Book Description

Author's abstract: It is important to study the cultivation of parasites in vitro for many reasons, such as to aid in developing antihelminthic drugs and vaccines, to eliminate the need for vertebrate hosts in parasite culture, and to more easily study the genetics and the biology of parasites. Trematodes have complex life cycles with multiple hosts which makes them difficult to grow in vitro. However, microphallid trematodes are excellent candidates for parasite in vitro cultivation because they are short lived and progenetic. The goal of my study was to optimize in vitro culture conditions for the microphallid trematode, Gynaecotyla adunca. I determined the optimal concentration of trypsin to be 0.5% in order to excyst the most metacercariae of G. adunca that were obtained from the green glands of the second intermediate host, the fiddler crab Uca pugnax. Hunter (1952) reported that G. adunca only self-fertilize, however, offered no evidence to support this claim. I decided to test G. adunca adult worms to either confirm or refute whether this is true or not. I also tested different culture conditions on adult G. adunca worms. I chose 3 parameters to evaluate in vitro cultivation experiments. To determine these parameters which included worm longevity, number of worms that produced eggs in utero, and the number of eggs deposited, the media DMEM and RPMI-1640 were compared to Hank's Balanced Salt Solution. Different sera were also tested including horse, new-born calf, and chicken, the best of which was tested at different concentrations. To test the viability of the eggs deposited by worms in culture, they were fed to the marsh snail Ilyanassa obsoleta. I also compared HBSS and DMEM plus 5% horse serum as the initial incubation conditions of the freshly excysted worms then observed them 24 hr later before adding them to culture to see if this affected egg production. G. adunca worms do self-fertilize. When worms were incubated alone, they showed signs of being fertilized. The percentage of worms with eggs in utero was greatest when worms were grown in DMEM. Worms lived longer and deposited more eggs when cultured in DMEM supplemented with 5% horse serum. Snails fed eggs from culture were not successfully infected. There was no significant difference on egg production between initially incubating the worms in HBSS and DMEM plus 5% horse serum within the 24 hr period between excystment and adding them to culture. Future studies will further refine in vitroculture conditions for G. adunca and investigate the best approach for snail infection.