Rev 3/16
Invertebrate Reproduction Exercise
Name ______________________________
Lab Partners: ______________________________________________________________
Introduction to Echinoderm Morphology:
In this exercise we examine the morphology and reproduction of a sea urchin. In fall, the species
we use may be the Short-Spined Urchin, Lytechinus variegatus; in spring, the species is more likely to
be the Purple Sea Urchin, Arbacia punctulata. Our ultimate goal is to observe gamete shedding by both
male and female sea urchins and the syngamy event between sperm and egg in vitro.
Sea urchins are members of Phylum Echinodermata and are in class, Class Echinoidea. All
echinoderms are marine and benthic as adults. They may be predators, detritivores, or filter feeders.
Adult echinoderms have pentamerous radial symmetry, with body parts in fives or multiples of five.
Radial symmetry is considered plesiomorphic among invertebrates. But, echinoderm embryonic
development includes larval stages with strong bilateral symmetry. This is evidence that adult
echinoderm radial symmetry may be a reversal homoplasy. Moreover, one group of echinoderms with
adults having nearly bilateral symmetry could represent a reversal homoplasy of a reversal homoplasy
(recall snakes with legs?)!
Another important echinoderm apomorphy is the water vascular system that, in most groups,
functions in support of the soft locomotory tube feet, but is also important in gas exchange, excretion,
and feeding (Fox, 2001). Because most echinoderms have lost cephalization (a key trait of its own),
classic anatomical terms (e.g. anterior, posterior, dorsal, ventral) of bilaterally symmetric organisms do
not apply. Instead the terms oral and aboral are used to refer to the benthos-facing mouth surface and
its opposite surface, respectively.
The test is another defining trait of sea urchins and is composed of calcareous ossicles (literally
“tiny bones”) held together by connective tissue and muscles. The test is complex, with pores, tube
feet, spines, and small pedicellariae (which clean the aboral surface of the test). In contrast to calcified
spines that cover much of the oral and aboral surfaces, tube feet and their related pores occur primarily
laterally and on the oral surface. Tube feet are soft and are connected directly to the water vascular
system. Stiff calcified spines and pedicellariae are protective defense structures. The ossicles are most
obvious in the center of the aboral surface. A look with a dissection microscope should reveal a circle
of more-or-less fused anal plates surrounding a central anal pore. Surrounding the anal plates is a
wreath of other ossicles; of these, the madreporite is the largest and most porous. It provides entry and
filtration for seawater into the water vascular system. The control of pressure in this system allows soft
tube feet to be limp or turgid, to shorten or to extend, and to attach or detach the suction cup tips of the
tube feet. Another of the ring of ossicles around the central anal plates are the medium-sized genital
plates; these each have a large pore (the gonopore) through which gametes (eggs or sperm) are shed
into the water column. Smaller ossicles, the ocular plates have small pore-like depressions that house
the photoreceptors.
On the oral surface, surrounded by spines and tube feet, you will find the centrally located mouth,
with the protruding tip of Aristotle’s lantern. This internal structure ending in (usually 5) teeth is
found only in sea urchins; other echinoderms may have vestigial remnants of the lantern’s structure.
The lantern consists of five bony internal jaws supported by connective tissue and muscles. The teeth
from the jaws protrude from the body cavity and are used to scrape food material (algae, detritus, etc.)
from hard surfaces.
Document © Ross E. Koning 1994. Permission granted for non-commercial instruction.
Koning, Ross E. 1994. Invertebrate Reproduction Exercise. Plant Information Website.
http://plantphys.info/organismal/labdoc/urchin.doc
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Lateral to the teeth, surrounding the jaws, is a very soft membrane, the peristomal membrane. The
test is open at the oral center for the teeth to protrude from within, and the space to the rim of the test is
filled by the soft connective tissues of the peristomal membrane. It will be into this soft tissue
surrounding the teeth and extending to the edge of the test that we will insert a hypodermic needle and
inject a chemical (KCl) to induce gamete release.
Echinoderm Reproduction:
When conditions are appropriate for spawning, males release a small portion of their sperm into the
water and wait for a response. If gravid females nearby respond by releasing eggs into the water
column, then the males in turn release the rest of their gametes. The result is a positive feedback loop
that, in a dense local population, rapidly picks up momentum until the water is thick with gametes!
This reproductive behavior is called broadcast spawning. Because of their limited mobility and the
vagaries of broadcast spawning, it is critical for adult sea urchins to find members of the opposite sex
and then, to respond rapidly to the presence of gametes in the water column. There are filter-feeding
predators that will use the planktonic gametes and early embryonic phases as food! Each sea urchin has
a gonopore in the medium-sized ossicles on its aboral surface that serves as the point of gamete
release.
