Early cleavage in echinoderms

Early development in echinoderm embryos follows a regular and predictable pattern. The first and second cleavages are equal and meridional (cut along the animal-vegetal axis of the egg). The third cleavage is equal and equatorial, i.e. perpendicular to the plane of the first two divisions, resulting in two tiers of 4 equal-sized cells. The nature of the fourth cleavage (from 8 to 16 cells) differs between echinoids and asteroids.

The first picture shows a side view of a 16-cell stage of the sea star Pisaster ochraceus (animal pole, marked by the two polar bodies, is at about two o’clock). As you can see here, all cells (12 of 16 are visible in this focal plane) are equal in size.

The second picture shows a side view of a 16-celled embryo of the sand dollar Dendraster excentricus (animal pole up). As in most other echinoids, the four cells of the animal pole tier divided equally and meridionally, producing a tier of eight equal-sized cells (called mesomeres). Four mesomeres are clearly visible in this focal plane. The blastomeres of the vegetal tier divided equatorially and unequally producing four smaller cells at the vegetal pole, called micromeres (two are visible), and four larger cells called macromeres (two are in focus). The micromeres of echinoids give rise to the larval skeletogenic cells. In comparison, asteroids, have no micromeres and no larval skeletal spicules (see a post by Nick Hayman).

Other differences in early development of the two classes are also apparent here. In the asteroids, developing oocytes in the adult ovary are arrested at prophase I. Meiosis is normally completed after fertilization (which traps the polar bodies inside the fertilization envelope). This is why you can see the polar bodies in the top picture. On the other hand, in echinoids oocytes complete meiosis in the ovary (prior to fertilization), thus no polar bodies can be seen inside the fertilization envelope. 

Development of the nudibranch Janolus fuscus

Nudibranchs are arguably some of the most flamboyant animals of the Pacific coast. Janolus fuscus (shown here), a sub-tidal species that is commonly found on the floating docks in Oregon, is no exception. Surprisingly, it’s brilliant and delicate morphology is a beautiful display of camouflage. Janolus fuscus is remarkably difficult to spot when it is on its primary food source, the bryzoan Bugula pacifica. Its distinctly lined cerata break up the nudibranch's outline, making the body less conspicuous. Its translucent flesh allows the nudibranch to blend in. The two characteristics seem gaudy until the nudibranch is viewed in it’s native habitat, where they act as a veil. Often, the egg masses of J. fuscus are more visible than the adults.

Nudibranchs are simultaneous hermaphrodites, so a mature adult can mate with any other mature adult of the species. Nudibranchs deposit eggs in characteristic gelatinous masses. An egg mass of J. fuscus depicted here is a cylindrical, jelly-filled cord with egg capsules

arranged in a single row, resembling white beads on a string.

As you can see, each capsule contains numerous embryos. The embryos undergo spiral cleavage and develop into trochophore larvae at around the forth day after egg deposition.

Two days later, the trochophores develop into veligers, with a shell and velum. Veligers hatch after about two weeks of development, and spend about six weeks in the plankton feeding on unicellular algae. Then the larvae settle on a bryzoan (preferably Bugula pacifica) and undergo metamorphosis to become a juvenile (e.g. loose the shell and become elongated). Unlike the larvae, the juveniles feed on bryzoan lophophores. The juveniles grow into mature adults, all the while camouflaged by the bryzoan upon which they depend.

M. Wolf. 2012. The reproductive ecology of a northeastern Pacific nudibranch, Janolus fuscus, with an examination of its endoparasitic copepod, Ismaila belciki, Biol. Bull. 222: 137–149.

Egg capsule and larva of the intertidal snail Littorina scutulata

Pictured here is the adult, egg case, and veliger larva of Littorina scutulata. L. scutulata is a small (up to 1.5 cm shell height) gastropod, common in the mid- to high-intertidal of the west coast  of North America (Kozloff and Price 1996, Reid 2007).  The shape of the penis, tentacle pattern, and characteristics of the egg capsule distinguish L. scutulata from its co-occurring sister species, L. plena.

