Fecundity of the zoonotic nematode Anisakis pegreffii cultivated in vitro

The zoonotic marine nematodes of the genus Anisakis Dujardin, 1845 are the causative agents of anisakiasis, a parasitosis that has been increasingly reported in Europe over the past decade due to the more frequent consumption of lightly processed or raw seafood. While the life cycle in the marine environment is relatively well-known, an in vitro life cycle has recently been established with the goal to serve as a model for a better understanding of the functional biology of the nematode and consequent devising of strategies for its detection and inactivation. However, the reproductive capacity of the nematode has not been investigated so far, although it is an important parameter for epidemiological modelling or risk assessment studies. To measure the fecundity of Anisakis pegreffii Campana-Rouget et Biocca, 1955, type I larvae were obtained from naturally infected blue whiting Micromesistius poutassou (Risso) from the Adriatic Sea (Croatia) and cultured to the adult stage in Schneider's insect Drosophila medium supplemented with 10% chicken serum (n = 30 in triplicate). Larvae reached stage 4 (L4) by day 4 post-incubation (dpi), followed by molting to the stage 5 (L5) after 15 days and transition to the adult stage, characterised by production and expulsion of eggs on day 17 dpi. The fecundity of the adults was quantified by the daily number of eggs expelled per female, as well as their hatchability. Eggs were detected from 17 to 133 dpi but started hatching only from 44 dpi. Over the next 51 days, the eggs typically hatched into L2 larvae within 5-7 days. Average fecundity peaked at 100 dpi with 44,125 eggs/day/female and a sex ratio of 1 : 2 to 1 : 3. Cumulative mortality of cultured animals reached 60, 50 and 53% for the triplicates at 133 dpi, whereupon the experiment was terminated as only unfertilised eggs were produced.