Anomalous morphologies in Ixodes scapularis feeding on human hosts

Cases of anomalous morphologies in the blacklegged tick, Ixodes scapularis, have been reported in both field-collected and human-biting specimen in the Northeastern and Midwestern United States, complicating the identification of this medically important tick species. We herein describe four cases of morphological anomalies in I. scapularis females exhibiting nanism and abnormally small genital apertures. We also report a female I. scapularis displaying slight asymmetry in the lower abdomen oriented toward the right side and an abnormal anal groove completely enclosing the anus. The identity of each specimen was confirmed using taxonomic keys, high resolution light and scanning electron microscopy imaging, and DNA sequencing of the 18S rRNA gene. All specimens described in this study were found parasitizing human hosts and were submitted to the Connecticut Agricultural Experiment Station-Tick Testing Laboratory in 2021 for species identification and pathogen screening. Here, we also discuss recent reports of teratological abnormalities in I. scapularis as well as likely causes for such deformities and potential implications.     

Tick immunity using mRNA,DNA and protein-based Salp14 delivery strategies

Guinea pigs exposed to multiple infestations with Ixodes scapularis ticks develop acquired resistance to ticks, which is also known as tick immunity. The I. scapularis salivary components that contribute to tick immunity are likely multifactorial. An anticoagulant that inhibits factor Xa, named Salp14, is present in tick saliva and is associated with partial tick immunity. A tick bite naturally releases tick saliva proteins into the vertebrate host for several days, which suggests that the mode of antigen delivery may influence the genesis of tick immunity. We therefore utilized Salp14 as a model antigen to examine tick immunity using mRNA lipid nanoparticles (LNPs), plasmid DNA, or recombinant protein platforms. salp14 containing mRNA-LNPs vaccination elicited erythema at the tick bite site after tick challenge that occurred earlier, and that was more pronounced, compared with DNA or protein immunizations. Humoral and cellular responses associated with tick immunity were directed towards a 25 amino acid region of Salp14 at the carboxy terminus of the protein, as determined by antibody responses and skin-testing assays. This study demonstrates that the model of antigen delivery, also known as the vaccine platform, can influence the genesis of tick immunity in guinea pigs. mRNA-LNPs may be useful in helping to elicit erythema at the tick bite site, one of the most important early hallmarks of acquired tick resistance. mRNA-LNPs containing tick genes is a useful platform for the development of vaccines that can potentially prevent selected tick-borne diseases.     

Immunization of guinea pigs with cement extract induces resistance against Ixodes scapularis ticks

As hematophagous parasites, many tick species are important vectors of medical and veterinary disease agents. Proteins found in tick saliva and midgut have been used with some success in immunizations of animal hosts against feeding ticks, and whole saliva has been used effectively in this capacity against Ixodes scapularis, the primary vector of tickborne pathogens in the United States. Tick saliva is a complex substance containing hundreds of proteins, and the identification of specific protective antigens is ongoing. We performed a series of experiments immunizing guinea pigs with extracts prepared from midgut or attachment cement collected from adult female I. scapularis followed by challenge with nymphs of the same species. Midgut extract did not induce protective immunity, while immunization with cement extract resulted in partial protection of hosts as evidenced by premature tick detachment and 34-41% reduction in tick engorgement weights. Proteomic characterization of I. scapularis cement was performed, demonstrating that the cement extract was compositionally different from tick saliva, and vitellogenin-like lipoproteins were the most abundant proteins in cement extract (>40%). Cement was also heavily enriched with lysozymes and defensins, including those originating from both the mammalian host as well as ticks. These results demonstrate that I. scapularis cement contains immunogenic components capable of stimulating host resistance against tick feeding. Because the cement is present at the tick-host interface for an extended period of time during the feeding process, these antigens present auspicious candidates for further evaluation and potential inclusion in an anti-tick vaccine.     

Human Babesiosis,Maine, USA, 1995–2011

We observed an increase in the ratio of pathogenic Babesia microti to B. odocoilei in adult Ixodes scapularis ticks in Maine. Risk for babesiosis was associated with adult tick abundance, Borrelia burgdorferi infection prevalence, and Lyme disease incidence. Our findings may help track risk and increase the focus on blood supply screening.     

