Monday, June 17, 2013

The life cycle of the Olympia oyster, Ostrea lurida

Coos Bay is home to two different species of oysters: Crassostrea gigas, a large species (typically up to 20 cm in shell height) which is native to Japan but reared commercially all over the world, and Ostrea lurida, a much smaller species (up to 6 cm in shell height), which is the only oyster native to the west coast of the United States (pictured here).  Both oysters are sequential hermaphrodites, although they differ in the regularity and seasonality of their sex changes. C. gigas is a broadcast spawner whose eggs are fertilized externally, while functionally-female O. lurida adults filter sperm from the water column and fertilize their eggs internally. While C. gigas larvae develop entirely in the plankton, O. lurida larvae are brooded in the adult’s mantle cavity until they reach an early veliger stage (which takes roughly ten days) and are then released into the plankton (Strathmann et al. 1987, and references therein).

Larvae at this stage are known as D-stage veligers because their flat-hinged shells look like a capital letter D.  In this picture the larva has extended its velum, the ciliated structure used for swimming and feeding. A green algal cell is visible in the larval gut. O. lurida veligers swim and feed in the plankton for a period ranging from one week to one month, depending largely on temperature. As the larva grows, it loses its resemblance to a capital D because the umbo becomes more prominent and triangular. The larva eventually develops an eyespot and is considered competent to settle and metamorphose when its shell measures about 0.3 mm.

O. lurida were harvested for a burgeoning West Coast market beginning in the late 19th century, but commercial stocks were depleted by the early 20th century.  Habitat degradation and, in some cases, predation by introduced species and disease further contributed to the decline of this estuarine species (Polson et al. 2009). Many estuarine scientists are interested in restoring the species along the West Coast, as oyster beds provide habitat for a variety of other species and help stabilize the estuarine shoreline (e.g. Jackson et al. 2001, Ruesink et al. 2005).

Jackson, J. B. C., Kirby, M. X., Berger, W. H., Bjorndal, K. A., Botsford, L. W., Bourque, B. J., Bradbury, R. H., Cooke, R., Erlandson, J., Estes, J.A., Hughes, T. P., Kidwell, S., Lange, C. B., Lenihan, H. S., Pandolfi, J. M., Peterson, C. H., Steneck, R. S., Tegner, M. J., and Warner, R. R. 2001. Historical overfishing and the recent collapse of coastal ecosystems. Science 293:629-638.

Polson, M. and Zacherl, D. 2009. Geographic distribution and intertidal population status for the Olympia oyster. Journal of Shellfish Res 28: 51-58.

Ruesink, J. L., Lenihan, H.S., Trimble, A.C., Heiman, K.W., Micheli, F., Byers, J.E., and Kay, M.C. 2005. Introduction of non-native oysters: Ecosystem effects and restoration implications. Ann Rev Ecol Evol Syst 36:643-689.

Strathmann, M.F., Kabat, A.R. and O’Foighil, D. 1987. Phylum Mollusca, Class Bivalvia. In Reproduction and Development of Marine Invertebrates of the Northern Pacific Coast: Data and Methods for the Study of Eggs, Embryos, and Larvae. Strathmann, M.  F. University of Washington Press. Seattle and London.

Friday, June 14, 2013

Gravity tells no lies

Haven't you ever wondered how larval invertebrates in the ocean orient themselves? The answer often lies in a small balance organ called the statocyst. A statocyst is a fluid-filled spherical capsule containing a small stone, or statolith, and sensory cells that detect the position of the statolith. Statocysts tell the organism which way is up, and, in some cases, how fast it is moving.

This is a recently hatched juvenile of the ctenophore (comb jelly) Beroe sp. At the aboral pole (up, opposite the mouth) there is a dome made of cilia - the equivalent of the statocyst capsule. Inside this dome you will note an aggregate of small marbles - that is the statolith. This aboral sense organ detects gravity and controls the movement of comb rows (ctenes) and the ctenophore’s orientation. The statolith rests on four tufts of support cilia, connected via ciliary grooves to the ctene rows. Tilting changes the gravitational pressure of the statolith on the support cilia, which ultimately controls the beating rate of of the ctenes. Differentially beating ctenes on one side allows the animal to turn and return to vertical position (Hyman 1940). 

This is a veliger larva of the nudibranch Diaulula sandiegensis (which hatched in the lab after we collected the egg mass off a dock in the Charleston marina about a month ago). At the anterior end (up) you will note a ciliated appendage - the velum, with which the larva swims. Below it there are two statocysts. Each contains a single statolith. Statocysts form during intracapsular development, in the late trochophore and early veliger stages in gastropod mollusks (Hyman 1967). 

