Ehrlichia chaffeensis Co-Opts Phagocytic Hemocytes for Systemic Dissemination in the Lone Star Tick, Amblyomma americanum.

INTRODUCTION: Hematophagous arthropods can acquire and transmit several pathogens of medical importance. In ticks, the innate immune system is crucial in the outcome between vector-pathogen interaction and overall vector competence. However, the specific immune response(s) elicited by the immune cells known as hemocytes remains largely undefined in Ehrlichia chaffeensis and its competent tick vector, Amblyomma americanum. METHODS: We utilized injection of clodronate liposome to deplete tick granulocytes combined with infection with E. chaffeensis to demonstrate their essential role in microbial infection. RESULTS: Here, we show that granulocytes, professional phagocytic cells, are integral in eliciting immune responses against commensal and pathogen infection. The chemical depletion of granulocytes led to decreased phagocytic efficiency of tissue-associated hemocytes. We demonstrate that E. chaffeensis can infect circulating hemocytes, and both cell-free plasma and hemocytes from E. chaffeensis-infected ticks can establish Ehrlichia infection in recipient ticks. Lastly, we provide evidence to show that granulocytes play a dual role in E. chaffeensis infection. Depleting granulocytic hemocytes increased Ehrlichia load in the salivary gland and midgut tissues. In contrast, granulocyte depletion led to a reduced systemic load of Ehrlichia. CONCLUSION: This study has identified multiple roles for granulocytic hemocytes in the control and systemic dissemination of E. chaffeensis infection.

Ticks without borders: Microbial communities of immature Neotropical tick species parasitizing migratory landbirds along northern Gulf of Mexico.

The long-distance, seasonal migrations of birds make them an effective ecological bridge for the movement of ticks. The introduction of exotic tick species to new geographical regions can lead to the emergence of novel tick-borne pathogens or the re-emergence of previously eradicated ones. This study assessed the prevalence of exotic tick species parasitizing resident, short-distance, and long-distance songbirds during spring and autumn at stopover sites in the northern Gulf of Mexico using the mitochondrial 12S rDNA gene. Birds were captured for tick collection from six different sites from late August to early November in both 2018 and 2019. The highest number of ticks were collected in the 2019 season. Most ticks were collected off the Yellow-breasted Chat (Icteria virens) and Common Yellowthroat (Geothlypis trichas), and 54% of the total ticks collected were from Grand Chenier, LA. A high throughput 16S ribosomal RNA sequencing approach was followed to characterize the microbial communities and identify pathogenic microbes in all tick samples. Tick microbial communities, diversity, and community structure were determined using quantitative insight into microbial ecology (QIIME). The sparse correlations for compositional data (SparCC) approach was then used to construct microbial network maps and infer microbial correlations. A total of 421 individual ticks in the genera Amblyomma, Haemaphysalis, and Ixodes were recorded from 28 songbird species, of which Amblyomma and Amblyomma longirostre was the most abundant tick genus and species, respectively. Microbial profiles showed that Proteobacteria was the most abundant phylum. The most abundant bacteria include the pathogenic Rickettsia and endosymbiont Francisella, Candidatus Midichloria, and Spiroplasma. BLAST analysis and phylogenetic reconstruction of the Rickettsia sequences revealed the highest similarities to pathogenic spotted and non-spotted fever groups, including R. buchneri, R. conorii, R. prowazekii, R. bellii, R. australis, R. parkeri, R. monacensis, and R. monteiroi. Permutation multivariate analysis of variance revealed that the relative abundance of Francisella and Rickettsia drives microbial patterns across the tick genera. We also observed a higher percentage of positive correlations in microbe-microbe interactions among members of the microbial communities. Network analysis suggested a negative correlation between a) Francisella and Rickettsia and, b) Francisella and Cutibacterium. Lastly, mapping the distributions of bird species parasitized during spring migrations highlighted geographic hotspots where migratory songbirds could disperse ticks and their pathogens at stopover sites or upon arrival to their breeding grounds, the latter showing means dispersal distances from 421-5003 kilometers. These findings strongly highlight the potential role of migratory birds in the epidemiology of tick-borne pathogens.