Woodchip borders at the forest ecotone as an environmental control measure to reduce questing tick density along recreational trails in Ottawa,Canada

Recent studies have highlighted the occurrence and distribution of Ixodes scapularis ticks infected with Borrelia burgdorferi sensu stricto (s.s.) around the city of Ottawa, Ontario, Canada, and the need for strategies to mitigate the risk of human exposure and infection. We conducted a field study from July to October 2018 to examine the effectiveness of ecotonal woodchip borders as an environmental control method to suppress the density of host-seeking ticks along recreational trails in Ottawa. We used an experimental design with ten 100-m trail replicates randomized to intervention or control groups, and monitored questing tick density at weekly intervals in mid-summer and early fall. We compared questing tick density between woodchip-treated and untreated trails using a mixed-effects Poisson regression model. Of the 138 I. scapularis ticks collected, there were 86 adult and nymphal ticks, 37 (43 %) of which were positive for B. burgdorferi s.s. A total of 58 Haemaphysalis leporispalustris were also collected. Mean combined adult and nymphal I. scapularis density was 1.15 (1.40 standard deviation; SD) per 100 m in the control group compared to 0.28 (0.56 SD) per 100 m in the intervention group, reflecting a 75 % reduction in questing tick density on trail replicates treated with woodchip borders (p < 0.001). An effect of the intervention was observed in both sampling periods. This study indicates that woodchip borders may be an effective strategy to suppress questing tick density along trail margins where recreational trail users are more active, thereby reducing the likelihood of tick encounters.     

Sheep experimentally infected with a human isolate of Anaplasma phagocytophilum serve as a host for infection of Ixodes scapularis ticks

Anaplasma phagocytophilum, first identified as a pathogen of ruminants in Europe, has more recently been recognized as an emerging tick-borne pathogen of humans in the U.S. and Europe. A. phagocytophilum is transmitted by Ixodes spp., but the tick developmental cycle and pathogen/vector interactions have not been fully described. In this research, we report on the experimental infection of sheep with the human NY-18 isolate of A. phagocytophilum which then served as a host for infection of I. scapularis nymphs and adults. A. phagocytophilum was propagated in the human promyelocytic cell line, HL-60, and the infected cell cultures were then used to infect sheep by intravenous inoculation. Infections in sheep were confirmed by PCR and an Anaplasma-competitive ELISA. Clinical signs were not apparent in any of the infected sheep, and only limited hematologic and mild serum biochemical abnormalities were identified. While A. phagocytophilum morulae were rarely seen in neutrophils, blood film evaluation revealed prominent large granular lymphocytes, occasional plasma cells, and rare macrophages. Upon necropsy, gross lesions were restricted to the lymphoid system. Mild splenomegaly and lymphadenomegaly with microscopic evidence of lymphoid hyperplasia was observed in all infected sheep. Female I. scapularis that were allowed to feed and acquire infection on each of the 3 experimentally infected sheep became infected with A. phagocytophilum as determined by PCR of guts (80–87%) and salivary glands (67–100%). Female I. scapularis that acquired infection as nymphs on an experimentally infected sheep transmitted A. phagocytophilum to a susceptible sheep, thus confirming transstadial transmission. Sheep proved to be a good host for the production of I. scapularis infected with this human isolate of A. phagocytophilum, which can be used as a model for future studies of the tick/pathogen interface.     

Identification of TROSPA homologue in Ixodes persulcatus Schulze,the specific vector for human Lyme borreliosis in Japan

The tick receptor for outer surface protein A (TROSPA) is an Ixodes scapularis (I. scapularis) receptor for Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease in North America. The blockade of TROSPA has been shown to reduce B. burgdorferi adherence to the I. scapularis gut in vivo. Thus, TROSPA is one of the potential targets for the development of vector–antigen-based vaccines to prevent the transmission of B. burgdorferi. The aim of this study is to identify the TROSPA gene in I. persulcatus Schulze, the specific vector for human Lyme borreliosis in Japan. The cDNA clone encoding the TROSPA-like sequence with 483 nucleotides was obtained from whole-body homogenates of fed nymphs of I. persulcatus. The putative amino acid sequence of I. persulcatus TROSPA was 88.2% and 87.8% identical to that of I. scapularis and I. ricinus, respectively. This finding will facilitate investigations on the role of I. persulcatus TROSPA and its interaction with Borrelia spp. and will have important implications on endeavors to develop a tick vaccine.     

