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Gene expression during the early stages of host perception and attachment in adult female Rhipicephalus microplus ticks

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Abstract

The cattle tick, Rhipicephalus microplus, is a serious pest of cattle, with significant economic consequences to the livestock industries of tropical and semitropical countries. Rhipicephalus microplus belongs to the Metastriata group of the Ixodidae family known as hard ticks. When adult hard ticks feed, mating has not yet occurred and an initial host attachment phase of 1–2 days is followed by a slow feeding phase that can last several days. Once mating occurs, feeding concludes with a rapid engorgement phase that is completed in 12–36 h. Our group’s interest in mining the genome and transcriptome of R. microplus for novel targets for development of tick control technologies led us to investigate the early transcriptional events occurring upon tick attachment and subsequent feeding. We placed newly molted unfed adult R. microplus females upon a bovine host and harvested the attached ticks after 3, 6, 12, and 24 h. We also placed a group of these ticks in a gas-permeable tube taped onto the side of the bovine host. These ticks were able to sense the host but unable to penetrate the tube to begin attachment and were ultimately harvested after 3 h. This study produced a comprehensive transcriptome from newly molted adult ticks and will provide a useful resource for studies of tick feeding and host perception and also assist genome annotation refinements.

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Acknowledgements

The authors wish to thank the late Ernie Retzel, National Center for Genome Resources for his inspiration and guidance at the inception of this research. This research has been supported through funding (FDG) from the U. S. Department of Agriculture, Agricultural Research Service (USDAARS) Project Nos. 6205-32000-031-00D and 3094-32000-036-00D. USDA is an equal opportunity employer.

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Authors and Affiliations

  1. Agricultural Research Service Knipling-Bushland U. S. Livestock Insects Research Laboratory, United States Department of Agriculture, 2700 Fredericksburg Rd, Kerrville, TX, 78028, USA

    Kylie G. Bendele, Felix D. Guerrero & Deanna M. Bodine

  2. National Center for Genome Resources, 2935 Rodeo Park Drive East, Santa Fe, NM, 87505, USA

    Connor Cameron

  3. Agricultural Research Service, Cattle Fever Tick Research Laboratory, United States Department of Agriculture, 22675 N. Moorefield Rd, Edinburg, TX, 78541, USA

    Robert J. Miller

Authors
  1. Kylie G. Bendele
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  2. Felix D. Guerrero
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  4. Deanna M. Bodine
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  5. Robert J. Miller
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Contributions

FDG conceived the study, participated in the cattle exposure experiments, conducted the RNA isolations, and drafted the manuscript. KGB participated in the cattle exposure experiments and led the bioinformatics for the gene expression analysis. CC led the transcriptome sequencing and the assembly of the Illumina reads. DMB conducted the RT-PCR experiments. RJM led the cattle exposure experiments. All coauthors participated in revision of the manuscript.

Corresponding author

Correspondence to Kylie G. Bendele.

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Conflict of interest: The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with ethical standards of the Institutional Animal Care and Use Committee for the Knipling-Bushland U. S. Livestock Insects Research Laboratory and Cattle Fever Tick Research Laboratory.

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Electronic supplementary material

Below is the link to the electronic supplementary material.

10493_2019_420_MOESM1_ESM.docx

Supplementary material 1 (DOCX 15459 kb) Supplementary File S1: Unigene fasta DNA sequences assembled from the R. microplus Unfed sequence reads

10493_2019_420_MOESM2_ESM.docx

Supplementary material 2 (DOCX 12504 kb) Supplementary File S2: Unigene fasta DNA sequences assembled from the R. microplus 3 Hr Fed sequence read

10493_2019_420_MOESM3_ESM.docx

Supplementary material 3 (DOCX 15454 kb) Supplementary File S3: Unigene fasta DNA sequences assembled from the R. microplus 3 Hr Frustrated sequence reads

10493_2019_420_MOESM4_ESM.docx

Supplementary material 4 (DOCX 25895 kb) Supplementary File S4: Unigene fasta DNA sequences assembled from the R. microplus Adult Female Pooled sequence reads

10493_2019_420_MOESM5_ESM.xlsx

Supplementary material 5 (XLSX 45 kb) Supplementary Table S1: Transcriptome assembly optimization results. CLC, Trinity, and Soapdenovo assemblers were utilized with varying kmer and bubble sizes to obtain optimal assemblies of the Unfed, 3 Hr Fed, 3 Hr Frustrated, and Adult Female Pooled transcriptomes. BUSCO analysis was also included in the ultimate determination of optimal assembly for submission to NCBI

10493_2019_420_MOESM6_ESM.xlsx

Supplementary material 6 (XLSX 8729 kb) Supplementary Table S2: Annotation associated with transcripts from the UnFed transcriptome. Blast2GO PRO version 5.1.13 was used to perform BlastX against the UniProt/SwissProt database with an e-value of 1.0E-10 and default blast parameters. BlastX, InterProScan, GO mapping, enzyme code assignment and KEGG analysis results are tabulated

