First Recognized Human Bite in the United States by the Asian Longhorned Tick, Haemaphysalis longicornis.

We present the case summary of the first human recognized to have been bitten by the Haemaphysalis longicornis tick in the United States, which occurred in New York State. Subsequent field studies confirmed that this tick was present in multiple geographic locations near the patient's residence, including on manicured lawns.

Assessment of Duration of Tick Feeding by the Scutal Index Reduces Need for Antibiotic Prophylaxis After Ixodes scapularis Tick Bites.

Doxycycline is recommended for persons with Ixodes scapularis tick bites in certain geographic areas, if the tick had fed for at least 36 hours. Based on the scutal index, over 40% of I. scapularis tick bites from patients seen at the Lyme Disease Diagnostic Center did not warrant antibiotic prophylaxis.

Multistate Infestation with the Exotic Disease-Vector Tick Haemaphysalis longicornis - United States, August 2017-September 2018.

Haemaphysalis longicornis is a tick indigenous to eastern Asia and an important vector of human and animal disease agents, resulting in such outcomes as human hemorrhagic fever and reduction of production in dairy cattle by 25%. H. longicornis was discovered on a sheep in New Jersey in August 2017 (1). This was the first detection in the United States outside of quarantine. In the spring of 2018, the tick was again detected at the index site, and later, in other counties in New Jersey, in seven other states in the eastern United States, and in Arkansas. The hosts included six species of domestic animals, six species of wildlife, and humans. To forestall adverse consequences in humans, pets, livestock, and wildlife, several critical actions are indicated, including expanded surveillance to determine the evolving distribution of H. longicornis, detection of pathogens that H. longicornis currently harbors, determination of the capacity of H. longicornis to serve as a vector for a range of potential pathogens, and evaluation of effective agents and methods for the control of H. longicornis.

Microbiome changes through ontogeny of a tick pathogen vector.

Blacklegged ticks (Ixodes scapularis) are one of the most important pathogen vectors in the United States, responsible for transmitting Lyme disease and other tick-borne diseases. The structure of a host's microbial community has the potential to affect the ecology and evolution of the host. We employed high-throughput sequencing of the 16S rRNA gene V3-V4 hypervariable regions in the first study to investigate the tick microbiome across all developmental stages (larvae, nymphs, adults). In addition to field-collected life stages, newly hatched laboratory-reared larvae were studied to determine the baseline microbial community structure and to assess transovarial transmission. We also targeted midguts and salivary glands due to their importance in pathogen maintenance and transmission. Over 100 000 sequences were produced per life stage replicate. Rickettsia was the most abundant bacterial genus across all sample types matching mostly the Ixodes rickettsial endosymbionts, and its proportion decreased as developmental stage progressed, with the exception of adult females that harboured a mean relative abundance of 97.9%. Laboratory-reared larvae displayed the lowest bacterial diversity, containing almost exclusively Rickettsia. Many of the remaining bacteria included genera associated with soil, water and plants, suggesting environmental acquisition while off-host. Female organs exhibited significantly different ß-diversity than the whole tick from which they were derived. Our results demonstrate clear differences in both α- and ß-diversity among tick developmental stages and between tick organs and the tick as a whole. Furthermore, field-acquired bacteria appear to be very important to the overall internal bacterial community of this tick species, with influence from the host bloodmeal appearing limited.

Variation in the Microbiota of Ixodes Ticks with Regard to Geography, Species, and Sex.

Ixodes scapularis is the principal vector of Lyme disease on the East Coast and in the upper Midwest regions of the United States, yet the tick is also present in the Southeast, where Lyme disease is absent or rare. A closely related species, I. affinis, also carries the pathogen in the South but does not seem to transmit it to humans. In order to better understand the geographic diversity of the tick, we analyzed the microbiota of 104 adult I. scapularis and 13 adult I. affinis ticks captured in 19 locations in South Carolina, North Carolina, Virginia, Connecticut, and New York. Initially, ticks from 4 sites were analyzed by 454 pyrosequencing. Subsequently, ticks from these sites plus 15 others were analyzed by sequencing with an Illumina MiSeq machine. By both analyses, the microbiomes of female ticks were significantly less diverse than those of male ticks. The dissimilarity between tick microbiomes increased with distance between sites, and the state in which a tick was collected could be inferred from its microbiota. The genus Rickettsia was prominent in all locations. Borrelia was also present in most locations and was present at especially high levels in one site in western Virginia. In contrast, members of the family Enterobacteriaceae were very common in North Carolina I. scapularis ticks but uncommon in I. scapularis ticks from other sites and in North Carolina I. affinis ticks. These data suggest substantial variations in the Ixodes microbiota in association with geography, species, and sex.