Lyme Disease,Virginia, USA, 2000–2011

Lyme disease, caused by the bacterium Borrelia burgdorferi and transmitted in the eastern United States by the black-legged tick (Ixodes scapularis), is increasing in incidence and expanding geographically. Recent environmental modeling based on extensive field collections of host-seeking I. scapularis ticks predicted a coastal distribution of ticks in mid-Atlantic states and an elevational limit of 510 m. However, human Lyme disease cases are increasing most dramatically at higher elevations in Virginia, a state where Lyme disease is rapidly emerging. Our goal was to explore the apparent incongruity, during 2000–2011, between human Lyme disease data and predicted and observed I. scapularis distribution. We found significantly higher densities of infected ticks at our highest elevation site than at lower elevation sites. We also found that I. scapularis ticks in Virginia are more closely related to northern than to southern tick populations. Clinicians and epidemiologists should be vigilant in light of the changing spatial distributions of risk.     

Increased diversity of zoonotic pathogens and Borrelia burgdorferi strains in established versus incipient Ixodes scapularis populations across the Midwestern United States

The center of origin theory predicts that genetic diversity will be greatest near a specie’s geographic origin because of the length of time for evolution. By corollary, diversity will decrease with distance from the origin; furthermore, invasion and colonization are frequently associated with founder effects that reduce genetic variation in incipient populations. The blacklegged tick, Ixodes scapularis, harbors a suite of zoonotic pathogens, and the geographic range of the tick is expanding in the upper Midwestern United States. Therefore, we posited that diversity of I. scapularis-borne pathogens across its Midwestern range should correlate with the rate of the range expansion of this tick as well as subsequent disease emergence. Analysis of 1565 adult I. scapularis ticks from 13 sites across five Midwestern states revealed that tick infection prevalence with multiple microbial agents (Borrelia burgdorferi, Borrelia miyamotoi, Babesia odocoilei, Babesia microti, and Anaplasma phagocytophilum), coinfections, and molecular genetic diversity of B. burgdorferi all were positively correlated with the duration of establishment of tick populations, and therefore generally support the center of origin – pathogen diversity hypothesis. The observed differences across the gradient of establishment, however, were not strong and were nuanced by the high frequency of coinfections in tick populations at both established and recently-invaded tick populations. These results suggest that the invasion of ticks and their associated pathogens likely involve multiple means of pathogen introduction, rather than the conventionally presented scenario whereby infected, invading ticks are solely responsible for introducing pathogens to naïve host populations.     

Isolation of Candidatus Rickettsia vini from Belgian Ixodes arboricola ticks and propagation in tick cell lines

Candidatus Rickettsia vini was originally detected in Ixodes arboricola ticks from Spain, and subsequently reported from several other Western Palearctic countries including Belgium. Recently, the bacterium was isolated in mammalian (Vero) cell culture from macerated male I. arboricola from Czech Republic, but there have been no reports of propagation in tick cells. Here we report isolation in a tick cell line of three strains of Ca. R. vini from I. arboricola collected from nests of great tits (Parus major) in Belgium. Internal organs of one male and two engorged female ticks were dissected aseptically, added to cultures of the Rhipicephalus microplus cell line BME/CTVM23 and incubated at 28 °C. Rickettsia-like bacteria were first seen in Giemsa-stained cytocentrifuge smears between 2 and 15 weeks later. Two of the isolates grew rapidly, destroying the tick cells within 2–4 weeks of onward passage in BME/CTVM23 cells, while the third isolate grew much more slowly, only requiring subculture at 4−5-month intervals. PCR amplification of bacterial 16S rRNA and Rickettsia gltA, sca4, ompB, ompA and 17-kDa genes revealed that all three isolates were Ca. R. vini, with 100 % identity to each other and to published Ca. R. vini sequences from other geographical locations. Transmission electron microscopy revealed typical single Rickettsia bacteria in the cytoplasm of BME/CTVM23 cells. The Ca. R. vini strain isolated from the male I. arboricola tick, designated Boshoek1, was tested for ability to grow in a panel of Ixodes ricinus, Ixodes scapularis and R. microplus cell lines and in Vero cells. The Boshoek1 strain grew rapidly, causing severe cytopathic effect, in the R. microplus line BME26, the I. ricinus line IRE11 and Vero cells, more slowly in the I. ricinus line IRE/CTVM19, possibly established a low-level infection in the I. ricinus line IRE/CTVM20, and failed to infect cells of any of four I. scapularis lines over a 12-week observation period. This study confirmed the applicability of the simple tick organ-cell line co-cultivation technique for isolation of tick-borne Rickettsia spp. using BME/CTVM23 cells.     

