Macro-parasites and micro-parasites co-exist in rodent communities but are associated with different community-level parameters.

Wildlife species are often heavily parasitized by multiple infections simultaneously. Yet research on sylvatic transmission cycles, tend to focus on host interactions with a single parasite and neglects the influence of co-infections by other pathogens and parasites. Co-infections between macro-parasites and micro-parasites can alter mechanisms that regulate pathogenesis and are important for understanding disease emergence and dynamics. Wildlife rodent hosts in the Lyme disease system are infected with macro-parasites (i.e., ticks and helminths) and micro-parasites (i.e., Borrelia spp.), however, there has not been a study that investigates the interaction of all three parasites (i.e., I. pacificus, Borrelia spp., and helminths) and how these co-infections impact prevalence of micro-parasites. We live-trapped rodents in ten sites in northern California to collect feces, blood, ear tissue, and attached ticks. These samples were used to test for infection status of Borrelia species (i.e., micro-parasite), and describe the burden of ticks and helminths (i.e., macro-parasites). We found that some rodent hosts were co-infected with all three parasites, however, the burden or presence of concurrent macro-parasites were not associated with Borrelia infections. For macro-parasites, we found that tick burdens were positively associated with rodent Shannon diversity while negatively associated with predator diversity, whereas helminth burdens were not significantly associated with any host community metric. Ticks and tick-borne pathogens are associated with rodent host diversity, predator diversity, and abiotic factors. However, it is still unknown what factors helminths are associated with on the community level. Understanding the mechanisms that influence co-infections of multiple types of parasites within and across hosts is an increasingly critical component of characterizing zoonotic disease transmission and maintenance.

Environment is associated with chytrid infection and skin microbiome richness on an amphibian rich island (Taiwan).

Growing evidence suggests that the origins of the panzootic amphibian pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are in Asia. In Taiwan, an island hotspot of high amphibian diversity, no amphibian mass mortality events linked to Bd or Bsal have been reported. We conducted a multi-year study across this subtropical island, sampling 2517 individuals from 30 species at 34 field sites, between 2010 and 2017, and including 171 museum samples collected between 1981 and 2009. We analyzed the skin microbiome of 153 samples (6 species) from 2017 in order to assess any association between the amphibian skin microbiome and the probability of infection amongst different host species. We did not detect Bsal in our samples, but found widespread infection by Bd across central and northern Taiwan, both taxonomically and spatially. Museum samples show that Bd has been present in Taiwan since at least 1990. Host species, geography (elevation), climatic conditions and microbial richness were all associated with the prevalence of infection. Host life-history traits, skin microbiome composition and phylogeny were associated with lower prevalence of infection for high altitude species. Overall, we observed low prevalence and burden of infection in host populations, suggesting that Bd is enzootic in Taiwan where it causes subclinical infections. While amphibian species in Taiwan are currently threatened by habitat loss, our study indicates that Bd is in an endemic equilibrium with the populations and species we investigated. However, ongoing surveillance of the infection is warranted, as changing environmental conditions may disturb the currently stable equilibrium.

Detection and Isolation of Rickettsia tillamookensis (Rickettsiales: Rickettsiaceae) From Ixodes pacificus (Acari: Ixodidae) From Multiple Regions of California.

The western black-legged tick (Ixodes pacificus) is the most frequently identified human-biting tick species in the western United States and the principal vector of at least three recognized bacterial pathogens of humans. A potentially pathogenic Rickettsia species, first described in 1978 and recently characterized as a novel transitional group agent designated as Rickettsia tillamookensis, also exists among populations of I. pacificus, although the distribution and frequency of this agent are poorly known. We evaluated DNA extracts from 348 host-seeking I. pacificus nymphs collected from 9 locations in five California counties, and from 916 I. pacificus adults collected from 24 locations in 13 counties, by using a real-time PCR designed specifically to detect DNA of R. tillamookensis. DNA of R. tillamookensis was detected in 10 (2.9%) nymphs (95% CI: 1.6-5.2%) and 17 (1.9%) adults (95% CI: 1.2-3.0%) from 11 counties of northern California. Although site-specific infection rates varied greatly, frequencies of infection remained consistently low when aggregated by stage, sex, habitat type, or geographical region. Four novel isolates of R. tillamookensis were cultivated in Vero E6 cells from individual adult ticks collected from Alameda, Nevada, and Yolo counties. Four historical isolates, serotyped previously as 'Tillamook-like' strains over 40 yr ago, were revived from long-term storage in liquid nitrogen and confirmed subsequently by molecular methods as isolates of R. tillamookensis. The potential public health impact of R. tillamookensis requires further investigation.

