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

Abstract

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.

Keywords: Pieris melete; cold tolerance; heat shock protein 70; heat tolerance; summer and winter diapause.

Figure 1

The alignment of amino acid sequences of PmHSC70, PmHSP70a, and PmHSP70b. The three signature sequences of HSP70 homologs are underlined. The conserved HSP70 protein domain (CL0108) is shown in the box. The final consensus sequence EEVD is at the C-terminus.

Figure 2

Phylogenetic neighbor-joining tree of PmHSC70, PmHSP70a, and PmHSP70b of Pieris melete and other insect HSPs using Mega X. The percentage bootstrap values obtained by 1000 repeated calculations were displayed at the nodes, and percentage values lower than 50% were collapsed. The PmHSC70, PmHSP70a, and PmHSP70b in our study were marked with a red solid circle. Lepidoptera, Coleoptera, Diptera, and Hymenoptera HSP70s from previous studies were labeled with a black solid circle, hollow rhombus, hollow circle, and hollow triangle, respectively.

Figure 3

Relative mRNA levels of PmHsc70, PmHsp70a, and PmHsp70b in ND, SD, and WD pupae of Pieris melete. The relative expression levels (Mean ± SE) are normalized to β-actin and 18S rRNA. Different letters above the bar indicate significant differences by ANOVA and post hoc Bonferroni multiple comparison test (p < 0.05).

Figure 4

Relative mRNA expression levels of PmHsc70 (a), PmHsp70a (b), and PmHsp70b (c) in response to low-temperature cryogenic chilling at 0 °C in ND, SD, and WD pupae of Pieris melete. The relative expression levels (Mean ± SE) are normalized to β-actin and 18S rRNA. Letters above the bar indicate significant differences by ANOVA and post hoc Bonferroni multiple comparison test (p < 0.05).

Figure 5

Relative mRNA expression levels of PmHsc70 (a), PmHsp70a (b), and PmHsp70b (c) in response to heat treatment in ND, SD, and WD pupae of Pieris melete. The relative expression levels (Mean ± SE) are normalized to β-actin and 18S rRNA. Different letters above the bar indicate significant differences by ANOVA and hoc Bonferroni multiple comparison test (p < 0.05).

Figure 6

Relative mRNA expression levels of PmHsc70 (a), PmHsp70a (b), and PmHsp70b (c) in response to 39 °C of heat stress in non-diapause (ND), summer diapause (SD), and winter diapause (WD) pupae of Pieris melete. The relative expression levels (Mean ± SE) are normalized to β-actin and 18S rRNA. Different letters above the bar indicate significantly differences by ANOVA and post hoc Bonferroni multiple-comparison (p < 0.05).

Figure 7

Relative expression analysis of PmHsc70, PmHsp70a, and PmHsp70b mRNA transcription levels in SD (a–c) and WD (d–f) pupae that were incubated at different temperatures during diapause development. The relative expression levels (Mean ± SE) are normalized to β-actin and 18S rRNA. Different letters or Greek alphabet (α, β, γ) above the bar indicate significant differences by ANOVA and post hoc Bonferroni multiple comparison test (p < 0.05) during diapause development at each temperature. The significant differences between 31 °C (SD), 4 °C (WD), and 18 °C (control) treatments are represented with an asterisk *. * p < 0.05, ** p < 0.01, and *** p < 0.001.