L. scutulata females typically lay transparent egg capsules shaped like a stack of two unequally sized disks. The egg capsules of L. plena females look similar, except that the upper and lower disk are equal in diameter (Hohenlohe 2002). The upper picture shows a side view of the L. scutulata egg capsule, in which the distinctive shape is evident. The lower picture focuses on the embryos, each of which develops in its own membrane (Buckland-Nicks et al. 1972).

The veligers hatch from the egg capsule about a week after egg laying, and measure about 160 micrometers long. They are competent to settle after spending four weeks in the plankton and reaching about 300-360 micrometers in length. The veliger shown here is about 210 microns long. One can see the velum and its two ciliary bands (the more prominent and more anterior prototroch, and the less prominent metatroch). Note also the larva’s paired eyes and statocysts. The statocyst is a balance organ that looks like a round vesicle with a marble inside. 


Reid, D. 2007. Littorina. In: Intertidal Invertebrates from Central California to Oregon. Carlton, James T. ed. University of California Press. Berkeley and Los Angeles, California. 2007.

Kozloff, E. N. and Price, L. H. 1996. Phylum Mollusca: Class Gastropoda. In: Marine Invertebrates of the Pacific Northwest. Kozloff, E.N. ed. University of Washington Press. Seattle and London.

Buckland-Nicks, J., Chia, F-S, and Behrens, S. 1972. Oviposition and development of two intertidal snails, Littorina sitkana and Littorina scutulata. Canadian Journal of Zoology. 51: 359-365

Hohenlohe, P. A. 2002. Life history of Littorina scutulata and Littorina plena, sibling gastropod species with planktonic larvae. Invertebrate Biology. 12 (1): 25-37.

Filopodia of mesenchymal cells in gastrulating echinoderms

These pictures show gastrulating echinoderm embryos.  I was fascinated by the role filopodial processes (small-thread-like-extensions) of mesenchymal cells play during echinoderm gastrulation.

The first two pictures show a 30-hour old gastrula of the sand dollar Dendraster excentricus. During echinoid gastrulation the first cells to enter the blastocoel (the spatial gel-filled cavity inside the embryo) are called primary mesenchyme cells. Then, the archenteron (the primary gut) buckles in (invaginates). Through a series of cell rearrangements called convergence and extension, the archenteron (a large tube visible here inside the embryo) elongates and extends towards the roof of the blastocoel. In the meantime the primary mesenchyme cells form two groups and begin to secrete calcareous larval spicules (two tri-radiate spicules are visible here on either side of the archenteron). Approximately two thirds of the way up, a second group of cells ingress into the blastocoel from the roof of the archenteron.  This is where the filopodia come in.

This image is a close up view of the tip of the archenteron, showing a secondary mesenchyme cell with a long conical projection reaching toward the roof of the blastocoel. These processes attach to the roof of the blastocoel and aid in the final extension of the archenteron.

This picture shows a 62 hour old gastrula of a sea star Pisaster ochraceous. Asteroids lack the primary mesenchyme cells, so the mesenchyme cells that ingress at the tip of the archenteron are the only mesenchyme they have. Note the lack of primary mesenchyme cells in the blastocoel and the numerous branched filopodia on the mesenchyme cells at the tip of the archenteron.  

The epitokes of Autolytus: swarming to mate

On April 24th, the spring 2013 Embryology class collected invertebrates in the large boat basin in Charleston. We scooped up rapidly swimming polychaete worms from the water's surface. These members of the Autolytus genus from the Syllidae family are benthic dwellers (benthos is Greek for “depths of the sea”). To reproduce, they undergo an incredible reproductive transformation known as epitoky (epitokos, Greek for “fruitful”). Depending on the species, either the entire worm or its posterior end develops chaetae for swimming and large eyes, and the gut is degenerated as well. This epitoke then swims up to swarm and mate. In cases where only a portion of the worm is transformed, the rest of the worm resumes its normal benthic life, but the epitokes usually die after spawning. For the subfamily Autolynae, the female epitokes are called sacconereis. We most often see bright orange epitokes, though the picture at left shows a brilliant blue-green sacconereis about 30 mm long.

Swarming in epitokes may be triggered by such factors as rising water temperatures and the lunar phase. The male circles the female, wrapping her in a mucous trail of sperm. The eggs are fertilized as they emerge, and either undergo planktonic development or, as is the case in the species shown here, collect in several brood sacs on the ventral side of the sacconereis. Even after larvae hatch from the brood sack, they hang on to the female. Can you see their eyes staring out?


