In vitro culture and confocal microscopy study of Maritrema gratiosum Nicoll, 1907 (Digenea): From metacercaria to ovigerous adult

This study set out to characterise the in vitro development, including musculature, of the microphallid parasite of the barnacle Semibalanus balanoides (Linnaeus, 1767), Maritrema gratiosum Nicoll, 1907 collected in Scotland. An in vitro culture model was developed to obtain ovigerous adults of M. gratiosum and their morphology was observed. Different media were tested and NCTC-109 was chosen as the best medium. The effects of different concentrations of serum upon adult longevity, size and egg production was measured. Survival for 10-days was achieved when flukes were cultured in NCTC-109 plus chicken serum and antibiotics. Forty percent chicken serum seemed to provide better results in terms of survival time and producing flukes with the largest body lengths. Both normal and abnormal eggs were observed from adults cultured in vitro. Confocal microscopy was undertaken to provide details of the development of the parasite's ultrastructure, including musculature, during the course of in vitro culture. While the musculature of M. gratiosum was similar to that of other microphallids, some additional novel structures were observed, most notably a ligament connecting pars prostatica and seminal vesicle and a racket-shaped excretory bladder. This study has provided greater insight into the biology M. gratiosum, and also developed a good in vitro model which might be applied to ecological or medical research in the future.