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

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.

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

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.

Origin, structure and functional transition of sex pheromone components in a false widow spider.

Female web-building spiders disseminate pheromone from their webs that attracts mate-seeking males and deposit contact pheromone on their webs that induces courtship by males upon arrival. The source of contact and mate attractant pheromone components, and the potential ability of females to adjust their web's attractiveness, have remained elusive. Here, we report three new contact pheromone components produced by female false black widow spiders, Steatoda grossa: N-4-methylvaleroyl-O-butyroyl-L-serine, N-4-methylvaleroyl-O-isobutyroyl-L-serine and N-4-methylvaleroyl-O-hexanoyl-L-serine. The compounds originate from the posterior aggregate silk gland, induce courtship by males, and web pH-dependently hydrolyse at the carboxylic-ester bond, giving rise to three corresponding carboxylic acids that attract males. A carboxyl ester hydrolase (CEH) is present on webs and likely mediates the functional transition of contact sex pheromone components to the carboxylic acid mate attractant pheromone components. As CEH activity is pH-dependent, and female spiders can manipulate their silk's pH, they might also actively adjust their webs' attractiveness.

Time- and tissue-specific antimicrobial activity of the common bed bug in response to blood feeding and immune activation by bacterial injection.

Unlike almost all hematophagous insects, common bed bugs, Cimex lectularius, are not known to transmit pathogens to humans. To help unravel the reasons for their lack of vector competence, we studied the time- and tissue-dependent expression of innate immune factors after blood feeding or immune activation through the intrathoracic injection of bacteria. We used minimum inhibitory concentration (MIC1) bioassays and the Kirby-Bauer protocol to evaluate antimicrobial peptide (AMP2) activity in tissue extracts from the midguts or 'rest of body' (RoB3) tissues (containing hemolymph and fat body AMPs) against Gram-positive and Gram-negative bacteria. We compared AMP activity between blood-fed female bed bugs and yellow fever mosquitoes, Aedes aegypti and determined how female and male bed bugs respond to immune challenges, and how long AMP gene expression remains elevated in bed bugs following a blood meal. Blood meal-induced AMP activity is 4-fold stronger in female bed bugs than in female mosquitoes. Male bed bugs have elevated AMP activity within 8 h of a blood meal or an intrathoracic injection with bacteria, with the strongest activity expressed in RoB tissue 24 h after the immune challenge. Female bed bugs have a stronger immune response than males within 24 h of a blood meal. The effects of blood meal-induced elevated AMP activity lasts longer against the Gram-positive bacterium, Bacillus subtilis, than against the Gram-negative bacterium Escherichia coli. Unravelling the specific immune pathways that are activated in the bed bugs' immune responses and identifying the bed bug-unique AMPs might help determine why these insects are not vectors of human parasites.

Cheese and cheese infusions: ecological traps for mosquitoes and spotted wing Drosophila.

BACKGROUND: Harnessing insect ecology for insect control is an innovative concept that seeks to exploit, among others, insect-microbe ecological interactions for improved control of pest insects. Microbe-produced cheese odour attracts several dipterans, including host-seeking mosquitoes, but this phenomenon has not been thoroughly explored for mosquito control. Here we tested the hypothesis that attraction of mosquitoes to cheese odour can be exploited as an ecological trap for mosquito control. RESULTS: In laboratory and/or field experiments, we show that (i) each of five cheese varieties tested (Raclette, Pecorino, Brie, Gruyere, Limburger) strongly attracts female Aedes aegypti and Culex pipiens; (ii) cheese infusions, or headspace odourant extracts (HOEs) of cheese infusions, significantly affect oviposition choices by mosquitoes, (iii) HOEs contain at least 13 odourants; (iv) in field settings, cheese infusions more effectively stimulate mosquito oviposition than positive bluegrass infusion controls, and also capture (by drowning) the spotted wing Drosophila, Drosophila suzukii; and (v) home-made cheese infusions modulate oviposition choices by mosquito females and affect the survivorship of their offspring larvae. CONCLUSION: Our data show that microbial metabolites associated with cheese are attractive to mosquito females seeking hosts and oviposition sites and are likely toxic to mosquito larvae. These microbes and their metabolites could thus be co-opted for both the attract, and the kill, function of 'attract & kill' mosquito control tactics. Implementation of customizable and non-conventional nutritional media as microbe-based ecological traps presents a promising concept which exploits insect ecology for insect control. © 2021 Society of Chemical Industry.