Here is another kind of balance organ in an ascidian tadpole larva. This tadpole was released by the colonial ascidian Distaplia occidentalis. In ascidian tadpoles the balance organ is called a statocyte, and occupies the bottom of a sensory vesicle, which also contains a light sensing organ - the ocellus or eye (Cloney et al. 2001). The statocyte contains a single melanin granule, the statolith. Both the statolith and the ocellus are visible on this picture. The ocellus is the black crescent shape, while the statolith is the black round shape underneath. These two organs are involved in the perception of environmental cues that drive ascidian tadpole behavior (Zega et al. 2006).

Cloney RA, Young CM, Svane I. (2001) Phylum Chordata: Urochordata. In:Atlas of Marine Invertebrate Larvae. Academic Press. New York. P. 567.

Hyman, L.H. (1940) Protozoa through Ctenophora. The Invertebrates. Vol 1. McGraw-Hill, New York. P. 665-8. 

Hyman, L. H. (1967) Mollusca. The Invertebrates. Vol VI. McGraw-Hill, New York. P. 471, 548, 583.

Zega G, Thorndyke MC, Brown ER (2006)  Development of swimming behaviour in the larva of the ascidian Ciona intestinalis J Exp Biol 209: 3405-12.

Wednesday, June 12, 2013

Brooding in cheilostome bryozoans

Bryozoa is a phylum of miniature, sessile, colonial invertebrates characterized by a crown of tentacles called the lophophore that facilitates ciliated filter feeding. You can see several individual zooids with extended lophophores in the picture below. Most bryozoans brood their embryos, but where and how they brood varies.

Bugula pacifica, a cheilostome commonly known as the spiral bryozoan, broods one embryo at a time inside shallow ovicells; specialized calcified brood chambers visible here as semi-circular structures attached to maternal zooids. The ovicell is produced jointly by the two neighboring zooids - the maternal zooid and the next distal zooid (Giese et al. 1947).

This is a close up view of a maternal zooid and an ovicell in Bugula pacifica. What looks like the head of a bird above the ovicell is a specialized zooid for defense called an avicularium. Ovicell and avicularium morphology vary within the genus, and can be used to key out species. B. pacifica has reduced ovicells, so reduced that a well developed embryo cannot be contained and bulges into the maternal zooid (Giese et al. 1947). Developing embryos in Bugula apparently receive extraembryonic nutrition from the maternal zooid (Giese et al. 1947). Ovicells of cheilostome bryozoans vary in morphology and development which suggests that they may have evolved independently many times in this group (Giese et al. 1947).

Schizoporella japonica pictured here, is another cheilostome bryozoan. It is often found encrusting mussel shells and other substrata in NE Pacific marinas. The ovicells are visible as small bumps on the surface of the colony, some with embryos (an orange mass inside) and some empty. S. japonica’s ovicells aren’t as intimate with the maternal zooids as in Bugula, and the embryos develop without the aid of extra-embryonic nutrition (Strathmann et al. 1987).  See a blog post by Dylan Cottrell about brooding of multiple genetically identical embryos (polyembryony) inside gonozooids in Crisia sp., a local stenolaemate.

Arthur Charles Giese, John S. Pearse, Vicki Pearse 1974. Phylum Bryozoa. In: Reproduction of Marine Invertebrates. Academic Press, University of California. Pp. 494-510

Megumi F. Strathmann 1987. Chapter 3: Bryzoa. In: Reproduction and Development of Marine Invertebrates of the Northern Pacific Coast. University of Washington Press. Pp. 116-158

Sunday, June 9, 2013

Hemigrapsus nudus: brooding mothers and their zoea

Adult Hemigrapsus  nudus can be found under rocks and in crevices of the mid-intertidal on the open coast and in estuaries, here found on the Portside rocks in the South Slough. This purple shore crab is distinctive for the spots on its chelae, or claws.
The female on the left measures 17 mm (11/16 in.) across. She is "in berry", in other words, carrying an egg mass. Crabs brood their eggs under the abdominal flap. As eggs emerge from the gonopore, they are inseminated by stored sperm from a prior encounter with a male. The eggs are attached to the setae (bristles) covering her pleopods, the abdominal appendages used for swimming during the final stage of the crab's pelagic larval life. Her broad abdominal flap forms a protective enclosure for the eggs. The female cares for the eggs, cleaning and aerating them, for up to four and one half months before they hatch as zoea larvae.

This H. nudus zoea hatched six days ago to swim and feed in the plankton and go through several larval molts before metamorphosing into a benthic juvenile. Two compound eyes look out with a multi-faceted view of its surroundings, the mosaic of images from many ommatidia, or eye-lets. A distinctive dorsal spine projects from the top of its carapace, and a rostral spine is located anterior to the eyes. These spines may discourage predation. Note a thin-walled sac at the base of the dorsal spine, the zoea's beating heart. The segmented abdomen ends in forked telson.

