Sunday, June 6, 2010

Blastomere Separation: Part 3

This Sunday, June 6, my experimental purple urchins will have their two-month birthday!! In my opinion, this is quite a feat. I feel like a proud parent! Of the 10-12 urchins I performed surgery on at the 4-cell stage, 3 have survived and appear to be developing normally. Taking pictures is a little nerve-racking. It's not getting them to smile for the camera that's tricky, but getting them ON and OFF the slide safely and without too much heat exposure from the microscope light. For this reason I waited to take more pictures. (I wasn't just trying to keep you in suspense!) But I couldn't wait any longer to share this next developmental milestone.
The two pictures to the left are basically the same image, however, one is taken using bright field microscopy (top) and the other - using dark field technique (bottom). At this time the larva was just over 7 weeks old. To orient you, this is a ventral view with the anterior to the right, posterior to the left, right side at the top, and the left side on the bottom. The large non-transparent circle in the center is the stomach and right below the stomach is the juvenile rudiment. This is the developmental milestone I am referring to above. The juvenile rudiment usually develops on the left side, except in rare cases, and will go on to become the juvenile urchin. It will develop tube feet and spines and eventually protrude from the larval body. The juvenile will reabsorb most of the larval body in a process known as metamorphosis, and will walk way on its tiny tube feet. At the most posterior end of the larva there is a thick ciliated band called the epaulette. It assists the larva in locomotion, and it if you look closely at both ends you can see the cilia.

This picture is of a different experimental urchin. This is a dorsal view which is why the juvenile rudiment is above the stomach rather than below it. Also, this picture was taken with a 4x magnification lens while the previous were taken with a 10x. (The larvae were approximately the same size). The larval arms aren't quite as straight as on the previous pictures - maybe it was waving to the camera. However, what is interesting about this larva is the size of the juvenile rudiment. It's about the same size, if not larger, than the stomach. It is not uncommon for larvae of the same age to have rudiments of different sizes. Despite the fact that our Embryology class is ending on June 7th, I will continue to rear these larvae. I hope my next pictures are an illustration of urchin metamorphosis!

See also my earlier posts on blastomere separations: Part 1   Part 2

Friday, June 4, 2010

Field Trip to South Cove

On May 3rd after listening to a lecture on fertilization ecology by Dr. Craig Young, the Embryology class piled into OIMB’s 15-passenger van and headed out to Cape Arago. Decked out in our rubber boots and rain gear, we descended the winding path from the road at the hill top to the rocky intertidal of South Cove. The tide was a -0.4 that day, exposing thousands of rocks and boulders to our searching eyes. Specifically, we were hunting for bryozoans, colonial “moss animals” that can often be found growing under overhanging rocks or encrusted on them. But while we were down there, we certainly weren’t going to pass up the chance to explore and find as many amazing organisms as we could!

Although the entire trip was informative and entertaining, I think the highlight for everyone was watching our professor, Dr. Svetlana Maslakova, crack open a sea urchin and eat the roe right out of it!

“A little salty, but good,” she said. Several of the students followed her example.

As the tide began to turn, and the rocks were covered once more by the sea, we climbed back up the hill, bryozoans in tow, to return to our classroom and study the embryology of this unique and fascinating phylum of animals.

Mudflat Field Trip

The morning of April 19, 2010, was a rainy one, but that didn’t stop the Embryology class at OIMB from heading out to the mudflats of the Coos Bay estuary during the low tide. Our mission: to find several kinds of worms by digging in the muddy sand. To enhance our studies of spiralian development, we wanted to find nemertean worms (Micrura and Cerebratulus) as well as the tube-dwelling polychaete Owenia, which has a unique larva called the mitraria. We also were keeping a look out for a tube-dwelling worm from another phylum, the phoronid Phoronopsis harmeri.

The going was slow as we slogged through the muck and picked through the mud, but after a couple hours of searching and digging, we had found at least a few specimens of every one of our target species. These we took back to the lab at OIMB, placed in flowing seawater tables, and studied over the next few weeks.

Plankton Tow

On the morning of Monday, April 12, the class climbed on board the 20-foot R/V Pugettia and set out for the Pacific Ocean to collect some plankton. Larry Draper, our boat captain, took us about a mile beyond the Coos Bay jetties, turned north to escape the river plume, and then cut the boat engine. We lowered our 153 µm-mesh plankton net into the water and allowed it to drift for several minutes. After we pulled the net out of the water, we dumped the contents of the cod end into a glass jar. When you held this up to the light, you could easily see thousands of tiny plants and animals floating and zooming around: plankton! We repeated this procedure inside the Coos Bay estuary so that we could compare the plankton between the two sites. The rest of the day was spent in the lab sorting through our catch with microscopes, observing the strange and beautiful creatures of the plankton community.

