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The hard tick Ixodes ricinus is an obligate hematophagous arthropod and the main vector for several zoonotic diseases. The life cycle of this three-host tick species was completed for the first time in vitro by feeding all consecutive life stages using an artificial tick feeding system (ATFS) on heparinized bovine blood supplemented with glucose, adenosine triphosphate, and gentamicin. Relevant physiological parameters were compared to ticks fed on cattle (in vivo). All in vitro feedings lasted significantly longer and the mean engorgement weight of F0 adults and F1 larvae and nymphs was significantly lower compared to ticks fed in vivo. The proportions of engorged ticks were significantly lower for in vitro fed adults and nymphs as well, but higher for in vitro fed larvae. F1-females fed on blood supplemented with vitamin B had a higher detachment proportion and engorgement weight compared to F1-females fed on blood without vitamin B, suggesting that vitamin B supplementation is essential in the artificial feeding of I. ricinus ticks previously exposed to gentamicin.

Current knowledge on immune cell interactions with Borrelia burgdorferi (Bb) derives mostly from studies done in vitro and ex vivo, which cannot assess host immunity to natural tick-delivered Bb within the complex architecture of host tissues. We report the first in vivo study on local and systemic immune responses to Bb during tick feeding on a surrogate reservoir host, in comparison with uninfected-tick and subcutaneously delivered Bb. We show that uninfected-tick and tick-transmitted Bb engaged mixed type-1/type-2/type-17 immune responses in the presence of anti-inflammatory IL-10, in contrast to a type-1 response induced by subcutaneously delivered Bb. Analyses of immune responses to tick-transmitted Bb in a reservoir host can enlighten immunity mechanisms that mediate persistence of Bb.

Background Hard tick infestation occurs naturally in humans, domestic animals, and livestock species. Upon feeding, ticks transmit a wide variety of pathogens that may result in serious diseases with severe public health and economic impacts. While tick-borne diseases significantly impact human and animal health and agricultural production worldwide, as ectoparasites, ticks can also cause serious tissue injury, tick paralysis, or exsanguination from mass infestation. Experimental tick feeding is necessary to study tick-borne diseases and effectively test novel vaccines and therapeutics. Such studies raise concerns about on-host tick containment. Classically, tick containment cells for feeding on animals are rigid, lidded containers that are adhered to the host’s skin with adhesive or tape. They are bulky and easily damaged. Methods Here, we describe the use of mesh packets, termed ‘teabags,’ containing 20 male and 5 female ticks each of both Amblyomma americanum and Dermacentor variabilis applied with surgical tape beneath harnesses on five dogs to allow tick feeding. Canine hematological and blood chemistry parameters were recorded before, during, and after tick feeding. Results Successful feeding for 7 days was observed for both tick species (21–24/25 A. americanum and 3–14/25 D. variabilis per dog). Statistically significant shifts were detected in canine host hematological and blood chemistry parameters during tick feeding, indicating that infestation with even small numbers of ticks affects the systemic hematological and blood chemistry parameters. Conclusions This new method is safe, humane, and effective and will improve the experimental design, containment, and safety of tick-feeding research across many host, parasite, and pathogen species. Graphical Abstract

Companion dogs are at risk of tick infestations. This paper describes cases of transfer of Dermacentor reticulatus ticks by dogs to apartments in eastern Poland, tick development in household conditions, and potential consequences for the residents. For the first time, the preoviposition and oviposition of D. reticulatus females removed from dogs or spontaneously detached from these hosts were studied in household conditions. Similar analyses were performed simultaneously in laboratory settings (25 °C and 75% RH). In the household characterized by a temperature range of 18.5–21.3 °C and 46.9–56% humidity, the preoviposition and oviposition periods with the development of D. reticulatus larvae lasted 20.8 ± 3.1 days and 29.9 ± 1.4 days, respectively. Greater numbers of eggs (2415.8 ± 983.1) were laid by females in the household than laboratory conditions. There were no statistically significant differences in the hatching success between both experiments. The study also provides the first description of infestation of a human by a partially engorged D. reticulatus female that had detached from dog’s skin. Ticks transferred by dogs can develop successfully in human homes. After feeding on dogs, females achieve high reproductive performance. This suggests that dogs may play an important role in the biology of this tick species in urbanized areas.

