Induction of triploidy in the turbot (Scophthalmus maximus): I. Ploidy determination and the effects of cold shocks

Abstract

The basis for induction of triploidy in the turbot by applying cold shocks shortly after fertilization (AF) was studied. Since this species exhibits a polymorphism in a number of nucleolar organizing regions (NOR), determination of the ploidy level through NOR analysis was first validated. Results showed that NOR analysis could discriminate well between diploid and triploid individuals whose ploidy level was verified karyotypically (n=44 chromosomes in diploids; n=66 in triploids). In diploids, the mean number of NOR per cell ranged from 1.10 to 1.85, whereas in triploids, it ranged from 1.50 to 2.35. However, histogram distribution of data on mean number of NOR per cell showed that the number of fish in the overlapping region (NOR number between 1.50 and 1.85) was very low. Cold-shocked fish with a NOR value >1.735 were considered triploids. The error in ploidy assessment using NOR analysis in the turbot was found to be consistently around 3% and always <5%. In this way, NOR analysis could be safely applied to monitor the effects of cold shocks on triploidy induction. Cold shocks were applied 5 min AF for 5, 10, 20 or 40 min at either 0°C, 2°C, or 4°C. Results showed that the number of triploids increased with lower shock temperatures and longer shock duration in the range from 5 to 20 min. In particular, cold shocks of 0°C applied during 20 min consistently resulted in ~90% triploid turbot (P<0.001). Shocks longer than 20 min (40 min) did not increase the number of triploid turbot in contrast to what has been found in other flatfish species. This is probably related to the higher pre-shock temperature at which turbot eggs are incubated. Survival, 1 day after hatching, was ~80% of the untreated controls and not different (P>0.05) from appropriate sham controls, indicating that lower survival is due to the effects of mechanical handling and stress during triploidy induction rather than the triploid condition per se. The highest triploid yield obtained in this study, not, vert, ~70%, is higher than the triploid yield obtained in other flatfishes