds, it would use its foot to push its operculum open, and evert its foot and velum. I took a series of pictures showing the veliger slowly evert its foot and part of its velum from the shell. Each picture also shows the hexagonal honeycomb-like texture of the shell, which I think looks cool. These larvae are fun to watch and I look forward to observing the continued development of my Calliostoma culture. See more pictures of Calliostoma veligers.

Development of sand dollar pluteus larva

On 03/29/2010, I started a culture of the sand dollar Dendraster excentricus. I observed its development from the raising of the fertilization envelope through the pluteus stage (several days later). The formation of the third arm pair, the postero-dorsal arms, was an especially exciting event.

One week after fertilization, I noticed a pair of buds between the post-oral (the longest pair) and the antero-lateral arms, and quickly realized that they were the postero-dorsal arm buds (top picture). I was impressed to see that the calcareous spicules which provide support for the arms were already present in the arm buds. I took a picture two days later using two polarizing filters to highlight the spicules (middle picture).

By two weeks after fertilization, the larvae in my culture had become 6- armed plutei (bottom picture). As I observed and drew this stage, I couldn’t quite figure out how the spicules in the postero-dorsal arm rods connected with the rest of the larval skeleton. I assumed that the spicules in the arms simply branched off the existing tri-radiate spicule, which projects branches into post-oral and antero-lateral arms, as well as the larval body. I was surprised to find out that these spicules are not connected to the rest of the skeleton.

Planuliform Nemertean Larvae

In the first of week of January I found egg strings of the nemertean worm Carcinonemertes errans in among the egg mass of a Dungeness crab (Cancer magister). These worms feed on the crabs eggs, acting like parasitic castrators to their hosts. The embryos within the worm egg strings began to hatch out after I agitated them a bit with my forceps.

The larvae that emerged were planuliform uniformly ciliated "blobs" as Svetlana calls them, only about 100 microns long. They contained lipid droplets and two eye spots. Each larva had cirri at both their posterior and anterior ends. These cirri, together with the cilia covering the body, were used for locomotion, helping the larvae swim in a distinctly zig-zag pattern.

I kept the larvae in large glass containers, changing the water every two days. By the time the larvae were three weeks old they had begun elongating and looking a lot more like young worms. The rudiments of many juvenile structures are present within the larvae, including the proboscis.

To date, I have several cultures of C. errans larvae, the oldest of which are nearly 50 days old. They have not begun to settle out yet, nor have they developed much further, which leads me to think that they might have a rather long planktonic life span or need more specific food and/or settlement cues that I have not yet exposed them to. I'll just have to keep trying new things. Isn't science great?

Pilidia in the plankton

On February 11, 2010 Marley Jarvis, a PhD student in Alan Shanks' lab at the OIMB, did a surface plankton tow off the F dock in the Charleston marina (Charleston, OR). She timed the tow to sample at high tide and used a 153 micron plankton net. She caught a lot of interesting animals, including several kinds of nemertean larvae. One of the most common morphotypes of nemertean larvae was a pilidium larva, which we named Pilidium megacephala because it has relatively large cephalic imaginal discs. Likely, this larva belongs to the genus Micrura. We are currently working to get the DNA sequence from it, to try to identify which species it belongs to.

Another kind of nemertean larva Marley found was also a pilidium, but of a different sort. Its morphology suggests that it belongs to the palaeonemertean genus Hubrechtella, or one of its relatives in the family Hubrechtiidae. It is characterized by relatively small lateral lappets displaced toward the posterior, a large anterior lobe, very large and conspicuous squamous epithelial cells with prominent nuclei, and lack of a muscle retractor, which in other pilidium larvae connects the apical organ to the esophagus. Our pilidium resembles the type of larva called Pilidium auriculatum described by Leuckart and Pagenstecker (1858) from the European waters. A similar pilidium from Swedish waters was identified as belonging to Hubrechtella dubia by Cantell (1969). Pilidium auriculatum type of larva had also been found in the Gulf of Mexico (George von Dassow, pers. comm.), in the Gulf Stream off the Atlantic Coast of Florida (Norenburg and Stricker, 2002) and in the Sea of Japan (Chernyshev, 2001). So far no hubrechtid nemerteans are known to occur on the West Coast of North America.

Cantell, C.-E. 1969. Morphology, development, and biology of the pilidium larvae (Nemertini) from the Swedish West Coast. Zool. Bidr. Uppsala. 38:61-112.

Chernyshev, AV. 2001. The larvae of unarmed nemerteans in Peter the Great Bay (Sea of Japan). Russian Journal of Marine Biology. 27(1): 58-61.

Leuckart, R. and A. Pagenstecker. 1858. Untersuchungen über niedere Seethiere. [Studies of lower marine animals]. In German. Arch. Anat. Phys. p. 569-588.

Norenburg and Stricker. 2002. Nemertea. Atlas of Marine Invertebrate Larvae, C. M. Young, M. A. Sewall, and M. E. Rice, eds. Academic Press, San Diego. Pp. 163–177.

Enterocoely in Chaetognaths

Marley Jarvis did a plankton tow at the F dock last week, and among the contents were many chaetognath embryos, almost certainly Sagitta elegans (because adults of these are common here, and a few small ones were in the same plankton sample). A couple were just finishing gastrulation. I made a time-lapse film of one of them overnight, and it so happened that it was a transverse view at about the middle of the embryo. This means that the formation of coeloms, as pouches that are pinched off of the archenteron, is easily visible in this movie.

At the beginning of the movie, the archenteron has folded all the way in. Primordial germ cells (four of them) emerge from the roof of the archenteron while the blastopore closes. The embryo begins to elongate along the anterior-posterior axis (not visible yet) as folds grow down from the roof of the archenteron. These drag the primordial germ cells with them. The coelomic compartments are the dorso-lateral chambers thus carved out by ingression of these folds. I am not clear on what becomes of the actual archenteron, because it's not visible in this film, and in embryos after this stage, the apparent archenteron is quite small. It may form by re-opening the space between the two ingressing folds.