Bao and Xu BMC Genomics 2011, 12:224 http://www.biomedcentral.com/1471-2164/12/224 3A), so these genes from the R library will help us to identify the genes associated with insect diapause if these differentially expressed genes in diapause-destined pupae are further characterized. A total of 150 sequences from the two libraries that were homologous to known genes were obtained. According to gene ontology analysis, most genes belonged to cellular pro- cess and metabolic process in the category of “biological process"; this implies that the insect brain at diapause initiation focuses on alteration of cellular and metabolic state. Signaling and transcriptional regulator activity also showed significant differences between the two libraries. Up-regulation of signaling genes and down-regulation of transcriptional regulators at diapause initiation indicate that signaling pathways are changed, global transcription levels are down-regulated, and diapause does require a unique gene expression regulatory mechanism. The quality and reliability of the two SSH libraries were validated by investigating gene expression differ- ence between diapause- and nondiapause-destined pupae. The two libraries were quite reliable, so the SSH method was useful to search for genes related to pupal diapause initiation. Subsequently, the expression pat- terns of four genes were detected by RT-PCR and Wes- tern blot analysis. All four genes were expressed higher at both the mRNA and protein levels during early pupal development in diapause-destined individuals than their nondiapause-destined counterparts. Apparently, these genes from the SSH library may reflect differential expression between diapause- and nondiapause-destined pupae for promoting diapause initiation. Based on the functions of the putatively up- and down-regulated genes (Table 1), we have proposed a possible mechanism for diapause initiation. Changes in metabolism and energy The brain of early diapause-destined pupae releases instructions to switch from development to diapause, so changes in metabolism and energy must be involved in the process. At diapause initiation, insects need to store energy and synthesize some specific compounds for cold hardiness, such as cryoprotectants. From the two SSH libraries, some transcripts function in metabolism and energy. Aldolase catalyzes the fourth step in glycolysis, which cleaves fructose 1, 6-bisphosphate and generates dihydroxyacetone phosphate and glyceraldehyde 3-phos- phate. Dihydroxyacetone phosphate is synthesized into glycerol, and glyceraldehyde 3-phosphate enters the gly- colytic pathway to generate energy. Enolase is an enzyme that catalyzes the ninth step of the glycolytic pathway, resulting in the formation of phosphoenolpyru- vate (PEP) and pyruvate. FBP catalyzes the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate, a key step between glycolysis and gluconeogenesis. To the Page 9 of 14 best of our knowledge, gluconeogenesis and glycolysis are coordinated so that one way is relatively inactive while the other is highly active. As shown in Figure 5A, the down-regulation of FBP and up-regulation of aldo- lase and enolase suggest that gluconeogenesis dimin- ished at diapause initiation, and glycerol biosynthesis is accelerated by glycolysis. Glycerol protects insects from cold stress. Meanwhile, the possible up-regulation of aldolase and enolase are responsible for generating pyru- vate, which is also elevated in S. crassipalpis during pupal diapause [9,26], and pyruvate enters the glycolytic pathway to generate energy. Aconitase and malate synthase, which participate in the TCA cycle, are down- regulated. This result implies that the down-regulated aconitase and malate synthase may directly repress the TCA cycle. In diapause pupae of the flesh fly, S. crassi- palpis, the TCA cycle is suppressed, and the metabolic intermediates from the TCA cycle are also reduced [9]. Therefore, inhibition of the TCA cycle and enhance- ment of glycolysis indicate that anaerobic metabolism is predominant at diapause initiation. In fact, respiration in diapause individuals is significantly lower than in nondiapause individuals [25], which is consistent with the decreased metabolic rate in diapause-destined indivi- duals, and inhibition of the TCA cycle in the brain helps diapause individuals save energy. Enhancement of anae- robic metabolism has also been reported in recent stu- dies of larval diapauses in the pitcher plant mosquito, Wyeomyia smitbii [27], and embryonic diapauses in the cricket, Allonemobius socius [28]. Additionally, three transcripts (HarDP-B843, HarDP- B170 and HarDP-B1356) for ATP generation were up- regulated at diapause initiation. ATP synthase f0 subunit 6 (HarDP-B843) plays a role in the production of ATP from ADP. Cytochrome c oxidase is a component of the respiratory chain in mitochondria. Cytochrome c oxidase subunit 2 (HarDP-B170) transfers electrons from cyto- chrome c to the bimetallic center of the catalytic subunit 1. Cytochrome c oxidase subunit 7C (COX7C, HarDP- B1356) is one of the nuclear-coded polypeptide chains of cytochrome c oxidase, the terminal oxidase in mito- chondrial electron transport. Such a change of cyto- chrome c oxidase subunits during diapause has been reported in C. pipiens [29]. These observations suggest that energy demand still high during pupal diapause initiation. As shown in Table 1A, the transcripts associated with lipid metabolism (apolipoprotein D and lipase) are down-regulated in diapause-destined pupal brain. Down-regulation of lipase has also been reported in early stage of diapause C. pipiens, but it is up-regulated in late diapause, suggesting that fatty acid-oxidation is suppressed in early diapause [30]. The down-regulation of apolipoprotein D and lipase implies that diapause