Optimization of Conditions for In Vitro Culture of the Microphallid Digenean Gynaecotyla adunca

Abstract

In vitro cultivation of digeneans would aid the development of effective treatments and studies of the biology of the parasites. The goal of this study was to optimize culture conditions for the trematode, Gynaecotyla adunca. Metacercariae of the parasite from fiddler crabs, Uca pugnax, excysted in trypsin, were incubated overnight to permit fertilization, and were cultured in different conditions to find those that resulted in maximum worm longevity and egg production. When cultured in media lacking serum, worms lived longer in Hanks balanced salt solution and Dulbecco's Modified Eagle medium/F-12 (DME/F-12) than in RPMI-1640 but produced the most eggs in DME/F-12. Worm longevity and egg production increased when worms were grown in DME/F-12 supplemented with 20% chicken, horse, or newborn calf serum but the greatest number of eggs was deposited in cultures containing horse or chicken serum. Horse serum was chosen over chicken serum due to the formation of a precipitate in chicken serum. The optimal concentration of horse serum with respect to egg production ranged from 5 to 20%. Infectivity of eggs deposited by worms in culture was tested by feeding eggs to mud snails, Ilyanassa obsoleta. None of these snails produced G. adunca cercariae.

Figure 1

In vitro egg deposition by the trematode Gynaecotyla adunca in Hanks balanced salt solution (HBSS) and 2 culture media maintained at 42 C in a gas phase of air. Asterisk indicates significantly different from worms cultured in HBSS. Each value is a mean ± 1 SE.

Figure 2

In vitro longevity of the trematode Gynaecotyla adunca in Hanks balanced salt solution (HBSS) and 2 culture media maintained at 42 C in a gas phase of air. Asterisk indicates significantly different from worms cultured in HBSS. Each value is a mean ± 1 SE.

Figure 3

In vitro egg deposition by the trematode Gynaecotyla adunca in DME/F-12 with and without 20% animal serum and maintained at 42 C in a gas phase of air. Asterisk indicates significantly different from worms cultured in serum-free DME/F-12 but not from each other. Each value is a mean ± 1 SE.

Figure 4

In vitro longevity of the trematode Gynaecotyla adunca in DME/F-12 with and without 20% animal serum and maintained at 42 C in a gas phase of air. Asterisk indicates significantly different from worms cultured in serum-free DME/F-12 but not from each other. Each value is a mean ± 1 SE.

Figure 5

Number of eggs deposited in culture after 10 days at 42 C by Gynaecotyla adunca in DME/F-12 supplemented with different concentrations of horse serum. The gas phase was air. Asterisk indicates significantly different from serum-free cultures but not from each other. Each value is a mean ± 1 SE.