LABORATORY 2: PHYLUM PORIFERA
The sponges are the first metazoans (multicellular animals) that we will study.
The principal features of phylum Porifera are listed below.
1. 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.
2. 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 (see S&S, p.45) 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.
3. 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 schleroprotein.
4. 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.
5. 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.
6. 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 chamgers
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.
7. Sponge taxonomy is based on skeletal composition. The four classes in phylun
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.
a) Class Calcarea - contains sponges having calcareous spicules with 1 to 4 rays.
(asconoid, syconoid, leuconoid)
b) 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)
c) Class Demospongiae - contains sponges having siliceous spicules (not 6-
rayed) and/or spongin fibers. (leuconoid)
d) Class Sclerospongiae - contains sponges having an internal skeleton of
siliceous spicules and spongin fibers plus an outer encasement of calcium carbonate.
Only six species from the Caribbean have been described to date. (leuconoid)
8. Sponges are capable of both sexual and asexual reproduction and they also have great
powers of tissue regeneration and reassociation. 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 (Fig. 2.7, p.55 of S&S). 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 (Fig. 2.8, p.55 of S&S).
In today's laboratory you will examine the structure of the sponge species
available and then perform experiments on the reassociation of porifera cells.
ASCONOID SPONGES
Asconoid sponges are the simplest and most primitive sponge architectural type
and are all relatively small due to their inefficient filtering system. Asconoid structure is
demonstrated in Leucosolenia. Obtain a small colony of Leucosolenia sponges and place
it in a dish filled with seawater. Examine it under a dissecting microscope. Does it
respond to a stimulus? Do you detect movement? To observe the filtering mechanism
of Leucosolenia, prepare a dilute suspension of carmine powder and seawater and then
gently place a drop of the suspension near the colony. Describe the water movement
through Leucosolenia. Where do the carmine particles enter? Where do they exit? Do
particles enter the sponge at the same velocity that they exit? Explain. Do you see
budding on your Leucosolenia colony? If so, where are the buds positioned? Examine
the colonial structure of Leucosolenia. Describe how you think the ultimate colonial
form develops from a single sponge tube.
From your Leucosolenia colony remove a single sponge tube for closer
examination and then rinse the colony in fresh seawater and return it to the holding tank.
Cut the sponge tube longitudinally into two halves (from osculum to base) and place the
two halves on a slide so that half the tube shows the inner surface and the other half
shows the outer surface. Add a drop of saltwater and cover with a coverslip. Examine
both surfaces under the compound microscope. Try to identify porocytes, pinacocytes,
and choanocytes, or evidence of their presence (refer to S&S, pp. 45-47 for diagrams).
Next, tease apart the sections of sponge with a dissecting needle and examine for spicules
and amoebocytes. Describe the shape and arrangement of the spicules. How are
spicules formed? Do you see any evidence of this in your preparation?
Examine the prepared slides of asconoid sponges. The staining of these slides will make
the cellular structures easier to identify.
SYCONOID SPONGES
Syconoid sponges are more complex than asconoid sponges. Syconoid sponges
look like large asconoid sponges, having a tubular shape, and each individual has a single
excurrent osculum. The body wall is thicker, however, and the spongocoel is lined with
pinacocytes. The choanocytes line finger-like chambers (radial canals), which permeate
the spongocoel (see pp. 50-51 in S&S). Because this arrangement provides a more
efficient pumping system than the asconoid design, syconoid sponges are larger than
asconoid sponges.
Obtain a single specimen of Scypha, a representative syconoid sponge. How does
the colonial form of Scypha compare to Leucosolenia? Place your Scypha individual in
a dish filled with seawater and examine it under the dissecting scope. Does it respond to
mechanical stimulation? How does its filtration system compare to Leucosolenia?
Section the Scypha and examine its body wall structure. Examine the mesoglea and
characterize the spicules of Scypha (p.52 of S&S).
The prepared slides of a second syconoid sponge, Grantia, clearly shows wall
structure, choanocytes, and spicules.