Host blood meal identity modifies vector gene expression and competency.

A vector's susceptibility and ability to transmit a pathogen-termed vector competency-determines disease outcomes, yet the ecological factors influencing tick vector competency remain largely unknown. Ixodes pacificus, the tick vector of Borrelia burgdorferi (Bb) in the western U.S., feeds on rodents, birds, and lizards. Rodents and birds are reservoirs for Bb and infect juvenile ticks, while lizards are refractory to Bb and cannot infect feeding ticks. Additionally, the lizard bloodmeal contains borreliacidal properties, clearing previously infected feeding ticks of their Bb infection. Despite I. pacificus feeding on a range of hosts, it is undetermined how the host identity of the larval bloodmeal affects future nymphal vector competency. We experimentally evaluate the influence of larval host bloodmeal on Bb acquisition by nymphal I. pacificus. Larval I. pacificus were fed on either lizards or mice and after molting, nymphs were fed on Bb-infected mice. We found that lizard-fed larvae were significantly more likely to become infected with Bb during their next bloodmeal than mouse-fed larvae. We also conducted the first RNA-seq analysis on whole-bodied I. pacificus and found significant upregulation of tick antioxidants and antimicrobial peptides in the lizard-fed group. Our results indicate that the lizard bloodmeal significantly alters vector competency and gene regulation in ticks, highlighting the importance of host bloodmeal identity in vector-borne disease transmission and upends prior notions about the role of lizards in Lyme disease community ecology.

Local Community Composition Drives Avian Borrelia burgdorferi Infection and Tick Infestation.

Globally, zoonotic vector-borne diseases are on the rise and understanding their complex transmission cycles is pertinent to mitigating disease risk. In North America, Lyme disease is the most commonly reported vector-borne disease and is caused by transmission of Borrelia burgdorferi sensu lato (s.l.) from Ixodes spp. ticks to a diverse group of vertebrate hosts. Small mammal reservoir hosts are primarily responsible for maintenance of B. burgdorferi s.l. across the United States. Nevertheless, birds can also be parasitized by ticks and are capable of infection with B. burgdorferi s.l. but their role in B. burgdorferi s.l. transmission dynamics is understudied. Birds could be important in both the maintenance and spread of B. burgdorferi s.l. and ticks because of their high mobility and shared habitat with important mammalian reservoir hosts. This study aims to better understand the role of avian hosts in tick-borne zoonotic disease transmission cycles in the western United States. We surveyed birds, mammals, and ticks at nine sites in northern California for B. burgdorferi s.l. infection and collected data on other metrics of host community composition such as abundance and diversity of birds, small mammals, lizards, predators, and ticks. We found 22.8% of birds infected with B. burgdorferi s.l. and that the likelihood of avian B. burgdorferi s.l. infection was significantly associated with local host community composition and pathogen prevalence in California. Additionally, we found an average tick burden of 0.22 ticks per bird across all species. Predator and lizard abundances were significant predictors of avian tick infestation. These results indicate that birds are relevant hosts in the local B. burgdorferi s.l. transmission cycle in the western United States and quantifying their role in the spread and maintenance of Lyme disease requires further research.

Grappling with the tick microbiome.