Ehrlichia chaffeensis co-opts phagocytic hemocytes for systemic dissemination in the Lone Star tick, Amblyomma americanum.

Hematophagous arthropods can acquire and transmit several pathogens of medical importance. In ticks, the innate immune system is crucial in the outcome between vector-pathogen interaction and overall vector competence. However, the specific immune response(s) elicited by the immune cells known as hemocytes remains largely undefined in Ehrlichi a chaffeensis and its competent tick vector, Amblyomma americanum . Here, we show that granulocytes, professional phagocytic cells, are integral in eliciting immune responses against commensal and pathogen infection. The chemical depletion of granulocytes led to decreased phagocytic efficiency of tissues-associated hemocytes. We demonstrate E. chaffeensis can infect circulating hemocytes, and both cell-free plasma and hemocytes from E. chaffeensis- infected ticks can establish Ehrlichia infection in recipient ticks. Lastly, we provide evidence to show granulocytes play a dual role in E. chaffeensis infection. Depleting granulocytic hemocytes increased Ehrlichia load in the salivary gland and midgut tissues. In contrast, granulocyte depletion led to a reduced systemic load of Ehrlichia . This study has identified multiple roles for granulocytic hemocytes in the control and systemic dissemination of E. chaffeensis infection.

Rickettsia parkeri hijacks tick hemocytes to manipulate cellular and humoral transcriptional responses.

Introduction: Blood-feeding arthropods rely on robust cellular and humoral immunity to control pathogen invasion and replication. Tick hemocytes produce factors that can facilitate or suppress microbial infection and pathogenesis. Despite the importance of hemocytes in regulating microbial infection, understanding of their basic biology and molecular mechanisms remains limited. Methods: Here we combined histomorphology and functional analysis to identify five distinct phagocytic and non-phagocytic hemocyte populations circulating within the Gulf Coast tick Amblyomma maculatum. Results and discussion: Depletion of phagocytic hemocytes using clodronate liposomes revealed their function in eliminating bacterial infection. We provide the first direct evidence that an intracellular tick-borne pathogen, Rickettsia parkeri, infects phagocytic hemocytes in Am. maculatum to modify tick cellular immune responses. A hemocyte-specific RNA-seq dataset generated from hemocytes isolated from uninfected and R. parkeri-infected partially blood-fed ticks generated ~40,000 differentially regulated transcripts, >11,000 of which were immune genes. Silencing two differentially regulated phagocytic immune marker genes (nimrod B2 and eater-two Drosophila homologs), significantly reduced hemocyte phagocytosis. Conclusion: Together, these findings represent a significant step forward in understanding how hemocytes regulate microbial homeostasis and vector competence.

Tick innate immune responses to hematophagy and Ehrlichia infection at single-cell resolution.

Introduction: Ticks rely on robust cellular and humoral responses to control microbial infection. However, several aspects of the tick's innate immune system remain uncharacterized, most notably that of the immune cells (called hemocytes), which are known to play a significant role in cellular and humoral responses. Despite the importance of hemocytes in regulating microbial infection, our understanding of their basic biology and molecular mechanisms remains limited. Therefore, we believe that a more detailed understanding of the role of hemocytes in the interactions between ticks and tick-borne microbes is crucial to illuminating their function in vector competence and to help identify novel targets for developing new strategies to block tick-borne pathogen transmission. Methods: This study examined hemocytes from the lone star tick (Amblyomma americanum) at the transcriptomic level using the 10X genomics single-cell RNA sequencing platform to analyze hemocyte populations from unfed, partially blood-fed, and Ehrlichia chaffeensis-infected ticks. The functional role of differentially expressed hemocyte markers in hemocyte proliferation and Ehrlichia dissemination was determined using an RNA interference approach. Results and discussion: Our data exhibit the identification of fourteen distinct hemocyte populations. Our results uncover seven distinct lineages present in uninfected and Ehrlichia-infected hemocyte clusters. The functional characterization of hemocytin, cystatin, fibronectin, and lipocalin demonstrate their role in hemocyte population changes, proliferation, and Ehrlichia dissemination. Conclusion: Our results uncover the tick immune responses to Ehrlichia infection and hematophagy at a single-cell resolution. This work opens a new field of tick innate immunobiology to understand the role of hemocytes, particularly in response to prolonged blood-feeding (hematophagy), and tick-microbial interactions.

