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

Jenna West,Oscar J Pung,Alexandra Mitchell

doi:10.1155/2014/382153

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.

Highlights

The ability to grow all of the life stages of a digenean in vitro would benefit development of anthelmintic drugs and vaccines [1] and facilitate research into the ecology, evolution, genetics, and behavior of these parasites [2,3,4,5]
There was a significant difference between worms cultured in serum-free Hanks balanced salt solution (HBSS), RPMI-1640, and DME/F-12 with respect to both the number of eggs deposited (ANOVA, df = 2, 21; F = 6.5, P = 0.006) and worm longevity (ANOVA, df = 2, 122; F = 56.8, P < 0.0001) but not the percentages of worms producing eggs in utero
Worms deposited the most eggs when cultured in DME/F-12 (Figure 1) and lived longer in HBSS and DME/F-12 than in RPMI 1640 (Figure 2)

Summary

Introduction

The ability to grow all of the life stages of a digenean in vitro would benefit development of anthelmintic drugs and vaccines [1] and facilitate research into the ecology, evolution, genetics, and behavior of these parasites [2,3,4,5]. Metacercariae of the parasite obtained from the grass shrimp second intermediate host, Palaemonetes pugio, mature into egg-depositing adults in our culture system. The objectives were to (1) optimize the reagents employed in the above protocol to maximize in vitro worm longevity and egg production of another microphallid, Gynaecotyla adunca, and to (2) determine if eggs deposited by the G. adunca in vitro were infectious. This is important as it will permit us to determine whether the successful laboratory cultivation of M. turgidus was due to an idiosyncrasy of that

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Discussion

Conclusion