This larva from one of the brood sacks of the above individual is a 500 µm long nectochaete, with chaetae bristling from three segments like legs on a cartoon stick figure. The apical tuft at its anterior end contains the sensory cells that may be involved in detecting the chemical cues during larval settlement. Once out of the brood sack, the larva may propel itself using several transverse bands of cilia. Three of the ciliary bands are visible here - one on its head, one just anterior to the first pair of chaetae, and one at the posterior end. The chaetae may also aid in locomotion. Four large ocelli (eye spots) are arrayed anterior to its large muscular pharynx, the clear oval shape anterior to the gut. The gut itself is filled with greenish yolk droplets packed into the egg by the mother. There will be no need for this nectochaete to feed during its brief life in the plankton. It will live on yolk reserves until settling out to begin its benthic existence.

Dorresteijn A & Westheide W. (2010). Reproductive strategies and developmental patterns in annelids. Dordrecht London: Springer.

Rouse G & Pleijel F. (2001). Polychaetes. Oxford New York: Oxford University Press.

Rouse G & Pleijel F. (2006). Reproductive biology and phylogeny of Annelida. Enfield, NH: Science Publishers.

Birefringent structures in marine invertebrates

If one treats light as a transverse wave, as per Maxwell’s equations, a beam of unpolarized light can be thought of as a collection of these waves oscillating in all planes (Bennett, 1950). A polarizer placed over this ray would then block the transmission of all but one plane of oscillation. Normally a second polarizer, called an analyzer, placed perpendicular to the first would then remove that last plane of light, allowing no transmission. However, if that light is first passed through an anisotropic medium, that is one that is highly ordered, it causes light to refract in a secondary, angled path in addition to the one that will be cancelled out by the analyzer. This double refraction is called birefringence (Inuoe, 1970). This effect can be seen in the luminescence of various calcareous structures of marine invertebrates.

The image at left is a four-week old pluteus larva of the sand dollar, Dendraster excentricus, that I cultured in the lab. The larval spicules of the pluteus, which are made primarily of calcite, an anisotropic material, are illuminated. The larva’s left postoral arm can be seen in the plane of focus, one of its six arms at this stage of development.
Here, the two rings of calcite larval ossicles of a doliolaria, the non-feeding planktonic larva of a sea cucumber, can be seen to be birefringent. These ossicles will eventually be embedded in the epidermis of the adult holothuroid, toughening the exterior of the animal.
 

This picture shows the tri-radiate calcite spicules of the sea sponge, Leucilla nuttingi. These three-pointed rods are imbedded in the organism’s mesohyl, and are characteristic of sponges in the class Calcarea. A section of the sponge’s exterior was excised and then allowed to dissolve in a solution of 8% bleach, leaving only its calcareous skeleton.

Bennett, HS. Methods Applicable to the Study of Both Fresh and Fixed Material, in McClung’s Handbook of Microscopial Technique, 3rd ed., Paul Hoeber, New York, 1950. pp. 591-677

Inoué, Shinya. An Introduction to Biological Polarization Microscopy. Program in Biophysical Cytology. Woods Hole, MA 1970.

Development of a polychaete metatrochophore

In the image at left is a metatrochophore larva of the polychaete Nephtys. I caught this larva on February 14, 2013 in a plankton tow taken off a dock in Charleston, OR. It has two ciliary bands, the anterior prototroch and the posterior telotroch, which help in locomotion, and 10 body segments. I wanted to observe the internal structures of this larva, so I fixed it with paraformaldehyde, stained it with fluorescent phalloidin, and cleared it in a mixture of benzyl benzoate and benzyl alcohol (Murray Clear). Because I stained it with phalloidin, which binds to filamentous actin, I was able to observe the details of muscular anatomy quite well.

You’ll notice two prominent, bright muscular bands along the sides of the larva. Many invertebrates have longitudinal and circumferential muscular bands that antagonize each other and control the shape and size of the animal; however, Nephtys lacks circumferential muscular bands (Clark and Clark 1960). Lateral to the bright longitudinal muscle band and running perpendicular to it are smaller muscles of the parapodia. These muscles control the lateral paddle-like projections of the polychaete body.

