A simple method to discriminate diapause from non-diapause pupae in large and small white butterflies, Pieris brassicae and P. rapae crucivora.

Differences in colour and shape have been used to discriminate diapause pupae from non-diapause pupae in butterflies. In the present study, we describe a simple discrimination method based on differences in the specific gravity of diapause and non-diapause pupae of large and small white butterflies, Pieris brassicae and Pieris rapae crucivora. When put into water, diapause pupae sink to the bottom (specific gravity is above 1.0), whereas non-diapause pupae float on the surface (specific gravity is below 1.0). Nuclear magnetic resonance microimaging revealed that this difference in specific gravity is due to a difference in the volume of an internal cavity located between the thorax and the abdomen in the pupae. The cavity appears quite early in development. We also examined the cavity in pupae of the unrelated swallowtail butterfly, Papilio xuthus, and found a similar cavity. However, in this species, the cavity is very small and, as a consequence, non-diapause as well as diapause pupae sink in water.