Chiton trochophore

These are pictures of a chiton trochophore larva, which hatched from an egg picked out from plankton and given to the Embryology class by Dr. Richard Emlet. Because it was colledcted from plankton rather than from a spawning event by a known adult, I don’t know what species this larva belongs to. A trochophore is the larva of a spiralian animal, in this case a polyplacophoran mollusk, or a chiton. The trochophore is characterized by the prototroch, a pre-oral ring of ciliated cells that propels the larva though the water. You can see the long prototroch cilia on both of these pictures. This chiton larva was very opaque, so I used a dark-field technique to make some structures, e.g. eye spots, become more apparent. Chiton trochophores have two eyes located ventro-laterally posterior to the prototroch. While in some trochophores the ciliated band is involved in feeding as well as locomotion, it is not the case in chiton larvae which are lecithitrophic, or non-feeding, and depend entirely on the yolk supplied in the egg.

The second picture is taken using bright-field microscopy, and shows a series of seven bumps on the dorsal side posterior to the prototroch. Each of those bumps will become one of the distinctive shell plates of the adult chiton. I could clearly see the lines that mark the plates run down the side of the trochophore as it swam around. I was told that later another plate will form anterior to the ciliated band to bring the total number to eight, the number found in adult chitons.

Molt of a larval barnacle

On Wednesday, April the 13th, our embryology class collected plankton from the mouth of the Coos Bay in Oregon. Amongst the microscopic organisms there were lots of beautifully transparent naupliar molts. Nauplius is the planktotrophic (feeding) or lecithotrophic (nonfeeding) larval stage of many crustaceans. Pictured here is a naupliar molt of a barnacle (characterized by the two frontal horns) in dark-field. I identified this one as likely belonging to either Balanus glandula or Balanus improvisus. In adult barnacles, thousands of fertilized eggs are held in pouches, called ovisacs. Nauplii hatch from the ovisacs. Once in the plankton, they grow and undergo a series of up to six molts shedding their exoskeleton. The molts are often found in the plankton; they retain the external features of the larva, so one can identify which stage and species it belongs to. This molt is a stage IV. Stage VI nauplius undergoes the final molt and metamorphoses into a bean-shaped nonfeeding cyprid larva, which later settles onto the substratum and develops into the adult barnacle. These two pictures are of the same molt, at slightly different focal planes, to show different features. The top picture shows the fronto-lateral horns, and the dorsal thoracic spine located at the posterior end. Also in focus is a pair of mandibles (posterior-most bifurcated appendages) and a pair of antennae (bifurcated appendages anterior to the mandibles). These appendages bear bristles called setae, and along with the antennulae (anterior-most appendages, in focus on bottom picture), are used for feeding and locomotion. The nauplius has a single naupliar eye which you cannot see in the molt. The two-pronged furcal ramus at the posterior end (bottom picture) is a feature commonly used to identify the nauplii of different species. One can also see the labrum, a posteriorly-directed fold of tissue which extends over the mouth, located in the center of the cephalic shield, and the two small spikes in between the antennulae, called frontal filaments.

Imaginal discs in the pilidium

On April 13th 2011 I found a pilidium larva in a plankton tow collected off the F dock in the marina in Charleston, OR. A pilidium is a planktonic larva of some nemertean worms, predominately from the order Heteronemertea. Pilidium larvae form eight juvenile rudiments which will eventually make up the adult worm (Maslakova 2010). These rudiments, called imaginal discs, fuse around the larval gut and form the juvenile worm. In the pilidium larva there are three pairs of imaginal discs, plus two unpaired rudiments (the dorsal disc, and the proboscis rudiment).

These two pictures show the same larva in two different focal planes. This particular larva has developed two pairs of imaginal discs and the proboscis rudiment. Larval anterior, marked by the apical plate, is up. In the first image one of the cephalic discs and one of the trunk discs is in focus. The cephalic discs (to the right on this picture) give rise to the head of the juvenile worm, including the cerebral ganglia. The trunk discs are to the left on this image, directly underneath the stomach, which the large round shape inside the pilidium larva. The trunk discs give rise to the vast majority of the trunk ectoderm of the juvenile. On the second image the proboscis rudiment is in focus - it is the small round blob underlying the pilidial epidermis almost directly above the cephalic discs (at about 2 o'clock in this picture). It will fuse with the cephalic discs and form the proboscis in the adult worm. Paired cerebral organ discs and an unpaired dorsal rudiment also form in the pilidium larva, but these are not yet developed in the photographed larva. The cerebral organ discs give rise to the cerebral organs (chemosensory structures), while the dorsal rudiment contributes to the formation of the adult ectoderm.

