Bao and Xu BMC Genomics 2011, 12:224 http://www.biomedcentral.com/1471-2164/12/224 individual insects can switch from direct development to arrested development. The cotton bollworm H. armigera (Har), an agricultu- rally important pest, enters pupal diapause for survival in winter. After pupation, the diapause-destined pupae will enter diapause within 7–8 days, because day 9 of pupae can not develop towards adults, even though these pupae are incubated in a suit conditions. The phy- siological characteristics of diapause are observed on day 3 pupae, such as low ecdysone titer and unmoved eyespots, so differentially expressed genes as diapause instructions may be issued at an earlier pupal stage. Thus, we focused on gene expression in day 1 and 2 of pupae. As the programmable center of diapause, the brain is the most important organ to release instructions for diapause initiation [6]. To understand the molecular mechanism of diapause initiation, we searched for differ- entially expressed genes during pupal diapause initiation in H. armigera by using SSH. Meanwhile, the differen- tially expressed genes in nondiapause individuals were also investigated to search those genes expressed at low level in diapause-destined individuals. Results General statistics from two SSH libraries Two subtracted cDNA libraries enriched in diapause- or development-correlative genes were constructed by using SSH. One was the F (forward) library expected to be enriched in diapause up-regulated cDNAs. The F library was obtained using the mRNAs from the dia- pause-destined pupal brains of days 1–2 after pupation as the “tester”, and mRNAs from the nondiapause pupal brains of days 1–2 as the “driver”. The other is the R (reverse) library to enrich cDNAs up-regulated in non- diapause-destined (or down-regulated in diapause-des- tined) pupal brain by reversing the “tester” and “driver” mRNAs. A total of over 1000 clones were randomly picked and subjected to colony PCR, and sequencing was carried out for 220 and 130 cDNA clones selected at random from the F and R libraries, respectively. After removing poor quality sequences, we finally obtained 194 unique sequences in the F library and 115 unique sequences in the R library. All the sequences (309) were compared against available databases to find similarities with known sequences. We carried out dynamic translation (blastx), and only matched sequences with an E-value lower than 10-03 were considered to be homologous sequences. Sequences with an E-value higher than 10-03 were labeled undescribed. Homologous sequences accounted for 38.7% of the sequences in the F library and 65.2% in the R library. The homologous sequences are shown in Additional File 1. Among the homologous sequences, 12 in the F library (12/75, 16.0%) and 18 in Page 2 of 14 the R library (18/75, 24.0%) lacked annotation. Addition- ally, the number of no mapping sequences was four in the F library and three in the R library, respectively. All of these data are summarized in Figure 1. Gene ontology analysis Most genes isolated from the two libraries have not been identified, so that we here define these genes as putatively up-regulated genes from the F library and putatively down-regulated genes from the R library as shown in Table 1. Gene ontology analysis was carried out using the blas- t2go program [12]. Sequences were classified into the three ontology categories: biological process (Figure 2 and Additional File 2), molecular function and cellular component (Additional File 3). In the category “biologi- cal process”, the most frequent process was cellular pro- cess (27% in the F library and 25% in the R library), followed by metabolic process (26% in the F library and 22% in the R library) and biological regulation (9% in the F library and 12% in the R library). In the category “molecular function”, binding was the most frequent activity (36% in the F library and 47% in the R library), followed by catalytic activity (31% in the F library and 34% in the R library). Significant differences were observed in structural molecular activity (20% in the F library and 6% in the R library) and transcription regula- tor activity categories (1% in the F library and 6% in the R library). In the last category, “cellular component” the most frequent activity was macromolecular complex (25% in the F library and 18% in the R library), and membrane-enclosed lumen (3% in the F library and 8% in the R library) appeared to be different between the two SSH libraries. Identification of differentially expressed transcripts To test the reliability of the two SSH libraries, we ran- domly selected 12 genes from each library to investigate their expression levels in diapause- and nondiapause- destined brains at the early pupal stage by RT-PCR. In the F library, 11 transcripts were expressed highly in diapause-destined individuals, with HarDP-C924 being the exception (Figure 3A). In the R library, 10 tran- scripts were expressed highly in nondiapause pupae, with HarNP-423 and HarNP-503 being the exceptions (Figure 3B). Furthermore, the levels of four transcripts from the F library were confirmed by Northern blot analysis. As shown in Figure 3C, their expression was higher in diapause-destined pupae. These results show that the two SSH libraries are reliable. Expression patterns at diapause initiation To obtain some clues about the functions of the genes from the SSH library, the expression patterns of four