Metatrochophore larva of Serpula columbiana

These pictures show a 14-day-old metatrochophore larva of the polychaete worm Serpula columbiana, which we raised in the Embryology class. The larval anterior is rotated up. These larvae have several segments with chaete (chitinous bristles), a prototroch (pre-oral ciliated band) which is used in locomotion and feeding, and a metatroch (a post-oral ciliated band). The first photo is a dorsal view. You can see two large black eye-spots marking the anterior end of the larva. The gut is reddish-brown, and the circle near the posterior end of the larva is the anus. Several chaete are also in focus.

The second photo is a lateral view, ventral to the left.  One can see the entire digestive system. The mouth opens ventrally between the prototroch and the metatroch, while the anus is dorsal. One can also see a small apical tuft at the anterior end of the larva. The large clear vesicle posterior to the anus is called the anal vesicle, but I do not know its function.



The third photo is a ventral view, which shows particularly well the long prototrochal cilia, the mouth, and the metatroch. The last photo is a close-up of the larva’s ciliated food groove located between the prototroch and the metatroch. The cilia of the prototroch and metatroch beat in opposite directions, and the larva feeds by trapping microscopic particles between these two ciliary bands, then moving them along the ciliated food groove to the mouth. This is referred to as the “opposed-band” feeding mechanism, which is characteristic of many trochophore-type larvae. See the trochophore stage of this species.

Class trip to the mudflats

The morning of the 4th of May, our Embryology class met an hour earlier than usual and descended into the mudflats behind the “Fisherman’s Grotto” restaurant in Charleston, OR. We are studying the development of spiralians, a large supra-phyletic group of protostome animals that undergo spiral cleavage. Today our goal was to collect worms of two spiralian phyla (nemerteans and annelids), and a representative of a third (non-spiralian) phylum - the Phoronida, to culture in class.

First, we collected the polychaete annelid, Owenia collaris. Owenia lives in a soft, flexible sandy tube and has a very interesting larva, called the mitraria, unique among the polychaetes. We also collected two species of nemerteans which have the pilidium larva: Cerebratulus cf. marginatus (the large brown worm in the palm of a student in the third picture), and Micrura alaskensis. Cerebratulus is very fragile, and easily breaks into many pieces, so we had to be very careful when extracting the worm from the mud. M. alaskensis is a small, pink nemertean that looks like a tiny thread in the mud. Though small, M. alaskensis is common, ranges from Alaska to northern California, and is easily raised to metamorphosis on a diet of unicellular algae.

Last but not least, we collected the phoronid worms, Phoronopsis harmeri (formerly known as Phoronopsis viridis). P. harmeri lives in a thick rigid sandy tube 15-20 cm long. Phoronids are commonly known as “horseshoe worms” because of the characteristic crown of feeding tentacles, called the lophophore, arranged in a horseshoe pattern around the mouth of the adult worm. P. harmeri, like most phoronids, has a unique planktotrophic larva, the actinotroch, which is characterized by a pre-oral hood, and a crown of tentacles used for feeding.

Coronate larva of bryozoan Schizoporella

This is a dark-field picture of a coronate larva of a bryozoan Schizoporella japonica. The bryozoan colony was collected together with a mussel off a boat dock in Charleston, OR. Seen in this picture is the apical disc (lighter-colored circle in the middle) and on both sides of it - red pigment spots. Coronate larvae are non-feeding. This larva is brooded in an ooecium which is a specialized zooid dedicated to the care of the larva. Normally, only one embryo is brooded in an ooeciam, but on a rare occasion two may be observed. Release of these larvae is triggered by light. Schizoporella colonies reproduce throughout the year, although in some colonies reproduction declines in October and November. The maturing embryos in ooecia are approximately 250 microns in diameter. Schizoporella larvae range in color from red-orange to orange. As the embryos mature, the color changes from red to orange. These larvae settle in low intertidal and sub-tidal regions. See an earlier post about these larvae by OIMB graduate student Kira Treibergs.

