Obtaining marine invertebrate embryos and larvae

The transformation from a single celled, spherical egg, to a complex animal, is one of the most fundamental puzzles in biology. Marine invertebrate embryos and larvae are particularly fascinating, and often surprisingly easy to obtain. Some of them were the first model systems in developmental biology, and they still have a huge amount to teach us about mechanisms of development, developmental evolution, and the ecology of development. M. Strathmann 1987 is an excellent reference for techniques for obtaining and culturing marine invertebrate embryos and larvae [1]. While it focuses on the species found in the northwest coast of the US, the techniques work for species from all over the world.


Gastropods will often lay egg masses or egg capsules containing embryos at various developmental stages if just left in a dish. The image below shows an opisthobranch gastropod, Haminaea vesicula (≈2 - 3 cm long), in the process of laying an egg mass (yellow ribbon) on the wall of an aquarium. The view is towards the foot of the animal. These snails — and their egg masses — are common in mudflats on the Washington State coast.


Searching in the intertidal, one can find lots of other kinds of egg masses or egg capsules (which have a tough, firm outer layer) from other species of gastropods


Some annelid worms also lay egg masses. For example, the polychaete Axiothella mucosa lays gelatinous, oval egg masses (≈4 cm long) tethered to the mud. An example is shown below: each white dot is an embryo, and the tether is visible to the upper right. The adult worm is shown on the right (roughly 5 cm long). These egg masses can be found on lots of mudflats in North Carolina.


Cnidarians and ctenophores:

Hydrozoan medusae (jellyfish) and ctenophores often spawn shortly after a change in light (dusk or dawn) so one can place a couple of adults in a dish with filtered seawater, and then pipette embryos or larvae out of the dish once they've spawned (in some species the embryos will be on the bottom; eggs of other species are buoyant, so they will float to the top).

Both cnidarians and ctenophores can be collected off docks using a dipper (a cup on the end of a stick). Place the cup near the animal, and let the water flow into the cup, gently carrying the animal into the cup. Some cnidarians have a strong enough sting to hurt, but many do not. Be sure you know which you have before handling.



At the right times of year, acorn barnacles embryos and larvae at different stages can be found inside the adult barnacle. If you live in cold temperate zones, embryos (S. balanoides) can be found from December until February. Amphibalanid = Balanus barnacles can be obtained from March until November in Warm Temperate to tropical areas (below cape Hatteras in the US). Embryos are collected by opening the barnacle and pulling out the eggs. One can break the barnacle off a rock or other surface, and reach in the back (if there is an opening), remove the tissues, and find the embryos, stuck to each other in tissues along the inside base of the barnacle. The following image shows an isolated, late stage embryo of Amphibalanus (=Balanus) amphitrite (~0.2 mm long), before it hatches:


To get swimming larvae, one can either take the approach above, scrape barnacles off a surface and put a small handful of them in a bucket with seawater, or place something like a small barnacle-covered branch or rock in a bucket with sea water. Add a couple of crushed barnacles and a little algae (if you have it), and put the bucket in a dark room with a light on the side (see video). Barnacles release their larvae when exposed to crushed barnacles and/or algae. If you don't get larvae in an hour, dump out the water, let the damp barnacle sit overnight, and try again the next day. The larvae will swim towards a light, where one can pipette them up. This is a nauplius larva of Balanus (=Amphibalanus) amphitrites:


Collection and maintenance of barnacle larvae for short periods is quite easy. If you put individual larvae into a vial containing about 4 mls of sea water and put them in a light place, some will grow to the settlement stage (the cyprid stage). Barnacle larvae are fragile. Don't try to change the water and don't let it evaporate. Culturing larvae though the six larval stages, to settlement-stage cyprid larvae, in numbers from 100s to thousands is very difficult (see video). Only one laboratory in the world raises barnacles in large numbers, and that laboratory provides barnacles research laboratories around the world. Here is a nice video by a high school student, Anna Kellner, that describes large-scale culture of barnacle larvae. If you would like to try and culture barnacle larvae contact ude.ekud|ttir#ude.ekud|ttir and set up to talk with Dr. Dan by Skype.


Echinoderms, especially sea urchins, are one of earliest model systems in developmental biology. Experiments in the mid-1800s and early 1900s were central to the discovery of fertilization, the development of notions of regulative development (a crucial step to our modern understanding of cell-cell signaling in development), and the role of chromosomes (reviewed in [2]). They continue to be important model systems for cell division, developmental patterning and morphogenesis, and developmental ecology and evolution (see http://worms.zoology.wisc.edu/urchins/SUmainmenu.html and [3] and [4] for reviews of a few aspects of these more recent advances).

