Characterization of Three Heat Shock Protein Genes in Pieris melete and Their Expression Patterns in Response to Temperature Stress and Pupal Diapause.

Heat shock protein 70 genes participate in obligatory pupal diapause in Pieris melete to survive unfavorable conditions. In this study, three full-length cDNAs of PmHsc70, PmHsp70a and PmHsp70b were identified, and their expression patterns in response to diapause and short-term temperature stresses were investigated. Summer and winter diapause were induced in the pupae and non-diapause individuals were used as a control. The pupae from each diapause group were subjected to either hot or cold conditions and the expression levels of the HSP genes were measured. Our results showed that up-regulation of PmHsc70 and PmHsp70b were detected both in summer and winter diapause, but not for PmHsp70a. Under cold stress, PmHsp70a and PmHsp70b were upregulated in summer and winter diapause, while heat shock significantly induced upregulation of all three genes. In non-diapause pupae, none of the genes responded to cold or heat stress. Furthermore, we found that incubation at 39 ∘C for 30 min was the most sensitive heat stress condition for PmHsc70 expression in summer diapause. On the other hand, the same temperature was effective for PmHsc70, PmHsp70a, and PmHsp70b expression in winter diapause. During summer diapause, expression of all three genes was upregulated in response to high-temperature acclimation at 31 ∘C, but only PmHsp70a and PmHsp70b were upregulated when acclimated to a low temperature of 4 ∘C in winter diapause. These results suggest that the PmHsc70, PmHsp70a, and PmHsp70b respond differently to pupal diapause and temperature stress, and that PmHsc70 is more sensitive to heat shock than to cold stress.

Identification and up-regulation of three small heat shock proteins in summer and winter diapause in response to temperature stress in Pieris melete.

Small heat shock proteins (sHSPs) are conserved proteins that play key roles in organismal adaptation to adversity stressors. However, little is known about sHSPs during summer diapause. Three sHSP genes: PmHSP19.5, PmHSP19.9, and PmHSP20.0 were identified and cloned from Pieris melete. Sequence alignment and phylogenetic analysis revealed that the three sHSPs have a typical, conserved α-crystallin domain. PmHSP19.5 and PmHSP20.0 were both upregulated in summer diapause (SD) and winter diapause (WD), compared to non-diapause (ND) pupae. All three sHSPs were upregulated and showed similar trends in response to thermal stress. The 0 °C chilling treatment slightly affected sHSP transcripts in ND pupae, whereas both PmHSP19.5 and PmHSP19.9 were upregulated and PmHSP20.0 was downregulated after chilling at 0 °C for 24-96 h in both SD and WD pupae. The transcripts of PmHSP19.5 and PmHSP19.9 were significantly induced at 31 °C for 30 d in SD and WD pupae. The PmHSP20.0 transcript gradually decreased during the SD and WD programs. This is the first time that sHSPs have been linked to both overwintering and summer diapause processes. These findings suggest that sHSPs are involved in both summer and winter diapause maintenance and play a possible key role in temperature stress.