Characterization of New Defensin Antimicrobial Peptides and Their Expression in Bed Bugs in Response to Bacterial Ingestion and Injection

Abstract

Common bed bugs, Cimex lectularius, can carry, but do not transmit, pathogens to the vertebrate hosts on which they feed. Some components of the innate immune system of bed bugs, such as antimicrobial peptides (AMPs), eliminate the pathogens. Here, we determined the molecular characteristics, structural properties, and phylogenetic relatedness of two new defensins (CL-defensin1 (XP_024085718.1), CL-defensin2 (XP_014240919.1)), and two new defensin isoforms (CL-defensin3a (XP_014240918.1), CL-defensin3b (XP_024083729.1)). The complete amino acid sequences of CL-defensin1, CL-defensin2, CL-defensin3a, and CL-defensin3b are strongly conserved, with only minor differences in their signal and pro-peptide regions. We used a combination of comparative transcriptomics and real-time quantitative PCR to evaluate the expression of these defensins in the midguts and the rest of the body of insects that had been injected with bacteria or had ingested blood containing the Gram-positive (Gr+) bacterium Bacillus subtilis and the Gram-negative (Gr–) bacterium Escherichia coli. We demonstrate, for the first time, sex-specific and immunization mode-specific upregulation of bed bug defensins in response to injection or ingestion of Gr+ or Gr– bacteria. Understanding the components, such as these defensins, of the bed bugs’ innate immune systems in response to pathogens may help unravel why bed bugs do not transmit pathogens to vertebrates.

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

“…In prior work, RT-qPCR revealed significant upregulation of CL-defensins and CL-prolixicins in response to ingestion of bacteria ( 69 ), supporting the results of this study. In summary, three defensin genes (CL- defensins 1, 2, and 3) were among the most up-regulated genes in pair-wise comparisons between midgut/controls and either midgut/Gr+ or midgut/Gr– samples (see 3.2; Figures 3 , 4 ; Supplementary Table S4 ).…”

“…Among these, defense- and immunity-associated genes were prominently up-regulated. Key genes include CL-defensin3 (LOC106661791), CL-defensin2 (LOC106661792), and CL-defensin1 (LOC106661793), which are modulated by the Imd and Toll pathways ( 68 , 69 ). The lysozyme-like gene (LOC106663700) aligns with the c-type lysozymes RpLys-A (EU250274) and RpLys-B (EU250275) from Rhodnius prolixus ( 70 ) that function in both digestion and immune defense by targeting bacterial cell walls ( 70 ).…”

“…Analyses of differential gene expression between midgut/control and midgut/Gr+ samples identified 3162 DE genes ( Figure 2C ; Supplementary Tables S2, S3 ), about as many DE genes as we found in analyses of differential gene expression between midgut/control and midgut/Gr– samples (see above). Among the prominently up-regulated genes were again defensins, Hsc70-3, and venom serine carboxypeptidase-like genes (LOC106668484), all of which are vital components of innate immunity ( 33 , 68 , 69 , 73 – 75 ). Concurrently, genes were accentuated that are integral to transport mechanisms, such as the sodium-dependent dopamine transporter gene (LOC106661545) and the acetyl-coenzyme A transporter 1 gene (LOC106662506).…”

Under the radar: differential responses of bed bugs to an entomopathogen, environmental bacteria, and a human pathogen

“…Annotations of the bed bug genome showed that, similar to other hemipteran insects, their immune repertoire is lacking in some canonical insect immune-related genes such as parts of the Imd pathway [14,23]. Along with this, bed bugs have a relatively low number of identifiable AMPs, with genes encoding three defensin AMPs and two diptericin-like putative AMPs [14,24]. Previous studies found that the three defensin AMPs are upregulated in response to various bacterial challenges, and another study found the two diptericin-like genes expressed in bed bug hemocytes [17,24].…”

“…Along with this, bed bugs have a relatively low number of identifiable AMPs, with genes encoding three defensin AMPs and two diptericin-like putative AMPs [14,24]. Previous studies found that the three defensin AMPs are upregulated in response to various bacterial challenges, and another study found the two diptericin-like genes expressed in bed bug hemocytes [17,24]. Along with this, several studies have shown that bed bug extracts have antimicrobial properties [25–27].…”