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Ticks are obligatory hematophagous ectoparasites that transmit pathogens among various vertebrates, including humans. The composition of the microbial and viral communities in addition to the pathogenic microorganisms is highly diverse in ticks, but the factors driving the diversity are not well understood. The tropical horse tick,Dermacentor nitens, is distributed throughout the Americas and it is recognized as a natural vector ofBabesia caballiandTheileria equi, the causal agents of equine piroplasmosis. We characterized the bacterial and viral communities associated with partially-fedD. nitensfemales collected by a passive survey on horses from field sites representing three distinct geographical areas in Colombia (Bolivar, Antioquia, and Cordoba). RNA-seq and sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene were performed using the Illumina-Miseq platform. A total of 356 operational taxonomic units (OTUs) were identified, in which the presumed endosymbiotic Francisellaceae/Francisellaspp. was predominantly found. Nine contigs corresponding to six different viruses were identified in three viral families: Chuviridae, Rhabdoviridae, and Flaviviridae. Differences in the relative abundance of the microbial composition among the geographical regions were found to be independent of the presence ofFrancisella-Like Endosymbiont (FLE). The most prevalent bacteria found on each region wereCorynebacteriumin Bolivar,Staphylococcusin Antioquia, andPseudomonasin Cordoba.Rickettsia-like endosymbionts, mainly recognized as the etiological agent of rickettsioses in Colombia were detected in the Cordoba samples. Metatranscriptomics revealed 13 contigs containing FLE genes, suggesting a trend of regional differences. These findings suggest regional distinctions among the ticks and their bacterial compositions.

Recent studies have provided strong evidence indicating that lone star tick bites are a cause of AGS (alpha-gal syndrome, also known as red meat allergy RMA) in humans. AGS is characterized by an increase in IgE antibody production against galactose-alpha-1,3-galactose (aGal), which is a common glycan found in mammalian tissue, except in Old World monkeys and humans. The main causative factor of AGS, the lone star tick (Amblyomma americanum), is broadly distributed throughout the east and midwest of the United States and is a vector of a wide range of human and animal pathogens. Our earlier glycomics study of the salivary glands of partially fed male and female ticks revealed relatively high levels of aGal epitopes. In this study, we found that partially fed males ofA. americanumon bovine blood, which engage in multiple intrastadial feedings, carry a large amount of aGal in the salivary glands. In our current work, we aimed to test whether ticks mediate the transmission of the aGal sensitizer acquired from nonhuman blood to humans in the intrastadial host switch (referred to as the “transmission” hypothesis). To test this hypothesis, we used an alpha-galactosyltransferase knockout mutant mouse (aGT-KO) model system infested with ticks that were unfed or partially fed on bovine blood. Based on the levels of total IgE and specific IgG and IgE antibodies against aGal after tick feedings, aGT-KO mice significantly responded to tick feeding and injection of aGal (Galα1-3Galβ1-4GlcNAc) conjugated to human serum albumin or mouse serum albumin (aGal-HSA or aGal-MSA) by increasing total IgE and aGal-specific IgE levels compared to those in C57BL/6 control mice. All of the treatments of aGT-KO mice involving the feeding of partially fed and unfed ticks functioned as sensitizers that increased the levels of specific IgE against aGal, with large individual variations. The data in this study do not support the “transmission” component of AGS, although they confirmed that aGT-KO mice can be used as a model for RMA studies.

Ixodid ticks are ectoparasites that feed exclusively on blood as their source of nutrients. Although ticks spend most of their life off the host, until now it has been assumed that the blood and the water vapor are the only sources of water to maintain water balance and prevent desiccation. Here we report for the first time that adult lone star ticks, Amblyomma americanum, also actively drink nutrient-free water, which greatly increases their survival. The volume of ingested water is greater in females than males (0.55 ± 0.06 vs 0.44 ± 0.07 µl) and most likely due to differences in tick size. Water uptake occurs through mouthparts and it can be later observed in the salivary glands and the midgut. We also exploited this behavior by adding a variety of inorganic compounds and microorganisms to water. Addition of inorganic salts to drinking water such as KH2PO4 + NaCl+KNO3 resulted in 100% tick mortality within 3 days. As a proof of concept for using the water drinking as a delivery route of toxic reagents for ticks, we also show that adding Pseudomonas aeruginosa to drinking water quickly leads to tick death. This tick behavior can be exploited to target important physiological systems, which would make ticks vulnerable to dehydration and microbial dysbiosis.

