LABORATORY: PHYLUM PORIFERA

BACKGROUND
The principal features of phylum Porifera are listed below so you can refer to this
background information throughout the lab. Exercises can be found on page 4.

a. While some sponges are radially symmetrical, the majority of sponges are
asymmetrical in body form. Sponges are considered to be at a cellular grade of
construction; that is, they have cellular differentiation (tissues) without cellular
coordination.

b. The outermost tissue layer of sponges is composed of cells called pinacocytes. In

some sponges this outer tissue layer is syncytial while in others the pinacocytes are all
distinctly separated from one another by cell membranes. The innermost tissue layer is
composed of cells called choanocytes or collar cells, which have flagella that beat to
produce water currents through the sponge body. Between these two tissue layers is a
gelatinous layer called the mesoglea (mesohyl). The mesoglea is not considered to be a
tissue since it contains a number of different kinds of independently functioning cells.
Each cell type in the mesoglea has a specific name, but the general term for all of these
wandering cells is amoebocyte.

c. Some of the amoebocytes in the mesoglea are specialized for secreting a skeleton.
The sponge skeleton may be composed of mineral spicules, spongin fibers or a
combination of these two, depending on the kind of sponge. Spicules may be calcareous
(composed of Ca CO3) or siliceous (composed of H2Si2O7). Spongin fibers are
composed of a sulfur-containing protein.

d. Water enters the body of a sponge by way of a number of minute incurrent pores or
ostia. Water leaves the body by way of one or more large excurrent pores or oscula.
Within the body of the sponge, the water may pass through a large cavity (the
spongocoel) through a system of canals and chambers, or through a combination of these
two.

e. Movement of water through the sponge body is accomplished by the beating of the
choanocyte flagella. The choanocyte cells line either a spongocoel or a number of small
chambers, depending on the sponge. A choanocyte cell consists of a nucleus, one or
more vacuoles, a long flagellum and a delicate, collarlike structure which surrounds the
base of the flagellum. Electron microscope studies show the collar of a choanocyte to be
composed of a circular arrangement of microvilli-like structures extending outward from
the cell body. The rotary motion of the flagellum forces solid food particles in the
incoming water to adhere to the outside surface of the collar. The streaming protoplasm
of the collar transfers the food to the collar base where ingestion can occur.
f. Sponges may be divided into three basic grades or types based upon the arrangement
of their water canal systems. Note that these grades or types are not taxonomic
groupings. The three types of sponges are described below and are shown
diagrammatically.

Asconoid Type - Water entering the sponge passes through ostia which are actually
openings within doughnut-shaped cells called porocytes, which are found only in
asconoid sponges. The water enters the large central cavity called the spongocoel,
which is lined with choanocytes. Water exits from the spongocoel through a single large
osculum.

Syconoid Type - Water enters the sponge through ostia, which are openings between
cells, rather than within cells as in asconoid sponges. Water then passes into radially
arranged incurrent canals, which lead to flagellated chambers lined with choanocytes.
Water leaves the flagellated chambers by way of excurrent canals that lead to the
spongocoel, which is lined a simple flat epithelium. Water exits from the spongocoel by
way of a single large osculum. Note that the body wall of syconoid sponges is thicker
than that of asconoid sponges and that the syconoid spongocoel is not lined by
choanocytes as is the asconoid spongocoel.

Leuconoid Type - The ostia of a leuconoid sponge are like those of a syconoid sponge.
These ostia lead into a complex system of canals and flagellated chambers that penetrate
the very thick, dense mesoglea. There is no spongocoel in a leuconoid sponge. Rather,
water reaches the oscula by way of large excurrent canals. The complex canal system of
leuconoid allows for greater surface area over which water may pass and consequently
creates an increased area for food and oxygen uptake and for waste removal. It is not
surprising, therefore, that leuconoid sponges are the largest in size of all the types and
that the vast majority of sponges are leuconoid.

g. Sponge taxonomy is based on skeletal composition. The three Clades (classes) in

larger clade (phylum( Porifera are listed below along with distinguishing characteristics
for each class. The grades of sponges found in each class are given in parenthesis,
although this is not distinguishing since there is overlap between the classes.