As a larva grows, its size (volume) will increase at a greater rate than its surface area, so the efficiency of locomotion by ciliary action alone decreases (Chia et al. 1984). My larva was about 700 µm long. The development of musculature can improve larval locomotion and extend pelagic larval residence times (Chia et al. 1984).

Chia F-S, Buckland-Nicks J, and Young CM. 1984. Locomotion of marine invertebrate larvae: a review. Can J Zool 62: 1205-1222.

Clark RB and Clark ME. 1960. The ligamentary system and segmental musculature of Nephtys. Q J Microsc Sci 101(2): 149-176. 

Confocal microscopy

This is a confocal projection of a chiton trochophore larva preserved with formaldehyde and stained with fluorescent phalloidin. Confocal microscopy allows one to collect a series of images of thin (e.g. 1 micron or less) optical sections from a small, fluorescently labeled specimen (like this ~200 micron long larva), excluding the out-of-focus light. One can then create a z-projection of the entire stack, as I did here.

Phalloidin binds to filamentous actin, so when it is fluorescently labeled it allows visualization of cell outlines and muscles, among other structures. The feature of interest on this image is what appears to be a transverse band of small cells about two thirds of the way to the anterior end (up) of the larva.  This is the prototroch. The prototroch is a transverse ciliary band anterior to the mouth, and is the defining feature of a trochophore larva. In chiton trochophores it is the main locomotory organ. 

The small, grid-shaped blocks that catch one's eye are not actually individual cells within the prototroch but rather a pattern of actin fibers near the surface of the prototroch cells. The cells of the prototroch are much larger.  One can visualize the outlines of the prototroch cells by removing the top few slices of the image stack (second picture). 

The image at left is a 10-micron-thick optical slice (in mid-sagittal plane) of a specimen, that was dehydrated through an isopropanol series and cleared in a mixture of benzyl benzoate and benzyl alcohol (Murray Clear) to visualize internal structures. Larval anterior is up, and ventral is to the left (marked by the position of the mouth). The bowling pin shape in the center is the body-wall musculature composed of longitudinal and circular fibers and located directly beneath the epidermis. These muscles act antagonistically to control the larva's locomotion and shape. The prototroch cells are visible here as the dark regions on either side of the larva, near the narrow neck of the "bowling pin." The mouth opens immediately posterior to the prototroch on the ventral side (left). The opening and the lumen of the foregut are also highlighted with phalloidin. Phalloidin labeling in combination with confocal microscopy is an excellent tool for studies of external and internal morphology of small embryos and larvae.

Development of a hoplonemertean

On February 4, 2013, I did a plankton tow off a dock in Charleston, OR and found a few embryos like the one pictured here. The embryo was about 180 microns across, and was surrounded by an inner chorion and an outer chorion about 360 microns in diameter. I kept the embryos, and after two days discovered that they turned into planuliform larvae, hatched from their chorions and were swimming around in the dish.

The planuliform larva is uniformly ciliated and has a prominent bundle of cilia called the apical tuft at the anterior end (12 o’clock on the second picture). Such larvae are found in nemerteans (phylum Nemertea, a.k.a. ribbon worms) from the orders Hoplonemertea and Palaeonemertea. 

After a few more days these larvae developed features that allowed me to identify them as belonging to the order Hoplonemertea (e.g. several pairs of subepidermal eyes visible on the third picture). This picture shows an 11-day old individual. At this point they mostly crawled on the bottom of the dish, and had developed many of the adult structures, such as the brain and proboscis, so they can be considered juveniles rather than larvae. Hoplonemertean metamorphosis (the transition from planktonic larva to benthic juvenile) is inconspicuous. The transition from swimming to crawling is accompanied by changes in the epidermis. Apparently many hoplonemerteans replace the larval epidermis composed of large, ciliated, cleavage-arrested cells with intercalating smaller cells of the definitive epidermis (Maslakova and von Döhren, 2009).

To witness this epidermal transition, I fixed some of my specimens in paraformaldehyde (with a touch of gluteraldehyde) and stained them with fluorescent phalloidin to visualize the outlines of the epidermal cells using a confocal microscope.

