Date of Award

5-2024

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

School

Biological, Environmental, and Earth Sciences

Committee Chair

Dr. Shahid Karim

Committee Chair School

Biological, Environmental, and Earth Sciences

Committee Member 2

Dr. Fengwei Bai

Committee Member 2 School

Biological, Environmental, and Earth Sciences

Committee Member 3

Dr. Yanlin Guo

Committee Member 3 School

Biological, Environmental, and Earth Sciences

Committee Member 4

Dr. Sarah Morgan

Committee Member 4 School

Polymer Science and Engineering

Committee Member 5

Dr. Ryan Smith

Abstract

The tick immune response consist of two main components: the cellular and humoral components. The cellular component consists of hemocytes that circulate in the hemolymph-filled body cavity. Tick hemocytes are functional analogs of the mammalian white blood cells and are involved in immune responses including phagocytosis. The Humoral component includes mainly soluble peptides that function as antimicrobial peptides (AMPs) and pattern recognition receptors. Although tick hemocytes partakes in the humoral responses, different tick tissues produce several humoral factors such as AMPs and lectins. We have a deep understanding of the different humoral effectors released across the tick tissues during tick-pathogen and tick-microbial interactions. However, our understanding of hemocyte-mediated responses and their effects on vectorial capacity in the different tick species remain limited.

This research investigates the functional role of hemocytes in responding to microbial infection and examines how they influence the outcome of tick-pathogen interactions in two different tick species. Morphological and fluorescent microscopic examination of hemocytes from Amblyomma maculatum and Amblyomma americanum ticks showed five hemocyte populations. These include granulocytes, plasmatocytes, prohemocytes, oenocytoids, and spherulocytes. Specifically, the granulocytes possess phagocytic capabilities, as evidenced by their ability to uptake green fluorescent beads following injection into the hemolymph. Additionally, when granulocyte population were depleted using clodronate liposome, both A. maculatum and A. americanum succumbed to increase mortality upon infection with Escherichia coli, Staphylococcus aureus, Ehrlichia chaffeensis (A. americanum) and Rickettsia parkeri (A. maculatum). The uptake of pathogen by hemocytes was assessed using the immunofluorescence technique and we demonstrated the intracellular presence of R. parkeri and E. chaffeensis in the cytoplasm of granulocytic hemocytes. These findings suggest that the pathogens utilize tick hemocyte for survival and dissemination inside of the ticks. Nimrod B2 and eater, which we identified from whole transcriptome sequencing of hemocyte from uninfected and R. parkeri-infected A. maculatum showed significant downregulation with infection. We characterized their role in phagocytosis of fluorescent beads using RNA interference and showed that they are potential markers of hemocyte phagocytosis. We further demonstrate that while phagocytic hemocytes are important in restricting E. chaffeensis load in tissues, it facilitate its systemic dissemination throughout the carcass. Lastly, we explored the cellular and transcriptomic diversity of A. americanum hemocytes in the presence of E. chaffeensis infection using single-cell sequencing. This technology allowed us to identify previously unidentified hemocyte population and their unique molecular markers. We identified fourteen distinct hemocyte populations and seven distinct differentiation lineages between uninfected and infected hemocyte. We functionally characterized four target genes; hemocytin, cystatin, fibronectin, and lipocalin, and demonstrate their role in hemocyte population changes, proliferation and Ehrlichia dissemination.

In conclusion, this study demonstrates that tick hemocyte in particular phagocytic hemocytes are essential in determining the outcome of tick-pathogen interactions.

ORCID ID

0000-0003-2390-4435

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