American Black Bears as Hosts of Blacklegged Ticks (Acari: Ixodidae) in the Northeastern United States.

Ticks and whole blood were collected from American black bears (Ursus americanus Pallas) between October 2011 and October 2012 across four counties in northwestern New Jersey, an area where blacklegged ticks (Ixodes scapularis Say) and their associated tick-borne pathogens are prevalent. Adult American dog ticks (Dermacentor variabilis Say) were the most frequently collected tick species in late spring, whereas adult and nymphal blacklegged ticks were found in both the late spring and fall months. Additionally, for blacklegged ticks, we determined the quality of bloodmeals that females acquired from black bears compared with bloodmeals from white-tailed deer (Odocoileus virginianus Zimmerman), the most important host for the adult stage of this tick species. Measures of fecundity after feeding on each host species were not significantly different, suggesting that the bloodmeal a female blacklegged tick acquires from a black bear is of similar quality to that obtained from a white-tailed deer. These results establish the American black bear as both a host and quality bloodmeal source to I. scapularis. Thus, black bears may help support blacklegged tick populations in areas where they are both present. In addition, samples of black bear blood were tested for DNA presence of three tick-borne pathogens. Anaplasma phagocytophilum Foggie and Babesia microti Franca were found in 9.2 and 32.3% of blood samples, respectively. All blood samples were quantitative polymerase chain reaction-negative for Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt, & Brenner. Although circulating pathogens were found in blood, the status of black bears as reservoirs for these pathogens remains unknown.

Assessing the impact of areal unit selection and the modifiable areal unit problem on associative statistics between cases of tick-borne disease and entomological indices.

The modifiable areal unit problem (MAUP) is a cause of statistical and visual bias when aggregating data according to spatial units, particularly when spatial units may be changed arbitrarily. The MAUP is a concern in vector-borne disease research when entomological metrics gathered from point-level sampling data are related to epidemiological data aggregated to administrative units like counties or ZIP Codes. Here, we assess the statistical impact of the MAUP when calculating correlations between randomly aggregated cases of anaplasmosis in New York State during 2017 and a geostatistical layer of an entomological risk index for Anaplasma phagocytophilum in blacklegged ticks (Ixodes scapularis Say, Acari: Ixodidae) collected during the fall of 2017. Correlations were also calculated using various administrative boundaries for comparison. We also demonstrate the impact of the MAUP on data visualization using choropleth maps and offer pycnophylactic interpolation as an alternative. Polygon simulations indicate that increasing the number of polygons decreases correlation coefficients and their variability. Correlation coefficients calculated using ZIP Code tabulation area and Census tract polygons were beyond 4 standard deviations from the mean of the simulated correlation coefficients. These results indicate that using smaller polygons may not best incorporate the geographical context of the tick-borne disease system, despite the tendency of researchers to strive for more granular spatial data and associations.

Spatiotemporal distribution of Borrelia miyamotoi (Spirochaetales: Spirochaetaceae) and coinfection with other tick-borne pathogens in host-seeking Ixodes scapularis (Acari: Ixodidae) from New York State, USA.

Blacklegged ticks (Ixodes scapularis Say, Acari: Ixodidae) were collected from 432 locations across New York State (NYS) during the summer and autumn of 2015-2020 to determine the prevalence and geographic distribution of Borrelia miyamotoi (Spirochaetales: Spirochaetaceae) and coinfections with other tick-borne pathogens. A total of 48,386 I. scapularis were individually analyzed using a multiplex real-time polymerase chain reaction assay to simultaneously detect the presence of Bo. miyamotoi, Borrelia burgdorferi (Spirochaetales: Spirochaetaceae), Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and Babesia microti (Piroplasmida: Babesiidae). Overall prevalence of Bo. miyamotoi in host-seeking nymphs and adults varied geographically and temporally at the regional level. The rate of polymicrobial infection in Bo. miyamotoi-infected ticks varied by developmental stage, with certain co-infections occurring more frequently than expected by chance. Entomological risk of exposure to Bo. miyamotoi-infected nymphal and adult ticks (entomological risk index [ERI]) across NYS regions in relation to human cases of Bo. miyamotoi disease identified during the study period demonstrated spatial and temporal variation. The relationship between select environmental factors and Bo. miyamotoi ERI was explored using generalized linear mixed effects models, resulting in different factors significantly impacting ERI for nymphs and adult ticks. These results can inform estimates of Bo. miyamotoi disease risk and further our understanding of Bo. miyamotoi ecological dynamics in regions where this pathogen is known to occur.