Ticks are hematophagous ectoparasites that transmit a wide range of pathogens. The lone star tick, Amblyomma americanum, is one of the most widely distributed ticks in the Midwest and Eastern United States. Lone star ticks, as other three-host ixodid ticks, can survive in harsh environments for extended periods without a blood meal. Physiological mechanisms that allow them to survive during hot and dry seasons include thermal tolerance and water homeostasis. Dermal fluid secretions have been described in metastriate ticks including A. americanum. We hypothesized that tick dermal secretion in the unfed tick plays a role in thermoregulation, as described in other hematophagous arthropods during blood feeding. In this study, we found that physical contact with a heat probe at 45 °C or high environmental temperature at ∼50 °C can trigger dermal secretion in A. americanum and other metastriate ticks in the off-host period. We demonstrated that dermal secretion plays a role in evaporative cooling when ticks are exposed to high temperatures. We find that type II dermal glands, having paired two cells and forming large glandular structures, are the source of dermal secretion. The secretion was triggered by an injection of serotonin, and the serotonin-mediated secretion was suppressed by a pretreatment with ouabain, a Na/K-ATPase blocker, implying that the secretion is controlled by serotonin and the downstream Na/K-ATPase.

The salivary gland of hematophagous arthropods is critical for blood meal acquisition, blood vessel localization, and secretion of digestive enzymes. Thus, there is significant interest in the regulation of salivary gland function and mechanisms driving the secretion of saliva and digestive proteins. We aimed to gain a broader understanding of the regulatory role of aminergic, cholinergic, and octopaminergic neuromodulators to saliva and protein secretion from the female A. aegypti salivary gland. Quantification of saliva after injection with neuromodulators showed that dopamine, serotonin, and pilocarpine increased the secretory activity of the salivary gland with potency rankings dopamine = serotonin > pilocarpine. No change in saliva secretion was observed with octopamine or ergonovine, which indicates the A. aegypti salivary gland may be regulated by dopaminergic, serotonergic, and cholinergic systems, but are not likely regulated by octopaminergic or tryptaminergic systems. Next, we studied the regulatory control of dopamine-mediated salivation. Data indicate extracellular calcium flux, but not neural function, is critical for dopamine-mediated salivation, which suggests epithelial transport of ions and not neuronal control is responsible for dopamine-mediated salivation. For regulation of protein secretion, data indicate dopamine or serotonin exposure facilitates amylase secretion, whereas serotonin but not dopamine exposure increased apyrase concentrations in the secreted saliva. General immunoreactivity to anti-rat D1-dopamine receptor antibody was observed, yet immunoreactivity to the anti-rat D2-receptor antibody was identified in the proximal regions of the lateral lobes and slight immunoreactivity in the distal portion of the lateral lobe, with no expression in the medial lobe.

Ticks are a growing concern to human and animal health worldwide and they are leading vectors of arthropod-borne pathogens in the United States. Ticks transmit a wide range of pathogenic microorganisms to humans and animals causing important health and economic impacts. Ticks are pool blood feeders that can attach to the host skin for days to weeks using their saliva to counteract the host defenses. Tick saliva, as in other hematophagous arthropods, contains pharmacological and immunological active compounds, which modulate local and systemic immune responses and induce antibody production. In the present study, we explore differences in the salivary gland extract (SGE) protein content of ticks, Amblyomma americanum, raised in a laboratory colony (CT) versus those found in the field (FT). Western-blot testing showed a set of different immunogenic bands for each antigen. Protein sequencing data from these bands revealed 39 proteins between 10 and 25 kDa from 19 CT and 20 FT. In addition, we measured human IgG antibodies against SGE from CT and FT in healthy individuals residing in Kansas. ELISA revealed that IgG antibody levels were higher when using the SGE from CT as compared to that from FT. Interestingly, antibody levels against both, LT and FT, were high in the warm months (May-June) and decreased in the cold months (September– November). In vitro testing using HUVEC, U937 and SH­SY5Y human cell lines showed higher induction of expression of inflammatory related genes when treating cells with SGE from FT. In conclusion, our results show that the human serum samples reacted more with the CT protein content whereas the FT saliva induced a higher in vitro immune response, causing more inflammation. We hypothesize that differences in the salivary proteins between CT and FT may explain the differential immune responses observed in this study. It is possible that the salivary proteins absent in CT but present in FT are required for tick fitness in nature and possibly have helped to adapt to specific hosts.

Tick salivary glands play critical roles in maintaining water balance for survival, as they eliminate excess water and ions during blood feeding on hosts. In the long duration of fasting in the off-host period, ticks secrete hygroscopic saliva into the mouth cavity to uptake atmospheric water vapor. Type I acini of tick salivary glands are speculated to be involved in secretion of hygroscopic saliva based on ultrastructure studies. However, we recently proposed that type I acini play a role in resorption of water/ions from the primary saliva produced by other salivary acini (i.e., types II and III) during the tick blood feeding phase. In this study, we tested the function of type I acini in unfed femaleIxodes scapularis. The route of ingested water was tracked after forced feeding of water with fluorescent dye rhodamine123. We found that type-I acini of the salivary glands, but not type II and III, are responsible for water uptake. In addition, the ingestion of water through the midgut was also observed. Injection or feeding of ouabain, a Na/K-ATPase inhibitor, suppressed water absorption in type I acini. WhenI. scapulariswas offered a droplet of water, ticks rarely imbibed water directly (5%), while some approached the water droplet to use the high humidity formed in the vicinity of the droplet (23%). We conclude that during both on- and off-host stages, type I acini in salivary glands of femaleIxodes scapularisabsorb water and ions.