The cat flea, Ctenocephalides felis, is an arthropod vector capable of transmitting several human pathogens including Rickettsia species. Earlier studies identified Rickettsia felis in the salivary glands of the cat flea and transmission of rickettsiae during arthropod feeding. The saliva of hematophagous insects contains multiple biomolecules with anticlotting, vasodilatory and immunomodulatory activities. Notably, the exact role of salivary factors in the molecular interaction between flea-borne rickettsiae and their insect host is still largely unknown. To determine if R. felis modulates gene expression in the cat flea salivary glands, cat fleas were infected with R. felis and transcription patterns of selected salivary gland-derived factors, including antimicrobial peptides and flea-specific antigens, were assessed. Salivary glands were microdissected from infected and control cat fleas at different time points after exposure and total RNA was extracted and subjected to reverse-transcriptase quantitative PCR for gene expression analysis. During the experimental 10-day feeding period, a dynamic change in gene expression of immunity-related transcripts and salivary antigens between the two experimental groups was detected. The data indicated that defensin-2 (Cf-726), glycine-rich antimicrobial peptide (Cf-83), salivary antigens (Cf-169 and Cf-65) and deorphanized peptide (Cf-75) are flea-derived factors responsive to rickettsial infection.

Fleas are morphologically unique ectoparasites that are hardly mistaken for any other insect. Most flea species that feed on humans and their companion animals, including the cat flea (Ctenocephalides felis), have medical and veterinary importance. Besides facilitating blood acquisition, salivary biomolecules can modulate pathogen transmission. Thus, dissection of salivary glands is essential for comprehensive studies on disease vectors like the cat flea. Herein, we present the pictorial dissection protocol assisting future research targeting individual flea organs, for revealing their roles in vector competence and physiology. We provide a comprehensive guide, allowing researchers, even with limited practical experience, to successfully perform microdissection for collecting cat flea salivary glands. Furthermore, the protocol does not require expensive, sophisticated equipment and can be accomplished with routinely available tools. We illustrated expected results with morphological changes of salivary glands upon blood feeding as well as fluorescently stained these organs.

Carnivorous plants recruit defense-related pathways for prey digestion, yet the cis-regulatory architecture underlying their expression remains poorly characterized. In addition to herbivore and fungal pathogen responses, chitinases play an important role in plant carnivory. We analyzed promoters of glycoside hydrolase family 19 (GH19) class I chitinase genes in the Droseraceae family, which are the most abundant chitinases associated with prey digestion. Promoters of newly identified putative class I chitinases found in Drosera spatulata, Drosera tokaiensis, and Aldrovanda vesiculosa were also examined. Core promoter features, cis-regulatory elements, transcription factor binding site (TFBS) profiles, de novo motifs, and promoter sequence divergence were assessed. Within the sampled promoter set, no single cis-element or transcription factor family-compatible pattern was shared by all carnivorous promoters while being absent from Arabidopsis reference promoter. Instead, Droseraceae promoters showed candidate ATG-relative positional configurations of shared stress-, defense-, and hormone-responsive elements, including a distal MYC/E-box-compatible–proximal W-box pattern, while an ordered DRE–W-box–ERE configuration occurred in six sundew promoters. The TFBS profiling revealed promoter-specific differences in the representation of loci compatible with bHLH-family models, WRKY, MYB, MYB-related, NAC, and HSF models. Together, these analyses identify candidate promoter-specific patterns and provide a foundation for further broader comparative and functional validation.

Rickettsial infections of the central nervous system (CNS) are manifested by severe neurological symptoms and represent a serious life-threatening condition. Despite the considerable health danger, only a few studies have been conducted focusing on the pathogenesis induced by Rickettsia sp. in CNS. To investigate the signaling pathways associated with the neurotoxic effects of rickettsiae, we employed an experimental model of cerebrocortical neurons combined with molecular profiling and comprehensive bioinformatic analysis. The cytopathic effect induced by Rickettsia akari and Rickettsia slovaca was demonstrated by decreased neuronal viability, structural changes in cell morphology, and extensive fragmentation of neurites in vitro. Targeted profiling revealed the deregulation of genes involved in the neuroinflammatory and neurotoxic cell response pathways. Although quantitative analysis showed differences in gene expression response, functional annotation revealed that the biological processes are largely shared between both Rickettsia species. The identified enriched pathways are associated with cytokine signaling, chemotaxis of immune cells, responses to infectious agents, interactions between neurons, endothelial and glial cells, and regulation of neuronal apoptotic processes. The findings of our study provide new insight into the etiopathogenesis of CNS infection and further expand the understanding of molecular signaling associated with neuroinvasive Rickettsia species.