Shotgun Fungus Lab

7/29/2019 Shotgun Fungus Lab

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PILOBOLUS: The ShotgunFungus

Materials you will need:

Fresh horse dungFinger bowls or 1 quart cartonsAluminum foilTapeColored acetateMoist paper towel

Introduction

Today's exercise asks you to contemplate adaptation to a habitat in a pasture by alowly but (to some eyes) charming member of the Class Zygomycetes. This fungus isrelated to the familiar bread mold (Rhizopus) and other small, inconspicuous whitefilamentous 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 abilityto 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 nameditself Pilobolus. You may have seen them on public television or at the modern dancesummer festival in Salt Lake City. One of the founders of the troop, from PrincetonUniversity, 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. Hischoreography 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 soggypasture?"

Pilobolus LIFE CYCLE

We will only consider asexual reproduction in Pilobolus.

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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 multicellularstructure that radiates out from the place where aspore germinated. The slimy growing fingers (calledmycelia) grow out from the spore over the horsedung, soaking up nutrients as they progress. A lightorange-colored swelling along the mycelium(singular for mycelia) is called a trophocyst. It is fromthis lump that the sporangium will develop.

A stalk grows straight upwards and develops a sporangium in which the blackspores develop. Usually, droplets of water are visible over both the subsporangial

swelling and the stipe (stalk that holds it up). During development, the subsporangialswelling, acting as a primitive lens, and the stipe function together in the orientation of thesporangium towards light. This is brought about by a growth response, which is triggeredwhenever light entering the subsporangial swelling is brought to focus at any point otherthan the orange-pigmented area at the top of the stipe.

Therefore, through light-trigged directional growth of the stipe, the sporangium isvery accurately aimed at the light source. On reaching maturity, the sporangium isviolently discharged ("shot") from the sporangiophore. This is accomplished by means ofa "water-squirt" mechanisms, which is capable of projecting the sporangium to a distance

of 1.8 m (almost 6 feet).

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Figure 2. Life cycle of Pilobolus

A sporangiophore (consists of stalk, subsporangial swelling and sporangium)

B discharge of sporangiophore (containing many spores)

C empty sporangiophore after shooting

D germinating spore

E growth of mycelia (threads)

F trophocyst forms

G beginning of stalk

H tall stalk with water droplets

I-K sporangium beginning to form

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On discharge, the sporangiophore is falls back against the dung, where, being no longerfunctional, it disintegrates (C in Fig. 2). However, the sporangium, by means of a ring ofmucilage at its base, will stick to any object it strikes. In nature this is often a nearby bladeof grass. Being so deposited the sporangia are in a good position to be eaten, along withthe 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, moistdung the spores within the sporangia germinate to give rise to the vegetative filamentsagain.

The development of the fungus results in synchronized maturation of thesporangia, 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 eatenby a horse. What stimuli would the fungi need to respond to?

Talk among yourselves regarding what powers of the fungus interest you. Someexamples 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. WillPilobolus

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 anoptimum distance where accuracy ofa shot is greatest?

5. Direction of light.

You will design a chamberthat is totally dark except forwindows that the fungi will chooseamong when discharging theirspores. The windows are small holescut in the foil. Colored acetatesquares are taped over these windows. Figure 3: A setup of a spore discharge experiment:

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Place dung in the bottom of a bowl on top of the moist paper towel. Make sure there is alarge enough gap between the dung and the windows for the fungus to take aim. Thedung 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 dungmoist so that the fungus will grow on the surface of the dung. Too much water is badbecause droplets form on the windows and the sporangia will cling to the droplet and notthe window

Fig. 4: Bowls ready to go

The fungi in your chamberswill discharge spores onto thewindows in 1 - 3 days. Please

check the progress of yourexperiment every day. Whena significant number ofsporangia have discharged,turn the foil bottom side upon a dissecting microscopeand count the numbers ofsporangia glued onto each ofthe windows.

Major hint regarding this experiment: You will want to put all your treatments in onebowl, e.g. a red window, a blue window, a yellow window. Why will it NOT WORK toput a different colored windows in each bowl? You will want to replicate yourexperiment. Three bowls treated exactly alike should do.

When your group has decided on your question and the hypothesis to be tested, clear itwith your T.A. and fill out the blanks below.

HA Research hypothesis

HO Null hypothesis

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dependent variable

independent variable

You will make your own datasheet and graphs.

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Documents

Shotgun Fungus Lab