S. A. Maslakova. 2010. Development to metamorphosis of the nemertean pilidium larva. Frontiers in Zoology 7:30

Zoea larva of a brachyuran crab

Among plankton sampled on April 16, 2011 by our Embryology class, I found a few crustacean larvae in the zoea stage. The top picture is a regular bright-field photomicrograph, and the bottom one is a dark-field image. A zoea is a larval stage in the development of crabs and other decapod crustaceans. This stage follows the nauplius stage, which (in most decapods) is passed in the egg, and precedes the post-larval planktonic megalopa stage. Zoea larvae swim using thoracic appendages, (maxillipeds and pereopods), which distinguishes them from both earlier and later stages of development. The nauplius larva uses cephalic appendages to swim (antennulae, antennae and mandibles), while the megalopa swims using abdominal appendages called pleopods. I watched my specimens swim—they would swim for a short while, stop, and then continue swimming in another direction.

Zoea larva of a brachyuran crab (shown here) has a dorsal spine, a rostral spine (the anterior-most spine), and two lateral spines, which all extend from the carapace. These spines are thought to aid the larva in directional swimming, and could also be used as a defense against predators. A zoea feeds using the endopodites (inner branch) of bi-ramous maxillipeds and, depending on the species, can be carnivorous, phytoplanktivorous or omnivorous. The zoea has two stalked compound eyes that are relatively large, compared to the rest of the body. You can see on these photos that the diameter of the eye is nearly one third of the diameter of the carapace (not including spines).  These photos also show two maxillipeds (the two long appendages visible between the rostral and lateral spines). The anterior-most maxilliped contains the endopodites that are used for feeding. In the adult, the maxillipeds (including a third pair, which is not present in this stage) are associated with the mouth. Sadly, the abdomen is turned, so only one of the developing pleopods can just barely be seen on the dark-field image.

Coronate larva of Bugula sp.

This is a dark-field photomicrograph of a coronate larva of the gymnolaemate bryozoan Bugula sp. Our instructor, Svetlana Maslakova collected colonies of this species from the docks in the Charleston Boat Basin and kept them in the dark until 11 am of the following day. Exposure to light stimulates release of brooded coronate larvae in many bryozoans, including Bugula. Coronate larvae are lecithotrophic (i.e. non-feeding), and there is no trace of a gut, mouth or anus. These larvae are round and opaque, and covered with ciliated epidermis, called corona ciliata. Cilia of corona ciliata propell coronate larva through the water. The larvae of Bugula sp. have two eye spots, which you can see on this picture, and are able to detect the direction from which the light is coming. These coronate larvae swim toward light, and they swim very fast! Aside from the two eye spots, and some pigment spots in the epidermis, and the ciliated epidermis, larvae of Bugula sp. have few features. The most prominent of them, is perhaps the vibratile plume - the tuft of long cilia on one side, which looks like a little flame (at about 1 o’clock on this picture). Vibratile plume is a part of the pyriform organ, a glandulo-sensory structure which plays a role in selection of substrate for metamorphosis.

Actinotroch larva from plankton

On April 13th 2011, our class collected plankton off the F dock in the Charleston marina. I was lucky enough to find an actinotroch larva pictured here. These photos show the same larva in lateral aspect in two different focal planes to emphasize different structures. The first photo is focused on the surface structures, the tentacles, in particular. The second photo is focused on the digestive tract. The larval anterior end is up. The actinotroch is a planktotrophic (feeding on plankton) larva of horseshoe worms (phylum Phoronida). This larva is characterized by a pre-oral hood (at the anterior end), a crown of tentacles posterior to the mouth, and a telotroch, a ring of long cilia at the posterior end. The larval tentacles are paired and located around the middle of the larva, the younger shorter ones near the dorsal midline, and the longest ones near ventral midline. These ciliated tentacles aid in swimming and feeding. The powerful telotroch consists of fused cilia and is used for locomotion. The preoral hood is fringed by a ciliated band. The hood plays a role in the transport of food to the mouth, and is distinctive of actinotrocha. The digestive tract of the actinotroch larva is relatively simple. The mouth located under the hood opens into the spacious stomach, the large oval shape that occupies much of the larval body. Stomach opens into the narrow cylindrical hindgut, which opens to the outside via an anus at the posterior end. After some weeks in the plankton the actinotroch metamorphoses into an adult sessile phoronid worm.

Life cycle of hydrozoan Obelia sp.

