When puparia of the onion maggot Delia antiqua were preexposed to 5°C for 5 days starting at different time points after pupariation, a large increase in survival after exposure to -20°C for 5 days was observed only when pre-exposure was initiated at 3-6 days after pupariation. The increase in cold hardiness was not associated with a large increase in the trehalose content of the puparia. The supercooling point of the puparia naturally decreased from -18 to -27°C in the first three days after pupariation, and pre-exposure to 5°C did not have an additional effect. Thus, factors responsible for the enhancement of cold hardiness by acclimation other than trehalose and supercooling point should be sought. The period of responsiveness to cold acclimation coincided with the time soon after head evagination, which corresponds to "pupation" in lepidopteran insects. The puparia appear to be physiologically flexible for a short time after head evagination, and able to adapt their physiology to the contemporary cold environment.
A sequence analysis of the mitochondrial cytochrome oxidase subunit II (COII) gene in Russian and Turkish maize-associated populations of Ostrinia nubilalis and a Slovenian population of O. nubilalis probably infesting maize revealed little diversity. This lack of diversity may have resulted from bottleneck event(s) when the maize-associated population of O. nubilalis expanded from small population(s) in association with the cultivation of maize in Europe ca. 500 years ago. In the genealogy of COII genes obtained in the present and previous studies, Eurasian samples were substantially differentiated from North American samples. Since the North American population of O. nubilalis came from Europe, our finding suggests that there is geographical differentiation in European maize-associated O. nubilalis, and that maize-associated populations of O. nubilalis expanded multiple times in Europe. Finally, a phylogenetic analysis of the COII gene did not support that O. nubilalis and O. furnacalis are the closest relatives within the O. furnacalis species group.