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We studied the parasitization by Anisakis spp. in European hake (Merluccius merluccius) of 40-52 cm from the Atlantic off north-west Africa and the Mediterranean off southern Spain. Infection parameters differed: The fish from the Atlantic showing a prevalence of 87.97% and a mean intensity of 4.69, while, in those from the Mediterranean, these were 41.27% and 1.73, respectively. In both samples the two-third larval stage types were isolated: Anisakis simplex sensu lato and Anisakis larvae type II, with prevalence of 85.71% and 30.83% in fish from the Atlantic and 41.27% and 1.59% for those from the Mediterranean, respectively. In both samples, the prevalence of larvae in viscera was clearly higher than in the muscle tissue. We also observed an increase in parasitization with increasing host length, those $46 cm having the highest prevalences (94.87% for those from the Atlantic and 58.33% for those from the Mediterranean; p<0.03).

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.

An epidemiological study was carried out on the anisakids in sardines (Sardina pilchardus) from the southern (Atlantic and Mediterranean) and eastern coasts of Spain. Length of fish was from 12.2-21.0 cm. The anisakids found were identified as the third larval stage (L3) of Hysterothylacium aduncum, with a total prevalence of 11.85%. Prevalence within the host was 9.64% in viscera and 4.69 % in muscle. The highest infection parameters were found in fish from the east coast (western Balearic Sea) with prevalence of 25.21%, mean intensity of 2.10, and mean abundance of 0.52. No worms of the genus Anisakis were found in the 359 sardines analyzed.

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