The first confocal image shows a 2-day old hoplonemertean planuliform larva – the same age as the live larva pictured above. You will notice that large cells dominate the epidermis, but small cells are visible in between. These small cells are the intercalating cells of the juvenile epidermis. The bottom image is a 4-day old larva, and you can see that the large cells of the larval epidermis are farther apart from each other. The small cells of the juvenile epidermis occupy more space in between. Eventually, cells of the larval epidermis will be either resorbed or sloughed off, leaving the cells of the definitive epidermis to cover the entire surface. 

Maslakova SA, von Döhren J. (2009) Larval development with transitory epidermis in Paranemertes peregrina and other hoplonemerteans. Biol Bull 216: 273-292

Observations of a magelonid nectochaete larva

In a plankton tow taken in Charleston, OR on February 20, 2013, I found several polychaete nectochaete larvae of Magelona sp., which I was able to identify by their long anterior tentacles. The tentacles apparently develop as extensions of the prototroch (larval ciliary band) and are suspected to be used in locomotion and feeding  (Wilson 1982). I noticed the tentacles were relaxed and extended when the larva was hanging suspended in the water column, but during rapid sinusoidal movements of the body, the larva coiled up its tentacles and kept them close to the body.

This picture is a close up of one of the tentacles. You will notice that they are covered by papillae. The papillae are present along the entire length of the tentacle but only on the dorsal side. Pairs of papillae appear to be at right angles to each other as they each emerge from the surface of the tentacle. It is suspected that the papillae may serve a sensory function by detecting vibrations in the surrounding water (Jones 1968).

This is a close up of the larval mouth (ventral view) - note its distinct triangular shape. Two eyespots are also noticeable near the anterior end of the larva. Toward the posterior (down), bundles of bristles emerge from paired chaetal sacs on each segment of the body.

Wilson DP. 1982. The larval development of three species of Magelona (Polychaeta) from localities near Plymouth. J Mar Biol Ass UK 62: 385-401

Jones ML. 1968. On the morphology, feeding, and behavior of Magelona sp. Biol Bull 134(2): 272-297.

Feeding mechanism of the actinotroch larva

This picture shows an unidentified actinotroch larva of a phoronid worm that I caught in a plankton tow taken off a dock in Charleston, OR on February 20, 2013. The photo is a lateral view, and the anterior end of the larva is up. Actinotroch larvae have an anterior hood that covers the mouth (a lobe-like structure seen at upper left) as well as a crown of tentacles located posterior to the mouth. You can see that the tentacles are longer ventrally and shorter dorsally. This is because tentacle pairs are added progressively with new tentacles forming mid-dorsally, so the mid-ventral tentacles are the oldest. The number of tentacles varies between species. This larva had ~ 20 tentacles. Tentacles are involved in feeding. A posterior ciliated band, called the telotroch (at about 6 o-clock), helps in locomotion.

The second picture is a close up of the tentacles showing the cilia. The tentacles are covered by motile cilia, which create the water flow, but also bear a row of non-motile latero-frontal cilia. These stiff latero-frontal cilia apparently serve as a mechanical sieve, detaining food particles as they flow past the larva (Riisgård, 2002). Once caught on the upstream side of the tentacle, a food particle can be transported up to the mouth by the beating frontal cilia (Riisgård 2002), a tentacle flick (Strathmann and Bone 1997), or rapid lifting of the preoral hood which creates negative pressure (Strathmann and Bone 1997).

I gave my actinotroch some unicellular algae Rhodomonas (from a lab culture), which is a good food for many ciliated marine invertebrate larvae. This picture was taken a day after food was added, and it appears that the larvae fed on Rhodomonas, judging from the accumulation of the reddish pigment (the color of Rhodomonas) in the stomach.

Riisgård HU. 2002. Methods of ciliary filter feeding in adult Phoronis muelleri (phylum Phoronida) and its actinotroch larva. Mar Biol 141: 75-87.

Strathmann RR and Bone Q. 1997. Ciliary feeding assisted by suction from the muscular oral hood of phoronid larvae. Biol Bull 193: 153-162.

