Thursday, June 9, 2011

Brachiolar arms in late-stage asteroid larvae

This is the brachiolaria larva of a starfish, Pisaster ochraceous,that I raised during the Embryology class. Larval anterior end is up, and you are looking at the ventral side. The brachiolaria is characterized by the presence of brachiolar arms and an adhesive disk, and the bottom image shows a close up of the pre-oral protuberance of the frontal lobe that bears these structures. The brachiolaria larva follows the bipinnaria larval stage in the forcipulates, a group of starfish.

The brachiolaria is the last larval stage in these asteroids. It is characterized by three brachiolar arms (the three stubby arm buds near the top of the larva in the first image and zoomed in on in the second image) which surround a central adhesive disk (the brown spot between the lower two brachiolar arms in both images). These appendages have sticky cells and are used to make contact with the substratum when the larva is competent to settle. Some other asteroids, that have lecithotrophic, or non-feeding development, skip the bipinnaria stage, and directly produce large yolky brachiolaria with three brachiolar arms and an adhesive disk also used for settlement and attachment.

Wednesday, June 8, 2011

Spermatophore of Phoronopsis harmeri

The first image shows the spermatophore of a phoronid worm Phoronopsis harmeri. A spermatophore is a package that contains multiple sperm. Sperm is produced by males inside the body coelom, then released through the nephridiopores at the anterior end, and shaped into these packages by the special spermatophoral gland located inside the crown of tentacles (called the lophophore) which surrounds the mouth. The spermatophores of this species are round, about 300 micron in diameter, and equipped with a cork-screw shaped transparent “sail”. The sail is easily dislodged from the capsule.

The second picture shows two spermatozoa (or sperm). The sperm in this species is unusual in that it is V-shaped. The nucleus is in one arm of the “V”, and the flagellum forms the other arm, the acrosome is at the apex of the “V”. After the spermatophore is released it floats in the water and lands on the lophophore of a female. Once there, the spermatophore somehow makes its way into the tentacular coelom of the female, and eventually into the body coelom where internal fertilization occurs. Typically development is arrested until the female releases the fertilized eggs into the water. Occasionally development is initiated inside the female coelom, and one can find gastrulae or even more advanced developmental stages. See a post by Phillip Warner showing a 6-day old actinotroch larva of this species reared in the lab, and a post by Svetlana Maslakova showing an advanced actinotroch of this species collected from plankton.

Tuesday, June 7, 2011

Polar lobe in Nassarius fossatus

Nassarius fossatus is a marine snail. Snails, or gastropods, belong to the Spiralia - a large group of animals with spiral cleavage. Nassarius fossatus has unequal cleavage, which means that one of the cells at the two-cell and four-cell stage is larger than the others. The first four cells in a spiralian embryo are denoted as A, B, C and D. The D cell is the largest in unequal spiral cleavage. There are several mechanisms by which unequal cleavage can be accomplished. Nassarius does this via the so-called polar lobe, which is shown in these pictures. A polar lobe is an anucleated protuberance which forms at the vegetal pole during first, second, and sometimes subsequent cell divisions. It then fuses with one of the cells, making it larger than the others.

The top picture shows polar lobe formation during the first cell division. One can see two polar bodies. Polar bodies are the tiny sister cells of the oocyte which are produced during meiosis, contain discarded DNA and mark the animal pole of the embryo (up in the first three pictures). The opposite pole of the embryo is the vegetal pole. The two cells at the animal pole are the first two blastomeres. What looks like a third cell at the vegetal pole is the polar lobe, which at this stage is nearly completely cinched off from either blastomere. Subsequently the polar lobe fuses with one of the blastomeres (second picture from top), so that by the end of the first cell division one of the blastomeres (called CD) is noticeably larger than the AB cell (third picture from top). Polar lobe also forms at the second cell division (not shown). At the four-cell stage blastomere D is the largest, blastomere C is the second largest, while A and B cells are about the same size (bottom picture). The first three pictures are lateral views, while the bottom picture is a polar view. It is the first time I have heard of and observed unequal spiral cleavage, and I think it is remarkable. I also liked these eggs because the egg capsules they are laid in are very beautiful when viewed under the dissecting microscope (see picture by Janelle Urioste).

Gastropod Egg Masses

I was intrigued by the diversity of egg masses among marine gastropods (snails and slugs). In our Embryology class I observed three species with different habits of laying their eggs, namely, the snails Nucella emarginata, and Nassarius fossatus, and a nudibranch (or sea slug) called Diaulula sandiegensis. N. emarginata (also known as the “dog winkle”) and N. fossatus (also known as the “giant western dog whelk”) belong to the group of marine prosobranchs and produce egg capsules which are leathery or hard, and tend to be attached to various substrata in the environment. The top picture shows two egg capsules laid in our sea table by Nucella emarginata. These capsules are shaped like a wine goblet, and are opaque and hard, and each contains numerous eggs. The middle picture shows a single egg capsule of Nassarius. These are more or less transparent, so the eggs are easily visible through the wall of the capsule. When well fed, these scavenger snails will lay egg capsules attached to the walls of sea table, or the hose as close to the surface of the water as they can get. These egg capsules are beautifully sculptured. The bottom picture is of the egg mass laid in our sea table by the nudibranch Diaulula sandiegensis. Opisthobranchs, in general, lay their eggs in gelatinous ribbon-like masses. These masses are often loosely attached to the substratum, and tend to be only a few eggs thick.

