In vitrocultivation ofAnisakis simplex:pepsin increases survival and moulting from fourth larval to adult stage

Abstract

This paper describes the in vitro cultivation of the 3rd-larval stage (L3) of Anisakis simplex to adulthood in a much simpler and easier to prepare medium than those described to date. The adult males obtained are between 3.8 and 6.5 cm long and the females between 4.5 and 8.0 cm. Some individually cultivated females laid eggs which had an average size of 44.4 x 50.5 microm. The culture conditions were as follows: medium RPMI-1640 supplemented with 20% heat-inactivated fetal bovine serum and 1% commercial pepsin, at pH 4.0 and a temperature of 37 degrees C, and in air atmosphere with 5% CO2. The pepsin was found to be the key to the success of the culture. The average survival of the worms in the culture increased from 50 to 88 days, due to the fact that the survival of the adults practically doubled (increasing by 1.9 times). Furthermore, the number of worms that completed the 4th moulting (M4) increased by 4.2 times, from 22.9 to 95.6%. This culture medium may facilitate, due to its simplicity, the study of anisakids, or at least of A. simplex, constituting another step towards achieving a complete in vitro life-cycle for these parasites.

Population Structure of Anisakis Simplex (Nematoda) in Harbor Porpoises Phocoena Phocoena Off Denmark

“…1). Although there is no information from field experiments, both laboratory infections in rats (Gibson, 1970) and in vitro studies of A. simplex (Grabda, 1976;Iglesias et al, 2001) indicate that L3s, and subadults in particular, are short-lived in comparison with L4s and adults, which conforms with the compositions of the component population and average infrapopulation observed in the present study. (Efron and Tibshirani, 1993).…”

In vitro development of the fish parasite Hysterothylacium aduncum from the third larval stage recovered from a host to the third larval stage hatched from the egg

“…Prior to cultivation, each larva was placed in an antibiotic-antifungal solution (80 mg gentamicin sulfate, 0.625 mg amphotericin B, 10 000 IU sodium penicillin G, 10 mg streptomycin sulfate and 4.5 ml Hanks' solution, for a final solution volume of 10 ml) and axenized as described elsewhere (Iglesias et al 1997, Iglesias et al 2001. Worms were cultured in sterile polystyrene 30 ml flasks.…”

“…This will facilitate morphological identifications, where required, and help to understand the different aspects of their biology and develop effective control measures, especially in cultured fish populations. We are currently working on the in vitro cultivation of anisakids, and we have individually cultured third stage larvae (L3) from fish to adulthood, including some anisakid egg-laying females (Iglesias et al 2001(Iglesias et al , 2002. This work consists of some observations on the in vitro development of L3 of Hysterothylacium aduncum from blue whiting Micromesistius poutassou, to the hatching of L3 from eggs obtained by the culture of clustered H. aduncum worms.…”

“…The worms were observed individually under an inverted microscope, and those that showed any kind of internal or external damage were discarded. They were then identified according to morphological features (Hartwich 1975, Petter & Maillard 1988, Petter & Cabaret 1995.Prior to cultivation, each larva was placed in an antibiotic-antifungal solution (80 mg gentamicin sulfate, 0.625 mg amphotericin B, 10 000 IU sodium penicillin G, 10 mg streptomycin sulfate and 4.5 ml Hanks' solution, for a final solution volume of 10 ml) and axenized as described elsewhere (Iglesias et al 1997, Iglesias et al 2001. Worms were cultured in sterile polystyrene 30 ml flasks.…”

“…Commercial pepsin was obtained from Probus (Spain). This commercial pepsin was analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine its purity (Iglesias et al 2001). …”