At nine days old, this crab zoea is close to undergoing a molt. The process of molting, called ecdysis, is characteristic of a large clade of animals (including arthropods and nematodes) known as the Ecdysozoa. A hard exoskeleton covering the body of these animals must be shed to accommodate growth. During successive molts of zoea, the abdomen adds new segments, and pleopods bud from them. Setae develop on the telson and the maxillipeds, the thoracic appendages present in newly hatched zoea. Hemigrapsus passes through five zoeal stages before becoming a megalopa. This stage resembles the adult form with stalked eyes and five pairs of pereopods (walking legs). The next ecdysis will take the megalopa to the juvenile stage and a benthic existence for the rest of its life.

Saturday, June 8, 2013

Planula of Proboscidactyla flavicirrata

The life cycle of the hydrozoan medusa, Proboscidactyla flavicirrata, is one that is typical of the hydrozoans, with full alternation of generations - i.e. with a fully developed medusa and polyp (see Ashley Choi's blog post, Life cycle of hydrozoan Obelia sp.). Freshly collected medusae readily release eggs or sperm (sexes are separate) in early morning the day after being collected. Fertilized eggs develop into planula larvae, depicted below.

This is a picture of the 5-day old planula larva of P. flavicirrata. As in other hydrozoans and scyphozoans, the planulae of P. flavicirrata are lecithotrophic - they are non-feeding, rather they depend on yolk reserves to reach metamorphosis. Planulae are ovoid in shape and uniformly ciliated. Many hydrozoan planulae develop muscles only after metamorphosis (S. Maslakova, pers. com.), but in my culture, planulae of P. flavicirrata were clearly contractile!
Believe it or not, this is the same individual as shown above. It has used its muscles to elongate.  Hydrozoan planulae have two types of epithelial muscle cells that are separated by mesoglea (Gröger 2001).

A close up view of the same individual shows the two cell layers: the outer ectodermis and the inner endodermis, separated by a thin extracellular layer of mesoglea. Myoepithelial cells in ectodermis and endodermis have muscle fibers (not detectable without special staining) that run along the mesoglea.

Gröger, H and V. Schmidt. 2001. Larval Development in Cnidaria: A Connection to Bilateria? gensis 29 (3):110-114.

Mills, C.E. and J.T. Rees. 2007. Key to the Hydromedusae. In "The Light and Smith Manual Intertidal Invertebrates from Central California to Oregon". Edited by J.T. Carlton. University of California press. Los Angeles. Pp. 137-150.

Wrobel, David and Claudia Mills. 1998. Pacific Coast Pelagic Invertebrates A Guide to the Common Gelatinous Animals. Monterey Bay Aquarium and Sea Challengers: California.

Thursday, June 6, 2013

Ascidian tadpole larvae: settlement and metamorphosis

Ascidians (class Ascidiacea), commonly known as sea-squirts, are a class of sessile, filter-feeding chordates who live solitarily or colonially inside an extracellular “tunic.” Although the adults have no overt chordate features, their free-swimming tadpole-like larvae have a tail supported by a notochord, homologous to the spinal cord in vertebrates, and a hollow nerve cord like that of other chordates (Sasakura et al. 2012).  The following pictures detail the process of larval settlement and metamorphosis in a colonial ascidian Distaplia occidentalis, a fouling organism on floating docks in NE Pacific marinas.

This is a lateral view of a recently released tadpole larva. Almost all colonial ascidians brood their larvae, and release them for a brief planktonic period (minutes to hours). The dark spot near the base of the notochord is the sensory vesicle, which contains both a light-sensing ocellus, and gravity-sensing organ called a statocyst (both pigmented). At the anterior end of the tadpole’s trunk (to the right) one can see two bulbous structures just under the tadpole’s epidermis. They are called adhesive papillae. They are everted at settlement to initially adhere the tadpole to the substratum.

Upon contact with whatever substratum the larva deems suitable for settlement, the tadpole rapidly (< 1 min) everts its papillae, the tips of which secrete an adhesive substance. This picture shows eversion of the adhesive papillae. A Distaplia tadpole has three adhesive papillae, and two are in focus here. The adhesive filamentous material seen radiating from the base of the papillae is secreted by the epidermal cells of the everted papillae (Cloney 1978, and previous studies by the same author cited therein).  A study by Flores and Faulkes (2008) suggests that ascidian tadpoles may be able to differentiate between different types of surfaces on which to settle (e.g. smooth vs. sandblasted), possibly due to the presence of mechanoreceptors in those epidermal cells.

After eversion of the papillae, the ampullae, large adhesive lobes which create a more permanent hold to the substratum, are everted. The ascidian begins to reabsorb its axial complex (the notochord, nerve cord, and associated musculature of the tail) and develops the respiratory and feeding structures that characterize the adult. This picture shows a recently metamorphosed juvenile, its ampullae (short root-like structures) attached to the substratum (the side of glass slide). The tail is reabsorbed within a few hours after settlement. The large dark shape inside is the remnant of the larval axial complex plus the developing branchial basket of the juvenile. The little dark spot at upper right of the dark shape is the degenerating sensory vesicle. Immediately above the sensory vesicle one can see one of the two developing siphons, through which the adult ascidian pumps water to breathe and feed.