Spawning urchins

Sea urchins and sand dollars can be spawned by injection of 0.5 - 0.53 M KCl into the body cavity (one injects 0.5 - 2 mL through the soft membrane around the mouth; volume depends on body volume). Use care when injecting, and dispose of the needle properly in a sharps container. Sea urchins are remarkably tolerant of injection with KCl, but humans are not. Do not inject yourself!

Inducing spawning. To induce spawning, one injects 0.5 - 0.53 M KCl into the urchin, through the soft membrane on the oral side. Photo courtesy of Dr. Karen Chan.

The gametes come out the top of the animal (white for sperm, other colors for eggs1). Females should be turned upside down in a beaker or cup of filtered seawater so that the gametes are shed into seawater. They will look slightly granular as they drift down in a thin stream to the bottom of the beaker.2 Eggs should not be clumpy: it is a sign of poor condition. If you check under the microscope, the eggs should be a uniform size and round3, and there should be few fragments and other debris. Once the eggs form a little pile, they can be cleaned (if necessary) by pipetting the pile gently (with a large bore transfer pipette) into clean seawater, or decanting off the old seawater and replacing with clean seawater. The sperm should be collected directly out of the male urchin, in air, without contact with seawater. The sperm should be used diluted (~1 small drop into about 50 mL seawater, mixed up, should work) before fertilization. Diluted sperm will only last a few minutes, however concentrated sperm can be stored, covered, at 4°C4 for a few days. Eggs should be used soon after spawning (within 30 minutes is best); they cannot be stored.

Fertilizing urchin eggs

To fertilize the eggs, add a few drops of dilute sperm suspension to the eggs and gently mix (best within a few minutes after collecting the gametes). Too much sperm causes severe developmental problems if multiple sperm fertilize the same egg5. Watch for the elevation of the fertilization envelope, which forms a very thin, transparent halo around the egg6. If few eggs fertilize, you can add more sperm.

Fertilization in the sand dollar Dendraster excentricus. Eggs were settled at the bottom of a 12 well plate, and a small amount of sperm suspension was added (when the water shakes at the beginning). Swimming sperm are visible around the large egg. Sperm-egg fusion triggers elevation of the fertilization envelope around the egg.
The image contrast and color balance were adjusted, and the frames were cropped and scaled, in Image J. The video was taken using an inverted microscope with a 20x objective.

Culturing embryos and larvae

After fertilization, the embryos should be diluted into a flat bottomed custard or petri dish of clean seawater. The eggs should be dilute enough that they do not pile on top of each other. They should be in a thin, single layer that does not cover the bottom of the dish. They should be kept at between 10°C and 30°C [13] (16 – 24°C seems ideal), and covered to reduce evaporation.

After hatching (about 1 day) they should be diluted further. For the first few days they can be kept at about 1 larva per 0.5 mL of seawater. As they get larger they should be kept more dilute (1 larva per 2 - 10 mL seawater). They should be fed 1 – 5 drops of algae culture (for a 50 mL dish) every 1 - 3 days. You should monitor them to make sure they are getting enough food but not too much. Their guts should appear to have material inside them. If the skeletal rods of their arms poke out of the epithelium, they are starving. However you also don't want to over feed them: the media should not be green after feeding and there should be nothing growing or settling on the bottom of the dish. The larvae should be gently transferred by wide-bore pipette to a new dish with clean seawater every few days.

Different species need to be maintained at different temperatures in order to permit normal development7.

Larvae of a sea urchin. (species unknown) Photo courtesy of Dr. Karen Chan.

Additional information

In addition to [1], other useful references for sea urchin methods include: http://celldynamics.org/celldynamics/downloads/methods/urchinlab.html (also see the gallery of amazing images)
http://www.stanford.edu/group/Urchin/contents.html (this site also has several lab exercise suggestions for classes)
http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Urchin/urchin_protocols.html for protocols.

Sea urchins can be collected with the appropriate fishing permits, and following all applicable local, state, and federal laws. Sea urchin habitat and collecting method will depend on the species. Some commercial suppliers are listed at http://www.stanford.edu/group/Urchin/suppliers.html (IGoR has no experience or relationship with any of these suppliers).

1. Strathmann, M.F., Reproduction and development of marine invertebrates of the northern pacific coast : Data and methods for the study of eggs, embryos, and larvae. 1987, Seattle: University of Washington Press. xii, 670 p.
2. Ernst SG. 2011. Offerings from an Urchin. Developmental Biology.358(2):285-94. http://dx.doi.org/10.1016/j.ydbio.2011.06.021 .
3. Gilbert, S.F., Developmental biology2010, Sunderland, Mass.: Sinauer Associates.
4. Wray, G.A., Echinoderms, in Embryology : Constructing the organism, S.F. Gilbert and A.M. Raunio, Editors. 1997, Sinauer Associates: Sunderland, MA. p. 309-329.

biology cnidarians ctenophores development echinoderms embryo invertebrates lab-techniques mollusks polychaetes

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