Secrets of a mollusk egg

I found a few of these interesting embryos in a plankton tow taken off a dock in Charleston, OR in January 2013. Under a dissecting microscope, I could see the embryo itself was greenish and about 250 microns across, but it seemed to be surrounded by a halo several hundred microns in diameter. When I sucked the embryo into my pipet, I could see the “halo” contacting the inside walls of the pipet. As it turns out, the “halo” is a jelly layer, a characteristic of some embryos that, among other things, helps protect it from microbes (Hellberg et al. 2012). Oregon Institute of Marine Biology research scientist George von Dassow identified the specimen as a gastropod mollusk (snail) embryo.

A closer look under a compound microscope revealed many fascinating things about this embryo. In the first photo, you’ll notice two small, clear cells at about 4 o'clock. These cells, called polar bodies because they mark the animal pole, are essentially the waste products of meiosis. In many invertebrate phyla, meiosis II is not completed until after fertilization, so the resulting polar bodies remain inside the chorion (the membrane surrounding the embryo inside the jelly).

In the next photo, you should notice two things: first, there seems to be a hole in the chorion at about 10 o'clock (a penetration point of sperm), and second, a sperm is hanging out just inside the chorion at about 7 o'clock. Other similar penetration points were visible in other parts of the chorion, indicating that multiple sperm were able to enter the chorion but did not fertilize the egg (because the embryo looks healthy). Polyspermy is actually a big problem for invertebrates, especially those that are free-spawning and have external fertilization (Franke et al. 2002). Polyspermy causes morphological abnormalities in development, so complex physiological mechanisms exist to prevent it. A close coupling exists between the species-specific surface proteins on sperm (e.g. lysin) and egg (vitellin envelope receptor for lysin), so interspecific hybridization is also prevented (Hellberg et al. 2012).

After a few days, the embryo had developed into a gastropod veliger larva about 400 microns long. The reticulated pattern on its shell suggests this veliger belongs to the turban snail genus Calliostoma, of which there are 5 species in Oregon (Goddard 2001). The larva uses the compound cilia of its velum (ciliated structure at 4 o'clock) to feed and move throught the water column.

Franke ES, Babcock RC, Styan CA. (2002) Sexual conflict and polyspermy under sperm-limited conditions: In situ evidence from field simulations with the free-spawning marine echinoid Evechinus chloroticus. American Naturalist 160(4): 485-496

Goddard JHR. 2001. Mollusca: Gastropoda. In: An Identification Guide to Marine Larval Invertebrates of the Pacific Northwest. Edited by Alan Shanks. OSU Press, Corvallis.

Hellberg ME, Dennis AB, Arbour-Reily A, Aagaard JE, Swanson WJ. (2012) The Tegula tango: A coevolutionary dance of interacting, positively selected sperm and egg proteins. Evolution 66(6): 1681-1694

Confirmed identity of wild-caught planktonic larvae

We recently confirmed the identity of two planktonic larvae that were each the subject of blog posts last spring.  Both larvae were cryopreserved after photographs were acquired and their DNA was extracted at the Oregon Institute of Marine Biology by a student in Svetlana Maslakova’s Marine Molecular Biology class this fall.

Nephtys sp. metatrochophore 

Metatrochophore larvae like this were found in April and May and we speculated that they were larvae of the polychaete genus Nephtys due to the red pigment bands along the prototroch and pygidium as well as the bright blue pigment lining the stomach which you can recall from this photograph (for full blog post see Nephtys sp. metatrochophore by Terra Hiebert).  

Jay Bowles, a student in the Marine Molecular Biology class, successfully amplified the 16S rDNA barcoding gene region for this larva with universal primers (Palumbi et al. 1991).  Using NCBI BLAST, we compared this sequence to those in GenBank and found that this larva is, in fact, the larva of Nepthys as its sequence matched other species within this genus with 97% sequence similarity.  The species level identify of this larva remains to be determined, as sequences did not match exactly, but we’ve confirmed our previous identification based on larval morphology.  

Holothuroid doliolaria 

I found this larva in a plankton sample taken from the Charleston marina docks on May 17th.  We speculated that it may be the larva of a sea cucumber (class Holothuroidea) because of its unique barrel-like shape and transverse ciliated bands (for full blog post see Holothuroid doliolaria by Terra Hiebert).  

