Christine P. Zolnik

Population genomics of the Anthropocene: urbanization is negatively associated with genome-wide variation in white-footed mouse populations.

Urbanization results in pervasive habitat fragmentation and reduces standing genetic variation through bottlenecks and drift. Loss of genomewide variation may ultimately reduce the evolutionary potential of animal populations experiencing rapidly changing conditions. In this study, we examined genomewide variation among 23 white‐footed mouse (Peromyscus leucopus) populations sampled along an urbanization gradient in the New York City metropolitan area. Genomewide variation was estimated as a proxy for evolutionary potential using more than 10 000 single nucleotide polymorphism (SNP) markers generated by ddRAD‐Seq. We found that genomewide variation is inversely related to urbanization as measured by percent impervious surface cover, and to a lesser extent, human population density. We also report that urbanization results in enhanced genomewide differentiation between populations in cities. There was no pattern of isolation by distance among these populations, but an isolation by resistance model based on impervious surface significantly explained patterns of genetic differentiation. Isolation by environment modeling also indicated that urban populations deviate much more strongly from global allele frequencies than suburban or rural populations. This study is the first to examine loss of genomewide SNP variation along an urban‐to‐rural gradient and quantify urbanization as a driver of population genomic patterns.

Duration of Exposure to Suboptimal Atmospheric Moisture Affects Nymphal Blacklegged Tick Survival

Abstract The biological processes affecting Ixodes scapularis Say survival are complex. Understanding these processes will be beneficial for predicting tick distribution and population dynamics. This research shows that the duration for which nymphal ticks are exposed to drying air is an important factor for their survival. Experimental analysis of variance results show that duration of exposure to dry air (duration) is as important as vapor pressure deficit (relative humidity) (duration, relative humidity, P < 0.0001). Ticks do not survive when exposed to dry air for long periods; however, the return of humid air within 4–8 h has as large a positive impact on tick survival, as does constant humid air. This experiment exposes nymphal ticks to conditions of suboptimal humidity for different durations and then returns them to saturated conditions that are more typical of daily relative humidity fluctuations experienced during summer in southern New England forests.

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 hosts 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 β‐diversity than the whole tick from which they were derived. Our results demonstrate clear differences in both α‐ and β‐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.

To beat or not to beat a tick: comparison of DNA extraction methods for ticks (Ixodes scapularis)

Background. Blacklegged ticks (Ixodes scapularis) are important disease vectors in the United States, known to transmit a variety of pathogens to humans, including bacteria, protozoa, and viruses. Their importance as a disease vector necessitates reliable and comparable methods for extracting microbial DNA from ticks. Furthermore, to explore the population genetics or genomics of this tick, appropriate DNA extraction techniques are needed for both the vector and its microbes. Although a few studies have investigated different methods of DNA isolation from ticks, they are limited in the number and types of DNA extraction and lack species-specific quantification of DNA yield. Methods. Here we determined the most efficient and consistent method of DNA extraction from two different developmental stages of I. scapularis—nymph and adult—that are the most important for disease transmission. We used various methods of physical disruption of the hard, chitinous exoskeleton, as well as commercial and non-commercial DNA isolation kits. To gauge the effectiveness of these methods, we quantified the DNA yield and confirmed the DNA quality via PCR of both tick and microbial genetic material. Results. DNA extraction using the Thermo GeneJET Genomic DNA Purification Kit resulted in the highest DNA yields and the most consistent PCR amplification when combined with either cutting or bead beating with select matrices across life stages. DNA isolation methods using ammonium hydroxide as well as the MoBio PowerSoil kit also produced strong and successful PCR amplification, but only for females. Discussion. We contrasted a variety of readily available methods of DNA extraction from single individual blacklegged ticks and presented the results through a quantitative and qualitative assessment.

No Observed Effect of Landscape Fragmentation on Pathogen Infection Prevalence in Blacklegged Ticks (Ixodes scapularis) in the Northeastern United States

Pathogen prevalence within blacklegged ticks (Ixodes scapularis Say, 1821) tends to vary across sites and geographic regions, but the underlying causes of this variation are not well understood. Efforts to understand the ecology of Lyme disease have led to the proposition that sites with higher host diversity will result in lower disease risk due to an increase in the abundance of inefficient reservoir species relative to the abundance of species that are highly competent reservoirs. Although the Lyme disease transmission cycle is often cited as a model for this “dilution effect hypothesis”, little empirical evidence exists to support that claim. Here we tested the dilution effect hypothesis for two pathogens transmitted by the blacklegged tick along an urban-to-rural gradient in the northeastern United States using landscape fragmentation as a proxy for host biodiversity. Percent impervious surface and habitat fragment size around each site were determined to assess the effect of landscape fragmentation on nymphal blacklegged tick infection with Borrelia burgdorferi and Anaplasma phagocytophilum. Our results do not support the dilution effect hypothesis for either pathogen and are in agreement with the few studies to date that have tested this idea using either a landscape proxy or direct measures of host biodiversity.

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

ABSTRACT 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.

