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Background It is well known that reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in the control of pathogens and microbiota in insects. However, the knowledge of the role of ROS and RNS in tick-pathogen and tick-microbiota interactions is limited. Here, we evaluated the immune-related redox metabolism of the embryonic cell line BME26 from the cattle tick Rhipicephalus microplus in response to Anaplasma marginale infection. Methods A high-throughput qPCR approach was used to determine the expression profile of 16 genes encoding proteins involved in either production or detoxification of ROS and RNS in response to different microbial challenges. In addition, the effect of RNAi-mediated gene silencing of catalase, glutathione peroxidase, thioredoxin and protein oxidation resistance 1 in the control of infection with A. marginale was evaluated. Results Infection with A. marginale resulted in downregulation of the genes encoding ROS-generating enzymes dual oxidase and endoplasmic reticulum oxidase. In contrast, the genes encoding the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, thioredoxin, thioredoxin reductase and peroxiredoxin were upregulated. The gene expression pattern in response to infection with Rickettsia rickettsii and exposure to heat-killed microorganisms, Micrococcus luteus , Enterobacter cloacae or S. cerevisiae was the opposite of that triggered by A. marginale challenge. The simultaneous silencing of three genes, catalase, glutathione peroxidase, and thioredoxin as well as the oxidation resistance 1 gene by RNAi apparently favoured the colonization of BME26 cells by A. marginale , suggesting that the antioxidant response might play a role in the control of infection. Conclusions Taken together, our results suggest that a general response of tick cells upon microbial stimuli is to increase ROS/RNS production. In contrast, A. marginale infection triggers an opposite profile, suggesting that this pathogen might manipulate the tick redox metabolism to evade the deleterious effect of the oxidant-based innate immune response.

Geopropolis, a complex natural product composed of propolis, wax, plant resins, and soil produced by Meliponine (stingless) bees, has traditionally been used for its therapeutic properties. Its chemically diverse composition and broad biological activities have recently attracted growing scientific interest. In this study, we characterized the physicochemical and immunomodulatory properties of a hydroalcoholic extract of geopropolis (HEG) from Melipona quadrifasciata (Mandaçaia). Physicochemical characteristics were determined by measuring moisture, ash, and wax content, and its bioactive constituents were identified by GC–MS. THP-1-derived macrophages were exposed to increasing HEG concentrations to assess cytotoxicity, and two sublethal doses were selected for immunomodulatory assays with or without LPS stimulation. Cytokine and chemokine secretion were quantified by CBA, and the expression of key immunoregulatory and angiogenic genes was evaluated by RT-qPCR. Chemical profiling revealed a high wax content and a predominance of di- and triterpenoids, largely derived from coniferous sources. In mccrophages stimulated with LPS, HEG at 31.25 and 62.50 µg/mL significantly reduced the secretion of pro-inflammatory mediators (IL-6, CCL2, CCL5, CXCL9, and CXCL10) while preserving cell viability. In unstimulated macrophages, HEG upregulated the expression of genes VEGFA and TGFB1 as well as the protein CXCL8, all of them associated with angiogenesis and tissue repair. These findings demonstrate that M. quadrifasciata geopropolis extract modulates macrophage activity, promoting a shift toward a reparative phenotype that integrates inflammatory resolution with pro-healing effects. These results underscore its pharmacological potential as a terpenoid-rich natural product with complementary anti-inflammatory and regenerative activities.

Sphingomyelinase D (SMase D), the main toxic component of Loxosceles venom, has a well-documented role on dermonecrotic lesion triggered by envenomation with these species; however, the intracellular mechanisms involved in this event are still poorly known. Through differential transcriptomics of human keratinocytes treated with L. laeta or L. intermedia SMases D, we identified 323 DEGs, common to both treatments, as well as upregulation of molecules involved in the IL-1 and ErbB signaling. Since these pathways are related to inflammation and wound healing, respectively, we investigated the relative expression of some molecules related to these pathways by RT-qPCR and observed different expression profiles over time. Although, after 24 h of treatment, both SMases D induced similar modulation of these pathways in keratinocytes, L. intermedia SMase D induced earlier modulation compared to L. laeta SMase D treatment. Positive expression correlations of the molecules involved in the IL-1 signaling were also observed after SMases D treatment, confirming their inflammatory action. In addition, we detected higher relative expression of the inhibitor of the ErbB signaling pathway, ERRFI1, and positive correlations between this molecule and pro-inflammatory mediators after SMases D treatment. Thus, herein, we describe the cell pathways related to the exacerbation of inflammation and to the failure of the wound healing, highlighting the contribution of the IL-1 signaling pathway and the ERRFI1 for the development of cutaneous loxoscelism.

In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a wellknown inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new molecular mechanism of resistance to pesticides.