Simone Michaela Simons

To validate an artificial feeding method for Ornithodoros brasiliensisticks, aligning with the 3Rs principles (Replacement, Reduction, and Refinement), through the qualitative and quantitative analysis of their salivary gland protein profiles. Fourth-instar nymphs (N4), adults, males, and adult females were divided into unfed and semi-fed (3 minutes) groups using either traditional feeding (control on rabbits; CEUA: 4130270323) or artificial feeding. Artificial feeding was performed using rabbit blood with either an artificial membrane (Parafilm®) or a natural membrane (mouse skin). Salivary gland extracts (SGE) were obtained by maceration and filtration (0.22 µm). Protein concentration was determined by UV spectrophotometry (NanoDrop, Thermo Fisher). Protein and peptide profiles were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC, 20A Prominence system, Shimadzu Co., Japan) using a C18 column, and by matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF/MS, Shimadzu Co., Kyoto, Japan). In traditional feeding, unfed ticks (N4 and females) exhibited higher protein concentrations than semi-fed ticks, whereas the opposite trend was observed in males. The artificial methods (Parafilm® and skin) showed similar protein concentration patterns to each other, except for the N4 stage on skin, where the semi-fed group surpassed the unfed group. MALDI-TOF/MS analysis revealed similar peptide profiles (m/z ratios) between the artificial feeding groups. However, marked differences were observed in both chromatographic and mass profiles when comparing traditional feeding with artificial feeding, indicating alterations in peptide expression resulting from the feeding method. Although artificial systems (Parafilm® and skin) yield consistent protein profiles between themselves, they alter the peptide composition of O. brasiliensis SGE compared to natural feeding. These results suggest that variations between the feeding methods used can lead to differences in tick peptide composition, indicating a potential similarity among peptides expressed during artificial feeding, alongside distinct differences between traditional and artificial feeding regimes. It should be noted that these results are preliminary, and further studies are required Grants # FAPESP No. 2024/23338-1; Butantan Fundation.

Ornithodoros rostratus is an argasid tick distributed in Argentina, Bolivia, Brazil, and Paraguay. The objective of this study is to preliminarily characterize the composition and diversity of the associated bacterial microbiome, expanding knowledge about its microbial community. Ticks were collected in the central area of the Brazilian Pantanal (Nhumirim Ranch), state of Mato Grosso do Sul, central-western Brazil. Specimens were processed individually and subjected to surface disinfection (3× 1% sodium hypochlorite, 3× 70% ethanol, 3× sterile distilled water). DNA extractions were performed on three male specimens and six negative controls, in which all steps of the protocol were conducted. The obtained DNA was used to construct libraries targeting the V3–V4 hypervariable region of the 16S rRNA gene, which were sequenced on the Illumina NovaSeq 6000 platform (2 × 250 bp). Amplicon sequence variants (ASVs) were inferred using the DADA2 pipeline, and taxonomic assignment was performed using the SILVA database (v. 138.2). High-throughput sequencing generated a total of 962,554 reads, with 325,034 from male samples and 637,520 from extraction controls. The average sequencing depth was 106,950 reads per library, with a standard deviation (SD) of 4,708. After quality control, an average of 98,006 ± 5,368 sequences per library were retained. The phylum Actinomycetota was more abundant in the male specimens (87.33% ± 12.16), while Bacillota and Pseudomonadota showed average relative abundances of 5.24% ± 3.13 and 3.69% ± 4.82, respectively. The most representative families and genera were Brevibacteriaceae 35.26% ± 27.53 (Brevibacterium - 35.25% ± 27.52) and Pseudonocardiaceae 27.43% ± 22.19 [(Saccharopolyspora - 23.68% ± 23.26) and Arthrobacter (7.98% ± 1.94)]. Brevibacterium can play an important role in blood digestion in hematophagous arthropods. Saccharopolysporais an environmental bacterium known for its ability to produce bioactive natural products, including antibiotics. Arthrobacter comprises soil species associated with invertebrates as insects and ticks, demonstrating ecological functions, as it has the ability to use pyridone as its sole carbon source and resistance to desiccation and starvation. Our studies are still being completed. Therefore, the results presented here represent preliminary data.