Ixodes scapularis and Ixodes pacificus are the predominant vectors of multiple human pathogens, including Borrelia burgdorferi, one of the causative agents of Lyme disease in North America. Differences in the habitats and host preferences of these closely related tick species present an opportunity to examine key aspects of the tick microbiome. While advances in sequencing technologies have accelerated a descriptive understanding of the tick microbiome, molecular and mechanistic insights into the tick microbiome are only beginning to emerge. Progress is stymied by technical difficulties in manipulating the microbiome and by biological variables related to the life cycle of Ixodid ticks. This review highlights these challenges and examines avenues to understand the significance of the tick microbiome in tick biology.

Host infection and community composition predict vector burden.

Lyme disease is the most prevalent vector-borne disease in the United States, yet critical gaps remain in our understanding of tick and host interactions that shape disease dynamics. Rodents such as deer mice (Peromyscus spp.) and dusky-footed woodrats (Neotoma fuscipes) are key reservoirs for Borrelia burgdorferi, the etiological bacterium of Lyme disease, and can vary greatly in abundance between habitats. The aggregation of Ixodes pacificus, the western black-legged tick, on rodent hosts is often assumed to be constant across various habitats and not dependent on the rodent or predator communities; however, this is rarely tested. The factors that determine tick burdens on key reservoir hosts are important in estimating Lyme disease risk because larger tick burdens can amplify pathogen transmission. This study is the first to empirically measure I. pacificus larval burdens on competent reservoir hosts as a function of community factors such as rodent diversity, predator diversity, and questing tick abundance. Rodents were live trapped at oak woodland sites to collect tick burdens and tissue samples to test for infection with Borrelia burgdorferi sensu lato. We found that N. fuscipes tick burdens were negatively correlated with predator diversity, but positively correlated with questing I. pacificus larvae. In addition, rodent hosts that were infected with B. burgdorferi sensu lato tend to have higher burdens of larval ticks. These results demonstrate that tick burdens can be shaped by variability between individuals, species, and the broader host community with consequences for transmission and prevalence of tick-borne pathogens.

A Beginner's Guide to Collecting Questing Hard Ticks (Acari: Ixodidae): A Standardized Tick Dragging Protocol.

Tick-borne diseases are emerging globally, necessitating increased research and coordination of tick surveillance practices. The most widely used technique for active collection of host-seeking, human-biting tick vectors is 'tick dragging', by which a cloth is dragged across the top of the vegetation or forest floor and regularly checked for the presence of ticks. Use of variable dragging protocols limits the ability of researchers to combine data sets for comparative analyses or determine patterns and trends across different spatial and temporal scales. Standardization of tick drag collection and reporting methodology will greatly benefit the field of tick-pathogen studies. Based on the recommendations of the Center for Disease Control and Prevention and other ecological considerations, we propose that tick dragging should be conducted to sample at least 750 m2 along linear transects when habitat allows in a manner that reduces bias in the sampled area, and report density of each tick species and life stage separately. A protocol for constructing a standard drag cloth, establishing linear transects, and drag sampling is presented, along with a downloadable datasheet that can be modified to suit the needs of different projects. Efforts to align tick surveillance according to these standard best practices will help generate robust data on tick population biology.

Parental Care Alters the Egg Microbiome of Maritime Earwigs.

Recruitment of beneficial microbes to protect offspring, often reducing the energetic costs of care, is now recognized as an important component of parental care in many animals. Studies on earwigs (order Dermaptera) have revealed that removal of females from egg tending increases mortality of eggs due to fungal infections, possibly caused by changes in the bacterial microbiome on the egg surface. We used a controlled female-removal experiment to evaluate whether female nest attendance in the maritime earwig, Anisolabis maritima, influences the bacterial microbiome on the egg surface. Further, we analyzed the microbiomes of mothers and their eggs to determine if there are a core set of bacteria transferred to eggs through female care. Microbiomes were analyzed using 16S rRNA bacterial DNA sequencing, revealing that bacterial operational taxonomic unit (OTU) richness and diversity were both significantly higher for female attended versus unattended eggs. The core microbiome of adult females contained bacteria which have the potential to carry anti-fungal characteristics; these bacteria were found in higher presence and relative abundance on eggs where females were allowed to provide care. These results demonstrate that female egg attendance significantly impacts the bacterial microbiome of A. maritima eggs, and identifies specific bacteria within the egg microbiome that should be investigated further for beneficial anti-fungal properties in this system.