Bartonella spp. Transmission by Ticks Not Established

Bartonella spp. infect humans and many animal species. Mainly because PCR studies have demonstrated Bartonella DNA in ticks, some healthcare providers believe that these microorganisms are transmitted by ticks. B. henselae, in particular, is regarded as being present in and transmissible by the Ixodes scapularis tick. The presence of a microbial agent within a tick, however, does not imply that the tick might transmit it during the course of blood feeding and does not confer epidemiologic importance. After a critical review of the evidence for and against tick transmission, we conclude that transmission of any Bartonella spp. by ticks, to animals or humans, has not been established. We are unaware of any well-documented case of B. henselae transmission by I. scapularis ticks.     

Repellency of novel catnip (Nepeta cataria) cultivar extracts against Ixodes scapularis and Haemaphysalis longicornis (Acari: Ixodida: Ixodidae)

Tick bites are a major public health concern due to the vector role that many tick species have in transmitting human pathogens. Synthetic repellents such as N?diethyl-meta-toluamide (DEET) remain the standard for repellency. Still, there is a need for natural commercial alternatives with similar or better properties than DEET. We evaluated the repellency of two extracts, CR3 and CR9, derived for newly developed catnip cultivars on two tick species, Ixodes scapularis and Haemaphysalis longicornis. Dose-response in vitro assays showed that CR3 and CR9 extracts have similar repellency properties to DEET. At a 20% concentration, both CR3 and CR9 extracts exhibited a repellency of 100%. Catnip extracts maintained their repellency properties for at least 8 h. In a two-choice assay, I. scapularis, but not H. longicornis, was more sensitive to CR3 than DEET. In addition, CR3 reduces the life span of I. scapularis, suggesting that it has an acaricidal effect on ticks. In summary, CR3 and CR9 catnip extracts are promising tick repellents that should be further developed, alone or in combination with other tick repellents, and tested for their use as tick repellents for humans.     

Risk maps for range expansion of the Lyme disease vector, Ixodes scapularis, in Canada now and with climate change

Background

Lyme disease is the commonest vector-borne zoonosis in the temperate world, and an emerging infectious disease in Canada due to expansion of the geographic range of the tick vector Ixodes scapularis. Studies suggest that climate change will accelerate Lyme disease emergence by enhancing climatic suitability for I. scapularis. Risk maps will help to meet the public health challenge of Lyme disease by allowing targeting of surveillance and intervention activities.

Results

A risk map for possible Lyme endemicity was created using a simple risk algorithm for occurrence of I. scapularis populations. The algorithm was calculated for each census sub-division in central and eastern Canada from interpolated output of a temperature-driven simulation model of I. scapularis populations and an index of tick immigration. The latter was calculated from estimates of tick dispersion distances by migratory birds and recent knowledge of the current geographic range of endemic I. scapularis populations. The index of tick immigration closely predicted passive surveillance data on I. scapularis occurrence, and the risk algorithm was a significant predictor of the occurrence of I. scapularis populations in a prospective field study. Risk maps for I. scapularis occurrence in Canada under future projected climate (in the 2020s, 2050s and 2080s) were produced using temperature output from the Canadian Coupled Global Climate Model 2 with greenhouse gas emission scenario enforcing 'A2' of the Intergovernmental Panel on Climate Change.

Conclusion

We have prepared risk maps for the occurrence of I. scapularis in eastern and central Canada under current and future projected climate. Validation of the risk maps provides some confidence that they provide a useful first step in predicting the occurrence of I. scapularis populations, and directing public health objectives in minimizing risk from Lyme disease. Further field studies are needed, however, to continue validation and refinement of the risk maps.     

