Comprehensive Analysis of the Manduca Sexta Immunotranscriptome

Abstract

As a biochemical model, Manduca sexta substantially contributed to our knowledge on insect innate immunity. The RNA-Seq approach based on massively parallel pyrosequencing was implemented in three studies to examine tissue immunotranscriptomes of this species. With the latest and largest focusing on highly regulated process- and tissue-specific genes, we further analyzed the same set of data using BLAST2GO to explore functional aspects of the larval fat body (F) and hemocyte (H) transcriptomes with (I) or without (C) immune challenge. Using immunity-related sequences from Drosophila melanogaster, Apis mellifera, and Bombyx mori, we identified 383 homologous contigs and compared them with those discovered based on relative abundance changes and BLASTX analyses. The major overlap of the two lists validated our previous research designed for gene discovery and transcript profiling in organisms lacking sequenced genomes. By concatenating the contigs, we established a repertoire of 232 immunity-related genes encoding proteins for pathogen recognition (16%), signal transduction (53%), and microbe killing (13%). We examined their expression levels along with attribute classifications and detected prominent differences in nine of the thirty level 2 GO categories, such as enzyme regulator (IC), cellular component organization (FH), signaling (FH), and extracellular region (IC). The increase in extracellular proteins (155% or 31,038 normalized read number) was consistent with the highly induced synthesis of defense molecules (e.g., antimicrobial peptides) in fat body after the immune challenge. We identified most members of the putative Toll, IMD, MAPK-JNK-p38, and JAK-STAT pathways and detected 1.1~1.8-fold increases in the first three and 30% average decrease in the fourth. The minor increases in the antibacterial and antifungal pathways led to dramatic elevations of transcripts for all antimicrobial peptides as well as some proteins involved in recognition, extracellular signaling, and cellular responses. Most importantly, this study sets the stage for on-going analysis of M. sexta immunogenome.