Actinotroch larva of Phoronopsis harmeri

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

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

Pteropod Limacina sp.

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

Blastomere Separation: Part Two

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

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

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

Hydrocoel in 8-Armed Ophiopluteus

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

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

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

Brooded Juvenile Brittle Star

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

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

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

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

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

Blastomere Separation in Purple Urchins

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

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

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

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

Bryozoan cyphonautes larva

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

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

Actinotroch larva of Phoronis pallida

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

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

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

Calliostoma veliger larva

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

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

Development of sand dollar pluteus larva

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

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

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

Planuliform Nemertean Larvae

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

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

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

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

Pilidia in the plankton

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

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

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

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

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

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

Enterocoely in Chaetognaths

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

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

Blog description

This blog is initiated by Dr. Svetlana Maslakova (Oregon Institute of Marine Biology, Charleston, OR, USA) to keep an illustrated log of field trips and laboratory observations of marine invertebrate embryos and larvae during the Spring 2010 Comparative Embryology and Larval Biology course taught at the OIMB.

We will post photographs from field trips (of habitats, people, and other animals), along with notes on which species we collected, and which of them turned out to be reproductive. We will try to keep a record of successful spawning events for each species, and post our observations on fertilization, embryonic and larval development, illustrated by microphotographs.

I expect each student to actively participate in creating the content, which will be reviewed by the instructor and TA for relevance, scientific accuracy and quality.

Other embryologists and larval biologists at the OIMB are welcome to participate!