PILOBOLUS: Fungus

The Shotgun

Materials you will need: Fresh horse dung Finger bowls or 1 quart cartons Aluminum foil Tape Colored acetate Moist paper towel Introduction Today's exercise asks you to contemplate adaptation to a habitat in a pasture by a lowly but (to some eyes) charming member of the Class Zygomycetes. This fungus is related to the familiar bread mold (Rhizopus) and other small, inconspicuous white filamentous organisms. Your opinion of these types of fungi probably run from "slimy" to "rotten" or just plain "blah". Pilobolus will change your mind. Pilobolus inhabits horse dung. It is a saprobe, meaning it must gain its nutrients as carbon compounds made by other organisms. It is well known for its ability to shoot off its spores in a violent manner (hence, "shotgun") in the direction of light. Others have been inspired by this lowly fungus. One of the most energetic, creative, innovative and just plain wacky modern dance groups active today has named itself Pilobolus. You may have seen them on public television or at the modern dance summer festival in Salt Lake City. One of the founders of the troop, from Princeton University, has a father who is a biologist. Most artists find biologists just a little odd. This dancer was inspired by what he saw under his father's microscope. His choreography often reflects the pulsing of jellyfish, the scurrying of ants, the suddenness of live birth. Your job today will be to put yourself in the position of the fungus and ask: "What adaptations will increase my chances of surviving and reproducing in this soggy pasture?" Pilobolus LIFE CYCLE We will only consider asexual reproduction in Pilobolus.

Figure 1. Closeup of the young fruiting body of Pilobolus S = sporangium SS = subsporangial swelling ST stipe WD = water droplet

Pilobolus (Figs. 1,2) consists of a multicellular structure that radiates out from the place where a spore germinated. The slimy growing fingers (called mycelia) grow out from the spore over the horse dung, soaking up nutrients as they progress. A light orange-colored swelling along the mycelium (singular for mycelia) is called a trophocyst. It is from this lump that the sporangium will develop.

A stalk grows straight upwards and develops a sporangium in which the black spores develop. Usually, droplets of water are visible over both the subsporangial swelling and the stipe (stalk that holds it up). During development, the subsporangial swelling, acting as a primitive lens, and the stipe function together in the orientation of the sporangium towards light. This is brought about by a growth response, which is triggered whenever light entering the subsporangial swelling is brought to focus at any point other than the orange-pigmented area at the top of the stipe. Therefore, through light-trigged directional growth of the stipe, the sporangium is very accurately aimed at the light source. On reaching maturity, the sporangium is violently discharged ("shot") from the sporangiophore. This is accomplished by means of a "water-squirt" mechanisms, which is capable of projecting the sporangium to a distance of 1.8 m (almost 6 feet).

Figure 2. Life cycle of Pilobolus

A B C D E F G H

sporangiophore (consists of stalk, subsporangial swelling and sporangium) discharge of sporangiophore (containing many spores) empty sporangiophore after shooting germinating spore growth of mycelia (threads) trophocyst forms beginning of stalk tall stalk with water droplets

I-K sporangium beginning to form

On discharge, the sporangiophore is falls back against the dung, where, being no longer functional, it disintegrates (C in Fig. 2). However, the sporangium, by means of a ring of mucilage at its base, will stick to any object it strikes. In nature this is often a nearby blade of grass. Being so deposited the sporangia are in a good position to be eaten, along with the grass, by a horse, cow, or other grazing animal. The sporangia and spores pass unharmed through the digestive system of the animal and are excreted. In the fresh, moist dung the spores within the sporangia germinate to give rise to the vegetative filaments again. The development of the fungus results in synchronized maturation of the sporangia, and spores for discharge at approximately noon the day they are ready. EXPERIMENTS WITH PILOBOLUS You will be given a lump of horse dung that is 4 days old (post horse). Use the diagram to identify the mycelia, trophocysts and developing stalks of the sporangiophore. Think about their world view: need to keep moist, need to find a horse, need to be eaten by a horse. What stimuli would the fungi need to respond to? Talk among yourselves regarding what powers of the fungus interest you. Some examples might be: 1. the effect of light on sporangiophore discharge'. Diffuse light? Direct light? Intense light? Dim light? Which will make the sporangia shoot straighter? farther? 2. Effect of the color of light. Will Pilobolus send more spores to blue light, yellow light, red light? Does it make a difference? 3. Gravity. Does Pilobolus respond to it?

4. Distance. Is there an optimum distance where accuracy of a shot is greatest? 5. Direction of light.

You will design a chamber that is totally dark except for windows that the fungi will choose among when discharging their spores. The windows are small holes cut in the foil. Colored acetate squares are taped over these windows. Figure 3: A setup of a spore discharge experiment:

Place dung in the bottom of a bowl on top of the moist paper towel. Make sure there is a large enough gap between the dung and the windows for the fungus to take aim. The dung should be at least 3 cm below the foil lid. Your TA will show you how to manipulate the light in a fingerbowl (or other container you may need) to test the ballistic properties of spores. It is important to keep the dung moist so that the fungus will grow on the surface of the dung. Too much water is bad because droplets form on the windows and the sporangia will cling to the droplet and not the window Fig. 4: Bowls ready to go The fungi in your chambers will discharge spores onto the windows in 1 - 3 days. Please check the progress of your experiment every day. When a significant number of sporangia have discharged, turn the foil bottom side up on a dissecting microscope and count the numbers of sporangia glued onto each of the windows. Major hint regarding this experiment: You will want to put all your treatments in one bowl, e.g. a red window, a blue window, a yellow window. Why will it NOT WORK to put a different colored windows in each bowl? You will want to replicate your experiment. Three bowls treated exactly alike should do. When your group has decided on your question and the hypothesis to be tested, clear it with your T.A. and fill out the blanks below.

HA Research hypothesis

HO Null hypothesis

dependent variable

independent variable

You will make your own data sheet and graphs.