iTRAQ-Based Comparative Proteomic Analysis of Larval Midgut From the Beet Armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) Challenged With the Entomopathogenic Bacteria Serratia marcescens

Abstract

Entomopathogenic bacteria Serratia marcescens is widely used as an environmentally friendly biocontrol agent against various pests, including Spodoptera exigua. Understanding the immune defense mechanism of S. exigua through comparative proteomic analysis can identify the key proteins expressed in response to the microbial infection. Here, we employed the as isobaric tags for relative and absolute quantification (iTRAQ) technique to investigate the effects of S. marcescens on the proteomic expression of S. exigua. Based on the molecular functional analysis, the differentially expressed proteins (DEPs) were mainly involved in the binding process and catalytic activities. Further bioinformatics analysis revealed important DEPs that played a crucial role in innate immunity of S. exigua with recognition (C-type lectin), melanization (propanol oxidase 3, serine protease, Serine-type carboxypeptidase activity, clip domain serine protease 4), antimicrobial activity (lysozyme, lysozyme-like, gloverin, cecropin B), detoxification (acetyl-CoA C-acetyltransferase, 3-dehydroecdysone 3-alpha-reductase, glucuronosyltransferase, glutathione S-transferase) and others. The Quantitative real-time PCR (qRT-PCR) results further indicated the significant upregulation of the immune-related genes in Spodoptera exigua following S. marcescens infection. To the best of our knowledge, this is the first iTRAQ based study to characterize S. marcescens mediated proteomic changes in S. exigua and identified important immune-related DEPs. The results of this study will provide an essential resource for understanding the host-pathogen interactions and the development of novel microbial biopesticides against various pests.

Keywords: DEPs; Serratia marcescens; Spodoptera exigua; host immune defense; iTRAQ.

FIGURE 1

Bioassay of S. exigua. (A) Larval mortality of fifth instar larvae of S. exigua post-treatment with different concentrations (CFU/ml) of S. marcescens. Each point represents mean ± SE of three replications from different sets of experiments of identical environmental conditions. Same letters above bars indicate no significant difference (P ≤ 0.05) according to a Tukey test. Y axis showed the mortality rate (%), and X-axis represent the induction time (h). (B) Probit analysis showing the lethal concentrations (LC50) of S. marcescens against fifth instars larvae of S. exigua. Y axis showed the mortality rate (%) at 72 h, and X-axis represent the concentration (log) of S. marcescens.

FIGURE 2

Differentially expressed proteins (DEPs) in S. exigua responding to S. marcescens. (A) Statistics of up- and downregulated DEPs in S. exigua. (B) Distribution of fold changes and molecular masses for DEPs. Red circles indicate upregulated proteins, and green circles indicate downregulated proteins.

FIGURE 3

Gene ontology (GO) analysis of all proteins in midguts identified by iTRAQ analysis. Shown above is the classification of these proteins in different categories based on biological process, molecular function, cellular component.

FIGURE 4

KEGG functional classification of the identified proteins in the midgut of S. exigua identified by iTRAQ analysis.

FIGURE 5

Quantitative real-time PCR (qRT-PCR) analysis of 21 genes (A–U for JNK, NOX1, Lectin, IMD, Relish, TAB, IKK, Defensin, Gioverin, Lebocin, Lysozyme, PGRP-LC, Moricin, Hemolin, Cecropin, CL1044, Unigene 14488, Unigene 19139, Unigene 4854, Unigene50 and Unigene 29765, respectively) of the midgut of S. exigua, which was fed with an artificial diet containing S. marcescens (1.0 × 10⁸ CFU/mL) for 24, 48, and 72 hours. L10 was used as an internal control. Error bars represent mean ± SD from three independent experiments. *p < 0.05, **p < 0.01. Same letters above bars indicate no significant difference (P ≤ 0.05).