Ticks represent a primary group of public health and global economic relevance due to their high vector competence. During hematophagy, they secrete pharmacologically active molecules via salivary glands. Thus, this study aims to investigate the modulatory potential of the Salivary Gland Extract (SGE) from the tick Ornithodoros brasiliensis (Acari: Argasidae) (Aragão, 1923) on microbial growth, identifying molecules capable of promoting or inhibiting the cellular proliferation of specific microorganisms. Salivary glands were obtained from semi-engorged specimens (CEUA No. 4130270323) and subjected to maceration. The resulting extract was sterilized under a laminar flow hood using 0.22 µm filters (MilliPore). Protein concentration was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific) at A280 nm. The SGE was evaluated against Staphylococcus aureus (ATCC 25923), Salmonella enterica (ATCC 14028), and Candida albicans (ATCC 10231). Antimicrobial activity assays were performed using broth dilution in Mueller- Hinton medium (for bacteria) or Sabouraud Dextrose broth (for yeasts), with absorbance measured at 595 nm in a Multiskan EX spectrophotometer (Thermo Fisher) after 20 hours of incubation at 37 °C. The SGE was subjected to reverse-phase high-performance liquid chromatography (RP-HPLC) using a Jupiter C18 column (20A Prominence, Shimadzu Co., Japan). Mass spectrometry analysis (Orbitrap Exploris 480), processed with PEAKS Studio 13 software, allowed the initial identification of proteins associated with the selected fractions. Protein sequences were retrieved and analyzed using BLASTp (Basic Local Alignment Search Tool for proteins) via the National Center for Biotechnology Information (NCBI) database. Microbiological assays demonstrated potent inhibitory activity against cellular growth for the three tested micr oorganisms: 100% inhibition of S. aureus in fractions F13 and I.3; 100% inhibition of S. enterica in fractions F2, F13, and I.3; and 50% and 70% inhibition of C. albicans in fractions F6 and I.3, respectively. Preliminary mass spectrometry analysis identified Arg r 1-like lipocalins (F2), acid-tailed salivary proteins (F13), hypothetical proteins (F6), and transporters involved in glycosylation processes (I.3). The presence of these proteins suggests a possible relationship between immunomodulatory mechanisms and the observed antimicrobial activity. Although detailed bioinformatic analysis is ongoing, the data obtained to date indicate that O. brasiliensis SGE constitutes a promising source of molecules with antimicrobial and biotechnological potential, justifying further investigations for the structural and functional characterization of the identified proteins Grants #2013/07467-1 and #2021/13939-0, São Paulo Research Foundation (FAPESP)

The coxal fluid of the tick species Ornithodoros brasiliensis (O. brasiliensis) was subjected to detailed chemical analysis using Instrumental Neutron Activation Analysis (INAA) and Energy Dispersive X-ray Fluorescence (EDXRF). These complementary techniques allowed the identification and quantification of key elements such as sodium (Na), chlorine (Cl), potassium (K), and zinc (Zn), along with others like sulfur (S), phosphorus (P), and iron (Fe). The study demonstrated the consistency between the two analytical methods and revealed significant data about the tick's physiological adaptations related to fluid excretion and blood meal processing. Additionally, the absence of heavy metals in the analyzed samples was confirmed. Understanding the composition of coxal fluid is vital for interpreting metabolic processes and exploring its toxicological potential, given the aggressive nature of O. brasiliensis, known to cause severe health impacts on humans and animals. This research contributes to advancing knowledge about tick biology and supports the development of public health measures against tick-borne hazards.

