Bao and Xu BMC Genomics 2011, 12:224 http://www.biomedcentral.com/1471-2164/12/224 Page 3 of 14 Figure 1 Gene distribution from two SSH libraries. In the F library, diapause-type pupae were used as the “tester”, and nondiapause pupae as the “driver” to detect highly expressed genes in diapause-type pupae. By contrast, the “tester” and “driver” were reversed in the R library to detect highly expressed genes in nondiapause-type pupae. The Arabic numerals represent the number of unique sequence at each step of the annotation process. NoBlast, no blast results; NoAnnot, no annotation; Annot, annotation. Data analysis and visualization of results were performed by the Blast2GO software. selected genes in the brain were investigated during early pupal development by RT-PCR and Western blot analysis. The four genes encoded ubiquitin-like protein smt3 (SUMO, HarDP-A589), Mn superoxide dismutase (MnSOD, HarDP-B1408), sericotropin (HarDP-B1016) and translated controlled tumor protein (TCTP, HarDP- A694), which were assessed by Northern blot analysis above (Figure 3C). All four mRNAs were expressed higher during early pupal development in diapause-des- tined individuals, especially SUMO from day 1 to day 2, MnSOD from day 0 to day 2, sericotropin from day 0 to day 1, and TCTP from day 1 to day 5 (Figure 4A). The four protein levels reflected their mRNA levels (Figure 4B). Apparently, high expression of these genes at the early pupal stage is likely to be associated with pupal diapause initiation. Metabolism and energy Nine genes, including four high- and five low-expression genes, were assigned to the metabolism and energy cate- gory (Table 1A). Two enzymes, aldolase (HarDP-C349) and enolase (HarDP-A209), which were up-regulated during diapause initiation, participate in glycolysis. In contrast, an enzyme fructose-1,6-bisphosphatase (FBP) (HarNP-1172), which is important in gluconeogenesis, was down-regulated at diapause initiation. Aconitase (HarNP-475) and malate synthase (HarNP- 491), which are important components of the tricar- boxylic acid (TCA) cycle, are down-regulated at dia- pause initiation. Additionally, a set of transcripts (HarDP-B843, HarDP-B170 and HarDP-B1356) encod- ing proteins involved in ATP generation were up-regu- lated at diapause initiation. ATP synthase f0 subunit 6