Antimicrobial peptide expression in the cockroach gut during enterobacterial infection is specific and influenced by type III secretion

Abstract

Omnivorous synanthropic cockroaches, such as the German cockroach (Blattella germanica), are reservoirs and vectors of enteric bacterial pathogens. A lifestyle conducive to frequent encounters with high loads of diverse bacteria may have led to the evolution of unique innate immune systems in these insects. The innate immune response of insects relies largely on generalized mechanisms to sense and eliminate foreign microbes. However, analyses of the genomes of common synanthropic cockroaches previously revealed a repertoire of pathogen associated molecular pattern (PAMP) receptors and antimicrobial peptides (AMPs) that is significantly expanded relative to most holometabolous insect models and vectors, supporting the intriguing possibility that cockroaches may encode enhanced recognition within their immune system and may possess an enhanced capacity to fine tune innate immune responses. Investigating how cockroaches respond to infection with enterobacteria provides the opportunity to expand our fundamental knowledge of the regulation of insect innate immunity in a context that is biologically and medically relevant. German cockroaches can harbor both Salmonella enterica serovar Typhimurium and Escherichia coli in their gut without experiencing pathogenesis. The former colonizes the gut and replicates while the latter persists only transiently. We hypothesized that differences in the innate immune response may contribute to or result from the difference in infection dynamics between the two enterobacteria. To test this hypothesis, we used qRT-PCR to analyze expression of five genes encoding representative AMPs (Attacins, Blattellicin, Defensins) in the gut of German cockroaches 1 and 24 h after ingestion of live or heat-killed enterobacteria. We found that robust AMP expression was induced in response to ingestion of a live wild-type strain of S. Typhimurium, but not in response to live E. coli, heat-killed S. Typhimurium, or a live mutant strain of S. Typhimurium lacking type III secretion systems. These results indicate that the cockroach immune system does not respond to stimulation with high levels of ingested bacterial PAMPs such as peptidoglycan. Rather, AMP expression in the gut appears to be induced by active bacterial colonization involving type III secretion. We speculate that this form of regulation may have evolved to prevent over activation of the immune system from frequent ingestion of innocuous, non-colonizing, or non-viable bacteria. While additional work is needed to delineate the molecular mechanisms underlying our observations, our findings provide significant novel insight into the immunological adaptation of cockroaches to life in septic environments as well as the factors that regulate bacterial pathogen transmission by these insects.

Identification of the Gene Repertoire of the IMD Pathway and Expression of Antimicrobial Peptide Genes in Several Tissues and Hemolymph of the Cockroach Blattella germanica

“…One of the analyzed insects was B. germanica , in which it was detected that hemocytes were located in the dorsal tergum and almost absent in the ventral sternum [ 49 ]. However, it has recently been reported that the expression of attacin-like_g1 , attacin_like_g2 and blattellicin_g1 genes in B. germanica gut is significantly increased after feeding with live Salmonella enterica serovar Typhimurium [ 50 ]. In this work, the expression of these three genes as well as defensin_g1-g2 was also detected at very low levels in control individuals by qRT-PCR (approximate range 10 −4 to 10 −5 ) [ 50 ].…”

“…However, it has recently been reported that the expression of attacin-like_g1 , attacin_like_g2 and blattellicin_g1 genes in B. germanica gut is significantly increased after feeding with live Salmonella enterica serovar Typhimurium [ 50 ]. In this work, the expression of these three genes as well as defensin_g1-g2 was also detected at very low levels in control individuals by qRT-PCR (approximate range 10 −4 to 10 −5 ) [ 50 ]. The levels of detection by this method compared to our RNAseq analysis were similar (in both cases relative to the expression of EF1A ), except for attacin-like_g1 , which was not detected at all in our samples ( attacin_like_g2 in the three gut sections: range 2 × 10 −3 to 1 × 10 −4 ; blattellicin_g1 in hindgut: 3 × 10 −4 ; defensin_g1-g2 in midgut: 8 × 10 −5 ).…”

Effects of copper and zinc oxide nanoparticles on German cockroach development, indoxacarb resistance, and bacterial load

“…Amplification conditions were as follows: 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 50 °C for 20 s, and extension at 72 °C for 30 s. Cycle threshold (CT) values were obtained for individual insects as a proxy for the bacterial load and analyzed by ANOVA with Dunnett's post hoc test and adjustment for multiple comparisons. The qPCR assays were performed at the University of South Dakota using the laboratory adapted American Cyanamid Orlando Normal strain of B. germanica from a colony maintained as previously described 30 …”

“…Two independent replicates were conducted for each nanoparticle, and in each replicate development curves were compared to the control using a Mantel–Cox log‐rank test. The development assays were performed at the University of South Dakota insectary facility using the laboratory adapted American Cyanamid Orlando Normal strain of B. germanica from a colony maintained as previously described 30 …”

Horizontal transmission of Salmonella Typhimurium among German cockroaches and its possible mechanisms

“…Groups of donor cockroaches were marked with color tags and orally infected with S. Typhimurium as described in our previous studies (Turner et al, 2022 ; Turner & Pietri, 2022 ). In brief, cockroaches were separated into experimental enclosures and starved of food and water for three days, which is sufficient to promote consistent feeding among individuals without causing mortality.…”