BACKGROUND: Rhipicephalus microplus is a hard tick species that has a high impact on cattle health and production in tropical and subtropical regions. Recently, ribosomal DNA and morphological analysis resulted in the reinstatement of R. australis as a separate species from R. microplus. Both feed on cattle and can transmit bovine pathogens such as Anaplasma and Babesia species. The current treatment with acaricides is becoming increasingly less effective due to the emergence of resistant tick strains. A promising alternative can be found in the form of anti-tick vaccines. The available commercial vaccines can be used to control tick infestation, but the lack of a knockdown effect (>90% reduction in tick numbers as seen with effective acaricides) hampers its widespread use, hence higher efficacious vaccines are needed. Instead of searching for new protective antigens, we investigated the efficacy of vaccines that contain more than one (partially) protective antigen. For screening vaccine formulations, a previously developed in vitro feeding assay was used in which R. australis larvae are fed sera that were raised against the candidate vaccine antigens. In the present study, the efficacy of the Bm86 midgut antigen and the cytosolic Subolesin (SUB) antigen were evaluated in vitro. RESULTS: Antiserum against recombinant Bm86 (rBm86) partially inhibited larval engorgement, whereas antiserum against recombinant SUB (rSUB) did not have any effect on feeding of larvae. Importantly, when larvae were fed a combination of antiserum against rBm86 and rSUB, a synergistic effect on signifcantly reducing larval infestations was found. Immunohistochemical analysis revealed that the rBm86 antiserum reacted with gut epithelium of R. australis larvae, whereas the antiserum against rSUB stained salivary glands and rectal sac epithelium. CONCLUSIONS: Combining anti-Bm86 and anti-subolesin antibodies synergistically reduced R. australis larval feeding in vitro. Rhipicephalus australis is a one host tick, meaning that the larvae develop to nymphs and subsequently adults on the same host. Hence, this protective effect could be even more pronounced when larvae are used for infestation of vaccinated cattle, as the antibodies could then affect all three developmental stages. This will be tested in future in vivo experiments.

Background/Objectives: Ticks evade host hemostasis and immunity in part by injecting serine protease inhibitors (serpins) into the host during feeding, yet the genomic organization of tick serpins has remained unresolved. To understand how ticks deploy these proteins, there is a need to elucidate their gene structure, arrangement and copy number in the genome. Methods: We annotated the recent Ixodes scapularis chromosome-level assembly and identified all the serpin genes to build a genome-wide atlas of serpin loci identifying the gene structure and duplication patterns. The gene expression of serpins during blood meal was also analyzed. Results: We identified 74 serpin genes across eight chromosomes and one unplaced scaffold, with a strongly non-random distribution dominated by chromosome 10, which harbored 67.6% of serpin genes in dense tandem clusters. Most genes were intronless and encoded secreted, N-glycosylated proteins, whereas a minority were conserved two-exon loci sharing a common splice junction. Pairwise amino acid comparisons revealed exact duplicates as well as very recent and divergent paralogs, indicating continued local duplication and diversification. Expression analysis across tissues and feeding time showed that serpin expression is structured primarily by organ and feeding stage, including a late feeding increase in midgut serpins that are predicted to inhibit trypsin-like proteases. Conclusions: This atlas provides a comprehensive description of I. scapularis serpins, provides a framework for understanding tick gene structure and function, prioritizes serpins as target candidates for tick control, and functions as a library for other serpin uses in medicine and industry.

Background Vector-borne diseases pose an increasing threat to global food security. Vaccines, diagnostic tests, and therapeutics are urgently needed for tick-borne diseases that affect livestock. However, the inability to obtain significant quantities of pathogen stages derived from ticks has hindered research. In vitro methods to isolate pathogens from infected tick vectors are paramount to advance transcriptomic, proteomic, and biochemical characterizations of tick-borne pathogens. Methods Nymphs of Rhipicephalus appendiculatus were infected with Theileria parva by feeding on a calf during an acute infection. Isolation of sporozoites was accomplished by feeding infected adult ticks on an in vitro tick feeding system. Sporozoite viability was tested using in vitro bovine lymphocytes. Results We isolated infectious T. parva sporozoites secreted into an in vitro tick feeding system. Infected adult R. appendiculatus ticks attached to and successfully fed on silicone membranes in the in vitro tick feeding system. Bovine blood in the receptacle was replaced with cell-free medium and the ticks were allowed to feed for 3 h to collect secreted T. parva sporozoites. Secreted sporozoites infected in vitro bovine lymphocytes, demonstrating that isolated sporozoites remained viable and infectious. Conclusions This work is the first to report the isolation of mature infectious T. parva sporozoites using an in vitro tick feeding system, which represents a significant step towards the development of a more efficient control strategy for T. parva . Isolation of infectious tick-stage parasites will facilitate the examination of the vector-pathogen interface, thereby accelerating the development of next-generation vaccines and treatment interventions for tick-borne pathogens. Graphical Abstract