First Detection of the Invasive Asian Longhorned Tick (Acari: Ixodidae) on Migratory Passerines in the Americas.

The Asian longhorned tick (Haemaphysalis longicornis Neumann), native to East Asia, was first reported in the United States in 2017 and is now established in at least 17 states. Haemaphysalis longicornis feeds on birds in its range outside of the United States, and migratory birds disperse this tick and tick-borne pathogens. However, early studies in the United States did not find H. longicornis on migrating passerine birds. The transport of the parthenogenetic H. longicornis on birds has the potential to greatly expand its range. We report the first discovery of H. longicornis on migratory passerine birds in the Americas.

Phylogeographic dynamics of the arthropod vector, the blacklegged tick (Ixodes scapularis).

BACKGROUND: The emergence of vector-borne pathogens in novel geographic areas is regulated by the migration of their arthropod vectors. Blacklegged ticks (Ixodes scapularis) and the pathogens they vector, including the causative agents of Lyme disease, babesiosis and anaplasmosis, continue to grow in their population sizes and to expand in geographic range. Migration of this vector over the previous decades has been implicated as the cause of the re-emergence of the most prevalent infectious diseases in North America. METHODS: We systematically collected ticks from across New York State (hereafter referred to as New York) from 2004 to 2017 as part of routine tick-borne pathogen surveillance in the state. This time frame corresponds with an increase in range and incidence of tick-borne diseases within New York. We randomly sampled ticks from this collection to explore the evolutionary history and population dynamics of I. scapularis. We sequenced the mitochondrial genomes of each tick to characterize their current and historical spatial genetic structure and population growth using phylogeographic methods. RESULTS: We sequenced whole mitochondrial genomes from 277 ticks collected across New York between 2004 and 2017. We found evidence of population genetic structure at a broad geographic scale due to differences in the relative abundance, but not the composition, of haplotypes among sampled ticks. Ticks were often most closely related to ticks from the same and nearby collection sites. The data indicate that both short- and long-range migration events shape the population dynamics of blacklegged ticks in New York. CONCLUSIONS: We detailed the population dynamics of the blacklegged tick (Ixodes scapularis) in New York during a time frame in which tick-borne diseases were increasing in range and incidence. Migration of ticks occurred at both coarse and fine scales in the recent past despite evidence of limits to gene flow. Past and current tick population dynamics have implications for further range expansion as habitat suitability for ticks changes due to global climate change. Analyses of mitochondrial genome sequencing data will expound upon previously identified drivers of tick presence and abundance as well as identify additional drivers. These data provide a foundation on which to generate testable hypotheses on the drivers of tick population dynamics occurring at finer scales.

Predicting spatio-temporal population patterns of Borrelia burgdorferi, the Lyme disease pathogen.

The causative bacterium of Lyme disease, Borrelia burgdorferi, expanded from an undetected human pathogen into the etiologic agent of the most common vector-borne disease in the United States over the last several decades. Systematic field collections of the tick vector reveal increases in the geographic range and prevalence of B. burgdorferi-infected ticks that coincided with increases in human Lyme disease incidence across New York State.We investigate the impact of environmental features on the population dynamics of B. burgdorferi. Analytical models developed using field collections of nearly 19,000 nymphal Ixodes scapularis and spatially and temporally explicit environmental features accurately explained the variation in the nymphal infection prevalence of B. burgdorferi across space and time.Importantly, the model identified environmental features reflecting landscape ecology, vertebrate hosts, climatic metrics, climate anomalies and surveillance efforts that can be used to predict the biogeographical patterns of B. burgdorferi-infected ticks into future years and in previously unsampled areas.Forecasting the distribution and prevalence of a pathogen at fine geographic scales offers a powerful strategy to mitigate a serious public health threat. Synthesis and applications. A decade of environmental and tick data was collected to create a model that accurately predicts the infection prevalence of Borrelia burgdorferi over space and time. This predictive model can be extrapolated to create a high-resolution risk map of the Lyme disease pathogen for future years that offers an inexpensive approach to improve both ecological management and public health strategies to mitigate disease risk.

