LABORATORY 2: PHYLUM PORIFERA



LABORATORY: PHYLUM PORIFERABACKGROUNDThe 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 anatomy2a. 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 laterDistribute 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. ................
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