Hematophagy and tick-borne Rickettsial pathogen shape the microbial community structure and predicted functions within the tick vector, Amblyomma maculatum.

Background: Ticks are the primary vectors of emerging and resurging pathogens of public health significance worldwide. Analyzing tick bacterial composition, diversity, and functionality across developmental stages and tissues is crucial for designing new strategies to control ticks and prevent tick-borne diseases. Materials and methods: Here, we explored the microbial communities across the developmental timeline and in different tissues of the Gulf-Coast ticks (Amblyomma maculatum). Using a high-throughput sequencing approach, the influence of blood meal and Rickettsia parkeri, a spotted fever group rickettsiae infection in driving changes in microbiome composition, diversity, and functionality was determined. Results: This study shows that the core microbiome of Am. maculatum comprises ten core bacterial genera. The genus Rickettsia, Francisella, and Candidatus_Midichloria are the key players, with positive interactions within each developmental stage and adult tick organ tested. Blood meal and Rickettsia parkeri led to an increase in the bacterial abundance in the tissues. According to functional analysis, the increase in bacterial numbers is positively correlated to highly abundant energy metabolism orthologs with blood meal. Correlation analysis identified an increase in OTUs identified as Candidatus Midichloria and a subsequent decrease in Francisella OTUs in Rickettsia parkeri infected tick stages and tissues. Results demonstrate the abundance of Rickettsia and Francisella predominate in the core microbiome of Am. maculatum, whereas Candidatus_Midichloria and Cutibacterium prevalence increase with R. parkeri-infection. Network analysis and functional annotation suggest that R. parkeri interacts positively with Candidatus_Midichloria and negatively with Francisella. Conclusion: We conclude that tick-transmitted pathogens, such as R. parkeri establishes infection by interacting with the core microbiome of the tick vector.

Rickettsia parkeri infection modulates the sialome and ovariome of the Gulf coast tick, Amblyomma maculatum.

The Gulf Coast tick, Amblyomma maculatum, is a vector of several tick-borne pathogens, including Rickettsia parkeri. The ability of R. parkeri to persist within the tick population through transovarial and transstadial transmission, without apparently harming the ticks, contributes to the pathogen's perpetuation in the tick population. Previous studies have shown that the R. parkeri load in A. maculatum is regulated by the tick tissues' oxidant/antioxidant balance and the non-pathogenic tick microbiome. To obtain further insights into the interaction between tick and pathogen, we performed a bulk RNA-Seq for differential transcriptomic analysis of ovaries and salivary glands from R. parkeri-infected and uninfected ticks over the feeding course on a host. The most differentially expressed functional category was of bacterial origin, exhibiting a massive overexpression of bacterial transcripts in response to the R. parkeri infection. Candidatus Midichloria mitochondrii and bacteria from the genus Rickettsia were mainly responsible for the overexpression of bacterial transcripts. Host genes were also modulated in R. parkeri-infected tick organs. A similar number of host transcripts from all analyzed functional categories was negatively and positively modulated, revealing a global alteration of the A. maculatum transcriptome in response to pathogen infection. R. parkeri infection led to an increase in salivary transcripts involved in blood feeding success as well as a decrease in ovarian immune transcripts. We hypothesize that these transcriptional alterations facilitate pathogen persistence and transmission within tick population.