Wednesday, June 2, 2010

Nudibranch veliger larva

Here are a few pictures of the veliger larva of the frosted nudibranch (a type of sea slug), Dirona albolineataIn the top picture, you can see two larvae inside an egg capsule. Many other capsules in the same egg mass held 6-8 larvae in each. The shell of the veliger protects the internal organs, e.g. the nervous system, digestive tract, and the retractor muscles, which allow the veliger to pull the velum and foot into the shell. A pair of statocysts (little capsules that work as balance organs) are visible as well, each with a statolith (a tiny granule) inside. One can also see the velum, which has two rounded lobes with long cilia used for locomotion in the water column once the veligers hatch from the egg capsules. 

The bottom picture shows a hatched veliger from the side, with a developed foot. On the back of the foot is the thin, visible operculum, which acts as a trap door, closing the shell opening when the velum and the foot are pulled in. When the velum is pulled in, the larva can’t swim and sinks to the bottom. Once the larval stage is complete, settlement cues in the environment induce metamorphosis  (transformation into juvenile stage). This includes permanent loss of the velum and shell (and other larval organs, like the muscle retractor), as the foot becomes the main locomotory organ of a sea slug.

Friday, May 28, 2010

Trochophore larva of the polychaete Serpula

On 26 April 2010, I fertilized eggs of the polychaete Serpula columbiana (red tube worm) from adults collected from the floating docks in Charleston, OR marina. These polychaete annelids live in calcareous coiled tubes and have a red plume of radioles (feathery tentacles) that aids in suspension and filter feeding. As with many other polychaetes, top-shaped trochophore larva is the first swimming stage. These trochophore larvae live in the plankton, and eventually settle and metamorphose into the adult worm. The trochophore larva is characterized by the presence of the prototroch, a preoral band of cilia, which beat rapidly to propell the larva through the water. These pictures show a 21-day-old trochophore larva of Serpula columbiana. The prototroch is clearly seen around the widest part of the larva. The apical tuft of cilia, located at the anterior end is partially in focus on the bottom photo (at about 8 o'clock). In the lateral view, the mouth is seen near the top and between the prototroch and metatroch (another circumferential band of cilia posterior to the mouth). The mouth leads to a heavily ciliated gut (brown in the photo). See pictures of a later developmental stage of this species.

Thursday, May 27, 2010

Twin juvenile rudiment in purple urchin larva

On March 29 2010, the first day of our Embryology class, we started our own cultures of sea urchin embryos. I have had the opportunity to observe the different stages of development of the purple urchin, Strongylocentrotus purpuratus. Last week while looking at this echinopluteus through a compound microscope I noticed something that I have not seen before. There were two juvenile rudiments growing inside the larva. Normally, a single rudiment develops on the left side of the larva. In this specimen however, there is one rudiment on the left side of the larval stomach (greenish oval shape roughly in the middle of the larval body) and another rudiment (with protruding tube feet) on the right side. This is a ventral view and the larval anterior is up.

A week later I checked on the twins to see how they were developing. The juvenile urchins have grown very large and the larval body mostly degenerated. Three of the remaining larval arms are visible on the upper right in the bottom picture. Because this specimen was so thick and non-transparent it was difficult to get a clear picture in the transmitted light. This picture  is taken using cross-polarized light to make the juvenile skeletal spicules glow. You can see the spines from the two different juvenile rudiments - some to the left and others to the right of the plane of bilateral symmetry of the larva (which cuts across from the upper right to the bottom left of the picture). The larger of the two rudiments in the first picture was more developed and is now mobile on its tube feet. The other rudiment has spines, but I could not see any tube feet stick out.

The case of twins in this situation intrigues me very much. I am a fraternal twin myself, which means that my twin sister came from a different fertilized egg. These urchin twins came from a single fertilized egg, but they are conjoined (share a single gut). They each have an oral side, but have no aboral side. Likely they will not survive much longer after metamorphosis (S. A. Maslakova, pers. communication).

Tuesday, May 25, 2010

Sabellaria cementarium larvae

While raising larval cultures in my embryology class I observed development of the polychaete Sabellaria cementarium which has a trochophore larva. The trochophore is an early developmental stage of marine animals such as annelids and mollusks. Trochophore larvae have a transverse ciliary band that assists in locomotion, sensory reception, and sometimes feeding. This ciliary band is called the prototroch. It separates the episphere, anterior region, from the hyposphere, posterior region. The mouth is located very close to the prototroch (on the downstream side). So that a current created by beating cilia on the prototroch brings food particles to the mouth.
The Sabellaria cementarium trochophore larvae have two large bundles of setae on the trunk. These setae are barbed and are longer than the larva itself. They are held close to the body when the larva is swimming (see another post by Kristina Sawyer), but can be fanned out when the larva stops moving. In 1984 J. Timothy Pennington and Fu-Shiang Chia observed Sabellaria cementarium larvae using their setae to prevent recognition and capture by predators, such as ctenophores (comb jellies). The barbed setae also might irritate the oral tissues of the predator and act as a deterrent. At the posterior end there is another ciliated band, which assist in swimming called the telotroch.