Occurrence of soil- and tick-borne fungi and related virulence tests for pathogenicity to Ixodes scapularis (Acari: Ixodidae).

Ixodes scapularis Say, the blacklegged tick, vectors Borrelia burgdorferi Johnson et al. 1984, the bacterium that causes Lyme disease, the most important vector-borne disease in the United States. Efforts to reduce I. scapularis populations are shifting toward the development of biological control methods. Currently, only a few entomopathogenic fungal species are considered virulent to ticks. We hypothesized that these species may not represent the most abundant local taxa that would be pathogenic to ticks in situ. To identify potential entomopathogenic fungi at a study site in Westchester County, New York, we sampled soils and ticks, extracted and amplified the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA), and compared sequences with those in GenBank. Over three sampling periods from June 2007 to May 2008, 70 fungal taxa were isolated and identified from soils (48 taxa) and ticks (27 taxa; 5 taxa were found both in soil and on ticks) collected in this study, encompassing species in 25 different genera. In laboratory bioassays, 15 fungal taxa were found to be significantly virulent, although none of these were previously considered common pathogens of I. scapularis. Two species, Hypocrea lixii Patouillard 1891 and Penicillium soppii K. M. Zalessky 1927, were tested in field trials by spraying suspensions on forested plots. Mean tick mortality was 71% after treatment with H. lixii, 58% after treatment with P. soppii, and 32% in the control plots. The complete diversity of entomopathogenic fungal species at this site is yet to be defined, but, in general, such fungi appear to be more common in forest habitats where I. scapularis resides than previously thought. Examination of intact fungal communities can provide information that serves as the foundation for site-specific biocontrol programs.

Optimal Collection Methods for Asian Longhorned Ticks (Ixodida: Ixodidae) in the Northeast United States.

The Asian longhorned tick, Haemaphysalis longicornis Neumann, is an invasive species in the United States. Since its earliest recorded presence in West Virginia in 2010, H. longicornis has been reported from 15 states. While its public health significance in the United States is unclear, globally it transmits pathogens that infect livestock and humans, causing economic losses and substantial morbidity. Management and control of H. longicornis requires knowledge of its biology, ecology, and distribution. Here, we address the need for effective collection methods for host-seeking H. longicornis as an important step for accurately assessing tick abundance and potential disease risk. The number of H. longicornis collected were compared across three collection methods (dragging, sweeping, CO2 traps) and three tick check distances (5 m, 10 m, and 20 m) were compared for dragging and sweeping. Field collections were conducted from June through August 2019 in Westchester County, New York, and ticks were grouped by life stage to assess collection method efficiency. Results indicated that implementing shorter (5 m) tick check distance was ideal for adult and nymphal collections. The dragging method proved better than sweeping for adult collections; however, there was no significant difference between the methods for nymphal collections, at any tick check distance evaluated. CO2 traps attracted H. longicornis, but additional research is necessary to devise an effective tick retaining method before the traps can be implemented in the field. The results are presented to inform and support H. longicornis surveillance and control programs across the nation.

Seasonal Activity of Haemaphysalis longicornis (Acari: Ixodidae) in Southern New York State.

The Asian longhorned tick, Haemaphysalis longicornis Neumann, is a species native to eastern Asia that has recently been discovered in the United States. In its native range, H. longicornis transmits pathogens that cause disease in humans and livestock. It is currently unknown whether H. longicornis will act as a vector in the United States. Understanding its seasonal activity patterns will be important in identifying which times of the year represent greatest potential risk to humans and livestock should this species become a threat to animal or public health. A study site was established in Yonkers, NY near the residence associated with the first reported human bite from H. longicornis in the United States. Ticks were collected once each week from July 2018 to November 2019. Haemaphysalis longicornis larvae were most active from August to November, nymphs from April to July, and adult females from June to September. This pattern of activity suggests that H. longicornis is capable of completing a generation within a single year and matches the patterns observed in its other ranges in the northern hemisphere. The data presented here contribute to a growing database for H. longicornis phenology in the northeastern United States. Potential implications of the short life cycle for the tick's vectorial capacity are discussed.