Circulation of Tick-Borne Spirochetes in Tick and Small Mammal Communities in Santa Barbara County, California, USA.

A diversity of Borrelia burgdorferi sensu lato (Johnson, Schmid, Hyde, Steigerwalt & Brenner) (Spirochaetales: Spirochaetaceae) genomospecies, including the Lyme disease agent, Borrelia burgdorferi sensu stricto (s.s.), have been identified in the western United States. However, enzootic transmission of B. burgdorferi s.l. in small mammals and ticks is poorly characterized throughout much of the region. Here we report prevalence of B. burgdorferi s.l. in small mammal and tick communities in the understudied region of southern California. We found B. burgdorferi s.l. in 1.5% of Ixodes species ticks and 3.6% of small mammals. Infection was uncommon (~0.3%) in Ixodes pacificus Cooley and Kohls (Acari: Ixodidae), the primary vector of the Lyme disease agent to humans in western North America, but a diversity of spirochetes-including Borrelia bissettiae, Borrelia californiensis, Borrelia americana, and B. burgdorferi s.s.-were identified circulating in Ixodes species ticks and their small mammal hosts. Infection with B. burgdorferi s.l. is more common in coastal habitats, where a greater diversity of Ixodes species ticks are found feeding on small mammal hosts (four species when compared with only I. pacificus in other sampled habitats). This provides some preliminary evidence that in southern California, wetter coastal areas might be more favorable for enzootic transmission than hotter and drier climates. Infection patterns confirm that human transmission risk of B. burgdorferi s.s. is low in this region. However, given evidence for local maintenance of B. burgdorferi s.l., more studies of enzootic transmission may be warranted, particularly in understudied regions where the tick vector of B. burgdorferi s.s. occurs.

Comparative vector competence of North American Lyme disease vectors.

BACKGROUND: Understanding the drivers of Lyme disease incidence at broad spatial scales is critical for predicting and mitigating human disease risk. Previous studies have identified vector phenology and behavior, host community composition, and landscape features as drivers of variable Lyme disease risk. However, while the Lyme disease transmission cycles in the eastern and western USA involve different vector species (Ixodes scapularis and Ixodes pacificus, respectively), the role of vector-specific differences in transmission efficiency has not been directly examined. By comparing the performance of traits involved in vector competence between these two species, this study aims to identify how vector competence contributes to variable Lyme disease risk. METHODS: We used a suite of laboratory experiments to compare the performance of traits related to vector competence for the two USA Lyme disease vectors. For each species, we measured the rate of attachment to a common rodent host, the engorgement weight, and the efficiency of pathogen acquisition (host to tick) and pathogen transmission (tick to host) from laboratory mice. In measuring pathogen acquisition and transmission, we used two different pathogen strains, one sympatric with I. scapularis and one sympatric with I. pacificus, to assess the importance of vector-pathogen coevolutionary history in transmission dynamics. RESULTS: We found I. pacificus had significantly higher host attachment success and engorgement weights, but significantly lower pathogen transmission efficiency relative to I. scapularis. Molting success and pathogen acquisition did not differ between these two species. However, pathogen acquisition efficiency was significantly higher for both sympatric vector and pathogen strains than the allopatric pairings. CONCLUSIONS: This study identified species-specific vector traits as a potential driver of broad scale variation in Lyme disease risk in the USA. In particular, the exceedingly low rates of pathogen transmission from tick to host observed for I. pacificus may limit Lyme disease transmission efficiency in the western USA. Further, observed variation in pathogen acquisition between sympatric and allopatric vector-pathogen strains indicate that vector-pathogen coevolutionary history may play a key role in transmission dynamics. These findings underscore the need to consider vector traits and vector-pathogen coevolution as important factors governing regional Lyme disease risk.

Patterns, Drivers, and Challenges of Vector-Borne Disease Emergence.

