Invertebrate Anatomy OnLine
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This is one of many exercises available from Invertebrate Anatomy OnLine , an Internet laboratory manual for courses in Invertebrate Zoology. Additional exercises can be accessed by clicking on the links to the left. A glossary and chapters on supplies and laboratory techniques are also available. Terminology and phylogeny used in these exercises correspond to usage in the Invertebrate Zoology textbook by Ruppert, Fox, and Barnes (2004). Hyphenated figure callouts refer to figures in the textbook. Callouts that are not hyphenated refer to figures embedded in the exercise. The glossary includes terms from this textbook as well as the laboratory exercises.
Although sponges are found in freshwater their greatest diversity is in marine habitats where they are often important members of benthic communities (Fig 5-3, 5-4). They are filter feeders specializing in bacteria-size particles, which they remove from the water column with great efficiency.
Sponges are an early experiment in the evolution of multicellularity and as such are collections of relatively independent cells (Fig 3-37). The cells retain their mobility and totipotent ability to redifferentiate and become other types of cells. The sponge body is mostly a connective tissue, the mesohyl, over which are applied epithelioid monolayers of cells, the outer pinacoderm and the inner choanoderm (Fig 5-5). The choanoderm is composed of flagellated collar cells, or choanocytes. The epithelioid layers may be syncytial or cellular. The body encloses internal water spaces, consisting of atrium and canals, continuous with the surrounding environment through incurrent ostia and excurrent oscula. The mesohyl includes several cell types as well as secreted skeletal elements such as protein fibers of collagen or spongin and mineral spicules composed of calcium carbonate (calcite) or silica (5-9).
Most aspects of sponge biology, including feeding, reproduction, and gas exchange, depend on a low pressure flow of water generated by the flagella of the choanoderm (Fig 5-13). Three grades of organization, asconoid, syconoid, and leuconoid, reflect the degree of elaboration of the choanoderm layer and mesohyl. In the asconoid plan the interior water space, or atrium, is large and unpartitioned (Fig 5-2A). In the syconoid plan the periphery of the atrium is divided into numerous small flagellated chambers with increased surface area for choanocytes (Fig 5-2C). In leuconoid sponges the atrium is replaced by a proliferation of mesohyl and a complex network of water channels and flagellated chambers (Fig 5-2D).
Porifera P, Cellularia sP, Calcarea C, Calcaronea sC, Leucosolenida O, Leucosoleniidae F (Fig 5-12A, 5-19)
Calcareous sponges have calcium carbonate spicules and collagen fibers but no spongin. The spicules are simple monaxons, triaxons, or tetraxons secreted extracellularly. Most calcareous sponges are small, seldom exceeding a few centimeters. All three grades of construction, asconoid, syconoid, and leuconoid, are present.
The asconoid grade of sponge organization is represented in this exercise by Leucosolenia . Members of this genus are small calcareous sponges that form colonies of intertwined tubular stolons produced by asexual budding.
1. With 40X magnification look at a commercially prepared wholemount. Living, preserved, or plastic embedded specimens can also be used. The colony consists of masses of tubular stolons about 1 mm in diameter from which arise short oscular tubes about 1-2 mm in diameter. The free end of each oscular tube bears a large opening, the osculum (Fig 1). The hollow interior of the stolons and oscular tubes is the atrium which connects with the exterior via the oscula.
The thin walls of the sponge are composed mostly of mesohyl with pinacoderm on the outside and choanoderm on the inside, lining the atrium (Fig 5-5). The mesohyl contains, among other things, calcareous spicules which can be seen projecting from the sponge surface. The walls are penetrated by microscopic pores, known as ostia, which admit water to the atrium. The flagellated choanocytes of the choanoderm generate a water current that enters the ostia, crosses the body wall, passes into the atrium, and then exits via an osculum.
1a. Place a small piece of sponge on a slide and add a drop or two of bleach. Let it sit until bubbles cease to form. The bleach oxidizes the organic material to carbon dioxide and water but leaves the mineral spicules intact. Add a coverslip, remove the excess bleach, and examine the preparation for spicules with the compound microscope at 40X. Go to higher power, if needed, to study the spicules (Fig 5-9E,F). These sponges possess abundant triaxons with three sharp rays. Monaxons, with a single pointed shaft, are also present in large numbers. An occasional tetraxon, with four points, may also be seen. Sketch the spicules you see.