Background The cattle fever tick, Rhipicephalus ( Boophilus ) microplus , is a vector of pathogens causative of babesiosis and anaplasmosis, both highly lethal bovine diseases that affect cattle worldwide. In Ecdysozoa, neuropeptides and their G-protein-coupled receptors play a critical integrative role in the regulation of all physiological processes. However, the physiological activity of many neuropeptides is still unknown in ticks. Periviscerokinins (CAP 2b /PVKs) are neuropeptides associated with myotropic and diuretic activities in insects. These peptides have been identified only in a few tick species, such as Ixodes ricinus , Ixodes scapularis and R. microplus, and their cognate receptor only characterized for the last two. Methods Expression of the periviscerokinin receptor ( Rhimi-CAP 2b R ) was investigated throughout the developmental stages of R. microplus and silenced by RNA interference (RNAi) in the females. In a first experiment, three double-stranded (ds) RNAs, named ds680-805, ds956-1109 and ds1102-1200, respectively, were tested in vivo. All three caused phenotypic effects, but only the last one was chosen for subsequent experiments. Resulting RNAi phenotypic variables were compared to those of negative controls, both non-injected and dsRNA beta-lactamase-injected ticks, and to positive controls injected with beta-actin dsRNA. Rhimi-CAP 2b R silencing was verified by quantitative reverse-transcriptase PCR in whole females and dissected tissues. Results Rhimi-CAP 2b R transcript expression was detected throughout all developmental stages . Rhimi-CAP 2b R silencing was associated with increased female mortality, decreased weight of surviving females and of egg masses, a delayed egg incubation period and decreased egg hatching ( P  < 0.05). Conclusions CAP 2b /PVKs appear to be associated with the regulation of female feeding, reproduction and survival. Since the Rhimi-CAP 2b R loss of function was detrimental to females, the discovery of antagonistic molecules of the CAP 2b /PVK signaling system should cause similar effects. Our results point to this signaling system as a promising target for tick control. Graphical Abstract

Background Rhipicephalus microplus is the vector of deadly cattle pathogens, especially Babesia spp., for which a recombinant vaccine is not available. Therefore, disease control depends on tick vector control. However, R. microplus populations worldwide have developed resistance to available acaricides, prompting the search for novel acaricide targets. G protein-coupled receptors (GPCRs) are involved in the regulation of many physiological processes and have been suggested as druggable targets for the control of arthropod vectors. Arthropod-specific signaling systems of small neuropeptides are being investigated for this purpose. The pyrokinin receptor (PKR) is a GPCR previously characterized in ticks. Myotropic activity of pyrokinins in feeding-related tissues of Rhipicephalus sanguineus and Ixodes scapularis was recently reported. Methods The R. microplus pyrokinin receptor ( Rhimi-PKR ) was silenced through RNA interference (RNAi) in female ticks. To optimize RNAi, a dual-luciferase assay was applied to determine the silencing efficiency of two Rhimi-PKR double-stranded RNAs (dsRNA) prior to injecting dsRNA in ticks to be placed on cattle. Phenotypic variables of female ticks obtained at the endpoint of the RNAi experiment were compared to those of control female ticks (non-injected and beta-lactamase dsRNA-injected). Rhimi-PKR silencing was verified by quantitative reverse-transcriptase PCR in whole females and dissected tissues. Results The Rhimi-PKR transcript was expressed in all developmental stages. Rhimi-PKR silencing was confirmed in whole ticks 4 days after injection, and in the tick carcass, ovary and synganglion 6 days after injection. Rhimi-PKR silencing was associated with an increased mortality and decreased weight of both surviving females and egg masses ( P  < 0.05). Delays in repletion, pre-oviposition and incubation periods were observed ( P  < 0.05). Conclusions Rhimi-PKR silencing negatively affected female reproductive fitness. The PKR appears to be directly or indirectly associated with the regulation of female feeding and/or reproductive output in R. microplus . Antagonists of the pyrokinin signaling system could be explored for tick control. Graphical abstract

The bacterial agent of Lyme disease, Borrelia burgdorferi, exists in an enzootic cycle by adapting to dissimilar mammalian and tick environments. The genetic elements necessary for host and vector adaptation are spread across a bacterial genome comprised of a linear chromosome and essential linear and circular plasmids. The promoter trap system, In Vivo Expression Technology (IVET), has been used to identify promoters of B. burgdorferi that are transcriptionally active specifically during infection of a murine host. However, an observed infection bottleneck effect in mice prevented the application of this system to study promoters induced in a tick environment. In this study, we adapted a membrane-based in vitro feeding system as a novel method to infect the Ixodes spp. vector with B. burgdorferi. Once adapted, we performed IVET screens as a proof of principle via an infected bloodmeal on the system. The screen yielded B. burgdorferi promoters that are induced during tick infection and verified relative expression levels using qRT-PCR. The results of our study demonstrate the potential of our developed in vitro tick feeding system and IVET systems to gain insight into the adaptive gene expression of the Lyme disease bacteria to the tick vector.