(1) Class Calcarea - contains sponges having calcareous spicules with 1 to 4

rays. (asconoid, syconoid, leuconoid)

(2) Class Hexactinellida - contains sponges having siliceous spicules with 6 rays.
These spicules are often fused to form a beautiful lattice-like cylinder, as in the so-called
Venus' flower basket. (syconoid)

(3) Class Demospongiae - contains sponges having siliceous spicules (not 6-

rayed) and/or spongin fibers. (leuconoid)
h. Sponges are capable of both sexual and asexual reproduction and they also have great
powers of tissue regeneration and re-association. Sexual reproduction is accomplished by
production of eggs and sperm, which unite to form a zygote. The zygote divides
repeatedly to produce a free-swimming larval form. Depending on the sponge, this larva
may be either a uniformly ciliated parenchymula larva or an amphiblastula larva,
which has flagella only at one pole. The larvae eventually settle and metamorphose into
the adult form.

Sponges may reproduce asexually by budding. In addition, all freshwater sponges and
some marine forms produce resistant overwintering bodies called gemmules. These
gemmules consist of aggregations of food laden amoebocytes surrounded by a resistant
covering. They are produced during periods of cold or drought and can survive to
produce a new sponge body when conditions improve .
Exercises.
Examine the specimens available. Exactly which species is available will
differ from year to year. Most are demosponges unless otherwise indicated.

Species that may be available in lab:

Microciona prolifera: a brick red encrusting sponge is the classic sponge used to
demonstrate cellular reaggregation. There appear to be significant biochemical
differences between the Gulf of Mexico form and Microciona prolifera at Woods Hole.
We will be working with the one from Gulf of Mexico this year. (Regeneration and
water circulation in and out of a sponge.)

Cliona spp.: These yellow sponges are lobular in shape and can form encrustations on
mollusk shells. They are capable of burrowing into the shell itself forming
interconnecting chamber that riddle the shell with small holes. (For sponge cell types.)

Dysidea camera: a purple bluish sponge that grows in sea grass beds, and has the unique
ability to extract nickel from the seawater and concentrate it in its tissues. (Use for
ecology unless directed otherwise.)

Halichondria spp. is a greenish or yellowish encrusting but not a burrowing sponge.

Green specimens house zoochlorellae, a symbionic algae. (NA)

Axinella polycapella: doesn't look like an ordinary sponge with its thick, fleshy orange
fingers rising from the aquarium floor. Axinella has a core of densely packed spicules.
(NA)

Leucosolinia spp.: A small encrusting asconoid sponge appearing as a pale mass with
irregular tubes rising up from a low spongy mass. Spicules are calcareous. (Slides only
this year.)

Scypha spp.: A small, tan, vase-shaped syconoid sponge. The opening of the sponge is
the "osculum" and is fringed by spicules. This is a common but easily overlooked sponge
as it usually only grows to a centimeter in height. You may be lucky enough to see
Amphiblastula larva present in the choanocyte chambers. (Slides only unless can find
some on filters Wednesday night.)

Spongilla spp.: North American freshwater sponges are demosponges in the taxon
Spongillidae. They usually form thin brown, or green if zoochlorellae are present, crusts
on submerged surfaces. Spongilla is common in clean natural waters but are
inconspicuous and rarely noticed. The skeleton is composed of spongin and siliceous
spicules of several types. (NA)

This year in lab we will be using the following sponges for the 4 exercises:
1. Life on a sponge _____________________

2. External anatomy_______________________

3.Internal anatomy_________________________

4.Totipotent cells____________________________

1. Life on a sponge. Dysidea camera is the usual best candidate for this exercise but
again this varies from year to year. It depends essentially on how soon the sponge is
collected before it is shipped. If collected right before it is shipped, all the creatures
associated with it in its natural habitat go for a ride. One year we had reproducing small
nudibanchs, various crustaceans, annelids, brittle stars, cnidarians, etc. We could have
that year spend the rest of the semester just photographing this one sponge.