Syngamy (aka: “fertilization”) is a two-step process. Once plasmogamy (the union of one sperm
and one egg) occurs, a vitelline coat (or vitelline envelope) is assembled on the outside of the egg cell
membrane to prevent polyspermy. It is vital for the vitelline coat to form immediately after penetration
of the egg cell membrane by the first sperm cell, as incorporation of additional sperm genomes are fatal
for the eventual embryo. While plants may tolerate polyploidy, animal embryos with even a small bit of
extra DNA may be developmentally disadvantaged (as in Down syndrome) or may die without further
embryonic development. After plasmogamy, the egg and sperm nuclei inside the cell join in
karyogamy. The two genomes combine inside the zygote cytoplasm to complete the process of
syngamy.
Developmental Biology:
After syngamy in echinoderms, the zygote undergoes cleavage, mitosis without change in total
volume of the embryo, ending in an embryo known as a morula consisting of a solid ball of diploid
cells. This ball enlarges forming a hollow blastocoel inside; the resulting single-layered embryo is
called a blastula.
Cell division and enlargement continues in the blastula and the outer layer invaginates into the
interior of the blastocoel forming a two-layered embryo known as the gastrula. The pore resulting
from the invagination is called the blastopore. This pore in animals eventually becomes either the
mouth or the anus; in echinoderms it becomes the anus, so sea urchins are deuterostomes; their mouth
(-stome) forms secondarily (deutero-). The new hollow area formed in gastrulation is the archenteron.
Between the outer layer (ectoderm) and the inner layer (endoderm) of the gastrula, a mesoderm is
formed. Thus the early embryo becomes triploblastic (three-layered). Also formed between ectoderm
and the internal tissues is a coelom (body cavity).
After the gastrula stage, further development results in other distinctive embryonic stages. These
include the famous and beautiful echinopluteus larva with its obvious bilateral symmetry. This
ciliated larva, like all of the earlier stages, is floating in the water column (planktonic), and this one is a
predator upon other members of the plankton community. The echinopluteus has long ciliated arms
supported by slender calcite rods.
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Laboratory Procedures
This laboratory exercise should allow your lab group to induce a sea urchin to release its gametes
by injection of concentrated salt. Each group will induce one animal and hopefully we have individuals
of both sexes among the lab groups so that both kinds of gametes can be mounted together in a
depression slide to observe both kinds of gametes and their syngamy in vitro.
Harvesting Gametes:
1. Load a syringe with 1.5 mL of 0.5 M KCl solution and inject it through the peristomal
membrane into the perioral area exterior to Aristotle’s lantern. Slant the needle from over the
mouth, through the membrane, and toward the interior of the test.
2. Gently swirl the animal to distribute the KCl solution inside the body cavity.
3. Place the animal with the aboral surface resting downward (injection site facing up!) in an
inverted dry Petri dish lid.
4. After a few minutes, some colorless (Lytechinus) or yellow (Arbacia), watery fluid will be
released from the gonopores and fall into the lid. This should be followed by a turbid, viscous
liquid containing the gametes.
a. If this secondary liquid is white, the urchin is male and you should immediately move
the animal to the dry Petri dish bottom to collect the sperm without any of the initial
liquid or other sea water contaminating them. Rinse and dry the dish lid, place it on the
dish bottom containing the sperm, and label it “dry sperm” for the class to use.
b. If this secondary liquid is bright yellow-orange-red, the urchin is female and you can let
the egg-laden liquid collect in the water of the Petri dish lid. Rinse these eggs with
some sea water into the Petri dish bottom. Rinse and dry the dish lid, place it on the
dish bottom containing the eggs, and label it “eggs” for the class to use.
5. When gamete release is complete, return your sea urchin (oral surface down) into the
designated container of sea water for the instructor to eventually euthanize.
Gamete and Syngamy Observations:
1. Using a pipette labeled “eggs,” add a few washed eggs and sea water to a depression slide. Be
sure to fill the depression full enough with sea water to be able to add a cover slip and avoid air
bubbles beneath it.
2. Using a pipette labeled “sperm,” lightly touch its tip to the mass of sperm. Stir the clinging
sperm into the eggs and sea water in the depression slide.
3. Add a cover slip to the wet mount and observe in the light microscope immediately.
4. You should initially find large true eggs with very tiny sperm swirling around them. As you
look around over a period of perhaps 20 minutes, you should find some eggs with a vitelline
coat. Among these, some have undergone plasmogamy but not karyogamy yet. Others may
have completed syngamy and thus are no longer eggs, but are zygotes instead.
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Laboratory Record:
In the space below sketch only the central areas of the oral and aboral surfaces of your sea urchin.
Label the sketches by connecting lines from the printed words that touch the corresponding structures.
Plan ahead (rotate the urchin before sketching!) to avoid crossing lines as much as possible. Make
each sketch large enough to distinguish all the structures clearly! Be sure to use your dissecting
microscope and any photograph provided at your station or what you may find through Google Image.
