Comparative Analysis of Adelphocoris suturalis Jakovlev (Hemiptera: Miridae) Immune Responses to Fungal and Bacterial Pathogens

Abstract

The wide-spread culture of transgenic Bt cotton resisting the infamous cotton bollworms has reduced the adoption of broad-spectrum insecticides to a large extent. Consequently, the non-targeted insect Adelphocoris suturalis Jakovlev has become a major cotton pest in China. Entomopathogenic microbes show promising results for controlling this pest in the future, but A. suturalis innate immune responses to these pathogens are poorly understood. Here, we used the entomopathogenic fungus Beauveria bassiana and the Gram-negative pathogenic bacteria Enterobactor cloacae to infect A. suturalis nymphs, followed by high throughput RNA-seq to analyze the immune transcriptomes of A. suturalis in response to the two pathogens. A total of 150 immunity-related genes were identified, including pattern recognition receptors, extracellular signal modulators, signal pathways (Toll, IMD, JNK, and JAK/STAT), and response effectors. Further quantitative real-time PCR analysis demonstrated that B. bassiana and E. cloacae were recognized by different receptors (GNBP and PGRP, respectively); activated Toll pathway and IMD pathway respectively; and both induced expression of the effector gene Defensin. However, melanization is suppressed in B. bassiana-infected nymphs. Collectively, this study provides a transcriptomic snapshot of the A. suturalis immune system, and at the genetic level, gains multifaceted insights of the immune response to fungal and Gram-negative bacterial pathogens. Ultimately this work pioneers the study of molecular mechanisms underlying immune interactions between A. suturalis and its pathogens and assists in the development of novel mitigation strategies to control this pest.

Keywords: Adelphocoris suturalis Jakovlev; Beauveria bassiana; Enterobactor cloacae; RNA-seq; immune system.

FIGURE 1

The survival curves and transcriptomic changes of A. suturalis nymphs injected with B. bassiana, E. cloacae or mock (Tween-80). (A) The survival curve of B. bassiana-infected nymphs; (B) The survival curve of E. cloacae-infected nymphs. The log-rank test was used to evaluate the significance of differences between groups (***p < 0.001).

FIGURE 2

Transcriptome analysis of A. suturalis nymphs in response to B. bassiana and E. cloacae infection. (A) Hierarchical clustering analysis of DEGs in the A. suturalis nymphs infected with B. bassiana, E. cloacae, and Tween 80 (p < 0.001, fold change > 2). The heatmap is divided into five clusters that are marked by different color boxes on the left; (B) Venn diagrams of DEGs in B. bassiana- or E. cloacae- challenged A. suturalis nymphs. The overlapping regions show genes that are regulated in both samples. The non-overlapping regions represent genes that are specifically regulated in corresponding sample. The directions of transcript level changes are indicated by upward- and downward- pointing arrows.

FIGURE 3

Go and KEGG analysis of DEGs. (A) Go (Gene ontology) enrichment and (B) KEGG pathway enrichment analysis of DEGs in the A. suturalis transcriptomes. Level 2 GO assignments are made in terms of cellular components, molecular functions, and biological processes.

FIGURE 4

A. suturalis immunity-related genes analysis. (A) Distribution of A. suturalis immunity-related genes in categories of recognition, signaling, regulation, and effectors; (B) Venn diagrams of immunity-related DEGs show that fungal and bacterial infections can regulate different immune-related genes. Overlapping regions show genes that regulated in both samples, while non-overlapping regions represent genes that are specifically regulated in corresponding sample. The directions of transcript level changes are indicated by upward- and downward- pointing arrows; and (C) Quantitative real-time PCR analysis of the A. suturalis immunity-related gene expression, the A. suturalis ribosomal protein 15S gene (RPS15) was used as an internal control. Data are normalized to the expression level in Tween 80-treated nymphs (*p < 0.05).