Differential Expression of Putative Ornithodoros turicata Defensins Mediated by Tick Feeding

Abstract

Additional research on soft ticks in the family Argasidae is needed to bridge the knowledge gap relative to hard ticks of the family Ixodidae; especially, the molecular mechanisms of Ornithodoros biology. Ornithodoros species are vectors of human and animal pathogens that include tick-borne relapsing fever spirochetes and African swine fever virus. Soft tick vector-pathogen interactions involving components of the tick immune response are not understood. Ticks utilize a basic innate immune system consisting of recognition factors and cellular and humoral responses to produce antimicrobial peptides, like defensins. In the present study, we identified and characterized the first putative defensins of Ornithodoros turicata, an argasid tick found primarily in the southwestern United States and regions of Latin America. Four genes (otdA, otdB, otdC, and otdD) were identified through sequencing and their predicted amino acid sequences contained motifs characteristic of arthropod defensins. A phylogenetic analysis grouped these four genes with arthropod defensins, and computational structural analyses further supported the identification. Since pathogens transmitted by O. turicata colonize both the midgut and salivary glands, expression patterns of the putative defensins were determined in these tissues 1 week post engorgement and after molting. Defensin genes up-regulated in the tick midgut 1 week post blood feeding were otdA and otdC, while otdD was up-regulated in the midgut of post-molt ticks. Moreover, otdB and otdD were also up-regulated in the salivary glands of flat post-molt ticks, while otdC was up-regulated within 1 week post blood-feeding. This work is foundational toward additional studies to determine mechanisms of vector competence and pathogen transmission from O. turicata.

Keywords: Ornithdoros turicata; antimicrobial peptide (AMP); argasid (soft) ticks; defensins; gene expression; immune response.

Figure 1

Alignment of mature tick defensin amino acid sequences. Amino acid sequences for the mature peptide of 10 tick defensins and our four putative defensins were obtained from NCBI and RACE sequencing, respectively. Alignment was performed using ClustalW in MEGAX 10.0.5. Shaded amino acids indicate amino acids that are shared among at least 50% of the sequences. Asterisks indicated the cysteines common to defensins. Numbers indicate the position of amino acids.

Figure 2

Predicted protein structure of defensins. In (A) are the protein structure of soft tick defensins O. moubata and the two most structurally similar O. turicata defensins, OtdA, OtdB. In (B) A. monolakensis defensin is shown with the most similar O. turicata defensins, OtdC and OtdD. Structures were predicted using MODELLER and visualized using SWISS-PDB viewer. Purple represents the alpha helix, green are the beta sheets, and the orange ball and stick regions are the disulfide bridges between cysteines C1–C4, C2–C5, and C3–C6. N represents the N terminus of the model.

Figure 3

Phylogenetic tree of mature arthropod defensins. Maximum likelihood tree showing mature protein region of defensins from ticks and insects, and defensin-like scorpion toxins. Bootstrap values (1,000 bootstraps) are shown at the nodes. Names highlighted in yellow represent the O. turicata defensins (OtdA, OtdB, OtdC, and OtdD) introduced in this manuscript. Dotted orange lines represent scorpion toxin, defensin-like sequences, and dotted green lines represent insect defensin sequences.

Figure 4

Expression analysis of putative O. turicata defensins in post-molt and fed midguts and salivary glands. Putative O. turicata defensins (A,B) expression was assessed in the fed and post-molt midguts and salivary glands. Defensin expression was normalized to β-actin expression and the relative log₂ fold change was calculated using the 2^−ΔCt method with post-molt (A) or fed (B) tissues as the control. Statistical significance was calculated between post-molt and fed tissue using the Student's t-test with Welch's correction. (P-value: ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001).

Figure 5

Model of pathogen colonization and defensin expression. Spirochetes and viruses are imbibed during the acquisition blood feeding and enter the midgut (red). Over the next 1–2 weeks a subset of the population migrates out of the midgut to colonize the salivary glands (yellow). At this time there is an upregulation (+ + +) of otdA and otdC in the midgut and otdC in the salivary glands. otdB is expressed (+) in the midgut. otdD is not expressed in the midgut (-) but transcript detected in the salivary glands with otdA and otdB. This characterizes early pathogen colonization of the tick. Persistent colonization occurs after the tick has molted and the pathogen remain in both the midgut (light red) and salivary glands. In the midgut otdA and otdB are expressed (+), otdC is not expressed (-), and otdD is upregulated (+ + +). In the salivary glands otdA and otdC are expressed (+), while otdB and otdD are upregulated (+ + +).