We have previously shown that the larvae of swallowtail butterfly, Papilio xuthus, exhibit substantial antibacterial activity in the hemolymph, upon challenging with bacterial lipopolysaccharide (LPS). Here we report the isolation and molecular characterization of several immune inducible genes that are specifically expressed by employing annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) from P. xuthus the larva. Using 120 arbitrary ACPs, we identified 24 differentially expressed genes (DEGs) that are up-regulated in response to injected LPS. Sequence analysis showed that 18 DEGs revelaed a high sequence similarity to the previously characterized genes of other insects, although 6 DEGs showed no significant similarity to any known genes. Among these inducible transcripts we found 8 putative immune-related genes including cecropin and attacin. Finally, we analysed the expression profiles of potential immune-related genes by RT-PCR and found all of them were considerably increased in the mRNA levels by LPS injection.

Protease from various sources have been studied biotecnologically. For biotechnological applications, one highly preferred enzyme is protease. There have been no reports of cloned genes encoding digestive proteases in the Laccotrephes japonenis, Ranatra unicolor, Muljarus japonicus. These insects are considered to be a predator of aquatic insects. RT-PCR was used to amplify cDNA fragments for digestive proteases from total RNA the hole body of the insects. The flanking sequences of the 5'- and 3'- end of the these genes were characterized by RACE-PCR. Sequence analysis showed that these genes contained complete ORF. The deduced amino acid sequences of these protease showed 62% identity to the serine protease of Creontiades dilutus, 58% to Lygus loneolaris trypsin-like serine proteinase , 54% to Triatonatoma infestans salivary trypsin. In the further study, to generate digestive protease, the DNA fragment coding for serine protease, trypsin-like serine protease were cloning into suttle vector pBACⅠ, and infected to Spodoptera frugiperda (sf9) insect cell. After that, we expect to carry out the proteolytic activity of these recombinant proteases. This is intended as a basis for future studies on the digestive protease in the insects.