This is a picture of a beautiful hydromedusa of the genus Obelia. In the center, you can see the manubrium - a little stalk at the tip of which the mouth opens. You can also see the four gonads. The medusa is the sexual stage of the hydrozoan life history! The four faint lines radiating from the manubrium toward the perimeter and underlying the gonads are the radial canals of the digestive system. They join the marginal ring canal and supply nutrients to the tentacles.

When the focal plane is just right, statocysts (balance organs) at the base of the tentacles are visible. You can see one of them at about 3 o'clock on this picture. Obelia is a classic example of alternation of generations in a hydrozoan life cycle. The medusa produces eggs and sperm. When fertilized, an egg develops into a simple non-feeding planula larva. The planktonic planula metamorphoses into a benthic polyp (hydroid), which buds off further polyps and forms a colony. Hydroid colonies have different kinds of zooids specialized for different functions.

This picture shows a small fragment of a colony, including a feeding zooid, its tentacles withdrawn into its goblet-shaped theca, at about 3 o’clock. A larger balloon-like structure at about 5 o’clock is the reproductive zooid, called
the gonangium. Gonangium includes gonophores (budding medusa-zooids), which you can see as round shapes inside the gonangium. When ready, the medusa buds off and swims away, as you can see in the bottom picture. Obelia's medusa was the most remarkable animal I have seen so far in this embryology class. I just wish to see its embryos someday.

Spicules or no spicules?

In our embryology class we have spent a lot of time raising and observing the development of echinoids and asteroids. The first two pictures show the planktotrophic, or feeding on plankton, larvae of these two classes of echinoderms. The top one is the pluteus larva of the sand dollar, Dendraster excentricus. Unlike other echinoderm larvae, the pluteus larva has long arms supported by calcareous skeletal rods, which you can see on this picture. A contiguous circumoral ciliated band spans the arms of the pluteus larva.

This picture shows bipinnaria larva of the ochre seastar, Pisaster ochraceus. The bipinnaria is characterized by a circumoral ciliary band divided into a pre-oral and post-oral loops, and lacks calcareous spicules. I was amazed at the difference in structure of these two feeding larvae, namely the presence and absence of the spicules. I did not know why these two related organisms had such different larvae. It turns out that this difference has a developmental explanation. Early cleavage in echinoids and asteroids is very similar. However, starting at the 16-cell stage, there is a subtle difference. In asteroids, the division from 8-cell-stage to 16-cell stage is equal, i.e. all 16 cells are of the same size. In most echinoids this division is unequal, i.e. it results in cells of different sizes.

The cells at the animal pole divide equally into eight cells, called mesomeres but the cells at the vegetal pole divide unequally into four large cells, called macromeres, and four small cells, called micromeres. The micromeres give rise to a population of cells in the early gastrula of echinoids, called primary mesenchyme. Primary mesenchyme cells ingress into the blastocoel before the primary gut starts to invaginate. In the third picture you can see the primary mesenchyme cells inside the blastocoel of a gastrula of D. excentricus. The primary mesenchyme cells go on to make the spicules in the pluteus larva. In the starfish the micromeres are absent, and there is no primary mesenchyme. Gastrulation in starfish begins with invagination of the primary gut, as you can see on the bottom picture of the early gastrula of P. ochraceus. The absence of the micromeres and the primary mesenchyme is causally related to the lack of larval spicules in the bipinnaria. That is amazing!

Field Trip to South Cove

The morning of April 20, 2011, we, students in the Embryology class at OIMB, made our way to the rocky intertidal at South Cove, located at the southernmost end of the Cape Arago Highway, near Charleston, OR.   We unloaded from the van with buckets, butter knives and small tubes, donned our rain gear, and descended the trail towards the beach.

Our task was to search throughout and beneath the boulder field, exposed by the low tide, for several kinds of organisms; specifically, several types of bryozoans. These included Crisia sp., Flustrellidra corniculata, and Dendrobaenia lichenoides.   Bryozoans are colonial “moss-animals” that can often be found growing under overhanging rocks or encrusted upon them. To enhance our study of mollusc development, we also looked for chitons, and the gastropod Calliostoma ligatum.

We tipped over rocks (and put them back where we found them), and after a couple hours of searching, we had found at least a few specimens of every one of our target species including one colony of the unusual, and rather uncommon on the intertidal, bryozoan Flustrellidra corniculata, which has a brooded lecithotrophic pseudocyphonautes larva (see post by Tony Dores).

