Activation of immune pathways in common bed bugs, Cimex lectularius, in response to bacterial immune challenges - a transcriptomics analysis

Abstract

The common bed bug, Cimex lectularius, is an urban pest of global health significance, severely affecting the physical and mental health of humans. In contrast to most other blood-feeding arthropods, bed bugs are not major vectors of pathogens, but the underlying mechanisms for this phenomenon are largely unexplored. Here, we present the first transcriptomics study of bed bugs in response to immune challenges. To study transcriptional variations in bed bugs following ingestion of bacteria, we extracted and processed mRNA from body tissues of adult male bed bugs after ingestion of sterile blood or blood containing the Gram-positive (Gr+) bacterium Bacillus subtilis or the Gram-negative (Gr-) bacterium Escherichia coli. We analyzed mRNA from the bed bugs' midgut (the primary tissue involved in blood ingestion) and from the rest of their bodies (RoB; body minus head and midgut tissues). We show that the midgut exhibits a stronger immune response to ingestion of bacteria than the RoB, as indicated by the expression of genes encoding antimicrobial peptides (AMPs). Both the Toll and Imd signaling pathways, associated with immune responses, were highly activated by the ingestion of bacteria. Bacterial infection in bed bugs further provides evidence for metabolic reconfiguration and resource allocation in the bed bugs' midgut and RoB to promote production of AMPs. Our data suggest that infection with particular pathogens in bed bugs may be associated with altered metabolic pathways within the midgut and RoB that favors immune responses. We further show that multiple established cellular immune responses are preserved and are activated by the presence of specific pathogens. Our study provides a greater understanding of nuances in the immune responses of bed bugs towards pathogens that ultimately might contribute to novel bed bug control tactics.

Keywords: antimicrobial peptides; bed bugs; immune transcriptomics; immunization; innate immunity; toll & Imd pathways; vectors & pathogens.

Figure 1

Venn diagrams and principal component analysis of treatment-dependent differentially expressed (DE) transcripts in two tissue types of bed bugs. (A–C) DE transcripts in midgut and rest of body (RoB: body minus head and midgut tissue) samples obtained from adult male bed bugs after ingestion of sterile blood (control treatment) (A) or blood laced with the Gram-negative (Gr–) bacterium Escherichia coli K12/D31 or the Gram-positive (Gr+) bacterium Bacillus subtilis subsp. spizizenii ATCC 6633 (B, C). Absolute numbers (and percentages of total transcripts) inside circles, and circle overlaps, represent the numbers (and percentages) of transcripts expressed exclusively in that circle, or circle overlap. (D) Principal component analysis (PCA) plots demonstrating treatment effects in midgut and RoB tissues. Expression levels were used to inform the PCA.

Figure 2

Volcano plots of treatment-dependent differentially expressed (DE) genes in bed bugs. Plots illustrate the global transcriptional change in pair-wise comparisons of midgut and rest of body (RoB: body minus head and midgut tissues) samples obtained from adult male bed bugs obtained after ingestion of sterile blood (control treatment) or blood laced with the Gram-negative (Gr–) bacterium Escherichia coli K12/D31 or the Gram-positive (Gr+) bacterium Bacillus subtilis subsp. spizizenii ATCC 6633. All genes are plotted and each data point represents one gene. The log2 Fold Change (log₂FC) of each gene is presented on the x-axis, and the log10 of its adjusted p-value is presented on the y-axis. Red points above the dotted x-axis represent genes (some of which annotated) that are significantly up- or down-regulated with an adjusted p value (padj) < 0.05 and a Log₂ Fold Change (log₂FC) > |1|. Blue dots represent genes with padj value < 0.05 and log₂FC < |1|. Green dots represent non-significant genes with log₂FC > |1|, and grey dots represent non-significant genes with log₂FC < |1|.

Figure 3

Treatment-dependent heatmap of most variable differentially expressed (DE) immune-related genes in bed bugs. Map data are based on six pair-wise comparisons of midgut and rest of body (RoB: body minus head and midgut tissue) samples obtained from adult male bed bugs after ingestion of sterile blood (control treatment) or blood laced with the Gram-negative (Gr–) bacterium Escherichia coli K12/D31 or the Gram-positive (Gr+) bacterium Bacillus subtilis subsp. spizizenii ATCC 6633. Genes are colored according to their Log₂ Fold Change (see legend) and clustered using Euclidean distances.

Figure 4

Treatment-dependent differential expression of Toll and Imd pathway components in bed bugs. Assignments of immunized components are based on data in pair-wise comparisons of midgut and rest of body (RoB: body minus head and midgut tissue) samples obtained from adult male bed bugs in response to ingestion of sterile blood (control treatment) or blood laced with the Gram-negative (Gr–) bacterium Escherichia coli K12/D31 and the Gram-positive (Gr+) bacterium Bacillus subtilis subsp. spizizenii ATCC 6633. Arrows pointing upward and downward indicate genes that are significantly up-regulated (log₂ Fold Change >1) and down-regulated (log₂ Fold Change < -1), respectively. Components in the midgut immunized by Gr– bacteria and Gr+ bacteria are marked by red and blue arrows, respectively.

Figure 5

Gene ontology (GO) enriched terms in midgut and RoB (Rest of Body: body minus head and midgut tissues) samples obtained from male adult bed bugs after ingestion of sterile blood (control treatment) or blood laced with the Gram-negative (Gr–) bacterium Escherichia coli K12/D31 or the Gram-positive (Gr+) bacterium Bacillus subtilis subsp. spizizenii ATCC 6633. (A, C, E) Dot plots showing GO terms from biological processes, molecular functions and cellular components ranked according to gene count. Gene ratio (% of total DE genes in a given GO term) and adjusted p-value are also plotted. (B, D, F) Ridgeplots showing the density of Log₂ Fold Changes (Log₂FCs) for core genes in each term.

Figure 6

KEGG (Kyoto Encyclopedia of Genes & Genomes) enriched terms in midgut and RoB (Rest of Body: body minus head and midgut tissues) samples obtained from male adult bed bugs after ingestion of sterile blood (control treatment) or blood laced with the Gram-negative (Gr–) bacterium Escherichia coli K12/D31 or the Gram-positive (Gr+) bacterium Bacillus subtilis subsp. spizizenii ATCC 6633. (A, C, E, G) Dot plots showing KEGG terms from biological processes, molecular functions and cellular components ranked according to gene count. Gene ratio (% of total DE genes in a given KEGG term) and adjusted p-value are also plotted. (B, D, F, H) Ridgeplots showing the density of Log₂ Fold Changes (Log₂FCs) for core genes in a term.