Jay successfully amplified the Cytochrome Oxidase Subunit I barcoding gene region from this larva, also using universal primers (Folmer et al. 1994).  Unlike the Nephtys sequence mentioned above, the sequence we acquired from this larva did not have a close match to any sequences in GenBank.  The closest match (87% similarity) was to the sea cucumber Acaudina molpadioides. Richard Emlet (pers. comm.) suspected that our larva may belong to the local holothuroid species, Paracaudina chilensis (rat-tail sea cucumber), based on its morphology and development, so we compared the larval COI sequence to that obtained from P. chilensis by Gustav Paulay (University of Florida, Florida Museum of Natural History).  The two sequences had only 4 mismatches over the 490 base pair region of overlap (0.8% divergence).  This level of divergence is low enough that we can be confident that our doliolaria larva belongs to P. chilensis.  

While pursuing the identification of this larva with DNA sequence data, we realized that we misinterpreted the madreporite as ossicles in the polarized light image you can see here.  Calcareous ossicles are found in sea cucumbers and are used for structure and support.  However,  as we now understand, they usually do not develop until much later stages (Richard Emlet, pers. comm.).  The madreporite, on the other hand, is a small calcareous disc that connects the water-vascular system to the outside, and is used to regulate internal water content in all echinoderms, including sea cucumbers. Madreporites have been documented in young Paracaudina sp. larvae (Richard Emlet, unpublished) and resemble the brighter spicule on the right in the photograph shown here.  In addition to the madreporite, the water vascular system of echinoderms includes another calcareous structure called the ring canal that helps to move water throughout the body.  We speculate that the ring canal can also be seen in this image and is the larger structure to the left of the madreporite. 

Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotech 3:294-299 

Palumbi S, Martin A, Romano S, McMillan WO, Stice L, Grabowski G. (1991) The simple fools guide to PCR Version 2.0.  Honolulu, HI: Department of Zoology Kewalo Marine Laboratory, University of Hawaii

MOCNESS

Last month I participated in an oceanographic research cruise near the Barbados Accretionary Prism. My job was to help with the MOCNESS (Multiple Opening/Closing Net and Environmental Sensing System), which is a net system used to sample plankton. The MOCNESS can collect independent plankton samples at specific depth intervals during a single deployment, this is accomplished by independently opening and closing the nets.

The MOCNESS has a rectangular frame that houses the environmental sensing system and controls the nine nets (MOCNESS can have between 6 and 20 nets). The cable connects the ship and MOCNESS and gives scientists real-time data so instrument adjustments can be made during sampling. The sensors were used to collect salinity, temperature, depth, and water flow measurements. We used 153 µm mesh nets and the speed of the MOCNESS through the water averaged 10m/min so that the small ciliated larvae would not get damaged. 

The pictures are of the MOCNESS after it had been recovered. The first image (taken by Svetlana Maslakova) is the science team transferring the samples from the cod ends to containers for sorting. The second image (taken by Svetlana Maslakova) is of myself and another graduate student setting up the nets in preparation for the next tow. The third image is a sample that was collected in one of the cod ends. We conducted nine MOCNESS tows, depths of the samples ranged from the surface to 4500 meters. These tows will allow us to look at the vertical distribution of deep sea larvae. 

Wiebe, P. H., K. H. Burt, S. H. Boyd and A. W. Morton. 1976. A multiple opening/closing net and environmental sensing system for sampling zooplankton. Journal of Marine Research 34:313-326

Wiebe, P. H., A. W. Morton, A. M. Bradley, R. H. Backus, J. E. Craddock, V. Barber, T. J. Cowles and G. R. Flierl. 1985. New developments in the MOCNESS, an apparatus for sampling zooplankton and micronekton. Marine Biology 87:313-323

Inarticulate Brachiopod Larvae

This June I participated in an oceanographic research cruise to Barbados. One of the goals of this expedition was to collect planktonic larvae of benthic marine invertebrates using a MOCNESS device. Among other things, we found larvae of inarticulate brachiopods (phylum Brachiopoda; class Inarticulata) pictured here. Brachiopods resemble bivalve molluscs (e.g. clams and mussels), but have dorsal and ventral valves rather than left and right. The valves are held together with muscles in inarticulate brachiopods, while those of articulate brachiopods are hinged. Brachiopods, as a phylum, are also characterized by the lophophore (a crown of tentacles surrounding the mouth). We collected two different kinds of inarticulate brachiopod larvae.