Cloney, R. A. (1978). Ascidian metamorphosis: review and analysis. In: Settlement and metamorphosis of marine invertebrate larvae. Chia, F.-S. and Rice, M.E. (eds).  Elsevier. New York. pp. 255-282.

Flores, A. R. and Faulkes, Z. (2008). Texture preferences of ascidian tadpole larvae during settlement. Marine and Freshwater Behaviour and Physiology. 41(3): 155-159.

Sasakura, Y., Mita, K., Ogura, Y., and Horie, T. (2012). Ascidians as excellent chordate models for studying the development of the nervous system during embryogenesis and metamorphosis. Development, Growth, and Differentiation. 54(3): 420-437.

Development of the hydrozoan Clytia gregaria

This is Clytia gregaria, formerly known as Phialidium gregarium. The instructor of our Embryology class collected many adult individuals of this hydromedusa from the plankton off F-dock in the Charleston Marina Complex. I like cnidarians and I wanted to follow the development in this species.
Adult C. gregaria is about 2 cm in diameter, and can be identified by its ruffled manubrium located in the center of the subumbrella (the underside of the bell). The four gonads are located along the radial canals of the digestive system (the thick white lines radially arranged on the underside of the bell). C. gregaria hydromedusae are present in the plankton from late spring to early fall.  Recently collected hydromedusae of this species readily release eggs and sperm (typically, at the crack of dawn the day after collection), fertilization and embryonic development is external.
This is a 4-cell stage. As you can see the cells are all the same size, so the cleavage is equal.  Clytia, like many hydromedusae, have transparent eggs which makes them convenient embryological study objects. The four little spheres (one inside each cell) in this picture are the cell nuclei, visible thanks to the clear cytoplasm of these eggs.
Cnidarians (and ctenophores) exhibit an unusual type of cell division illustrated here. It is called unilateral cleavage, and means that cleavage furrow forms at one pole of the cell and progresses to the other pole, so the cells appear heart-shaped in mid-cleavage, as you can clearly see on this picture of a ~16-celled embryo. The site of initiation of first embryonic cleavage defines the oral end of the developing embryo.
The embryo develops into a planktonic planula larva shown here. The planula larva is characteristic of most cnidarians. The hydrozoan planula is uniformly ciliated, oval-shaped, usually somewhat opaque, and lacking any appendages or defined gut (they do not feed). Hydrozoan planulae usually spend a short period in the plankton (days), then settle and undergo metamorphosis into a benthic polyp stage, the asexual generation in the life cycle of a hydrozoan.

Gastrulation and early actinotroch of Phoronopsis harmeri

This is a side view of a blastula of the phoronid, a.k.a. horseshoe worm, Phoronopsis harmeri. It is about 22 hours after egg release (developing at ambient sea temperature).  Eggs in this species are fertilized inside the body cavity, but initiate their development after spawning (or being dissected, as is the case here).  Note the spacious cavity called blastocoel inside the blastula. The surface of the blastula is ciliated, each cell bearing a single cilium. Longer cilia at the animal pole (up) indicate position of the future apical sensory organ. 

This is a gastrula stage. It is 24 hours old.   The circle of cells inside the blastocoel is the developing gut of the embryo, otherwise known as the archenteron.  The archenteron opens to the outside via the blastopore which will later develop into the mouth. Cells clustered at one end of the archenteron are the mesoderm cells which will form the muscles and coelomic sacs in the larva. 

This is a young actinotroch larva that is ready to feed on microscopic algae. It has a complete gut with mouth and anus. The mouth opens under the anterior hood and leads into a spacious vestibule, which leads into the gut. Note a thickened region of epidermis directly overlaying the vestibule - this is the apical sense organ. Sandwiched between the apical sense organ and the vestibule is a thin-walled sac - this is the anterior coelomic cavity, the protocoel. The gut proper has two distinct compartments - the stomach, which occupies most of the actinotroch’s body, and the short hindgut that opens via an anus at the posterior end. The actinotroch will later develop a crown of tentacles posterior to the mouth which will assist in capturing food. 

Procuring gametes from echinoderms

Echinoderms, sea urchins and sea stars in particular, are classical objects of embryological studies because it is relatively easy to obtain their gametes. Here I will describe two standard techniques used to obtain embryonic cultures of echinoids, exemplified by the sand dollar Dendraster excentricus, and asteroids, exemplified by the ochre sea star Pisaster ochraceus.

Here you can see how we collect sperm from a spawning male of the sand dollar. Injection of ~1 ml of 0.5M KCl into the body cavity of the sand dollar induces spawning by causing strong contractions of the gonad.
If the specimen has ripe gonads, after a few moments gametes (sperm or eggs) begin to emerge from the five small openings called gonopores on the aboral (opposite side from the mouth) surface. Sperm is cream colored, and eggs are pink. To collect eggs, we inverted a spawning female (oral end up) over a beaker full of filtered seawater. The eggs collect at the bottom of the beaker. 

