Based on these data, −11.5 and −12.5 °C were designated as the DTemps for juvenile and

mature larvae, respectively. Survival of larvae exposed to the DTemp for 8 h increased following prior acclimation to −5 °C for 1 h, and gradual cooling (+4 °C to the ABT-263 in vitro DTemp at 0.2 °C min−1), but not after acclimation for 1 h at 0 °C (Fig. 3). The highest survival was seen after gradual cooling for both juvenile (74%) and mature (83%) larvae. This was significantly different from their survival after direct transfer to the DTemp (F1,4 = 26.156, P < 0.05; F1,4 = 48.400, P < 0.05, respectively). Under all treatments, the strength of the RCH response was not significantly different between juvenile and mature larvae (P > 0.05 Tukey’s multiple range test). RCH lowered the lower lethal temperature (LLT) by 2.5 and 6.5 °C in mature and juvenile larvae, respectively (Fig. 4). Survival Obeticholic Acid ic50 ⩾80% at the DTemp (−12.5 °C) was also extended by at least 14 h in mature larvae following RCH and some individuals even survived 48 h under the same treatment (Fig. 5). Mature larvae acclimated to a model Signy Island thermoperiod (+6 to −1 °C over a 24 h cycle) exhibited increased survival of the DTemp for 8 h (Fig. 6). However, this was not significant (P > 0.05 Tukey’s multiple

range test). Survival was also not significantly different within or between −1 and +6 °C conditioned groups across all 3 days tested (P > 0.05 Tukey’s multiple range test). In contrast, mature larvae acclimated to a model Anchorage Island thermoperiod (+4 to −3 °C over a 24 h cycle) showed significantly higher survival of the DTemp for 8 h following removal at −3 °C after 2 d (F1,4 = 8.915, P < 0.05) and 3 d

(F1,4 = 9.291, P < 0.05) ( Fig. 7). There was a significant decline in cold tolerance during the warming phase at +4 °C on day 2, but cold tolerance was regained during the subsequent cooling phase on day 3 ( Fig 7) The tolerance accrued over 3 d was maintained during the day 3 warming phase, with significantly higher survival exhibited at the DTemp when larvae were Progesterone removed at 4 °C on day 3 (F1,4 = 11.560, P < 0.05). The mean SCP of mature larvae following RCH (0.2 °C min−1) was −5.54 °C. While slightly lower, this was not significantly different to the mean SCP of larvae cooled at 1 °C min−1 (−5.07 °C) and larvae directly transferred to the DTemp (−5.73 °C) (table 1, P > 0.05 Tukey’s multiple range test). Juvenile larvae cooled at 0.2 °C min−1 (SCP: −7.29 °C) also showed no significant difference in their SCP when compared with those directly transferred to the DTemp (SCP: −5.86 °C) ( Table 1, P > 0.05 Tukey’s multiple range test). The difference in survival between mature larvae that were held frozen at −7 °C for 4 min (20% survival) or frozen for 1 h 4 min (13% survival) was not statistically significant (F1,4 = 0.308, P > 0.05), indicating that RCH was not induced after the organisms froze.