Larvae of the first kind were collected from relatively shallow depths (900-0 m). In this image, one can see the large circular larval shell valves, as well as the embryonic shell (the half circle at the posterior portion of the larval shell, tinged blue). The lophophoral tentacles are retracted in the first image, but extended in the second.

These tentacles form progressively during the larval life from short tentacle buds on either side of a longer unpaired anterior median tentacle (at about 12 o’clock on the second image). Consequently, the number of tentacles reflects larval age. This larva has 10 pairs of tentacles. In some species, a lophophore with this many tentacles indicates larval competence for settlement (Pennington and Stricker, 2002). One can also see that the tentacles of the lophophore are ciliated. Cilia are used for larval feeding and swimming (Rudwick 1970). This particular type of larva is produced by members of the superfamily Lingulacea which contains only two extant genera: Lingula and Glottidia (Pennington and Stricker 2002).

Larvae of another kind were collected from deeper waters (1600-900 m). Characteristically, they lack an embryonic shell, and have a pair of larval chaetae. Presence of chaetae in this larva suggests that it belongs to the inarticulate superfamily Discinacea (Pennington and Stricker 2002). Chaetae are usually thought of as a feature unique to annelid worms, but some brachiopod larvae and adults also have chaetae, which are similar structurally and developmentally to those of annelids. 

Pennington, J T and S A Stricker. 2002. Phylum Brachiopoda. In: Atlas of Marine Invertebrate Larvae. Edited by Craig M Young. Academic Press. 

Rudwick, M J S. 1970. Living and Fossil Brachiopods. Hutchinson & Co, London.

Fan worm larva

On May 10th, 2012 I waded through the calm waters of Middle Cove at Cape Arago State Park, trailing a 153μm plankton net behind me.  While sorting through the plankton I found a polychaete larva from the family Serpulidae, subfamily Spirorbinae.  Serpulids belong to an order of annelids, Sabellida, commonly known as fan worms.  Adult Spirorbins live in small calcareous tubes approximately 2-5 mm in diameter typically attached to undersides of intertidal rocks. Adults are hermaphroditic and brood embryos inside their tube, then release lecithotrophic larvae that are only briefly planktonic (Strathmann 1987).

This metatrochophore larva has a prominent ciliated band anterior to the mouth, called the prototroch, which is used for swimming. At the anterior (up) is the apical tuft, and at the posterior end there is another cilary band, called telotroch. What helped us to identify this larva is the prominent collar located posterior to the prototroch (Kupriyanova et al. 2001). The collar is the widest portion of the larval body. This larva had  5-6 segments posterior to the collar, and three pairs of ocelli. 

To my surprise, when I went to photograph the larva a few days later, I could not find it at first.  No longer content with the planktonic life, it had settled on the bottom of the culture dish and built a calcareous tube (left). The tube is smooth, non transparent, and coils to the right (dextral). The characteristics of the tube helped us to further classify this specimen as likely belonging to the genus Circeis (Blake and Ruff 2007). 

You can see on the subsequent photograph that it got quite a bit longer in 4 days. The juvenile worm is caught peaking out of it’s tube in the first picture. One can discern a radiolar crown of tentacles used for feeding and respiration (Blake and Ruff 2007), red eyespots, and an operculum.  When disturbed the animal quickly retreats back into its tube, and shuts the operculum (below).

Blake JA, and Ruff RE.  2007. Polychaeta.  In: The Light and Smith Manual:  Intertidal Invertebrates from Central California to Oregon 4th Edition.  Edited by James Carlton. University of California Press, Berkeley.  

Kupriyanova, E.K., E. Nishi, H.A. ten Hove, & A.V. Rzhavsky 2001. A review of life history in serpulimorph polychaetes: ecological and evolutionary perspectives. Oceanography and Marine Biology: an Annual Review 39:1-101.

Strathmann, Megumi F. 1987.  Phylum Annelida, class Polychaeta. In: Reproduction and Development of Marine Invertebrates of the Northern Pacific Coast.  United States: University of Washington Press.