There are several ways to get fertilizable oocytes from sea stars. One way is to inject the body cavity with 1-methyl adenine (1-MA) solution (about 1 ml of 100 μM 1-MA in distilled water per 100 ml of body volume). 1-MA, also known as maturation-inducing substance, stimulates spawning, ovulation (release of oocytes from follicles), and oocyte maturation (completion of meiosis) in sea stars.  The advantage of this method is that the adult remains intact. The disadvantage is that it will have completely spawned out, and won’t be useful for future embryological experiments for at least a few months. In order to use the same individual several times, one can take advantage of the sea star’s regenerative abilities. Instead of injecting 1-MA into the body of the intact individual, dissection of just a piece of ovary or testis is sufficient. For example, Patiria miniata, the bat star, tolerates biopsy very well, then promptly heals, so one can re-use the same individual many times. One can use a biopsy tool to cut a small window at the base of an arm, and pull a small piece of gonad out. However, some species, like Pisaster ochraceus do not heal well after biopsy, and heal much better if an entire ray is severed to remove gonads (apparently, the cutting of the radial nerve stimulates regeneration).

As shown here, one can use a razor blade to cut the ray and reveal paired gonads within the body cavity. Ovaries in P. ochraceus are pink or orange-ish, while testes are creamy white. Some oocytes thus dissected complete meiosis spontaneously and can be fertilized, however, most need some help in the form of 1-MA. Incubation of dissected ovaries with 1 μM of 1-MA in filtered sea water for about an hour stimulates ovulation and oocyte maturation.

Development of the ctenophore Beroe

Below are pictures of early embryos of the ctenophore Beroe sp. Three adults were collected around Charleston docks near OIMB in mid-May and shed strings of ~ 220 µm eggs in the lab. 

Ctenophore embryos are beautifully clear, and possess a unique type of development. Like cnidarians, ctenophores have unilateral cleavage, which means that the cleavage furrow cuts in from one pole of the egg and gradually proceeds to the other side (see a video of cleaveage in another species of Beroe by George von Dassow). Ctenophore cleavage is stereotypical (all embryos exhibit the same series of highly ordered divisions), and the same cells consistently give rise to certain structures in the adult. Ctenophores exemplify mosaic development, which means that cell fates are determined early in development by inheritance of different regions of the egg’s cytoplasm which possess different properties. As a result, with few exceptions, when specific blastomeres are damaged, they are not replaced; and the embryo lacks the structures these blastomeres normally give rise to (Martindale & Henry 1999). 

This is a gastrula of the same species of Beroe. Ctenophores undergo gastrulation by a variety of mechanisms simultaneously, including epiboly, delamination, invagination, and involution. At the end of gastrulation large cells, called macromeres, end up surrounded by small cells called micromeres (see another video by George von Dassow). 

These embryos developed into little juvenile Beroe, pictured here, with beautiful red pigment granules in the epidermis. Another curious fact about the development of Beroe is that apparently, at least one species, Beroe ovata, can tolerate naturally occurring polyspermy (Carré & Sardet 1984). Polyspermy is a condition in which more than one sperm enter the egg. In most animals polyspermic embryos develop abnormally. However, Carre & Sardet (1984) demonstrated that in Beroe ovata the female pronucleus appears to preferentially select a sperm pronucleus with which to bind. The female pronucleus visits each sperm pronucleus, returning to the site of formation of polar bodies in between visits, and finally fuses with one of them (see video on Christian Sardet's website). The other male pronuclei are destroyed and do not participate in development which proceeds normally. 
Carré, D., Sardet, C. (1984). Fertilization and Early Development in Beroe ovata. Developmental Biology 105: 188-195. 

Martindale, M.Q., Henry, J.Q. (1999). Intracellular fate mapping in a basal metazoan, the ctenophore Mnemiopsis leidyi, reveals the origins of mesoderm and existence of indeterminate cell lineages. Developmental Biology 214: 243-257.

Tuesday, June 4, 2013

Brachiolaria larva of the seastar Mediaster aequalis

Mediaster aequalis is a bright red asteroid, three to seven inches in diameter, found subtidally off the coast of Oregon. M. aequalis has a breeding season from late March through May and spawns large (~1.2mm) yolky oocytes that are opaque and bright orange (Birkeland et al. 1971). In order to get the oocytes and sperm out we biopsied adult M. aequalis. Pieces of dissected ovary were placed into a solution of 1-methyl adenine (1-MA) for an hour to induce oocyte maturation. Oocytes were then fertilized with a dilute suspension of sperm. Several of the fertilized eggs developed.

I took some pictures of 35-day old brachiolaria larvae. As in other species of seastars with large yolky eggs, Mediaster lacks a feeding bipinnaria larva, and develops via a non-feeding pelagic brachiolaria. As you can see here these yolky non-feeding brachiolariae have three brachiolar arms (stubby appendages at upper right). These are used for attachment to substratum during metamorphosis. The juvenile seastar develops at the opposite (morphologically posterior) end. 

