PITTKIT-SeaUrchinDevelopment-Protocol&LectureSlides

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Sea Urchin

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Habitat of Sea Urchins and

Other Echinoderms

View the YouTube video

Army of Sea Urchins

(http://www.youtube.com/watch?v=D3W4OCnHyCs)

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Timing of Injections

We schedule your shipment of sea urchins to arrive the

day prior to your classroom activity.

Teachers will typically inject the urchins three times, and

harvest gametes twice:

•_{Once on the day the shipment arrives, usually after school, to}

harvest gametes. This takes about two hours, along with unpacking the shipment.

•_{Once on the morning of the classroom activity, before school,}

to harvest gametes.

•_{Additional urchins can be injected during each class period to}

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Unpacking The Shipment

From the brown jug of artificial seawater, pour about an inch

of seawater into each of seven plastic tubs.

Cut open the plastic bags containing the urchins and place

3-4 urchins into each of the tubs.

This segregation into small groups prevents the spontaneous spawning of any urchin from triggering the spawning of all the other urchins in the same water.

Fill a separate plastic tub with Pacific Ocean seawater from

the shipment.

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Injecting Urchins

Place one urchin on an open petri dish with its mouth

facing up.

Fill a syringe with 0.5M KCl:

1.5ml for the larger purple urchins 0.5ml for the smaller brown urchins

Inject the urchin through the soft tissue surrounding the

“lantern of Aristotle” (the urchin’s mouth).

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Gametes of the purple sea urchin

Strongylocentrotus purpuratus

Turn the urchin over

(mouth down) on the

petri dish. Within 1-3

minutes you will see

yellow eggs or white

sperm oozing from

the gonopores.

If no gametes are seen

within 5 minutes,

re-inject the urchin.

Eggs

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Harvesting Eggs

Select a beaker slightly smaller than the urchins. Fill the

beaker to near the brim with artificial seawater from the

clear bottle.

Once eggs are evident, rest the female urchin inverted on

top of the beaker so that it touches the seawater. The

released eggs with settle to the bottom of the beaker.

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Harvesting Sperm

Set a 20µl micropipetter to 20µl, or a 200µl micropipetter to

40µl.

Place the pipet tip in the mass of sperm and

slowly

draw up

the viscous liquid. Move the tip around as you slowly

release the plunger to keep it in the liquid.

Because the liquid is viscous and will clog the pipet tip if left

there more than a minute, frequently transfer small

amounts of the sperm to a microtube. Change the tip often.

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Timing of Fertilizations

You will typically set up at least two sets of fertilizations:

•_{In the afternoon the shipment arrives. This will yield} late-stage embryos (morula, blastula, gastrula) for the following day’s classes to observe.

•_{First thing in the morning of the class day. This will produce} early stage embryos (2-, 4-, 8-cell and morula) for the

students to observe. Use either the previous day’s gametes (check that these produce a high fraction of fertilized eggs) or freshly harvested gametes (best, if time permits).

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Diluting Eggs

In a 15ml screw-cap plastic tube (labeled with

the date and time), dilute the collected eggs

1:10

in artificial seawater from the clear bottle

1000µl eggs in 10ml seawater

View the diluted eggs under the microscope. At

100X magnification (10X objective x 10X ocular),

each field should have about 10-20 eggs. If

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Diluting Sperm

In a 15ml screw-cap plastic tube (labeled with the date and time), dilute the collected sperm 1:1000 in artificial seawater from the clear bottle

10µl sperm in 10ml seawater

The diluted sperm sample should look only slightly milky. If it is too opaque, it should be further diluted.

View the diluted sperm under the microscope. At 100X magnification (10X objective x 10X ocular), you should see many very small sperm moving, but with space between

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Setting Up Fertilizations

In a 15ml screw-cap plastic tube (labeled with

the date and time), mix 2ml of diluted eggs with

2ml of diluted sperm.

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Observing Fertilization Samples

On a three-well depression slide, place the following:

•_{Left well: Unfertilized eggs}

•_{Center well: Fertilization from that morning (early-stage embryos)}

•_{Right well: Fertilization from the previous afternoon (late-stage}

embryos).

After observing each sample, add one drop of diluted sperm

to the left well and quickly observe fertilizations as they

occur.

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Two-Cell Stage with Fertilization

Membrane (Two Hours Post-Fertilization)

After this point,

division occurs

approximately

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Blastula Gastrula

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