1b. Draw some 8% HCl under the coverslip. Keep your focus on an individual spicule while you do this and watch its response to the acid. Hydrochloric acid and calcium carbonate react to form soluble calcium chloride and gaseous carbon dioxide so that the spicule disappears. Silicon does not react with HCl and remains unaffected. Based on your results do you think Leucosolenia has calcareous or siliceous spicules? _______________
Porifera P, Cellularia sP, Calcarea C, Calcaronea sC, Sycettida O, Sycettidae F (Fig 5-17A)
2. Use a dissecting microscope to examine a specimen of the syconoid sponge Sycon (Grantia and Scypha are older names for this sponge. Material from biological supply companies may be sold under any of these names.) covered with tapwater in a small culture dish. Look for young individuals, or buds, growing from a larger individual. The basal end of the sponge is flattened where it attaches to the substratum. At the apical end is an osculum surrounded by a collar of very large, protruding monaxon spicules. The body surface bristles with emergent spicules.
Figure 1. The calcareous asconoid sponge , Leucosolenia . Porifera57L.gif
2a. Use low power of the compound microscope to study a prepared slide of Sycon in longitudinal section and find the atrium, osculum, and the body wall (Fig 2). (If you are using cross sections instead of longitudinal, you will not see the osculum.)
Figure 2. The calcareous syconoid sponge , Sycon . Porifera58L.gif
Look closely at the thick body wall and determine that it is composed of alternating choanocyte chambers (= flagellated chambers) and incurrent canals (= inhalant canals) with intervening mesohyl. The flagellated chambers are lined with choanocytes and open to the atrium via pores called apopyles. Note how this arrangement expands the flagellated surface area in comparison with that of an asconoid sponge such as Leucosolenia. The incurrent canals connect with the outside via ostia. Incurrent canals do not open directly into the atrium, rather into the adjacent choanocyte chambers via tiny, inconspicuous openings known as prosopyles. Water passes through the ostia into the incurrent canals. From here it moves through the prosopyles into the choanocyte chamber. Small particles, such as bacteria, are removed from the water by the choanocytes. The filtered water then passes through the apopyles into the atrium and out the osculum.
The angle of the plane of section makes a great difference in the appearance of these structures and their relationships to each other. Be sure you understand how water flows through these animals. How is the water current generated? What roles does this water current play in the life of the sponge?
Some of the slides may have oocytes, with large conspicuous nuclei, in the mesohyl. Amphiblastula larvae (Fig 5-16B) may be present in the choanocyte chambers on some of the slides. The small cells at one end of the amphiblastula are monociliated, and will become the first choanocytes of the adult. (Unlike Sycon, most sponges have another type of larva, the parenchymella (Fig 5-16C). A parenchymella is a solid ball of cells [= stereoblastula] ciliated over its outer surface.)
2b. Ask the instructor for a small piece of Sycon. Place it on a clean slide and add a drop of bleach. After the sponge disappears and most of the bubbles have dispersed, affix a coverslip and examine the spicules with the compound microscope. There are three types of spicules in this species; monaxons, triaxons and tetraxons. Sketch each of the spicules you find. Use 8% HCl to test the composition of the spicules. Of what are they composed? ________________
Porifera P, Cellularia sP, Demospongiae C (Fig 5-17A)
Most of the world's sponges are demosponges and all large sponges belong to this taxon. Demosponges are always leuconoid, the grade of construction which permits large size by allowing expansion of the choanoderm surface area. Spicules are usually present and are always siliceous, often diverse and complicated. Spongin is usually present but a few demosponges lack both spongin and spicules. Demosponge spicules are secreted intracellularly.
Look at the demonstrations of dry skeletons of bath sponges. These sponges are prized for the bath and car care because their well-developed spongin skeleton lacks spicules of any type. The examples you see have been treated to remove all tissues so that only the protein skeleton remains. No mineral skeleton was ever present.
Feel the texture of the sponge and consider its suitability for washing your car or your body. Would it be so suitable if it contained millions of tiny glass needles?