The many small bumps (clearly visible in the bottom picture) cover the juvenile rudiment and develop into adult spines. The larger bumps of the juvenile rudiment are the developing rays of the seastar. Once the juvenile rudiment and the brachiolar arms are formed (which may be as early as 9-10 days) the larva is capable of metamorphosing. M. aequalis will only settle and metamorphose on a suitable substratum, such as a tube of the annelid Phyllochaetopterus. If no suitable substratum is available, larvae can delay metamorphosis for as long as 14 months (Birkeland et al. 1971). 

Birkeland, C., Chia, F-S., Strathmann, R.R. 1971. Development, substratum selection, delay of metamorphosis and growth in the seastar, Mediaster aequalis stimpson. Biological Bulletin. 141:1, 99-108.

Saturday, June 1, 2013

Planktonic larvae of the polychaetes Nephtys and Capitella

I collected the two polychaete larvae pictured here from a plankton sample taken off a dock in Charleston, OR on May 8, 2013. The thing that caught my eye was the deep blue color in the lining of the gut of the larva shown immediately below. 

This blue pigmentation along with a dome shaped episphere, a pair of eyes, and two transverse ciliary bands (anterior prototroch and posterior telotroch) separated by 10 body segments suggests that this larva (called metatrochophore) belongs to a polychaete from the family Nephtyidae, likely the genus Nephtys. Twelve species of nephtyids are reported from the NE Pacific, but the larval development has been described only for one of them, Nephtys caeca (Crumrine 2001). 

Jay Bowles, a student in the Molecular Marine Biology class at the OIMB,  matched a similar-looking larva to the genus Nephtys using DNA sequence data (see blog post Confirmed identity of wild-caught planktonic larvae by Terra Hiebert). Nephtys larvae like this one are planktotrophic, their gut has a spacious lumen, and one can sometimes see diatoms and other food particles inside (S. A. Maslakova, pers. comm.).

The morphology of this other larva suggests that it belongs to the polychaete family Capitellidae, likely the genus Capitella. There are eight species of capitellids in the NE Pacific, but the development is described only for two of them. Although this larva fits the brief description of larval Capitella capitata in Crumrine (2001), it may also belong to another capitellid species whose development has not been described. This larva, like the one pictured above, is also propelled by two transverse ciliary bands, the anterior prototroch and posterior telotroch. There are several segments bearing developing chaetae (none protrude at this stage) between the two ciliary bands. The segment posterior to the telotroch, called pygidium is covered with short cilia. The two eyes are at the same level as the prototroch. The shiny marbles inside are the lipid droplets filling the cells of the gut. These lipid droplets are likely inherited from the egg, - i.e. this larva is likely non-feeding (lecithotrophic). See my other blog post about lecithotrophy vs. planktotrophy.  

Crumrine, L.  2001.  Polychaeta. In: An Identification Guide to the Larval Marine Invertebrates of the Pacific Northwest.  Edited by Alan Shanks.  OSU Press, Corvallis.

Mass spawning of beached hoplonemertean Nipponnemertes bimaculata

The tide was low at 6 am on May 8th as our Embryology class descended into South cove at Cape Arago, OR. As we made our way to the tide pools we noticed a few live Nipponnemertes bimaculata, bright orange ribbon worms (nemerteans), laying among the floatsam and kelp washed up onto the sandy beach of the cove. The two characteristic brown spots on the head are responsible for the specific epithet (“bimaculata” means “two spots”). Svetlana, our instructor and an expert on the biology of nemerteans, thought it strange; these are usually subtidal and are only occasionally found under rocks intertidally, but are not generally known to get stranded on sandy beaches. Puzzled, we continued on to the tide pools to collect bryozoans (our study subjects that day). 

When we made our way back to the sandy beach at about 9 am we found the beach littered with stranded N. bimaculata. We soon realized that most of them were releasing gametes! Hundreds of these worms got stranded in an unheard-of, for hoplonemerteans, mass spawning event. N. bimaculata and other members of the family Cratenemertidae are unusual among monostiliferan hoplonemerteans because they can swim by undulating their body. Svetlana hypothesized that the worms (normally benthic) were swimming and spawning en mass, when the change in tide and currents got them stranded. 

We collected several of the worms of each gender, placing males and females in separate bags as well as in a bag together in order to observe embryonic and larval development, which has not been previoulsy described. The males were rather orange and released white sperm, while the females were somewhat darker (brownish or greenish) due to the color of the eggs inside them, You can see both males and females on this picture. Sexes cannot be easily distinguished once the worms release gametes. On our way back to OIMB we checked the sandy beaches of Middle cove at Cape Arago, and the nearby Sunset Bay, but they were devoid of nemerteans. This may be either because the suitable adult habitat is lacking (e.g. kelp holdfasts) or because the hydrodynamics are different in these coves.