Orally delivered fipronil-laced bait reduces juvenile blacklegged tick (Ixodes scapularis) burdens on wild white-footed mice (Peromyscus leucopus)

While the topical acaricidal treatment of rodent pathogen reservoirs has been readily explored over the past several decades, oral systemic acaricidal treatment is only recently gaining traction as an alternative approach to the management of ticks and tick-borne pathogens. Recent laboratory tests have shown promise in the effectiveness of this systemic strategy against the blacklegged tick (Ixodes scapularis) and a Canadian field evaluation was recently published, but no recent field data from the United States yet exist. With this research, we sought to field deploy a commercially available fipronil-laced bait (Kaput® Flea Control Bait, Scimetrics LLC., Wellington, CO, USA; 0.005% fipronil; Environmental Protection Agency Reg. No. 72500-28), in an alternate use targeting white-footed mice (Peromyscus leucopus) to determine bait acceptance and potential impacts to juvenile I. scapularis burdens. Bait was readily accepted by wild P. leucopus and other rodent reservoirs. An ad libitum distribution strategy as well as placing smaller volumes of fipronil-laced bait within individual Sherman traps both resulted in significant reductions (57-94%) in juvenile I. scapularis burdens as compared to control over two years. The oral delivery of systemic acaricides shows promise in reduction of I. scapularis burdens on P. leucopus and should be further explored to determine effectiveness on host-seeking tick abundances, associated pathogen infection, and potentially incorporated into integrated tick management programs.     

Potential Role of Deer Tick Virus in Powassan Encephalitis Cases in Lyme Disease–endemic Areas of New York,USA

Powassan virus, a member of the tick-borne encephalitis group of flaviviruses, encompasses 2 lineages with separate enzootic cycles. The prototype lineage of Powassan virus (POWV) is principally maintained between Ixodes cookei ticks and the groundhog (Marmota momax) or striped skunk (Mephitis mephitis), whereas the deer tick virus (DTV) lineage is believed to be maintained between Ixodes scapularis ticks and the white-footed mouse (Peromyscus leucopus). We report 14 cases of Powassan encephalitis from New York during 2004–2012. Ten (72%) of the patients were residents of the Lower Hudson Valley, a Lyme disease–endemic area in which I. scapularis ticks account for most human tick bites. This finding suggests that many of these cases were caused by DTV rather than POWV. In 2 patients, DTV infection was confirmed by genetic sequencing. As molecular testing becomes increasingly available, more cases of Powassan encephalitis may be determined to be attributable to the DTV lineage.     

Predicting distributions of blacklegged ticks (Ixodes scapularis), Lyme disease spirochetes (Borrelia burgdorferi sensu stricto) and human Lyme disease cases in the eastern United States

Lyme disease is the most commonly reported vector-borne disease in the United States (US), with approximately 300,000 -to- 40,000 cases reported annually. The blacklegged tick, Ixodes scapularis, is the primary vector of the Lyme disease-causing spirochete, Borrelia burgdorferi sensu stricto, in high incidence regions in the upper midwestern and northeastern US. Using county-level records of the presence of I. scapularis or presence of B. burgdorferi s.s. infected host-seeking I. scapularis, we generated habitat suitability consensus maps based on an ensemble of statistical models for both acarological risk metrics. Overall accuracy of these suitability models was high (AUC = 0.76 for I. scapularis and 0.86 for B. burgdorferi s.s. infected-I. scapularis). We sought to compare which acarological risk metric best described the distribution of counties reporting high Lyme disease incidence (≥10 confirmed cases/100,000 population) by setting the models to a fixed omission rate (10%). We compared the percent of high incidence counties correctly classified by the two models. The I. scapularis consensus map correctly classified 53% of high and low incidence counties, while the B. burgdorferi s.s. infected-I. scapularis consensus map classified 83% correctly. Counties classified as suitable by the B. burgdorferi s.s. map showed a 91% overlap with high Lyme disease incidence counties with over a 38-fold difference in Lyme disease incidence between high- and low-suitability counties. A total of 288 counties were classified as highly suitable for B. burgdorferi s.s., but lacked records of infected-I. scapularis and were not classified as high incidence. These counties were considered to represent a leading edge for B. burgdorferi s.s. infection in ticks and humans. They clustered in Illinois, Indiana, Michigan, and Ohio. This information can aid in targeting tick surveillance and prevention education efforts in counties where Lyme disease risk may increase in the future.     