With the instructor's permission, remove a tiny piece of one of the bath sponges, place it in a drop or two of fresh bleach on a microscope slide and allow digestion to take place. When bubbling subsides cover, the liquid with a coverslip and examine for spicules. Do you find any? ___________
Freshwater Sponge, Spongilla
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 is inconspicuous and rarely noticed. The skeleton is composed of spongin and siliceous spicules of several types.
Recently collected, living Spongilla may be available for your examination. Use the dissecting microscope to study such a sponge in a fingerbowl of lake water. Note that the sponge is very thin crust, only about 1-2 mm thick. At high power, examine the edges of the sponge where you should see spicules emerging from the mesohyl (Fig 5-6). Try to observe the interior of the sponge. What grade of construction are these sponges?
Remove a small piece of the sponge and transfer it to a slide. Use bleach to oxidize the organic material, apply a coverslip, and observe the spicules with the compound microscope. What shape are the spicules? Use 8% hydrochloric acid to test the composition of the spicules. Are the results of the test consistent with what you expect of demosponges? __________________
Look for spherical yellow gemmules in the mesohyl (Fig 5-15). If you find some, use your fine forceps and minuten nadeln to remove a few and place them two microscope slides. On one slide cover the gemmules with bleach and set the slide aside. After the organic material has been digested affix a coverslip and observe the special spicules, the gemmoscleres, which are characteristic of gemmules. Gemmoscleres have a short shaft with what looks like a tiny umbrella at each end. Place a coverslip on your second gemmule slide and continue to the next exercise.
With the compound microscope study a slide of the overwintering stages, or gemmules, of a freshwater sponge (Fig 5-15). You may use the slide you made or one that has been commercially prepared.
Gemmules are formed in the fall when water temperatures begin to drop. Each consists of a thick outer layer of specialized spicules, the gemmoscleres, a thinner layer of spongin inside that, and a core of undifferentiated archeocytes known as thesocytes. One end of the spherical gemmule is a small plugged opening, the micropyle. If the gemmule is oriented with the micropyle facing you, it will be easily seen as a small circular opening. In the spring when conditions improve, the thesocytes migrate out of the micropyle and establish a new sponge.
If living or dried sponges are present in the laboratory, use bleach to digest small pieces on slides and examine for spicules. There is far more variety in spicule types among demosponges than in calcareous sponges (Fig 5-9D). Look for large spicules, known collectively as megascleres, and for much smaller spicules, known as microscleres. The microscleres may have bizarre shapes. Of what are demosponge spicules composed? Verify this with 8% hydrochloric acid.
Boring sponges, Cliona
Look at the demonstration of living or preserved boring sponge, Cliona. These sponges excavate tunnels in calcareous substrata such as mollusc shells, limestone, or coral rubble (Fig 5-14A). The sponge burrows into and lives inside the shell, rock, or coral and communicates with the surrounding seawater via small papillae which protrude through small circular pores in the shell (Fig 5-14B). Some papillae are incurrent ostia and some are excurrent oscula. All are small, about 1 mm in diameter.
If boring sponges are not present in your laboratory perhaps there are some shells they once inhabited. Look at one of these shells and note the abundant small openings penetrating its surface. The shell looks as if it had been shot with birdshot. You may have collected such shells on a trip to the beach and wondered what made the holes. If permitted, break the shell or look at an already broken shell to see the tunnels inside.
This process, known as bioerosion, is accomplished by special archeocytes, the etching cells, which chemically remove tiny chips of calcium carbonate and release them into the excurrent canals (Fig 5-14B). Boring sponges are one reason that old mollusc shells do not accumulate in the sea.
Without exposing it to air, cut the branches of a healthy specimen of redbeard sponge, Microciona prolifera, or another species (Hymeniacidon, Lissodendoryx, etc), into sections about 5 mm in length. Place several such sections in the center of a 15 cm square of silk bolting cloth (or a cotton handkerchief) and fold the corners to the center to make a pouch. Immerse the pouch in a 10cm culture dish about half full of cool, clean, aerated sea water. Holding the pouch closed with one hand, repeatedly squeeze the sponge (through the cloth) using the wide flat arms of a pair of formalin-free forceps. Clouds of sponge cells (they will be red, if using Microciona) will settle from the bag to the bottom of the dish.