Juvenile Sand Dollar

On May 7th, I was able to see the end product of metamorphosis of the sand dollar Dendraster excentricus in my culture. It was so exciting to finally see a juvenile sand dollar after watching it go through different stages of development starting from fertilization (at the end of March), and be able to photograph and document the transformation. This first photo shows the secondary podia (tube feet) quite well. They are the longer, more transparent “limbs” coming off the body with cross-containing circles at the tips. At this stage and from this angle, it is more difficult to see the primary podia. Tube feet are used primarily for movement and attachment.

The spines of the sand dollar are the darker “limbs” with no circles at the tips. There are large and small spines, which are homologous to the interambulacral and ambulacral spines in sea stars. The sand dollar juveniles also have two extra large spines marking the posterior end of the animal. These extra long spines can be seen more clearly in the two dark-field photos at the bottom right portion of the juvenile urchin. The middle photo shows the spines of the sand dollar clearly. In the bottom photo, one can see black network-like pigment cells on the aboral (the opposite of oral, which is facing down) side of the juvenile.

Feeding in echinopluteus larva

Planktonic larvae of the sand dollar, Dendraster excentricus, can remain in the water column for various amounts of time from a few weeks up to two months (Emlet, 1986). They have cilia, that help them feed and move in the water. These pictures show six-armed pluteus larva of D. excentricus from ventral side (where the mouth opens). Larval mouth is facing us. It is surrounded by a circumoral (= around the mouth) ciliated band, stretched out on the larval arms. The cilia in the ciliated band direct food, such as microscopic algal cells, into the mouth. The top picture shows the post-oral (= posterior to the mouth) portion of the ciliated band stretched between the two post-oral larval arms. The second picture is of the same larva, but in a different focal plane, showing the pre-oral portion (anterior to the mouth) of the ciliated band. The third picture shows the same larva, in a different (deeper) focal plane. I am now focussing on the larval gut. From the mouth the food particles are directed into the esophagus (the anterior portion of the larval gut). The mouth (upper left) and esophagus together make up the bulb-like shape. Mouth is the “head” of the bulb, and esophagus is the narrower portion. 
The esophagus is surrounded by a layer of circular muscles. Peristaltic constrictions of these muscles force the food particles toward the stomach (the middle portion of the larval gut, separated from the esophagus by a cardiac sphincter). Musculature in the esophagus helps open the cardiac sphincter to allow food to enter the stomach (Burke, 1981). The stomach is the large oval shape occupying majority of the space inside the body of the pluteus larva. The sphincter is the lentil-like shape between the esophagus and the stomach.

Thursday, May 20, 2010

Early spicule development in Dendraster exentricus

On April 11, 2010 I observed the early spicule formation in one-day-old embryos of the sand dollar, Dendraster excentricus. The embryos hatched from their fertilization envelopes and have nearly completed gastrulation, you can see the invaginated primary gut (archenteron) almost touching the roof of the blastocoel (the space between the outer and inner layer of cells in the embryo). Particularly noticeable were the progeny of the micromeres, which are the small cells at the vegetal pole at the 16 cell stage. These cell give rise to the primary mesenchyme cells, which ingress into the blastocoel, and secrete the calcareous spicules, which form the larval skeleton.

By using cross-polarized light (one polarizing filter placed above the specimen and one below), I was able to visualize the spicules in stark contrast. With this technique, the light that passes through the first polarizer is blocked by the second, and the only structures that remain bright are those that rotate the plane of polarized light e.g. various crystalline structures - in this case skeletal spicules, composed of calcium carbonate. This highlights the spicules on a dark background. The first two spicules in urchin larvae form at the base of the archenteron, one on each side, where the primary mesenchyme cells are concentrated. The initial spicule is tri-radiate. The three branches grow and form the postoral and antero-lateral arm rods, and the body rod of the pluteus larva.

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.

Wednesday, May 19, 2010

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, 2003The 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 26th, 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.

Sunday, May 16, 2010

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.

Friday, May 14, 2010

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).

Thursday, May 13, 2010

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".

Tuesday, May 11, 2010

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.