Universal and differential transcriptional regulatory pathways involved in the preparation of summer and winter diapauses in Pieris melete.

Much progress has been made in understanding the environmental and hormonal systems regulating winter diapause. However, transcriptional regulation of summer diapause is still largely unknown, making it difficult to understand an all-around regulation profile of seasonal adaptation. To bridge this gap, comparison RNA-seq to profile the transcriptome and to examine differential gene expression profiles between non-diapause, summer diapause, and winter diapause groups were performed. A total number of 113 million reads were generated and assembled into 79,117 unigenes, with 37,492 unigenes categorized into 58 functional gene ontology groups, 25 clusters of orthologous group categories, and 256 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. KEGG analysis mapped 2108 differentially expressed genes to 48 and 67 pathways for summer and winter diapauses, respectively. Enrichment statistics showed that 11 identical pathways similarly overlapped in the top 20 enriched functional groups both related to summer and winter diapauses. We also identified 35 key candidate genes for universal and differential functions related to summer and winter diapause preparation. Furthermore, we identified some genes involved in the signaling and metabolic pathways that may be the key drivers to integrate environmental signals into the summer and winter diapause preparation. The current study provided valuable insights into global molecular mechanisms underpinning diapause preparation.

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.

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.

Expression of alternative developmental pathways in the cabbage butterfly, Pieris melete and their differences in life history traits.

The seasonal life cycle of the cabbage butterfly, Pieris melete is complicated because there are three options for pupal development: summer diapause, winter diapause, and nondiapause. In the present study, we tested the influence of temperature, day length, and seasonality on the expression of alternative developmental pathways and compared the differences in life history traits between diapausing and directly developing individuals under laboratory and field conditions. The expression of developmental pathway strongly depended on temperature, day length, and seasonality. Low temperatures induced almost all individuals to enter diapause regardless of day length; relatively high temperatures combined with intermediate and longer day lengths resulted in most individuals developing without diapause in the laboratory. The field data revealed that the degree of phenotypic plasticity in relation to developmental pathway was much higher in autumn than in spring. Directly developing individuals showed shorter development times and higher growth rates than did diapausing individuals. The pupal and adult weights for both diapausing and directly developing individuals gradually decreased as rearing temperature increased, with the diapausing individuals being slightly heavier than the directly developing individuals at each temperature. Female body weight was slightly lower than male body weight. The proportional weight losses from pupa to adult were almost the same in diapausing individuals and in directly developing individuals, suggesting that diapause did not affect weight loss at metamorphosis. Our results highlight the importance of the expression of alternative developmental pathways, which not only synchronizes this butterfly's development and reproduction with the growth seasons of the host plants but also exhibits the bet-hedging tactic against unpredictable risks due to a dynamic environment.

Molecular characterization of three Hsp90 from Pieris and expression patterns in response to cold and thermal stress in summer and winter diapause of Pieris melete.

Heat shock proteins (Hsps) have been linked to stresses and winter diapause in insects, but whether they are components of summer diapause is still unknown. In this study, complementary DNAs of Hsp90 from Pieris melete, Pieris rapae and Pieris canidia named PmHsp90, PrHsp90 and PcHsp90, respectively, were cloned and sequenced. The deduced amino acid sequence consisted of 718 amino acid residues with a putative molecular mass of 82.6, 82.6 and 82.7 kDa, respectively. The amino acid sequences contained all of the five conserved signature motifs in the Hsp90 family and a bHLH protein folding activity region. The differential expression pattern of PmHsp90 in response to summer diapause and winter diapause, which are related to heat/cold stress, was investigated. Cold stress induced Hsp90 up-regulation in summer and winter diapause pupae, but not in non-diapause individuals. Heat shock up-regulated PmHsp90 gradually with an increase in temperature in summer diapause, and PmHsp90 was rapidly up-regulated in winter diapause. After 30 min heat shock at 39°C, substantial up-regulation of PmHsp90 transcript levels were observed both in summer and winter diapause. However, in non-diapause a relatively stable expression was found under different durations of 39°C heat shock. Compared to the optimal treatment of 18°C for diapause development, a high temperature acclimation of 31°C induced PmHsp90 up-regulation in summer diapause, whereas a low temperature acclimation of 4°C induced up-regulation in winter diapause. The current results indicate that Hsp90 may play an important role in response to heat/cold stress both in summer and winter diapause.