Term of Award

Summer 2009

Degree Name

Master of Science in Biology (M.S.)

Document Type and Release Option

Thesis (restricted to Georgia Southern)

Department

Department of Biology

Committee Chair

Oscar Pung

Committee Member 1

Lance Durden

Committee Member 2

Dana Nayduch

Abstract

The in vitro cultivation of digenetic trematodes would greatly contribute to the development of effective vaccines, control measures, and the understanding of the basic biology of these parasites. Previous experiments in our laboratory determined that metacercariae of the trematode Microphallus turgidus (Leigh) mature into adults and deposit eggs infectious to hydrobiid snails when cultured in air at 42°C in RPMI-1640 medium supplemented with 20% horse serum. The purpose of the present study was to vary culture conditions (i.e., serum and glucose concentrations, gas phase, and incubation time prior to cultivation) in order to optimize worm survival and production of viable, infective eggs. To do so, excysted metacercariae from grass shrimp, Palaemonetes pugio (Holthuis), were incubated in HBSS for 0-48 h in conical bottom tubes and then cultured in 24-well plates (5 worms/well) at 42°C. Worms produced the greatest number of eggs when incubated for 24 h in a conical bottom tube prior to cultivation. Serum concentrations of 20-40% were optimal with respect to both number of eggs produced and worm survival. Varying glucose concentrations from 0.5-2% had no effect on egg production or worm survival, although 3% glucose decreased the number of eggs deposited. Worms deposited more eggs and survived longer when cultured in 5% CO2 rather than air. Worms cultured in anaerobic conditions died sooner and produced fewer eggs, than worms in 5% CO2 or air, even when the medium was supplemented with glucose. None of the treatments had a dramatic effect on the percentage of normal, embryonated eggs. Finally, the effect of optimal worm culture conditions on infectivity of deposited eggs to hydrobiid snails after incubation for 10 days at 30ºC was tested. However, only one snail became infected. In conclusion, M. turgidus produced more eggs and survived longest when incubated 24 h in conical bottom tubes and then cultured in RPMI-1640 containing at least 20% horse serum in a gas phase of 5% CO2. No conclusion can be made concerning the infectivity of eggs cultured in these conditions at this time.

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