With a Pasteur pipet, transfer a drop of the sediment from the bottom of the dish to a clean microscope slide, affix a coverslip, and find a concentration of cells with the scanning lens of your compound microscope. Once you have located a group of cells, use 400X to examine them. Several types of cells are present but at this magnification you will not be able to distinguish most of them from each other although a few are identifiable. All are small, about 10 µm. The most common and conspicuous cells are amoeboid, more or less spherical, reddish cells with densely granular cytoplasm. Many of these are archeocytes (undifferentiated cells) but some are lophocytes (collagen secreting cells), sclerocytes (spicule-secreting cells), spongocytes (spongin-secreting cells) and other amoeboid cells. Pick one cell and watch it closely for the appearance of short, thick or long, slender pseudopods. The pseudopods form and change shape VERY slowly. You must be patient and watch closely for a minute or so before you will notice any change.
There should be many choanocytes also. They can be recognized by the flagellum but the collar will not be apparent. In fresh preparations, the flagella may be active but it will soon cease to undulate. The apical, or flagellar, end of the cell is thick and the basal end is elongate but this is not the normal shape of choanocytes.
Other cells are present in the preparation but they will not be identified here. Most are hyaline with few, if any, granular inclusions.
As you watch, you may see the amoeboid cells begin to aggregate and within a few minutes they may form clusters of cells. Gradually other types of cells will join the clusters. You should be able to see pseudopodia form at the periphery as the clusters move over the slide. Soon the small aggregates join each other but they continue to recruit individual cells also. These are the initial steps of the process that can (in running seawater) eventually culminate in the reconstruction of a complete, functioning sponge from the disassociated cells.
7. Use a 1% solution of toluidine blue in seawater to visualize flow in living sponges. Almost any sponge can be used for this. Apply the dye (with the same density as seawater) gently to ostia on the surface of the sponge in the vicinity of an osculum. It is not necessary that you see the ostia; simply place the dye on the surface of the sponge. Watch as the pigment appears (quickly) in the canals just below the surface of the sponge and then watch closely for its appearance at the osculum. It will not be as easily seen as it emerges from the osculum since it is diluted with uncolored water from ostia of other parts of the sponge.
Porifera P, Symplasma sP, Hexactinellida C (Fig 5-17A)
Hexactinellids, or glass sponges, are so named because of the siliceous composition of their skeletons and because of their characteristic hexactine spicules with six points. The tissues of glass sponges are syncytial (Fig 5-7).
Admire the bleached skeleton of Euplectella , the Venus flower basket, a well known species of glass sponge (Fig 5-4A). Euplectella is a syconoid sponge that lives in deep water (up to 5000 m.) on soft bottoms. These sponges are tubular with a spacious atrium in the center. The large osculum is covered by a grid of fused spicules. Note the intricate arrangement of spicules that form the skeleton. Most are hexactines fused together to form a rigid skeleton. The basal end of the sponge has very long specialized monaxonal root spicules that anchor the animal in the sediment.
*Hyphenated call-outs, such as this one, refer to figures in Ruppert, Fox, and Barnes (2004). Those without hyphenation refer to figures embedded in this exercise.
Bergquist PR. 1978. Sponges. Univ. California Press, Berkeley. 268p.
Harrison FW, DeVos L. 1991. Porifera. pp. 29-89 in F.W. Harrison & J.A. Westfall (eds). Microscopical Anatomy of Invertebrates, vol. 2. Wiley-Liss, New York.
Penney JT. 1932. A simple method for the study of living fresh-water sponges. Sci. 75:341.
Ruppert EE, Fox RS, Barnes RB. 2004. Invertebrate Zoology, A functional evolutionary approach, 7 th ed. Brooks Cole Thomson, Belmont CA. 963 pp.
Simpson T. 1984. The Cell Biology of Sponges. Springer-Verlag, New York. 585p.
Wilson HV. 1911. Development of sponges from dissociated tissue cells. Bull. Bur. Fisheries, 30:1-30, pls. 1-5.
Small culture dish or Syracuse dish
Slides: Leucosolenia wholemount, Scypha longitudinal (or cross) sections, Spongilla gemmules,
Preserved specimens: Leucosolenia, Scypha,
Dropper bottles of: fresh bleach, 8 % hydrochloric acid, 1 % toluidine blue in seawater,
Other specimens: Prepared skeletons of Euplectella, plastic mounts of boring sponge,bath sponges
Living specimens: Spongilla, Microciona or other marine sponge.