Spionid nechtochaete

This is a picture of a larva of spionid polychaete (Fam. Spionidae, class Polychaeta, phylum Annelida). It was collected in a plankton tow off the F dock in Charleston, OR on April 13, 2011. Spionids have a small prostomium (segment anterior to the mouth) without antennae. Adult spionids are tube-dwelling worms that feed by extending two grooved tentacles called palps out of their tube into the water. On this picture you can see the primordial palps - the two appendages at the anterior end of the larva (up) as well as four eye spots. Some species of spionids shed eggs directly into the seawater; others keep eggs in their tubes during the early development. In spionids that brood only a few embryos develop, the others are called “nurse” eggs, and are eaten by the developing embryos. Spionids hatch at the stage of 3 setigers (segments bearing chaete). They can spend up to two months in the plankton, but usually they settle within three weeks. This larva already has over a dozen setigers. It is referred to as the nectochaete larva. In the plankton spionid larvae feed on unicellular green and golden-brown algae. Pigment derived from algal food is what likely colors the gut - the long tan-colored tube inside the larva you can see on this picture. Seen in the second picture is a close up of the segments to show the transverse ciliated bands found on many of the segments. These ciliated bands are used to propel the larva through the water.

Mitraria larva of the polychaete Owenia collaris

The polychaete worm Owenia collaris has a beautiful mitraria larval stage that we have been able to culture in lab. The following pictures show a week-old mitraria larva oriented with its anterior end up. The mitraria larva is transparent and has a through gut, a long equatorial ciliated band, and a bundle of very long larval chaetae (bristles).

The first picture taken at lower magnification shows the general shape of the larva, the entire digestive tract, the ciliated band (golden-brown ring that spans the broadest portion of the larval body) and the chaetae. The large brownish oval in the center of the larval body is the stomach, which is connected to the pink intestine (hindgut), on one side, and the greenish esophagus (foregut) on the other. The mouth opens below the ciliated band (on the right in this picture) and leads into the foregut. The intestine opens via the anus below the ciliated band near the center of the larval body.


The middle picture is a close-up view, highlighting some of the internal features such as the stomach and the foregut. The mitraria is a planktotrophic larva, and we have been feeding it unicellular algae, Rhodomonas and Dunaliella, whose pigments color the larval gut. The apical organ can be seen at the apex of the larval body from which a tuft extends.

The bottom picture is a ventral view of the larva, which showcases the paired chaetal sacs from which the chaetae protrude. This image also shows the round pink anus just anterior to the chaetal sacs. In the next three weeks or so, this larva will develop an invagination between the mouth and anus, which will form the trunk of the juvenile worm!

Actinotroch larva of Phoronopsis harmeri

This photo shows a six-day-old actinotroch larva of the phoronid worm Phoronopsis harmeri. Our class collected the adults from a mudflat just north of the Charleston bridge in Charleston, OR. Our instructor Dr. Svetlana Maslakova started a culture the week before our phoronid day, so that we had the opportunity to observe advanced developmental stages. Phoronopsis harmeri have separate sexes. The fertilization is internal, so that when one dissects a female, the eggs that fall out of the body coelom are already fertilized.

On this picture one can see the hood (at the anterior end of the larva, which is oriented up). One can also see the developing tentacular ridge (looks like a bump on the ventral side, which is to the right). Otherwise one can clearly see the gut, which consists of several regions. The mouth, which opens under the hood leads into the vestibulum, which is the large cavity under the hood. The vestibulum leads into the esophagus (or foregut), which looks like a wide tube with thick but colorless walls. The esophagus leads into the stomach. The stomach occupies the majority of the space inside the trunk of the larva and is colored orange-brown. The pigment is derived from the algal food. The stomach leads into the midgut (a short colorless region at the posterior end of the stomach), which in turn leads to the proctodaeum (or hindgut), a short pink-colored tube which opens via the anus at the posterior end of the body. See a more advanced larva of this species collected in plankton.

Veliger larvae of molluscs

Many gastropod (snails and their allies) and bivalve (clams, mussels, oysters and scallops) molluscs pass through a trochophore larval stage before developing into veliger larvae. Veligers are planktonic larvae of many bivalve and gastropod molluscs characterized by a shell, foot, and velum (a lobed, ciliated structure used for swimming and feeding). The velum is derived from the prototroch - a pre-oral ciliated band in the trochophore larva. A dorsal shell gland secretes the shell of the veliger. The shell of a bivalve veliger is bi-valved while the shell of a gastropod veliger resembles a spiraled snail shell.

The first image shows a veliger of the marine snail Nassarius fossatus in polarized light (hence the funny color). The spiraled shell of the veliger is in focus with the bi-lobed ciliated velum extended from the shell. A secondary (post-oral) ciliated band known as the metatroch is also visible below the main ciliated band of the velum.

