Bao and Xu BMC Genomics 2011, 12:224 http://www.biomedcentral.com/1471-2164/12/224 individuals first utilize sugar as energy and store lipid for use during long diapause periods. Stress resistance During the long overwintering phase, diapause pupae must encounter various stress challenges. The expres- sions of some specific genes are evoked in response to environmental stress [22], and stress resistance is impor- tant for the survival of diapause individuals. Hsp70 func- tions as a molecular chaperone to protect cellular proteins from denaturation in many species, including Diptera, Lepidoptera, Coleoptera and Hymenoptera [13]. In addition, a small hsp, Hsp21.4 identified by proteo- mic analysis, is more abundant in the brain of H. armi- gera pupae at diapause initiation [11]. Thus, the up- regulated Hsp70 at diapause initiation plays a role in cold-hardiness for overwintering (Figure 5B). Moreover, up-regulation of Hsp has also been reported under short day-length conditions, and Hsp up-regulation could represent a molecular exaptation to diapause [31]. Ferritin is the primary iron storage protein, and it functions in scavenging oxygen radicals [32,33]. Ferritin and ferritin light chain are up-regulated at diapause initiation, as reported in Nasonia [34] and in S. crassi- palpis [8]. MnSOD is also up-regulated at diapause initiation. In Caenorhabditis elegans, MnSOD partici- pates in the regulation of both longevity and dauer for- mation (a type of developmental arrest) as a physiological redox signaling modulators [35]. GST and bombyrin also have antioxidant function and are up- regulated in the brain, as reported in proteomic analysis of H. armigera [11]. Oxidative stress can damage tissues and cellular components during diapause. Therefore, the up-regulation of transcripts of antioxidant proteins will protect diapause individuals from oxidative stress (Fig- ure 5B). Rad23 is a nucleotide excision repair (NER) gene that, functions in DNA repair and protein degradation [36]. Integrator complex subunit 3, which is also called SOSS-A, is involved in sensing ssDNA and maintaining genome stability [14]. up-regulation of genes related to DNA repair (NER and SOSS-A) in diapause has not been reported previously. However, it is possible that DNA lesions occur under extreme environmental condi- tions during diapause, and the integrity of DNA is cru- cial for re-starting the development into an adult when diapause is terminated. Therefore, these up-regulated genes at diapause initiation mainly respond to stress resistance for insect survival in rigorous environmental conditions (Figure 5B). Signaling pathways Genes involved in signaling pathway were also found in the SSH library (Table 1C). Akt is an essential Page 11 of 14 component of the insulin signaling pathway for glucose uptake to synthesize sugar and also as an activator of the target of rapamycin (TOR) pathway to increase pro- tein synthesis [37]. Insect organs and tissues need to accumulate a large store of sugar as energy and other substances, such as antifreeze agents glycerol and sorbi- tol, for use during a long diapause phase. Therefore, enhanced Akt transcription reflects increased sugar metabolism in diapause-destined pupal brain, and Akt participates in the regulation of energy reserves and in response to environmental stress at the onset of dia- pause (Figure 5C). Calmodulin signaling, which is involved in the regula- tion of neuronal development and plasticity [38], is down-regulated at diapause initiation in H. armigera [11]. In this study, CaMK II, which modulates synaptic plasticity, learning, and memory [38], was down-regu- lated. ArgK was also down-regulated at diapause initia- tion, and high expression of ArgK, which is a developmental signal, was closely correlated with pupal development (data not shown). Thus, down-regulation of CaMK II and ArgK may cause developmental arrest at diapause initiation (Figure 5C). Cell cycle During diapause, the cell cycle is arrested in the embryo of B. mori [39] and in the brains of S. crassipalpis [40] and Chymomyza costata [41]. Cyclin-dependent kinase 8 is a kinase partner of cyclin C, interacts with the large subunit of RNA polymerase II, and then participates in the regulation of the G1/S transition of mitosis [42]. More than 97% of the brain cells become arrested in the G0/G1 phase in the diapause pupae of S. crassipalpis [40]. Proteomic analysis of Sitodiplosis mosellana has found a strong up-regulation of inhibitor of nuclear fac- tor kappa-B kinase interacting protein isoform 2 (IKIP2) during diapause, which contributes to inhibiting cell division during diapause [43]. Therefore, cyclin-depen- dent kinase 8 and five other transcripts down-regulated in the brain at diapause initiation may cause cell cycle arrest, inducing the insect to enter diapause (Figure 5C). Transcription and translation Transcription and translation are two major energetic costs in cellular development. To reduce energy con- sumption, many genes are silenced during diapause [22]. In this study, several genes involved in the regulation of transcription and translation were identified (Table 1E). The down-regulation of transcription factor PLAG1 may result in the modulation of downstream target genes [44]. The down-regulation of elongation factor 1 delta indicates that translation is also suppressed at diapause initiation. In addition, some transcripts of proteins involved in transcription were up-regulated at diapause