RNA interference-based characterization of Caspar, DREDD and FADD genes in immune signaling pathways of the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae)

Kakeru Yokoi,Ken Miura,Daiki Kato,Wataru Ito

doi:10.14411/eje.2022.003

Abstract

We previously demonstrated that two immune signaling pathways, Toll and IMD, were concomitantly activated in the model beetle Tribolium castaneum by challenges to their immune system by several species of microbes, including Gram-positive and -negative bacteria as well as yeast. This contrasts with the Drosophila immune system in which more specific pathway activation depending on the type of microbe is well established. We suggest that the activation of an indiscriminate immune pathway in T. castaneum is due in part to an unselective recognition of pathogen-associated molecular patterns by the extracellular sensing modules of the two pathways. In order to obtain a more detailed understanding of the T. castaneum immune pathway, we investigated whether potential components of the T. castaneum IMD pathway, Caspar, DREDD and FADD, are involved in immune reactions triggered by microbial challenges. A sequence analysis of these three genes with the orthologues of other species, including insects, mouse and human, indicated that T. castaneum Caspar, DREDD and FADD functioned as immune signal transducers, which are usually induced by microbial challenges. However, these genes were not induced by microbial challenges. To establish whether these genes are involved in immune reactions, we used RNA interference-mediated knockdown of these genes to assess the microbial induction levels of the representative read-out antimicrobial peptide genes of the respective classes. The results indicated that these genes encode the canonical constituents of the IMD pathway of this beetle. DREDD and FADD influenced the induction of Toll-dependent antimicrobial peptide genes, providing novel crosstalk points between the two immune pathways, which appears to support indiscriminate pathway activation in T. castaneum. Furthermore, the phenotypes of DREDD or FADD knockdown pupae challenged by the two model bacterial pathogens correlated with AMP gene induction in the respective knockdowns, indicating that these intracellular factors contributed to antibacterial host defenses.

Highlights

The insect immune system is solely composed of innate immune reactions that utilize germ line-encoded receptors to detect the invasion of several distinct pathogens (Hultmark, 2003; Ferrandon et al, 2007; Lemaitre & Hoffmann, 2007)
The sequences of T. castaneum orthologues for Caspar, DREDD and FADD (Tc-Caspar, Tc-DREDD, and Tc-FADD, respectively) were obtained from gene set data based on T. castaneum genome data (Zou et al, 2007; Tribolium Genome Sequencing Consortium, 2008), and their deduced amino acid sequences were subjected to phylogenetic analyses (Supplementary Fig. 1) together with the related sequences of other organisms [Aedes aegypti (Linnaeus), Anopheles gambiae (Giles), D. melanogaster, Bombyx mori (Linnaeus), Apis mellifera (Linnaeus), Nilaparvata lugens (Stål), mouse (Linnaeus) and human] (Supplementary Fig. 1)
We examined the genes coding for potential receptor-proximal components of the IMD pathway in the model beetle T. castaneum, namely, Caspar, DREDD and FADD, the products of which are constituents of the IMD pathway of Drosophila (Kleino & Silverman, 2014)

Summary

Introduction

The insect immune system is solely composed of innate immune reactions that utilize germ line-encoded receptors to detect the invasion of several distinct pathogens (Hultmark, 2003; Ferrandon et al, 2007; Lemaitre & Hoffmann, 2007). When recognized, invading pathogens are attacked by a battery of innate immune responses. Intensive studies on the model organism Drosophila melanogaster (Meigen) over the past two decades have delineated the paths and factors that uphold the AMP production response as one of the hallmarks of insect immunity. Extracellular pathogen recognition leads to the activation of two signaling pathways in Drosophila, the Toll and IMD pathways (Valanne et al, 2011; Kleino & Silverman, 2014). The Toll pathway is mainly activated by either Lys-type peptidoglycan associated with most Grampositive bacteria or fungal β-1,3 glucan, and the recognition of these two pathogen-associated molecular patterns (PAMPs) in circulation is mainly executed by peptidoglycan recognition protein SA (PGRP-SA) and Gram-negative binding protein 3, respectively (Gobert et al, 2003; Gottar et al, 2006). PAMP binding to these receptors subsequently activates a downstream serine protease cascade, which leads to the proteolytic activation of the Toll ligand Spät-

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