Vector-borne diseases are emerging at an increasing rate and comprise a disproportionate share of all emerging infectious diseases. Yet, the key ecological and evolutionary dimensions of vector-borne disease that facilitate their emergence have not been thoroughly explored. This study reviews and synthesizes the existing literature to explore global patterns of emerging vector-borne zoonotic diseases (VBZDs) under changing global conditions. We find that the vast majority of emerging VBZDs are transmitted by ticks (Ixodidae) and mosquitoes (Culicidae) and the pathogens transmitted are dominated by Rickettsiaceae bacteria and RNA viruses (Flaviviridae, Bunyaviridae, and Togaviridae). The most common potential driver of these emerging zoonoses is land use change, but for many diseases, the driver is unknown, revealing a critical research gap. While most reported VBZDs are emerging in the northern latitudes, after correcting for sampling bias, Africa is clearly a region with the greatest share of emerging VBZD. We highlight critical gaps in our understanding of VBZD emergence and emphasize the importance of interdisciplinary research and consideration of deeper evolutionary processes to improve our capacity for anticipating where and how such diseases have and will continue to emerge.

Comparative Microbiome Profiles of Sympatric Tick Species from the Far-Western United States.

Insight into the composition and function of the tick microbiome has expanded considerably in recent years. Thus far, tick microbiome studies have focused on species and life stages that are responsible for transmitting disease. In this study we conducted extensive field sampling of six tick species in the far-western United States to comparatively examine the microbial composition of sympatric tick species: Ixodes pacificus, Ixodes angustus, Dermacentor variabilis, Dermacentor occidentalis, Dermacentor albipictus, and Haemaphysalis leporispalustris. These species represent both common vectors of disease and species that rarely encounter humans, exhibiting a range of host preferences and natural history. We found significant differences in microbial species diversity and composition by tick species and life stage. The microbiome of most species examined were dominated by a few primary endosymbionts. Across all species, the relative abundance of these endosymbionts increased with life stage while species richness and diversity decreased with development. Only one species, I. angustus, did not show the presence of a single dominant microbial species indicating the unique physiology of this species or its interaction with the surrounding environment. Tick species that specialize in a small number of host species or habitat ranges exhibited lower microbiome diversity, suggesting that exposure to environmental conditions or host blood meal diversity can affect the tick microbiome which in turn may affect pathogen transmission. These findings reveal important associations between ticks and their microbial community and improve our understanding of the function of non-pathogenic microbiomes in tick physiology and pathogen transmission.

Drivers and patterns of microbial community assembly in a Lyme disease vector.

Vector-borne diseases constitute a major global health burden and are increasing in geographic range and prevalence. Mounting evidence has demonstrated that the vector microbiome can impact pathogen dynamics, making the microbiome a focal point in vector-borne disease ecology. However, efforts to generalize preliminary findings across studies and systems and translate these findings into disease control strategies are hindered by a lack of fundamental understanding of the processes shaping the vector microbiome and the interactions therein. Here, we use 16S rRNA sequencing and apply a community ecology framework to analyze microbiome community assembly and interactions in Ixodes pacificus, the Lyme disease vector in the western United States. We find that vertical transmission routes drive population-level patterns in I. pacificus microbial diversity and composition, but that microbial function and overall abundance do not vary over time or between clutches. Further, we find that the I. pacificus microbiome is not strongly structured based on competition but assembles nonrandomly, potentially due to vector-specific filtering processes which largely eliminate all but the dominant endosymbiont, Rickettsia. At the scale of the individual I. pacificus, we find support for a highly limited internal microbial community, and hypothesize that the tick endosymbiont may be the most important component of the vector microbiome in influencing pathogen dynamics.

Students as ecologists: Strategies for successful mentorship of undergraduate researchers.

Guiding undergraduates through the ecological research process can be incredibly rewarding and present opportunities to break down barriers to inclusion and diversity in scientific disciplines. At the same time, mentoring undergraduate researchers is a complicated process that requires time and flexibility. While many academics receive extensive guidance on how to be successful in research endeavors, we pay much less attention to training in mentorship and working collaboratively with undergraduate students. This paper seeks to provide a framework for successfully collaborating with undergraduates including initial recruitment, development of a contract, fostering student ownership of research projects, and submission of a polished manuscript.