Novel ITS1 Fungal Primers for Characterization of the Mycobiome

The mycobiome constitutes all the fungal organisms within an environment or biological niche. The fungi are eukaryotes, are extremely heterogeneous, and include yeasts and molds that colonize humans as part of the microbiome. In addition, fungi can also infect humans and cause disease. Characterization of the bacterial component of the microbiome was revolutionized by 16S rRNA gene fragment amplification, next-generation sequencing technologies, and bioinformatics pipelines. Characterization of the mycobiome has often not been included in microbiome studies because of limitations in amplification systems. This report revisited the selection of PCR primers that amplify the fungal ITS1 region. We have identified primers with superior identification of fungi present in the database. We have compared the new primer sets against those previously used in the literature and show a significant improvement in read count and taxon identification. These primers should facilitate the study of fungi in human physiology and disease states. ABSTRACT Studies of the human microbiome frequently omit characterization of fungal communities (the mycobiome), which limits our ability to investigate how fungal communities influence human health. The internal transcribed spacer 1 (ITS1) region of the eukaryotic ribosomal cluster has features allowing for wide taxonomic coverage and has been recognized as a suitable barcode region for species-level identification of fungal organisms. We developed custom ITS1 primer sets using iterative alignment refinement. Primer performance was evaluated using in silico testing and experimental testing of fungal cultures and human samples. Using an expanded novel reference database, SIS (18S-ITS1-5.8S), the newly designed primers showed an average in silico taxonomic coverage of 79.9% ± 7.1% compared to a coverage of 44.6% ± 13.2% using previously published primers (P = 0.05). The newly described primer sets recovered an average of 21,830 ± 225 fungal reads from fungal isolate culture samples, whereas the previously published primers had an average of 3,305 ± 1,621 reads (P = 0.03). Of note was an increase in the taxonomic coverage of the Candida genus, which went from a mean coverage of 59.5% ± 13% to 100.0% ± 0.0% (P = 0.0015) comparing the previously described primers to the new primers, respectively. The newly developed ITS1 primer sets significantly improve general taxonomic coverage of fungal communities infecting humans and increased read depth by an order of magnitude over the best-performing published primer set tested. The overall best-performing primer pair in terms of taxonomic coverage and read recovery, ITS1-30F/ITS1-217R, will aid in advancing research in the area of the human mycobiome. IMPORTANCE The mycobiome constitutes all the fungal organisms within an environment or biological niche. The fungi are eukaryotes, are extremely heterogeneous, and include yeasts and molds that colonize humans as part of the microbiome. In addition, fungi can also infect humans and cause disease. Characterization of the bacterial component of the microbiome was revolutionized by 16S rRNA gene fragment amplification, next-generation sequencing technologies, and bioinformatics pipelines. Characterization of the mycobiome has often not been included in microbiome studies because of limitations in amplification systems. This report revisited the selection of PCR primers that amplify the fungal ITS1 region. We have identified primers with superior identification of fungi present in the database. We have compared the new primer sets against those previously used in the literature and show a significant improvement in read count and taxon identification. These primers should facilitate the study of fungi in human physiology and disease states.

Applications of a Climatic Water Budget to Tick-Borne Disease Research

Tools initially developed for analyzing climate or the environment are often used subsequently by scientists in more applied areas of research. The Thornthwaite—Mather (T-M) water budget equation was primarily developed to calculate a water budget, which, of course, had obvious uses for irrigation purposes. But John Russell Mather hoped his work would be used in a variety of applications, and fortunately he lived to see it used even by his son in the arena of vector-borne diseases. Our team is screening various climatic and environmental variables to identify relevant determinants of tick abundance in an effort to predict levels of disease risk both spatially and temporally in the northeastern United States. Identifying relevant climatic and environmental variables delimiting tick habitat at a high resolution has led us to consider a variety of tools designed for environmental applications. We used the Thornthwaite—Mather water budget technique to predict soil moisture under forest canopies, the primary habitat for the tick vectors of Lyme disease bacteria. To date, the exact controls of spatio-temporal tick distribution are not yet confirmed, but we now believe that whichever variables are shown to most precisely delimit the extent of tick distributions over large areas can be identified by vegetation and other moisture-based variables using satellite sensor data.

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.

Population genomics of the Anthropocene: urbanization reduces the evolutionary potential of small mammal populations

Urbanization results in pervasive habitat fragmentation and reduces standing genetic variation through genetic drift. Loss of genome-wide variation may ultimately reduce the evolutionary potential of animal populations experiencing rapidly changing conditions. In this study, we examined genome-wide variation among 23 white-footed mouse (Peromyscus leucopus) populations sampled along an urbanization gradient in the New York City metropolitan area. Genome-wide variation was estimated as a proxy for evolutionary potential using more than 10,000 SNP markers generated by ddRAD-Seq. We found that genome-wide variation is inversely related to urbanization as measured by percent impervious surface cover, and to a lesser extent, human population density. We also report that urbanization results in enhanced genome-wide differentiation between populations in cities. There was no pattern of isolation by distance among these populations, but an isolation by resistance model based on impervious surface significantly explained patterns of genetic differentiation. Isolation by environment modeling also indicated that urban populations deviate much more strongly from global allele frequencies than suburban or rural populations. This study is the first to examine evolutionary potential along an urban-to-rural gradient and quantify urbanization as a driver of population genomics patterns.Urbanization results in pervasive habitat fragmentation and reduces standing genetic variation through bottlenecks and drift. Loss of genome-wide variation may ultimately reduce the evolutionary potential of animal populations experiencing rapidly changing conditions. In this study, we examined genome-wide variation among 23 white-footed mouse (Peromyscus leucopus) populations sampled along an urbanization gradient in the New York City metropolitan area. Genome-wide variation was estimated as a proxy for evolutionary potential using more than 10,000 SNP markers generated by ddRAD-Seq. We found that genome-wide variation is inversely related to urbanization as measured by percent impervious surface cover, and to a lesser extent, human population density. We also report that urbanization results in enhanced genome-wide differentiation between populations in cities. There was no pattern of isolation by distance among these populations, but an isolation by resistance model based on impervious surface significantly explained patterns of genetic differentiation. Isolation by environment modeling also indicated that urban populations deviate much more strongly from global allele frequencies than suburban or rural populations. This study is the first to examine loss of genome-wide SNP variation along an urban-to-rural gradient and quantify urbanization as a driver of population genomic patterns.