Pyrosequencing-based expression profiling and identification of differentially regulated genes from Manduca sexta, a lepidopteran model insect with unknown genome sequence

Although Manduca sexta has significantly contributed to our knowledge on a variety of insect physiological processes, the lack of its genome sequence hampers the large-scale gene discovery, transcript profiling, and proteomics analysis in this biochemical model species. Here we report a new experimental approach based on massively parallel pyrosequencing, which allows us to categorize transcripts based on their relative abundances and to discover process- or tissue-specifically regulated genes simultaneously. We obtained 1,821,652 reads at an average length of 289 bp per read from fat body (F) and hemocytes (H) of naïve (C) and microbes-injected (I) M. sexta larvae. Most (92.1%) of these reads were assembled into 19,020 contigs and, for each contig, numbers of the assembled reads from groups CF, IF, CH, and IH were separately normalized for ratio calculations. Using RAIF/CF and RAIH/CH, we identified 528 contigs whose relative abundances (RAs) increased at least 5 and 8 folds in fat body and hemocytes, respectively, after the immune challenge. Polypeptides encoded by these contigs include immunity-related proteins such as antimicrobial peptides and molecules with no known sequence homology but participating in defense in new ways. In addition, we discovered contigs that reduced to less than 1/10 of the control levels in either tissue. Using RACF/CH and RAIF/IH, we found 250 and 161 contigs that were preferentially expressed in fat body (ratio 100) and hemocytes (ratio 0.025), respectively. These are candidates for exploring tissue-specific gene regulation before and after the injection of microbes. Furthermore, we integrated data from our previous study and generated a sequence database to support future gene annotation and proteomics analysis in M. sexta. In summary, we have successfully established a combined approach for gene discovery and expression profiling in organisms lacking known genome sequences.