Examine large chunks or clumps of the species under the dissecting scope chosen by your
instructor for this exericse. Check with your lab instructor for material that may have
fallen off the sponges during the journey that may contains living specimens that may
have been saved for you. Examine the sponges and other material and catalog the
number and other types of organisms you see that either feed or live on these sponges.
This is a videotape exercise using your dissecting scopes.

THE REST OF THE EXERCISES ON SPONGES SHOULD BE SAVED FOR

THE LAST HOUR AND HALF OF LAB. OBSERVE LIVING CTENOPHORES
AND CNIDARIANS AVAILABLE BEFORE ATTEMPTING THESE
ACTIVITIES.

2: External anatomy

2a. Obtain a small sample of species to be observed and place it in a dish filled with
seawater. Describe the overall shape of the sponge. Any symmetry apparent? Do
you see budding on any of the species? If so, where are the buds positioned?

2b. Examine it under a dissecting microscope. To observe the filtering mechanism of the
sponge, prepare a dilute suspension of carmine powder or dye and seawater, and then
gently place a drop of the suspension near the colony. Where do the carmine particles
enter? Where do they exit? Do particles enter the sponge at the same velocity that they
exit?
Record your observations, videotaping your observations if possible. Do not spend a
lot of time on this exercise. Sometimes it works, sometimes, it does not.
When you are done, simply gently pour off old water containing dye and add new
seawater. Keep the sponge underneath seawater at all times.

3. Internal anatomy.

3a. Obtain a specimen. Scrape a very small section on to a slide. Add seawater and
macerate as best you can. If you cannot see different cell types under the microscope,
simply differ this exercise until after you have prepared specimens for aggregation
studies. Use the following diagrams to aid your identifications. Sponge cells are very,
very small. Your photographs should be taken using 40X or 100X (oil) lenses. Try to
label choanocytes and pinacocytes found. Label spicules; any cell around a spicule is a
scleocyte. Amebocytes are the smallest of the cells and will only be identified if you will
notice some very small cells moving.
3b. FOR NEXT WEEK OR IF YOU HAVE TIME (AFTER COMPLETING ALL
EXERCISES AND OBSERVING CTENOPHORES AND ALL LIVING
CNIDARIANS). Examine the prepared slides of Grantia and Scyha sponges available.
The staining of these slides will make the cellular structures easier to identify. Try to
identify porocytes, pinacocytes, and choanocytes.

Students in the past have found longitudinal sections easier to photograph and label, but
slides vary in quality and again you may have to examine one or two before you decide
which to photograph and label.

4. Totipotent cells and re-associating cells.

Prepare plastic dishes to house re-associating sponge by scaring them with a teasing
needle. The instructors will change the media daily.
Next week you will put your small dishes under rocks in the large tank. Re-association
will not progress beyond the cells forming small clumps unless the cells are introduced
into tanks where the developing sponge is constantly exposed to re-circulating water.

Your lab instructor may decide to do this as a class demonstration or assign some
students the task of preparing dissociated sponge material.

Keep the sponge under water throughout this procedure. Separate a small fragment of
sponge about 2-4 cm. long. Use initially specimens of Microciona prolifera. Place in a
double layer of cheesecloth in a large finger bowl. Force the sponge through the cloth
into sterile seawater. It should consist of individual and very small clumps of cells.

Place a sample of the dissociated cells suspension on a few drop slides to examine later

Distribute the dissociate sponge cells into the petri dishes containing sterile seawater.
Unfortunately, there is no way to avoid some contamination and often feeding protists
and bacteria will overpopulate your dishes of re-associating cells. One or two usually
make it to the stage in which clumps of cells are large enough and somewhat attached to
the dish so they can be put into the tanks without being forced out of the dish by the
circulating water.

A small piece of sponge should yield enough cells to feed 2-4 petri dishes. Each table
should claim one of these petri dishes and include a few picture of the initial
suspension under low and higher power (around 80 x) in their journals.

Place a drop of the suspension you saved on a slide and examine it under l00X and 400x.
Try to identify different types of cells if you could not with the sponge scrapings.