Anaplasma marginale is a Gram-negative, obligate intraerythrocytic bacterium that causes bovine anaplasmosis. While hard ticks of the genera Dermacentor and Rhipicephalus can be biological vectors, transmitting this pathogen via saliva during blood meals, blood-sucking insects, and fomites play a role as mechanical vectors. Little is known about the interaction between Anaplasma marginale and Argasidae ticks. Among soft ticks, Ornithodoros fonsecai (Labruna and Venzal) and Ornithodoros brasiliensis Aragão inhabit environments surrounding localities where many cases of bovine anaplasmosis have been reported. Ticks of the species O. fonsecai parasitize bats, while O. brasiliensis can parasitize different vertebrate species. Therefore, the present study aimed to feed third-instar nymphs artificially (N3) of O. fonsecai and O. brasiliensis using blood samples obtained from a calf naturally infected with A. marginale and rabbit blood added to A. marginale-containing bovine erythrocytes, to investigate the ability of these nymphs to acquire, infect and transstadially perpetuate this agent. For the artificial feeding system, adapted chambers and parafilm membranes were used. Nymphs of both tick species were submitted to different replications weighed before and after each feeding. Blood samples and molted ticks were submitted to DNA extraction, quantitative real-time PCR for the msp1β gene to detect A. marginale DNA, while a semi-nested polymerase chain reaction for the msp1α gene was performed for genotyping. Using calf blood naturally infected with A. marginale, among the three artificial feeding replications performed with O. fonsecai and O. brasiliensis nymphs, the DNA of A. marginale was detected in both nymphs after 30–50 days of molting. For artificial feeding with rabbit blood added to bovine erythrocytes containing A. marginale, the DNA of this pathogen was also detected in both nymph species. As for the assay for the msp1α gene, strains were found Is9; 78 24-2; 25; 23; α; and β. It was concluded that nymphs (N3) of O. fonsecai and O. brasiliensis could feed artificially through a parafilm membrane using blood from calves and rabbits infected by A. marginale. The DNA of A. marginale was detected in nymphs fed artificially of both tick species studied after molt. However, further studies are needed to confirm transstadial perpetuation in other instars and their host transmission capacity.

Ornithodoros fonsecai is a species of argasid tick endemic to Brazil, described in the “São Miguel” cave located in the municipality of Bonito, state of Mato Grosso do Sul, central-western region of Brazil. The artificial feeding technique makes it possible to study the biology of hematophagous arthropods using artificial or natural membranes, as well as different types of blood and anticoagulants. Thus, the aim of the present study was to feed artificially O. fonsecai second instar (N2) nymphs with rabbit blood using parafilm membrane and the anticoagulant Alsever. Ninety percent of the total N2 nymphs engorged and molted to N3 nymphs between 27 and 30 days after feeding, indicating that the use of this anticoagulant is efficient for artificially feeding O. fonsecai N2 nymphs under laboratory conditions.

Specific blood coagulation inhibitors from hematophagous organisms, with different structures and novel mechanism of action, have been described and they represent promising agents for the treatment of a variety of human diseases related to coagulation and cancer. In our lab, the salivary glands transcriptome of the adult Amblyomma cajennense tick was previously characterized by expressed sequence tags (EST). A transcript that codes for a tissue factor pathway inhibitor (TFPI)-like protein with unique structure was found, and the recombinant form of this protein was named Amblyomin-X. This protein was able to inhibit the factor Xa amidolytic activity and the activation of factor X by the extrinsic tenase complex (FVIIa/TF). Herein, it was performed functional and structural evaluation of Amblyomin-X. The CD assay and molecular dynamics simulations revealed that Amblyomin-X is structurally stable and the naturally unfolded regions as well as the presence of three disulfide bridges in its Kunitz-type domain seem to sustain its inhibitory activity. Regarding the electrostatic potential mapping on the Kunitz-type region, the pattern of charged residues was not quite the same in comparison to human TFPI-1 and TFPI-2, pointing out there might be distinct functional and structural features, which are going to be experimentally exploited.