Regeneration in bipinnaria II

In May I surgically bisected several bipinnaria larvae of the starfish Pisaster ochraceous. I made the cuts across the esophagus (anterior to the coeloms), separating the preoral lobe and the mouth from the rest of the larva (see pictures). I wanted to see if the fragments would indeed regenerate, as described in the literature.

The anterior and posterior fragments were cultured together and observed 13 days post-surgery. I observed two distinct morphotypes in the culturing vessel representing the anterior and posterior fragments. The first photo shows one of the posterior fragments 13 days after bisection. You can see that these fragments healed and regenerated the preoral lobe and the mouth. The fragments that I interpreted as being anterior, healed, but did not appear to regenerate 13 days after bisection (see bottom picture). This is surprising because bipinnaria larvae of the same species are apparently capable of regenerating their anterior ends under similar experimental conditions (Vickery et. al. 2002). I continued to monitor these fragments for several more weeks to see if the anterior ends would finally regenerate. As of six weeks post-surgery, I found two (out of original 15) anterior fragments that did not appear to regenerate.

Vickery, M. S., Vickery, M. C. L., McClintock, J. B. 2002. Morphogenesis and Organogenesis in the Regenerating Planktotrophic Larvae of Asteroids and Echinoids. Biol. Bull. 203: 121–133

Monday, June 6, 2011

Pseudocyphonautes of Flustrellidra

These pictures are of the bryozoan Flustrellidra coniculata and its pseudocyphonautes larva. The pseudocyphonautes resembles the planktotrophic cyphonautes larva of some bryozoans in that it has a bivalve shell. But it is lecithotrophic (non-feeding) and is brooded. The top picture shows the bean-shaped pseudocyphonautes larva using a dark-field technique. One can distinguish the cilia of the corona ciliata along one edge of the larva. One can also see a ridge on the opposite side which corresponds to the shell margin. The clear outer coating is the shell that protects the larva’s interior structures.

The middle picture shows a small section of the colony (with zooid lophophores retracted). The three white oval shapes out of focus are the larvae which I have dissected. Just beneath the surface of the colony one can see a few yellowish-white masses. These are the ovicells, specialized zooids for brooding eggs and larvae. I extracted the pseudocyphonautes larvae by puncturing the ovicells with a pair of fine forceps and squeezing gently to push them through the hole.

The bottom picture shows a complete Flustrellidra colony, with my palm for scale. This is the largest upright bryozoan colony I have ever seen. It does not look like a bryozoan at all. In the field I would have easily mistook this animal for some sort of alga or a sponge!

Thursday, June 2, 2011

Brooded ophiuroid juveniles

Amphopholis squamata is a common small intertidal ophiuroid (brittle star) in the Pacific NorthWest. Unlike many larger species of ophiuroids which produce large numbers of eggs, free-spawn them and have pelagic development, this small species produces few eggs at a time and broods its young in specialized pouches, called the genital bursa, located in the central disc. The juveniles may be removed from the brooding adult by opening the genital bursa from the oral side. The juveniles dissected from a single adult are often at different developmental stages and of different size. They may range from fertilized egg to a juvenile ready for self-sufficiency. The different stages may be found within a single brooding pouch. These are dark-field photomicrographs of two juveniles dissected from a single adult taken at the same magnification. The top picture is of a small juvenile that was still connected to the brood pouch of the adult brittle star, so that it may receive necessary nutrients for development. Because the embryos receive nutrition from the mother, they grow inside the brood pouches. The bottom picture shows a larger juvenile which has developed arms.

Raising larval cultures

In this Embryology class we raise embryos and larvae to gain a better understanding of how organisms develop. It is not hard to raise larvae, but it must be done with care. All of the species we work with are local marine invertebrates, collected either by us during the class field trips, or by our instructor ahead of time. The first step is to procure gametes, and the technique varies by species. Once we have fertilized eggs, we culture them in filtered sea water in clear glass custard bowls that hold 100-200 ml. The bowls are set in a sea table with flowing sea water, so the cultures are kept at ambient sea temperature. We label cultures using clothespins and colored tape (top picture).

Every other day the water must be exchanged using reverse filtration. A small plastic beaker with a mesh bottom (mesh size of 50-100 microns) is set in the culture bowl, and water is removed with a turkey baster, while the embryos or larvae remain in the bowl as you can see one of the students do in the middle picture. Bacteria and detritus go through the mesh together with the water. You must choose an appropriate mesh size so that the larvae are not going through it and make sure you leave a small amount of water on the bottom with larvae, so they are not crushed.

The next step is to transfer your larvae into a clean bowl and fill with filtered seawater. Some of our cultures need food to grow and develop. We have been feeding them a mixture of two kinds of unicellular algae, Rhodomonas lens (looks red) and Dunaliella tercioleta (looks green), which our teaching assistant grows in the lab in 0.5 liter glass flasks shown on the bottom picture.

Monday, May 30, 2011

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!

Sunday, May 29, 2011

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.

Saturday, May 28, 2011

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.

Monday, May 23, 2011

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.

Wednesday, May 18, 2011

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.

Tuesday, May 17, 2011

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.

Tuesday, May 10, 2011

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.

Monday, May 9, 2011

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