Recently Evolved Francisella-Like Endosymbiont Outcompetes an Ancient and Evolutionarily Associated Coxiella-Like Endosymbiont in the Lone Star Tick (Amblyomma americanum) Linked to the Alpha-Gal Syndrome.

Background: Ticks are hematophagous arthropods that transmit various bacterial, viral, and protozoan pathogens of public health significance. The lone star tick (Amblyomma americanum) is an aggressive human-biting tick that transmits bacterial and viral pathogens, and its bites are suspected of eliciting the alpha-gal syndrome, a newly emerged delayed hypersensitivity following consumption of red meat in the United States. While ongoing studies have attempted to investigate the contribution of different tick-inherent factors to the induction of alpha-gal syndrome, an otherwise understudied aspect is the contribution of the tick microbiome and specifically obligate endosymbionts to the establishment of the alpha-gal syndrome in humans. Materials and Methods: Here we utilized a high-throughput metagenomic sequencing approach to cataloging the entire microbial communities residing within different developmental stages and tissues of unfed and blood-fed ticks from laboratory-maintained ticks and three new geographical locations in the United States. The Quantitative Insights Into Microbial Ecology (QIIME2) pipeline was used to perform data analysis and taxonomic classification. Moreover, using a SparCC (Sparse Correlations for Compositional data) network construction model, we investigated potential interactions between members of the microbial communities from laboratory-maintained and field-collected ticks. Results: Overall, Francisellaceae was the most dominant bacteria identified in the microbiome of both laboratory-raised and field-collected Am. americanum across all tissues and developmental stages. Likewise, microbial diversity was seen to be significantly higher in field-collected ticks compared with laboratory-maintained ticks as seen with a higher number of both Operational Taxonomic Units and measures of species richness. Several potential positive and negative correlations were identified from our network analysis. We observed a strong positive correlation between Francisellaceae, Rickettsiaceae, and Midichloriaceae in both developmental stages and tissues from laboratory-maintained ticks, whereas ovarian tissues had a strong positive correlation of bacteria in the family Xanthobacteraceae and Rhizobiaceae. A negative interaction was observed between Coxiellaceae and Francisellaceae in Illinois, and all the bacteria detected from ticks from Delaware were negatively correlated. Conclusion: This study is the first to catalog the microbiome of Am. americanum throughout its developmental stages and different tissue niches and report the potential replacement of Coxiellaceae by Francisellaceae across developmental stages and tissues tested except in ovarian tissues. These unique and significant findings advance our knowledge and open a new avenue of research to further understand the role of tick microbiome in tick-borne diseases and develop a holistic strategy to control alpha-gal syndrome.

An Exploratory Study on the Microbiome of Northern and Southern Populations of Ixodes scapularis Ticks Predicts Changes and Unique Bacterial Interactions.

The black-legged tick (Ixodes scapularis) is the primary vector of Borrelia burgdorferi, the causative agent of Lyme disease in North America. However, the prevalence of Lyme borreliosis is clustered around the Northern States of the United States of America. This study utilized a metagenomic sequencing approach to compare the microbial communities residing within Ix. scapularis populations from northern and southern geographic locations in the USA. Using a SparCC network construction model, we performed potential interactions between members of the microbial communities from Borrelia burgdorferi-infected tissues of unfed and blood-fed ticks. A significant difference in bacterial composition and diversity was found between northern and southern tick populations. The network analysis predicted a potential antagonistic interaction between endosymbiont Rickettsia buchneri and Borrelia burgdorferi sensu lato. The network analysis, as expected, predicted significant positive and negative microbial interactions in ticks from these geographic regions, with the genus Rickettsia, Francisella, and Borreliella playing an essential role in the identified clusters. Interactions between Rickettsia buchneri and Borrelia burgdorferi sensu lato need more validation and understanding. Understanding the interplay between the microbiome and tick-borne pathogens within tick vectors may pave the way for new strategies to prevent tick-borne infections.