Insights into the evolution and drug susceptibility of Babesia duncani from the sequence of its mitochondrial and apicoplast genomes.

Babesia microti and Babesia duncani are the main causative agents of human babesiosis in the United States. While significant knowledge about B. microti has been gained over the past few years, nothing is known about B. duncani biology, pathogenesis, mode of transmission or sensitivity to currently recommended therapies. Studies in immunocompetent wild type mice and hamsters have shown that unlike B. microti, infection with B. duncani results in severe pathology and ultimately death. The parasite factors involved in B. duncani virulence remain unknown. Here we report the first known completed sequence and annotation of the apicoplast and mitochondrial genomes of B. duncani. We found that the apicoplast genome of this parasite consists of a 34 kb monocistronic circular molecule encoding functions that are important for apicoplast gene transcription as well as translation and maturation of the organelle's proteins. The mitochondrial genome of B. duncani consists of a 5.9 kb monocistronic linear molecule with two inverted repeats of 48 bp at both ends. Using the conserved cytochrome b (Cytb) and cytochrome c oxidase subunit I (coxI) proteins encoded by the mitochondrial genome, phylogenetic analysis revealed that B. duncani defines a new lineage among apicomplexan parasites distinct from B. microti, Babesia bovis, Theileria spp. and Plasmodium spp. Annotation of the apicoplast and mitochondrial genomes of B. duncani identified targets for development of effective therapies. Our studies set the stage for evaluation of the efficacy of these drugs alone or in combination against B. duncani in culture as well as in animal models.

Evidence for transmission of the zoonotic apicomplexan parasite Babesia duncani by the tick Dermacentor albipictus.

Babesiosis is a potentially fatal tick-borne zoonotic disease caused by a species complex of blood parasites that can infect a variety of vertebrates, particularly dogs, cattle, and humans. In the United States, human babesiosis is caused by two distinct parasites, Babesia microti and Babesia duncani. The enzootic cycle of B. microti, endemic in the northeastern and upper midwestern regions, has been well characterised. In the western United States, however, the natural reservoir host and tick vector have not been identified for B. duncani, greatly impeding efforts to understand and manage this zoonotic disease. Two and a half decades after B. duncani was first described in a human patient in Washington State, USA, we provide evidence that the enzootic tick vector is the winter tick, Dermacentor albipictus, and the reservoir host is likely the mule deer, Odocoileus hemionus. The broad, overlapping ranges of these two species covers a large portion of far-western North America, and is consistent with confirmed cases of B. duncani in the far-western United States.

Reduced skin bacterial diversity correlates with increased pathogen infection intensity in an endangered amphibian host.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) infects the skin of amphibians and has caused severe declines and extinctions of amphibians globally. In this study, we investigate the interaction between Bd and the bacterial skin microbiome of the endangered Sierra Nevada yellow-legged frog, Rana sierrae, using both culture-dependent and culture-independent methods. Samples were collected from two populations of R. sierrae that likely underwent Bd epizootics in the past, but that continue to persist with Bd in an enzootic disease state, and we address the hypothesis that such "persistent" populations are aided by mutualistic skin microbes. Our 16S rRNA metabarcoding data reveal that the skin microbiome of highly infected juvenile frogs is characterized by significantly reduced species richness and evenness, and by strikingly lower variation between individuals, compared to juveniles and adults with lower infection levels. Over 90% of DNA sequences from the skin microbiome of highly infected frogs were derived from bacteria in a single order, Burkholderiales, compared to just 54% in frogs with lower infection levels. In a culture-dependent Bd inhibition assay, the bacterial metabolites we evaluated all inhibited the growth of Bd. Together, these results illustrate the disruptive effects of Bd infection on host skin microbial community structure and dynamics, and suggest possible avenues for the development of anti-Bd probiotic treatments.

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing.

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.