The greenish ~ 265 micron eggs are loosely connected by jelly when released, are very delicate despite the protection of the chorion, and easily rupture when handled. Most of the eggs in our culture disintegrated after the first few days, but a few developed into planktonic planuliform larvae, one of which is depicted here - a week and two days after fertilization. Note the two large reddish eyes and conspicuous lipid droplets. Most hoplonemertean larvae have several pairs of relatively smaller eyes (e.g. see blog posts by  Jenna Valley and Kirstin Meyer). The larva depicted here is 460 micron long.

Brooding copepods

Copepoda, with approximately 12,000 described species, is one of largest taxa of crustaceans and a large contributor in most plankton samples. This is particularly true of calanoid copepods, as most species in this group live entirely planktonic lifestyles.  I collected the brooding calanoid copepod pictured here from a plankton sample taken in the Charleston boat basin. The anterior-most and longest appendages (first antennae) extend out of the frame of this picture. Some copepods release eggs into the plankton, but, as is the case here, many species carry their eggs in sacks attached to the abdomen. Note the two ovisacs laterally along the abdomen (the narrow posterior portion of the body) posterior to the thorax (the broad portion of the body) and anterior to the caudal ramus (the forked tail). 

The second picture shows a close up view of the ovisacs. Inside the very thin transparent membrane of each ovisac (barely visible here) are 15-25 large eggs (each 70-80μm in diameter). The ovisacs are secreted by the epithelial cells lining the oviducts and are attached to the genital segment (the 6th thoracic segment fused to the 1st abdominal segment). Copepods in general are dioecious (meaning that they have separate sexes). During copulation males release spermatophores (sperm packages) and glue them to the female abdomen. Spermatozoa from the spermatophores then move into the female reproductive tracts and are stored there until they are needed. The eggs are fertilized internally on their way to the ovisacs where they will be brooded until they hatch as nauplii. 

The nauplius larva pictured here hatched from just such a brood. It is about 270μm long. The nauplius hatches and swims using three pairs of head appendages: the first and second antennae, and the mandibles (from anterior to posterior respectively). The reddish blotch between the two first antennae is the naupliar eye. Copepod larvae go through six naupliar stages followed by five copepodid larval stages, molting between each stage. The mature adult no longer molts, and thus cannot grow. 

Thursday, May 30, 2013

Two types of Aeolidia papillosa larvae

This is a picture of an adult nudibranch Aeolidia papillosa, found on the Charleston docks near OIMB. As with many other nudibranchs, A. papillosa package their eggs a few per egg capsule, the capsules embedded in a gelatinous ribbon, which are deposited as egg masses (pictured below). Early development is encapsulated, and embryos begin to move inside the capsule, before they hatch as shelled veligers (bottom picture).

Many marine invertebrates are characterized by a particular type of development e.g. either lecithotrophic or planktotrophic (see Jon Gienger's blog post, Planktotrophy versus lecithotrophy). Interestingly,  Aeolidia papillosa veligers hatching from the same egg capsule can be polytypic: some released as yolk-laden lecithotrophic larvae, and others as yolk-free planktotrophic larvae (Williams, 1980). Williams (1980) also noted that larvae that hatched without yolk reserves were, paradoxically, larger than those released with yolk reserves, although both types of larvae developed from uniformly small eggs. A simple explanation might be that because these two types of larvae develop within the same egg capsule, it is possible that the yolk-laden (slower developing) larvae are prematurely released from the egg capsule by their yolk-free (faster-developing) siblings.  However, yolk-laden larvae hatched from egg capsules that did not contain any yolk-free larvae. What’s more, smaller larvae were apparently less likely to feed on unicellular algae (e.g. Chlorella, Dunaliella) than their larger siblings.  Both yolk-free and yolk-laden veligers were present in the egg masses I looked at, in addition to yolk-laden trochophores, indicating that larvae were still developing.

One possible explanation for this polytypic development may be bet-hedging (varying strategy to increase the overall chances of offspring survival and success).  Lecithotrophic larvae are expected to survive to metamorphosis better than the planktotrophic under conditions of scarce food, whereas planktotrophic larvae may be more successful when phytoplakton is abundant. Producing both types of larvae may be advantageous when phytoplankton has spatially and temporally patchy distribution (Williams, 1980).

Williams, L.G. (1980). Development and feeding of the larvae of the nudibranch gastropods Hermissenda crassicornis and Aeolidia papillosa. Malacologia 20:99–116.

Reproduction in monogonont rotifers

This is a marine rotifer from the genus Synchaeta (according to a key in Wallace & Snell 2010). Rotifers are a phylum of small, mostly fresh-water invertebrates. This adult individual is 0.7 mm long. When I found it in the plankton sample, it was carrying two egg capsules attached to its foot (a little stalk at posterior end of the organism-see photo). While I watched it, one of the egg cases ruptured, releasing a fully formed and seemingly completely functional miniature (~ half a millimeter long) rotifer. The empty case remained attached to the foot of the adult, while the newly hatched rotifer (shown below) began swimming immediately.   