In the second image is a veliger larva of the jingle shell, Pododesmus cepio. This larva has a bivalved shell. One valve is in focus while the other valve is located on the opposite side of the larva. The velum of this larva is pulled into the shell in this image, however its cilia can be just barely seen protruding beyond the edge of the shell.

Brooding seastar, Leptasterias hexactis

One of the organisms I had the opportunity to observe in my Embryology class was the small sea star Leptasterias hexactis. As the species’ name suggests it has six arms, rather than five, as is common for asteroids. Even more interesting is how Leptasterias reproduces. Typically, sea stars spawn by raising themselves up on their arms and releasing hundreds of thousands of small eggs into the surrounding water from the gonopores located on the aboral surface, which is the surface you would see if you were looking at a sea star clinging to a rock. Leptasterias, on the other hand, have their gonopores on the oral surface, which is the side that contains the mouth, and what most people would consider the “bottom” of the sea star. The female Leptasterias produces a relatively small number of eggs (a few hundred) and broods her eggs, which is to say instead of releasing her eggs into the water, she holds them under the oral surface.

The top picture shows the oral view of an individual with a brood (the large yellowish mass in the center). The eggs are large (almost a millimeter in diameter) and yolky and the development is lecithotrophic (i.e. non-feeding, or yolk dependent). After about 40 days the juveniles have tube feet, and the entire brooding period is about two months (Chia 1966). The mother does not feed during brooding. The two bottom pictures show an advanced juvenile of Leptasterias, which has been living independently for a week or two. The middle picture shows the oral side (mouth in middle) and the bottom picture shows the aboral side. The protrusions ending with little bulb-like suckers seen under the central disk and arms are the tube feet, which the juvenile uses for locomotion.

Chia, F.S. 1966. Brooding behavior of a six-rayed starfish, Leptasterias hexactis. Biological Bulletin 130:304-315.

Spiral cleavage in Calliostoma

These pictures show three views of a 16-celled embryo of the marine gastropod Calliostoma ligatum which displays equal spiral cleavage. Spiral cleavage is a stereotypical cleavage pattern present in many phyla, including Nemertea, Annelida, and Mollusca. Collectively, these phyla are referred to as spiralians. Because of how conserved their early development is scientists can identify homologous body parts of these animals! Spiral cleavage got its name from the way the blastomeres are arranged after the four-cell stage. The four cells divide at oblique angles with respect to the animal-vegetal axis of the egg.

The division from the 4 to 8 cells is typically dextral (clockwise), the next is sinistral (counter-clockwise). Dextral and sinistral divisions alternate creating the “spiral” pattern. Because the position of cells is well conserved, cells receive names in spiralian embryos. The 8 cells in focus on the top image (animal view), are the daughters of the first-quartet micromeres.

The second image (vegetal view) shows the four macromeres in focus. The cells are not necessarily named for their size (although the mircomeres are smaller in this particular embryo) but rather location; the micromeres (and their descendants) are at the animal pole, while the macromeres are always at the vegetal pole.

The bottom image (lateral view) shows the stereotypical “twist” of the blastomeres. You can follow a lineage of cells by zig-zagging up from the vegetal pole. The most in-focus (and largest) cell is the macromere. The cell at 1 o’ clock is its sister, the second-quartet micromere. The two cells above and to the left (at 11 and 12 o’ clock) are the daughters of the first-quartet micromere (generated in the previous division from 4 to 8 cells) in the same quadrant. See veliger larva of Calliostoma ligatum.

Juvenile spicules in bipinnaria

During the 4th week of development of my Evasterias troschelii (a starfish) larvae, I noticed several spicules near the larval stomach. These calcareous structures will eventually become incorporated into the skeleton of the adult asteroid. The spicules began forming near the posterior end of the bipinnaria larva, which is the first larval stage of asteroids. The top picture shows a dark-field view of portion of the larval body (anterior end up). The dark triangle near the top is the larval mouth. The opaque tube right below is the esophagus, which is connected to the stomach via a sphincter. The stomach is the large upside down pear shape at the bottom of the image. You can see six dichotomously branching spicules overlaying the larval stomach. The transparent sack on either side of the stomach and esophagus is the larval coelom (body cavity). The bottom picture is a close up of the same individual. I noticed that most commonly the spicules start off looking like the third one from the top and subsequently grow and branch to create an intricate and beautiful pattern.

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!