Tick-Borne Pathogens Shape the Native Microbiome Within Tick Vectors.

Ticks are blood-feeding arthropods and transmit a variety of medically important viral, bacterial, protozoan pathogens to animals and humans. Ticks also harbor a diverse community of microbes linked to their biological processes, such as hematophagy, and hence affect vector competence. The interactions between bacterial and/or protozoan pathogens and the tick microbiome is a black-box, and therefore we tested the hypothesis that the presence of a protozoan or bacterial pathogen will alter the microbial composition within a tick. Hence, this study was designed to define the microbial composition of two tick species, Hyalomma (H.) anatolicum and Rhipicephalus (R.) microplus. We used a combination of PCR based pathogen (Anaplasma marginale and Theileria species) and symbiont (Wolbachia species) identification followed by metagenomic sequencing and comparison of the microbial communities in PCR positive and negative ticks. A total of 1786 operational taxonomic units was identified representing 25 phyla, 50 classes, and 342 genera. The phylum Proteobacteria, Firmicutes, Actinobacteriota, and Bacteroidota were the most represented bacteria group. Alpha and beta diversity were not significantly affected in the presence or absence of Theileria sp. and A. marginale as see with H. anatolicum ticks. Interestingly, bacterial communities were significantly reduced in Theileria sp. infected R. microplus ticks, while also exhibiting a significant reduction in microbial richness and evenness. Putting these observations together, we referred to the effect the presence of Theileria sp. has on R. microplus a "pathogen-induced dysbiosis". We also identify the presence of Plasmodium falciparum, the causative agent of human malaria from the microbiome of both H. anatolicum and R. microplus ticks. These findings support the presence of a "pathogen-induced dysbiosis" within the tick and further validation experiments are required to investigate how they are important in the vector competence of ticks. Understanding the mechanism of "pathogen-induced dysbiosis" on tick microbial composition may aid the discovery of intervention strategies for the control of emerging tick-borne infections.

Laboratory colonization by Dirofilaria immitis alters the microbiome of female Aedes aegypti mosquitoes.

BACKGROUND: The ability of blood-feeding arthropods to successfully acquire and transmit pathogens of medical and veterinary importance has been shown to be interfered with, or enhanced by, the arthropod's native microbiome. Mosquitoes transmit viruses, protozoan and filarial nematodes, the majority of which contribute to the 17% of infectious disease cases worldwide. Dirofilaria immitis, a mosquito-transmitted filarial nematodes of dogs and cats, is vectored by several mosquito species including Aedes aegypti. METHODS: In this study, we investigated the impact of D. immitis colonization on the microbiome of laboratory reared female Ae. aegypti. Metagenomic analysis of the V3-V4 variable region of the microbial 16S RNA gene was used for identification of the microbial differences down to species level. RESULTS: We generated a total of 1068 OTUs representing 16 phyla, 181 genera and 271 bacterial species. Overall, in order of abundance, Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the most represented phylum with D. immitis-infected mosquitoes having more of Proteobacteria (71%) than uninfected mosquitoes (56.9%). An interesting finding in this study is the detection of Klebsiella oxytoca in relatively similar abundance in infected and uninfected mosquitoes, suggesting a possible endosymbiotic relationship, and has been previously shown to indirectly compete for nutrients with fungi on domestic housefly eggs and larvae. While D. immitis colonization has no effect on the overall species richness, we identified significant differences in the composition of selected bacterial genera and phyla between the two groups. We also reported distinct compositional and phylogenetic differences in the individual bacterial species when commonly identified bacteria were compared. CONCLUSIONS: To the best of our knowledge, this is the first study to understand the impact of a filarial infection on the microbiome of its mosquito vector. Further studies are required to identify bacteria species that could play an important role in the mosquito biology. While the microbiome composition of Ae. aegypti mosquito have been previously reported, our study shows that in an effort to establish itself, a filarial nematode modifies and alters the overall microbial diversity within its mosquito host.