Interestingly, rotifers can reproduce both sexually and asexually. One class, Bdelloidea, appears to lack males altogether (Wallace & Snell 1991). Sexes are separate in the Class Monogononta, to which the marine genus Synchaeta belongs. For the majority of the year, females produce offspring asexually by generating diploid embryos that develop without fertilization into females. Under certain conditions though, females produce haploid eggs. As in certain social insects (e.g. bees), if these eggs remain unfertilized, they develop into haploid males. If fertilized, diploid eggs can remain in a diapause (resting state) for up to 20 years (Fradkin 2007), and eventually develop into females who feed and grow before becoming sexually mature (Ricci & Melone 1998). These resting eggs allow the population to outlive adverse conditions (e.g. desiccation, freezing etc.). Haploid males are sexually mature at birth, do not feed and live a short life (usually about half as long as females) with the apparent sole purpose of fertilizing females via hypodermic insemination (Ricci & Melone 1998). Another fascinating fact is that most rotifers have constant cell numbers as adults (~ 1000) (Fradkin 2007). In other words, no cell divisions take place after embryogenesis is completed, and growth is accomplished solely by enlarging existing cells. In contrast, humans start out as one cell, but end up with trillions in the adult body, the vast majority of which are born post-embryonically. Additionally, in some asexually reproducing rotifer genera, generational clones have progressively shorter life spans (i.e. daughters live a shorter life than their mothers, grand-daughters live even shorter, and so on), which eventually leads to extinction of the line (see King 1969 for a review).

A dearth of information about development, distribution, and ecology in NE Pacific rotifers leave members of this phylum prime candidates for future research.

Fradkin, S.C. (2007). Rotifera. Light's manual; intertidal invertebrates of the central California coast. 4th ed. pp 280-282. J.T. Carlton (ed.). University of California Press.

King, C.E. (1969). Experimental studies of aging in rotifers. Experimental Gerontology 4: 69-79.

Wallace, R.L., Snell T.W. (2010). Rotifera. In: Ecology and classification of North American freshwater invertebrates. 1st ed. pp 173-235. J.H. Throp and A.P. Covich, eds. Academic Press.

Ricci, C., Melone, G. (1998). Dwarf males in monogonont rotifers. Aquatic Ecology 32: 361-365.

Wednesday, May 29, 2013

Early development and spermatophore of the phoronid Phoronopsis harmeri

Phoronopsis harmeri is a member of Phoronida, a relatively small phylum of sessile, tube-building marine worms. Phoronids release sperm in packets called spermatophores, which probably drift in water currents before landing on the lophophore of another individual. In P. harmeri and some other species, these spermatophores are equipped with a mucoid spiral “sail” (Zimmer 1997). The spermatophore of P. harmeri, sail included, is pictured here. 

There is some controversy regarding the early cleavage pattern of phoronids. While some researchers have reported radial cleavage, others have reported spiral cleavage, biradial cleavage, and “derived spiral” cleavage (reviewed by Pennerstorfer and Scholtz, 2012). In spiral cleavage, the blastomeres of the early embryo divide along a plane that is oblique to the animal-vegetal (A/V) axis, and the cells are not stacked directly on top of each other but are offset from one another. In contrast, blastomeres undergoing typical radial cleavage divide either parallel or perpendicular to the A/V axis, and the cells stack directly on top of one another (Ruppert et al. 2004). Pennerstorfer and Scholtz (2012) quantified the number of embryos exhibiting spiral-like cleavage in Phoronis muelleri and the degree of tilt of the plane of division. They concluded that, beginning with the third cleavage, early cell division in P. muelleri tends to occur in a spiral pattern, although there is a high amount of variability among embryos of this species.

Pictured at left are examples of the 8-cell and 16-cell stage of P. harmeri, whose early development I observed while taking the Embryology course at OIMB. The sister cells in the 8-cell embryo (side view) appear to be nearly on top of one another, so the third cleavage (at least in some embryos) of this species is not obviously spiral. However, the four polar blastomeres in the 16-cell stage pictured here are noticeably offset from their four sisters, though not every embryo in our class culture looked like this at the 16-cell stage. At least some embryos in this culture exhibited alternating (dextral vs. sinistral) divisions (S. Maslakova, pers. comm.), as in typical spiralians. Is early development in P. harmeri also somewhat spiral?

Pennerstorfer. M. and Scholtz, G. 2012. Early cleavage in Phoronis muelleri (Phoronida) displays spiral features.Evolution and Development. 14(6): 484-500.

Rupert, E., Fox, R., and Barnes, R. 2004. Invertebrate Zoology: A Functional Evolutionary Approach. 7 ed. Brooks/Cole.

Zimmer, R. 1997. Phoronids, Brachiopods, and Bryozoans: The Lophophorates. In Embryology: Constructing the Organism. Gilbert, S. and Raunio, A. Eds. Sinauer Associates, Inc. Sunderland, MA.