LEUCONOID SPONGES
Leuconoid sponges are by far the most complex architectural-type of sponge. Most
leuconoids are colonial, and although individual oscula can be distinguished, it is difficult
to separate individual members of the colony. The vast majority of sponges are
leuconoid. Examine the external and internal anatomy of the leuconoid available in the
laboratory (probably Microciona). How does its filtration system differ from the
asconoid and syconoid sponges you examined? Examine the body wall structure (see
S&S, p. 51). Characterize the spicules.
SPICULE COMPOSITION AND STRUCTURE
The systematics of sponges are based primarily on the composition and structure of
spicules rather than on architectural plan. Spicules are composed of either calcium
carbonate or silicon dioxide, and the skeleton may consist entirely of collagenous fibers
(spongin) or a combination of spicules and spongin. See the introduction of this exercise
(#8) for the characteristics of the four classes of Porifera. The class membership of
sponges is easily determined using the following tests:
1. The organic matrix of sponges (spongin) dissolves when boiled in 5% sodium
hypochlorite solution. Place small pieces of sponge tissue in 1-2 ml of the sodium
hypochlorite solution in a testtube to carry out this test. Boil the mixture for a couple of
minutes by placing the test tube in a beaker of boiling water. After cooling, examine
under the compound microscope. Are spicules present? This technique is also used to
remove spongin from spicules to examine spicule form.
2. The inorganic chemical nature of spicules is determined by drawing acetic acid or
dilute hydrochloric acid under the cover slip of a wet mount of spicules. Calcium
carbonate spicules dissolve when treated in this manner, while silicon dioxide spicules
are not influenced by the treatment.
3. Examination of the shape of spicules found in a sponge are also important taxonomic
characteristics (see S&S, p. 52, for representative spicule morphology).
Determine the class membership of the sponges available in the laboratory.
REASSOCIATION
Sponges have remarkable powers of regeneration. A complete sponge can
regenerate from only a handful of cells. In the natural environment this means that when
a sponge is disturbed by a predator or physical disturbance, remaining fragments are able
to form new individuals and colonies. The impressive regeneration ability of sponges is
due to the loose organization of cells in individuals. Individual cells and unorganized
clusters of cells are able to reassociate and organize into new individuals. In today's
laboratory we will examine the reassociation phenomenon of sponges.
Procedure: Obtain a few milliliters of the suspension of Microciona cells
available in the laboratory. This suspension was prepared by pressing pieces of fresh
Microciona through a silk bolting cloth into seawater. Examine a drop of the suspension
under a compound microscope. It should consist of small clumps of cells.
Prepare a series of dilutions in seawater from the original suspension so that you
have samples of decreasing densities. Make your dilutions 100%, 50%, 25%, and 10%
solutions of the original suspension. Fill 2 small fingerbowls two-thirds full of cool
seawater; place a Syracuse watch glass on the bottom of each fingerbowl and put two
slides on each watch glass. Before placing the slides on the watch glasses, individually
number each slide. Using a Pasteur pipet, gently dispense equal aliquots ( 2 drops) of
each suspension onto the slides. Place two treatments (dilutions) on each slide and have
all four dilutions represented in each fingerbowl. Be very careful not to allow mixing of
the dilutions on the slides. Be sure you record the positions of the different dilutions on
each slide. Clearly label your fingerbowls and have one stand at 4C and the other at
room temperature for 24 hours. After 24 hours, gently remove the slides, cover with a
coverslip, and examine under a microscope. Systematically count the number of
aggregates in each dilution and compare the size distribution of aggregates in each
dilution. Record all your data and observations. How do cell density and temperature
influence aggregation? What form do the aggregates take? How are different cell types
arranged in the aggregates?
Place a fresh drop of the Microciona suspension on a slide and examine it during
the course of the laboratory under l00X. Do you see any evidence of aggregation?
Following the procedures outlined above for examining reassociation in
Microciona, prepare similar solutions of a suspension of the cells of two sponge species
(Microciona & Haliclona). Prepare a fingerbowl incubating the four interspecific
dilutions and let it stand for 24 hours at 15C. Compare these results with the
Microciona suspension. What has happened to the cells of the two species? How might
segregation of the two cell types have occurred? What other experiments could be
performed to test this phenomenon further?