THE ORDER MONOBLEPHARIDALES
GONAPODYA PROLIFERA IN THE CLASSROOM
Isolate M#6 of Gonapodya prolifera grows on mPmTg agar and demonstrates the following features of the genus: subumbellate branching of the pseudoseptate mycelium, scalariform hyphal contents, proliferation of sporangia and gametangia, oogonia containing one to many oospheres (female gametes) that leave the oogonium propelled by the antheridial flagella and form oospores in the medium. Hyphae of G. prolifera are narrower than those of G. polymorpha and may be 200—500 µm long when grown on seeds. They are more regularly pseudoseptate than G. polymorpha. Older hyphae are often misshapen and distorted with uneven swellings.
The available isolates of G. prolifera seldom form reproductive structures but, if they do, look for the following features of the species: siliquiform zoosporangia with long drawn out tips that proliferate freely, antheridia and oogonia that occur together in subumbellate clusters and both proliferate freely. Clusters of gametangia and zoosporangia are subtended by cantenulate swellings separated by pseudoseptations. The narrow siliquiform antheridia are smaller than zoosporangia and contain numerous small antherozoids. The oogonia are wide at the basal end and contain several large oospheres. Zygotes and mature oospores may form inside the oogonium, but usually the female gametes emerge and are fertilized outside the oogonium. The antheridial flagella propells the zygotes away and mature oospores form in the medium some distance from the point of origin. A hyaline membrane surrounds the round oospores.
CAUTION - Use sterile technique at all times. Sterilize all glassware and media by autoclaving before use.
LONGTERM MAINTENANCE – If desired, cultures can be maintained on mPmTG agar slants stored in a refrigerator and transferred every three months.
PREPARATION TIME - Start “stock plate A” 23 days before scheduled class. Consider this Day 0.
DAY 0 - Transfer a piece of the stock culture to an mPmTG agar plate, stock plate A, and incubate 7 days at room temperature (22 C) to increase stock. (Note: If wishing to maintain this isolate in culture, it is advisable to make new stock culture slants at the same time.)
DAY 7 - Using stock plate A, with a sterile transfer needle cut the newly grown thallus into small slivers and transfer the slivers to 2 new mPmTG agar plates, stock plates B, for further increase. Incubate 7 days at room temperature (22 C), one in light and one in dark.
Day 14 - Using stock plates B, with a sterile transfer needle move the pieces of agar aside, cut the agar containing each new thallus into small pieces and transfer the pieces into 150 mL Erlenmeyer flasks containing 50 mL of T/2 broth. Incubate 5 days, one in light, one in dark, checking daily for the presence of spores. If spores are found, immediately inoculate Petri plates with cover slips in T/2 broth and incubate both light and dark for 5 to 7 days for class.
DAY 19 - If spores were not found earlier, wash the thalli by pouring the contents of the flasks into sterile wire baskets and place the baskets in Petri dishes of sterile DW for 10 minutes. With sterile forceps lift the wire baskets, empty the Petri dishes, refill with sterile DW and replace the wire baskets. Let stand in both light and dark over night.
DAY 20 - Remove DW and replace with sterile 1/3 PW.
DAY 22 - If no response is found, remove the sterile 1/3 PW and replace with unsterile 1/3 PW.
DAY 23 - Check the thalli daily for production of zoosporangia or gametangia.
SCHEDULE ADJUSTMENTS - Incubation time on agar plates is adjustable.
RECIPES - mPmTG agar for plates and slants
0.4 g peptonized milk
0.4 g tryptone
2 g glucose
10 g agar for plates, 12 g for slants
1000 mL distilled water
T/2 broth for flasks and Petri dishes with cover slips
5 g tryptone
1000 mL distilled water
1/3 PW - Collect water from a quiet, weedy area of the nearest small pond and
dilute, 1 part pond water to 2 parts distilled water. Autoclave 1/3 pond water in screw-capped bottles
Keep unsterilized pond water in refrigerator
MATERIALS NEEDED - In addition to screw cap test tubes for agar slants (for long term maintenance), you will need sterile plastic Petri dishes for agar plates, 150 mLErlenmeyer flasks (screw caps preferred), glass Petri dishes and wire baskets for washing cultures, glass Petri dishes for cover slip cultures, microscope slides and cover slips..
REFERENCES - The following papers may provide useful information.
Johns, Robert M. and R.K. Benjamin. 1954. Sexual reproduction in Gonapodya. Mycologia 46: 201—208.
Karling, J. S. 1977. Chytridiomycetarum iconographia. Lubrecht & Cramer.
Sparrow, F. K. 1960. Aquatic Phycomycetes. 2nd ed. Univ.
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