Some factors which influence the in vitro maintenance of Anisakis simplex (Nematoda)

Several culture media as well as some factors that may affect the in vitro development of the nematode Anisakis simplex Rudolphi, 1809 have been studied. After testing six media and four temperatures, the conditions for the in vitro culture selected were as follows: RPMI-1640 + 20% (v/v) heat-inactived fetal bovine serum or Meyer's M3 (without agar) media, at 37 degrees C, under 5% CO2 in air atmosphere, and renewal of the medium twice a week. The average survival rates of the larvae were significantly increased when the pH of the culture medium was increased (from 4.0 to 7.2) or decreased (from 7.2 to 4.0) after L3 to L4 moulting. The length of the larvae at the onset of culture affected the survival and moulting of themselves, but these were culture medium-dependent. On the other hand, we have observed that several L3 and L4 were attached, by means of a brown unknown substance apparently secreted by themselves, to the bottom of the substratum. Frequently, when a larva was spontaneously detached, a "cap" of the brown substance blocked, apparently, its mouth. The possible absorption of nutrients through the L3 larvae cuticle of A. simplex is discussed.