Echinus rudiment formation

Shown here is the echinopluteus larva of the purple sea urchin, Stronglyocentrotus purpuratus (top image), and a sand dollar Dendraster excentricus (bottom image). Larval anterior is up. In both pictures, you can see the juvenile sea urchin or sand dollar developing inside the larval body. The rudiment of the juvenile, called the echinus rudiment develops in a pouch called a vestibule, which forms as an unpaired epidermal invagination on the left side of the larval body. The left coelomic sac contributes to the formation of the juvenile. The top picture shows the three-week old larva of S. purpuratus from the ventral side. This larva does not yet have a well-developed echinus rudiment, but the invagination can be seen clearly to the right and above the stomach, which is the large dark oval shape in the middle of the larva.

The three-week old D. excentricus larva shown here has a much more advanced juvenile rudiment than the S. purpuratus larva. The larva is shown from ventro-lateral view, so that the juvenile rudiment is facing you. The juvenile rudiment is quite large and takes up most of the posterior portion of the larva, obscuring the larval stomach, which barely peaks out from behind the juvenile rudiment on the left. By the late pluteus stage, the juvenile is almost fully developed within the vestibule, and will have acquired juvenile spines and skeletal structures. The juvenile also has five podia, or tube feet, on the oral side of the juvenile, which is the side facing away from the larval stomach. Juvenile tube feet can be extended out of the vestibule and retracted again. This D. excentricus juvenile has developed podia, which appear as five lobes on the right. When ready to settle, the juvenile extends its podia from the vestibule, contacts the substrate, and walks away, having re-absorbed much of the larval body.

Coelom formation in bipinnaria

We are raising bipinnaria larvae of the sea star, Pisaster ochraceus in our Embryology class at OIMB! I was particularly interested in the development of coelomic sacs in the bipinnaria. These pictures show three different stages of development of coelomic sacks with larval anterior oriented up.

The top picture is of a 2-day old late gastrula, and the elongated cylinder inside the gastrula is the archenteron, or primary gut. You may notice that the tip of the archenteron is slightly T-shaped. This is because during gastrulation, before the archenteron makes contact with the oral epithelium, two coeloms form as pouches from the tip of the archenteron, during a process called enterocoely.

The coeloms bud off from the archenteron forming a sac on the left and right side of the gut as you can see in 4-day old bipinnaria larva in the middle picture.

The bottom picture is a dorsal view of a 12-day old bipinnaria with well-developed coelomic sacs. It is interesting to note the larger size of the left coelom because the two coeloms have different roles in development. The left coelom, called the hydrocoel, is connected to the dorsal epithelium via a hydropore canal, and opens to the environment via a small round hole, called the hydropore, which you can see as a small dark shape to the upper left of the larval stomach (the upside-down pear-shaped structure occupying the posterior portion of the larva). The hydrocoel will form the water-vascular system of the adult sea star. I look forward to continuing to watch the development of the Pisaster ochraceus larvae as well as the development of their coelomic sacs!

Regeneration in bipinnaria

The top picture on the left shows a bipinnaria larva of the starfish Pisaster ochraceus. One interesting characteristic of this organism, and the starfish in general, is its ability to regenerate missing body parts both as a larva and as an adult. I read that bisected starfish larvae have been observed to regenerate to form complete larvae within 12-14 days (Vickery and McClintock, 1998). I wanted to see it myself, so on April 13, 2011 I surgically bisected several bipinnarias across the middle, separating the anterior from the posterior portion. The two bottom pictures show the divorced anterior and posterior portions of the bisected larva. I took these pictures within 5 minutes of the surgery. If you look closely you can already see that each fragment is closing the wound! Amazing!

Cutting the larvae was difficult, because these bipinnarias keep swimming around. In addition to tracking and anticipating their movements, I had to be careful not to break the tip of the glass needle I used to make a cut. I pulled pipettes using a Sutter Micropipette Puller. They are so sharp, their ends so miniscule, that they cannot be seen with the naked eye. I broke 5 needles while cutting 15 individuals!

Finally, towards the end of the procedure, I started to get the hang of trapping the larva and cutting it without poking myself or breaking the needle! After the bisection, the two pieces of the larva swam away as if nothing had happened! I will follow the regeneration of these larvae, taking pictures as they develop.  I hope to witness organogenesis, the formation of organs, until the larvae are complete once again!

Vickery, MS and McClintock, JB. 1998. Regeneration in metazoan larvae. Nature. 394: 140

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

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.

Nudibranch veliger larva

Here are a few pictures of the veliger larva of the frosted nudibranch (a type of sea slug), Dirona albolineata. In 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.

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.

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).

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 7^th, 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.

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.