Optimization of tetraploid induction in Pacific oysters, Crassostrea gigas, using first polar body as a natural indicator

Abstract

Tetraploid Crassostrea gigas were first successfully produced in 1993 by inhibiting the first polar body of eggs from triploids that had been fertilized with sperm from diploids (Guo and Allen method). However, attempts to repeatedly produce high yields of tetraploids were inconsistent. Because of these uncertainties, we examined some of the fundamental aspects of tetraploid production in an attempt to optimize tetraploid induction using the Guo and Allen method. Varying the duration of the treatment to inhibit polar body 1 (PB 1) of triploid eggs had clear effects on ploidy of progeny. Short treatments (15–35 min after fertilization — about half the period of meiosis 1 in triploid eggs) yielded tetraploid and heptaploid cells. Long treatments (7–43 min — about three quarters of the period of meiosis I in triploid eggs) yielded only heptaploid cells among the embryos. Tetraploid induction was most consistent when treatments were accomplished on eggs from individual triploid females rather than pooled from a number of females, and when treatments were metered according to biological landmarks. That is, eggs from individual triploids were fertilized and 0.5 mg/l cytochalasin B (CB) added after 10 min. A subsample of the fertilized eggs was kept aside untreated. When 50% of the untreated eggs showed PB 1 extrusion (as judged by microscopic examination of dividing, untreated eggs), the CB treatment was discontinued. In eight treatments based on these “biological criteria,” proportions of tetraploids ranged from 13% to 92% after 8 days for an average of 55%, and seven of eight replicates went through metamorphosis and settlement. At settlement, the percentage of tetraploids ranged from 7% to 96%, averaging 45%. Average survival in all the replicates at 8 days was 4.4%, which is acceptable considering tetraploid progeny are destined for use as brood stock.