Spatio-temporal variation in environmental features predicts the distribution and abundance of Ixodes scapularis.

Many species have experienced dramatic changes in both geographic range and population sizes in recent history. Increases in the geographic range or population size of disease vectors have public health relevance as these increases often precipitate the emergence of infectious diseases in human populations. Accurately identifying environmental factors affecting the biogeographic patterns of vector species is a long-standing analytical challenge, stemming from a paucity of data capturing periods of rapid changes in vector demographics. We systematically investigated the occurrence and abundance of nymphal Ixodes scapularis ticks at 532 sampling locations throughout New York State (NY), USA, between 2008 and 2018, a time frame that encompasses the emergence of diseases vectored by these ticks. Analyses of these field-collected data demonstrated a range expansion into northern and western NY during the last decade. Nymphal abundances increased in newly colonised areas, while remaining stable in areas with long-standing populations over the last decade. These trends in the geographic range and abundance of nymphs correspond to both the geographic expansion of human Lyme disease cases and increases in incidence rates. Analytic models fitted to these data incorporating time, space, and environmental factors, accurately identified drivers of the observed changes in nymphal occurrence and abundance. These models accounted for the spatial and temporal variation in the occurrence and abundance of nymphs and can accurately predict nymphal population patterns in future years. Forecasting disease risk at fine spatial scales prior to the transmission season can influence both public health mitigation strategies and individual behaviours, potentially impacting tick-borne disease risk and subsequently human disease incidence.

Environmental Determinants of Aedes albopictus Abundance at a Northern Limit of Its Range in the United States.

Aedes albopictus is a vector of arboviruses with high rates of morbidity and mortality. The northern limit of Ae. albopictus in the northeastern United States runs through New York state (NYS) and Connecticut. We present a landscape-level analysis of mosquito abundance measured by daily counts of Ae. albopictus from 338 trap sites in 12 counties during May-September 2017. During the study period, the mean number of Ae. albopictus caught per day of trapping across all sites was 3.21. We constructed four sets of negative binomial generalized linear models to evaluate how trapping methodology, land cover, as well as temperature and precipitation at multiple time intervals influenced Ae. albopictus abundance. Biogents-Sentinel (BGS) traps were 2.78 times as efficient as gravid traps and 1.49 times as efficient as CO2-baited CDC light traps. Greater proportions of low- and medium-intensity development and low proportions of deciduous cover around the trap site were positively associated with increased abundance, as were minimum winter temperature and March precipitation. The cumulative precipitation within a 28-day time window before the date of collection had a nonlinear relationship with abundance, such that greater cumulative precipitation was associated with increased abundance until approximately 70 mm, above which there was a decrease in abundance. We concluded that populations are established in Nassau, Suffolk, and New York City counties in NYS; north of these counties, the species is undergoing population invasion and establishment. We recommend that mosquito surveillance programs monitoring the northward invasion of Ae. albopictus place BGS traps at sites chosen with respect to land cover.

Molecular Characterization of Haemaphysalis Species and a Molecular Genetic Key for the Identification of Haemaphysalis of North America.

Haemaphysalis longicornis (Acari: Ixodidae), the Asian longhorned tick, is native to East Asia, but has become established in Australia and New Zealand, and more recently in the United States. In North America, there are other native Haemaphysalis species that share similar morphological characteristics and can be difficult to identify if the specimen is damaged. The goal of this study was to develop a cost-effective and rapid molecular diagnostic assay to differentiate between exotic and native Haemaphysalis species to aid in ongoing surveillance of H. longicornis within the United States and help prevent misidentification. We demonstrated that restriction fragment length polymorphisms (RFLPs) targeting the 16S ribosomal RNA and the cytochrome c oxidase subunit I (COI) can be used to differentiate H. longicornis from the other Haemaphysalis species found in North America. Furthermore, we show that this RFLP assay can be applied to Haemaphysalis species endemic to other regions of the world for the rapid identification of damaged specimens. The work presented in this study can serve as the foundation for region specific PCR-RFLP keys for Haemaphysalis and other tick species and can be further applied to other morphometrically challenging taxa.

First glimpse into the origin and spread of the Asian longhorned tick, Haemaphysalis longicornis, in the United States.

