Identification of immunity-related genes in the larvae of Protaetia brevitarsis seulensis (Coleoptera: Cetoniidae) by a next-generation sequencing-based transcriptome analysis.

Larvae of all kinds of beetles live on the ground, where they frequently encounter pathogenic microorganisms such as bacteria, fungi, viruses, and parasitoids. Therefore, it is conceivable that beetles have well-developed immune systems. Despite the diversity of beetle species, the immune systems of only a few beetles have been studied, including the weevil Sitophilus zeamais, the mealworm beetle Tenebrio molitor, and the burying beetle Nicrophorus vespilloides (Zou et al. 2007, Anselme et al. 2008, Vogel et al. 2011, Dobson et al. 2012, Vogel et al. 2014). Generally, the insect immune system consists of two arms, the cellular and the humoral immune response, and is a well-organized defensive system against various pathogens despite the lack of an acquired immune response, as seen in mammals (Hoffmann 2003). Many immune-related genes are upregulated in response to microbial challenge and antimicrobial peptides (AMPs) are ultimately synthesized through Toll and Immune deficiency (Imd) pathways in insects (Lemaitre and Hoffmann 2007, Bang et al. 2012, Kwon et al. 2014b). Approximately 20, 10, and 30 genes were identified as AMP genes in Drosophila melanogaster, Anopheles gambiae, and Bombyx mori, respectively (De Gregorio 2001, Tanaka et al. 2008, Lee et al. 2013). AMPs have been characterized and isolated from many insect species. In particular, coleoptericin-like proteins (coleoptericin, acaloleptin, holotricin, and rhinocerosin), which are found in coleopterans, were characterized in Holotricia diomphalia, Zophobas atratus, Allomyrina dichotoma, Acalolepta luxuriosa, and Stitophilus oryzae (Bulet et al. 1991, Lee et al. 1994, Sagisaka et al. 2001, Imamura et al. 2009, Login et al. 2011). By contrast, cellular immune responses are mediated by insect blood cells (hemocytes), which are activated via pattern recognition receptor (PRR)-linked signal transduction pathways and conduct cellular processes that result in pathogen killing, such as phagocytosis, encapsulation, and nodulation. Transmembrane or extracellular PRR pathways (Toll receptors, C-type lectins [CTLs], and peptidoglycan recognition proteins [PGRPs]) are activated by Spatzle in the presence of microbe-associated molecular recognition patterns (MAMPs; microbial peptidoglycan, lipopolysaccharides, β-glucans, lipoproteins, CpG dinucleotides, or flagellin) expressed by pathogens (Shelby and Popham 2012). The confusion still exists regarding hemocyte categorization because hemocyte types vary greatly depending on the insect species (Gupta 1985, Kwon et al. 2014a). Although there has been some debate on the characterization of insect blood cell types, plasmatocytes and granulocytes are considered the key players in cell-mediated immunity (Kwon et al. 2014a). As discussed earlier, it is reasonable to assume that many genes are associated with the cellular and humoral immune response in insects. Recently, genome-wide analysis was applied for the identification of immune-related genes and study of the molecular basis of host-microorganism interactions. In particular, whole genome mRNA sequencing (RNA-seq or transcriptome sequencing) provides comprehensive insight into the immune gene repertoire of nonmodel insects, and this technology has become a powerful tool for the analysis of differential gene expression (Liu et al. 2014, Vogel et al. 2014). This study focused on high-throughput RNA sequencing (RNA-seq) of the immune response in white-spotted flower chafers, Protaetia brevitarsis seulensis (Kolbe). This insect was very easy to maintain under laboratory conditions, and the average duration of the larval stage (the period the insect spends on the ground) is over 60 days under constant conditions. In addition, the cellular immune system in this insect is very well-developed, as we reported (Kwon et al. 2014a). Many of the components of the immune system, including the Toll pathway, the Imd pathway, and AMPs, have not been identified or studied in this insect. Therefore, we examined the differentially expressed genes (DEGs) between untreated and immunized larvae by Illumina RNA-seq digital expression profiling (de novo transcriptome assembly [TA]). More than 70,000 putative transcripts were assembled, and >30,000 were annotated to known databases. These results were validated by comparison with our previous results on humoral and cellular immune response-related genes, which revealed a comprehensive overview of gene expression changes, including additional identification of immune-related genes.