An ecological approach to predict areas with established populations of Ixodes scapularis in Quebec,Canada

Public health management of Lyme disease (LD) is a dynamic challenge in Canada. Climate warming is driving the northward expansion of suitable habitat for the tick vector, Ixodes scapularis. Information about tick population establishment is used to inform the risk of LD but is challenged by sampling biases from surveillance data. Misclassifying areas as having no established tick population underestimates the LD risk classification.We used a logistic regression model at the municipal level to predict the probability of I. scapularis population establishment based on passive tick surveillance data during the period of 2010-2017 in southern Quebec. We tested for the effect of abiotic and biotic factors hypothesized to influence tick biology and ecology. Additional variables controlled for sampling biases in the passive surveillance data.In our final selected model, tick population establishment was positively associated with annual cumulative degree-days > 0°C, precipitation and deer density, and negatively associated with coniferous and mixed forest types. Sampling biases from passive tick surveillance were controlled for using municipal population size and public health instructions on tick submissions. The model performed well as indicated by an area under the curve (AUC) of 0.92, sensitivity of 86% and specificity of 81%.Our model enables prediction of I. scapularis population establishment in areas which lack data from passive tick surveillance and may improve the sensitivity of LD risk categorization in these areas. A more sensitive system of LD risk classification is important for increasing awareness and use of protective measures employed against ticks, and decreasing the morbidity associated with LD.     

Tick abundance and diversity are substantially lower in thinned vs. unthinned forests in the New Jersey Pinelands National Reserve,USA

Forest thinning is a management tool used in the New Jersey Pinelands and elsewhere to improve forest health and resilience, mitigate wildfire risk, and manage for wildlife. Forest thinning leads to warmer drier microclimates, which have been shown in both field and laboratory studies to reduce tick survival and reproduction. To directly assess the effects of forest thinning on the abundance and diversity of ticks and on the prevalence of tick-borne human pathogens, we sampled ticks weekly from March to November 2021 at three replicated pairs of thinned and unthinned forest sites composed primarily of pitch-pine, shortleaf pine, and various oak species. We characterized microclimate in the understory and forest floor at each sampling plot by deploying multiple data loggers to monitor temperature and relative humidity throughout the study period. As expected, we found that thinned plots were significantly drier and warmer than unthinned plots. We also found that average questing tick abundance was 92% lower in thinned as compared with unthinned plots. Of the three main tick species collected in unthinned plots (Amblyomma americanum, Ixodes scapularis, and Dermacentor albipictus) only A. americanum and a single I. scapularis were collected in thinned plots. Prevalence of Ehrlichia species in A. americanum did not differ between treatments, and the sole I. scapularis collected in a thinned plot was infected with Borrelia burgdorferi sensu lato. However, the significant and much lower tick abundance in thinned plots indicates a lower risk of human-tick encounters. Our results add to the growing evidence that landscape and forest management can reduce local tick abundance, thereby reducing tick-borne disease risk.     

The identification of tick autophagy-related genes in Ixodes scapularis responding to amino acid starvation

Ticks are obligate hematophagous arthropods and must tolerate starvation during off‐host periods. Macroautophagy (hereafter autophagy) is a well-conserved self-eating mechanism of cell survival and is essential for recycling cellular contents during periods of starvation, stress, and injury in organisms. Although the genome sequence of Ixodes scapularis (Say) is available, the characteristics and functions of autophagy-related gene families remain largely unknown. To advance our understanding of autophagy in I. scapularis, we used comprehensive genomic approaches to identify Atg genes. Homologues of 14 Atg genes were identified, and their protein motif compositions were predicted. Phylogenetic analysis indicated that ATGs in I. scapularis were evolutionarily closely related to their homologues in Haemaphysalis longicornis and Rhipicephalus microplus ticks. Expression patterns of Atg genes differed across tick developmental stages. Immunofluorescence results by monodansylcadaverine (MDC) staining indicated that autophagy was activated after amino acid starvation treatments in I. scapularis embryo-derived cell lines ISE6 and IDE8. Subsequently, the expression of key Atg genes involved in autophagy pathway in both cell lines were examined. In ISE6 cells, the expression levels of three Atg genes (Atg4B, Atg6 and Atg8A) increased significantly after amino acid starvation; similarly, four Atg genes (Atg4A, Atg4B, Atg6 and Atg8B) were upregulated in IDE8 cells in response to starvation. In parallel, the MDC and lysotracker staining results indicated that autophagy was triggered after amino acid starvation treatments in R. microplus embryo-derived cell line BME26. Our observations showed that Atg family genes are highly conserved in ticks and function in autophagy pathway induced by amino acid starvation. These results also provide valuable insight for further autophagy-related research as a new strategy for blocking the transmission of tick-borne pathogens.