Established populations of Asian longhorned ticks (ALT), Haemaphysalis longicornis, were first identified in the United States (US) in 2017 by sequencing the mitochondrial cytochrome c oxidase subunit I (cox1) 'barcoding' locus followed by morphological confirmation. Subsequent investigations detected ALT infestations in 12, mostly eastern, US states. To gain information on the origin and spread of US ALT, we (1) sequenced cox1 from ALT populations across 9 US states and (2) obtained cox1 sequences from potential source populations [China, Japan and Republic of Korea (ROK) as well as Australia, New Zealand and the Kingdom of Tonga (KOT)] both by sequencing and by downloading publicly available sequences in NCBI GenBank. Additionally, we conducted epidemiological investigations of properties near its initial detection locale in Hunterdon County, NJ, as well as a broader risk analysis for importation of ectoparasites into the area. In eastern Asian populations (China/Japan/ROK), we detected 35 cox1 haplotypes that neatly clustered into two clades with known bisexual versus parthenogenetic phenotypes. In Australia/New Zealand/KOT, we detected 10 cox1 haplotypes all falling within the parthenogenetic cluster. In the United States, we detected three differentially distributed cox1 haplotypes from the parthenogenetic cluster, supporting phenotypic evidence that US ALT are parthenogenetic. While none of the source populations examined had all three US cox1 haplotypes, a phylogeographic network analysis supports a northeast Asian source for the US populations. Within the United States, epidemiological investigations indicate ALT can be moved long distances by human transport of animals, such as horses and dogs, with smaller scale movements on wildlife. These results have relevant implications for efforts aimed at minimizing the spread of ALT in the United States and preventing additional exotic tick introductions.

Isolation of entomopathogenic fungi from soils and Ixodes scapularis (Acari: Ixodidae) ticks: prevalence and methods.

Entomopathogenic fungi are commonly found in forested soils that provide tick habitat, and many species are pathogenic to Ixodes scapularis Say, the blacklegged tick. As a first step to developing effective biocontrol strategies, the objective of this study was to determine the best methods to isolate entomopathogenic fungal species from field-collected samples of soils and ticks from an Eastern deciduous forest where I. scapularis is common. Several methods were assessed: (1) soils, leaf litter, and ticks were plated on two types of media; (2) soils were assayed for entomopathogenic fungi using the Galleria bait method; (3) DNA from internal transcribed spacer (ITS) regions of the nuclear ribosomal repeat was extracted from pure cultures obtained from soils, Galleria, and ticks and was amplified and sequenced; and (4) DNA was extracted directly from ticks, amplified, and sequenced. We conclude that (1) ticks encounter potentially entomopathogenic fungi more often in soil than in leaf litter, (2) many species of potentially entomopathogenic fungi found in the soil can readily be cultured, (3) the Galleria bait method is a sufficiently efficient method for isolation of these fungi from soils, and (4) although DNA extraction from ticks was not possible in this study because of small sample size, DNA extraction from fungi isolated from soils and from ticks was successful and provided clean sequences in 100 and 73% of samples, respectively. A combination of the above methods is clearly necessary for optimal characterization of entomopathogenic fungi associated with ticks in the environment.

Transient influence of blood meal and natural environment on blacklegged tick bacterial communities.

Blacklegged ticks (Ixodes scapularis) spend the majority of their life cycle off host, typically in woodland habitat, but require a blood meal at each of three life stages (larva, nymph, adult) to reach maturity and reproduce. Blood feeding usually lasts for several days each time and as blood is imbibed, a range of known pathogens from the host may also be acquired. Using next generation sequencing of 16S rRNA gene amplicons, we examined the influence of host blood meal on the internal bacterial community within nymphal blacklegged ticks across host-seeking, feeding, blood meal digestion, and after molting into the adult stage. Results demonstrate bacterial community structuring across host and ticks with 287 taxa found exclusively in ticks, suggesting the field environment plays a significant role in shaping the internal tick microbiome. A decrease in bacterial diversity was noted from unfed nymphs through feeding/digestion and after molting into adults, suggesting that bacterial species are lost during the corresponding physiological changes. The similarity in biochemical pathways across the different tick categories suggests that the loss of bacterial taxa does not mirror a large change in microbial function. Ticks likely lose bacterial taxa after feeding, but continual exposure to bacteria from the field environment counters this loss.