A culture technique for experimental studies of embryonic development in the pond snail lymnaea stagnalis

The gastropod mollusc Lymnaea stagnalis has long played an important role in strengthening our knowledge of developmental biology. However, experimental studies of this and related species have been limited because its embryos develop within egg capsules, which prevent the passage of large molecules and puncturing the capsules results in disrupted development and eventual death. Therefore, long-term experiments involving administration of substances, cell ablations or dye injections have been impossible. The present study explores conditions for culturing embryonic L. stagnalis outside the capsule. The most effective method consisted of raising embryos within capillary tubes and bathed in their natural capsule fluid. Additional experiments were also performed to test the extent to which the capsule fluid could be replaced with artificial media. Results revealed that certain artificial media could sustain prolonged survival of the embryos but that inclusion of a proportion of natural capsule fluid was essential for development to proceed past a mid-veliger stage. Therefore, a range of concentrations of the capsule fluid was tested and it was found that a minimum of 30% natural capsule fluid diluted with either an artificial culture medium or distilled water permitted normal development. These results suggest that the capsule fluid may contain a factor(s) which is required for normal development in this species. We also tested the possibility that such a factor is a growth factor. However, solutions of nerve growth factor (β-NGF), transforming growth factor (TGF-β1) and epidermal growth factor (EGF) were not sufficient to support embryonic development.