Oral Surface
Aboral Surface (rotate so madreporite is at the bottom)
peristomal
membrane-
-anal plate
teeth-
-anus
spines-
-genital plate
tube feet-
-gonopore
ossicles
-madreporite
(draw a line to
the closest
example of the
structure)
-ocular plate
Sea urchin adult symmetry is bilateral radial and is a(n) apo- plesio- -morphic character state
because we observe that the echinopluteus larva have bilateral radial symmetry.
The teeth observed on the aboral oral surface of the adult sea urchin
are an extension of this internal structure __________________________________________ .
Sea urchins lack legs, so what external structures help them locomote? __________________________
How many of these structures are found on a sea urchin? one
a few
many
Are these structures hard or soft? hard soft
The gametes are released from gonopores on the aboral
Which are not ossicles? anal plate
anus
oral surface of the adult sea urchin.
genital plate
gonopore
ocular plate
madreporite
Thought Question: What are four biotic or abiotic factors that might increase the chances of syngamy
actually happening in the ocean in the case of sea urchins reproducing by broadcast spawning? Be sure
the polarity (more vs. less) of your factor is specified correctly to increase the chances!
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Make sketches of what you observed in the microscope in depression slides in the laboratory. Be sure
your sketches are drawn to correct relative size scale, and are fully labeled! You should, again, sketch
the objects large enough to adequately show each structure. Draw a line from each printed label word
over to the corresponding sketch, and be sure that the line touches the corresponding structure.
Avoid crossing lines.
Gametes Prior to Syngamy
egg
\
Between Plasmogamy and
Karyogamy
After Syngamy
vitelline coat
vitelline coat
\
cell membrane
\
cell membrane
/
/
/
\
egg nucleus
sperm
\
sperm nucleus
/
zygote nucleus
The gametes of sea urchins are: anisogamous
isogamous
oogamous
Literature Resources
Anonymous. Undated. Sea Urchins and Sand Dollars. Gulf Specimen Marine Lab Website.
http://www.gulfspecimen.org/SeaUrchin.html (12-12-2012)
Fox, R. 2001. Invertebrate Anatomy On line. Lander University Website.
http://webs.lander.edu/rsfox/invertebrates/strongylocentrotus.html (1-7-2011)
Mazur, J. E. and J. W. Miller. 1971. A description of the complete metamorphosis of the sea urchin
Strongylocentrotus variegatus cultured in synthetic sea water. Ohio J. Science. 72: 30-36.
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Observing Syngamy in Chlamydomonas reinhardtii
Chlamydomonas reinhardtii is an isogamous freshwater alga with a unisexual life cycle. Two strains (CC-620 R3
NM mt+ and CC-621 NO mt-) are of opposite mating type. These strains were inoculated into separate flasks of 50 mL of
Tris Acetate Phosphate Medium (TAP medium) and cultivated at room temperature in bright light with shaking at 180 rpm
for five days. This medium was buffered with Tris/Acetic Acid and mono/di basic Phosphate buffer at pH 7.0 and provided
macro and microelements suitable for both photoautotrophic and heterotrophic growth. The resulting liquid culture was a
strong medium-green color.
The Chlamydomonas cells were spun down at 3000G for 3 minutes, the rich, nutrient supernatant was decanted,
and the cells were resuspended in 25 mL of 10 mM HEPES pH 7.2 buffer. This buffer contained essentially neither mineral
nutrients nor acetate for growth. The cells were cultured overnight in bright light with rocking agitation to starve them and
induce gamete expression.
Laboratory Procedures
The two starved Chlamydomonas cultures are provided in separate centrifuge tubes. There is a separate,
labeled pipette to use for each culture. Be sure to use the correct pipette for each culture!
Make a wet mount consisting of one drop of CC-620 and one drop of CC-621 starved cultures on a
microscope slide under a cover slip and observe microscopically over about an hour or so. As usual,
use the lowest magnification objective to focus on the edge of the cover slip. Then move to higher
magnification.
Initially all of the cells should be free-swimming individual cells.
You will not be able to distinguish the CC-620 from the CC-621 cells. Why not?
Because they are: anisogamous
isogamous
As time passes, look for syngamy events; the most obvious will be karyogamy
oogamous
plasmogamy
Make large sketches and label both completely. Notice the order of printed label words and rotate your
paper before sketching to avoid crossing lines extending from the printed words to the sketch. Please
do NOT write your own label words! Be sure that your label lines touch the corresponding structure!
Be careful to draw the organelles to their correct relative sizes to each other.
A single Chlamydomonas cell
Two mating Chlamydomonas cells
(hint: rotate your image so the flagella are at the top)
(connect lines to just one partner)
-flagella-eyespot-cell wall-cell membrane-nucleus-chloroplast-
Overall the cells have this color:___________, found in this organelle:_________________________ .
This overall color is likely due to the presence of this chemical pigment: _______________________ .
You may need to adjust the light intensity and the iris diaphragm to be able to see the flagella. A phasecontrast microscope may be available to observe flagella more easily. IF there is time in the class
period to observe a zygote, also sketch that.
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