Naturwissenschaften (2009) 96:827–834 Materials and methods Insects Laying females insert their eggs deeply into the bark, which complicates egg counting because dissecting the bark often leads to egg destruction. Therefore, no reliable method could be used to count the numbers of living eggs on the woody canes. S. titanus egg populations were collected as described in Caudwell et al. (1970). Two-year-old grape- vine woody canes were collected in an organic vineyard 60 km east of Bordeaux (Castillon-la-Bataille) where important populations of S. titanus were observed for successive years and during the preceding summer. Canes were extensively collected after pruning during the winter. The occurrence of eggs was checked by removing the bark of sample of 20 canes selected at random under micro- scope. Egg hatchings were obtained by placing wood pieces (20–25 cm long) inside plastic hatching cages (50 × 38× 36 cm) in a climatic chamber under a 16:8 (L/D) photoperiod, at 23 ±1 °C, and 65–70% RH. To avoid egg desiccation, a 1-cm layer of vermiculite (Efisol, France) was placed below the eggs and was humidified with distilled water every 7 days. In order to harvest neonate larvae, six cutting leaves of Cabernet-Sauvignon cultivar, maintained in a glass tube with water, were added to the cage ca. 20 days after eggs were removed from the cold room. Leaves were replaced when they began to wither. In each hatchery cage, larvae were gently removed each day from the lower side of leaves with a mouth aspirator and the number of larvae hatched, and this was computed as hatching eggs. Observation ended when no more hatching occurred during 1 week. Cold temperature durations In this study, we set a temperature threshold of 5 °C and tested the effect of time spent at this temperature on the hatching dynamics of the eggs. Wood canes were kept at 5 °C before hatching for varying durations (0, 20, 28, 36, 49, and 63 days) in the conditions as described above. The canes were collected during the first week January 2006 and 2007. Repeatability between replicates was gained by pooling batches of ca. 14 kg of canes, which were then shared randomly in eight samples of 1.6–1.7 kg each. Using this method, batches are expected to bear equivalent amounts of eggs. We recorded the day of the first hatching, the total number of hatchings per day, the median, and the total duration of hatching for each batch of grapevine woody canes. 829 Winter simulation In order to avoid the effects of climatic conditions imposed upon eggs before cane collection, we used two environmen- tal finely regulated climatic chambers (PR-25T, ESPEC) to simulate different winter conditions. We simulated the daily mean temperature recorded during the 2000–2001 winter in the Burgundy (Beaune) and Bordeaux areas (Castillon-la- Bataille) each under a constant short day photoperiod 8L:16D. The former conditions were typical of a 93-day cold winter (298.2 degree-day) and the latter of a mild winter (821.4 degree-day; Fig. 2). Two-year-old grapevine canes bearing eggs were collected on mid-November, and 12 kg of canes was split into six hatching cages as described above. Three of them were exposed to a cold winter and the other three to a mild winter. After 3-month simulations, hatching cages were placed in climatic chamber as described above. Statistical analysis The effect of cold temperature was analyzed using the Spearman correlation test. Hatching dynamics were compared with the Kaplan–Meier survival method, and pair compar- isons were performed with the log rank and Gehan–Wilcoxon tests. The log-rank test accounts for all events throughout the period of observation, while the Gehan–Wilcoxon test puts more weight onto early events (Pyke and Thompson 1986). The former allows a general study of the dynamics, whereas the latter test is better to compare precocity of hatchings. Profiles of the hatching dynamics were compared with the Spearman correlation test. All statistical analyses were performed using Statistica 6.0 (Stat Soft). Results Hatching dynamics The egg cohort exposed to “Burgundy” winter hatched in a shorter period of time compared to those exposed to the Fig. 2 Temperature program of simulated Burgundy (cold) and Bordeaux (mild) winters