10493_2019_420_MOESM7_ESM.xlsx

Supplementary material 7 (XLSX 8342 kb) Supplementary Table S3: Annotation associated with transcripts from the 3 Hr Fed transcriptome. Blast2GO PRO version 5.1.13 was used to perform BlastX against the UniProt/SwissProt database with an e-value of 1.0E-10 and default blast parameters. BlastX, InterProScan, GO mapping, enzyme code assignment and KEGG analysis results are tabulated

10493_2019_420_MOESM8_ESM.xlsx

Supplementary material 8 (XLSX 9300 kb) Supplementary Table S4: Annotation associated with transcripts from the 3 Hr Frustrated transcriptome. Blast2GO PRO version 5.1.13 was used to perform BlastX against the UniProt/SwissProt database with an e-value of 1.0E-10 and default blast parameters. BlastX, InterProScan, GO mapping, enzyme code assignment and KEGG analysis results are tabulated

10493_2019_420_MOESM9_ESM.xlsx

Supplementary material 9 (XLSX 11202 kb) Supplementary Table S5: Annotation associated with transcripts from the Adult Female Pooled transcriptome. Blast2GO PRO version 5.1.13 was used to perform BlastX against the UniProt/SwissProt database with an e-value of 1.0E-10 and default blast parameters. BlastX, InterProScan, GO mapping, enzyme code assignment and KEGG analysis results are tabulated

10493_2019_420_MOESM10_ESM.xlsx

Supplementary material 10 (XLSX 53 kb) Supplementary Table S6: Listing and rankings of the top 100 BP, CC, and MF GO terms for the Unfed, 3 Hr Fed, 3 Hr Frustrated, and Adult Female Pooled transcriptomes. Terms are also shown in relation to their ranking in the Adult Female Pooled transcriptome

10493_2019_420_MOESM11_ESM.xlsx

Supplementary material 11 (XLSX 480 kb) Supplementary Table S7: : OrthoVenn analysis cluster lists of translated transcripts for Unfed, 3 Hr Fed and 3 Hr Frustrated. The table includes a separate tab for each set of cluster lists found during OrthoVenn analysis of Unfed, 3 Hr Fed and 3 Hr Frustrated translated contigs from assembled transcriptomes. Each row is a protein cluster determined by the OrthoVenn program

10493_2019_420_MOESM12_ESM.xlsx

Supplementary material 12 (XLSX 7746 kb) Supplementary Table S8: Differential gene expression analysis data for Unfed vs. 3 Hr Fed. Using the EdgeR package 3.4.0-based algorithm of the CLC Genomics Workbench 8.0.1, we mapped reads to the Adult Female Pooled transcriptome and show expression data for each transcript. The table includes read counts from each transcriptome replicate and data generated from the EDGE test of Unfed vs. 3 Hr Fed, including fold-change, and p-values

10493_2019_420_MOESM13_ESM.xlsx

Supplementary material 13 (XLSX 9535 kb) Supplementary Table S9: Differential gene expression analysis data for Unfed vs. 3 Hr Frustrated. Using the EdgeR package 3.4.0-based algorithm of the CLC Genomics Workbench 8.0.1, we mapped reads to the Adult Female Pooled transcriptome and show expression data for each transcript. The table includes read counts from each transcriptome replicate and data generated from the EDGE test of Unfed vs. 3 Hr Frustrated, including fold-change, and p-values

10493_2019_420_MOESM14_ESM.xlsx

Supplementary material 14 (XLSX 7724 kb) Supplementary Table S10: Differential gene expression analysis data for 3 Hr Fed vs. 3 Hr Frustrated. Using the EdgeR package 3.4.0-based algorithm of the CLC Genomics Workbench 8.0.1, we mapped reads to the Adult Female Pooled transcriptome and show expression data for each transcript. The table includes read counts from each transcriptome replicate and data generated from the EDGE test of 3 Hr Fed vs. 3 Hr Frustrated, including fold-change, and p-values

10493_2019_420_MOESM15_ESM.pptx

Supplementary material 15 (PPTX 447 kb) Supplementary Figure S1: Pie charts showing unigenes with BlastX hits. Each pie chart slice displays number and % of total of the Unfed, 3 Hr Fed, 3 Hr Frustrated and Adult Female Pooled transcriptome unigenes that have a BlastX hit with associated GO terms, have a BlastX hit without associated GO terms, have no BlastX hit but have an InterProScan entry, and have no BlastX hit and no annotation terms

10493_2019_420_MOESM16_ESM.pptx

Supplementary material 16 (PPTX 4224 kb) Supplementary Figure S2: Bar graphs showing E-values for each transcriptome’s BlastX hits. For each of the Unfed, 3 Hr Fed, 3 Hr Frustrated, and Adult Female Pooled transcriptome, E-Value (represented by 1e-X with X indicated on the Y axis) is plotted vs. the number of unigenes with a BlastX hit at that specific E-value

10493_2019_420_MOESM17_ESM.pptx

Supplementary material 17 (PPTX 1509 kb) Supplementary Figure S3: Bar graphs showing the top 35 most common species showing as the top BlastX hit for the Unfed, 3 Hr Fed, 3 Hr Frustrated, and Adult Female Pooled transcriptomes

10493_2019_420_MOESM18_ESM.pptx

Supplementary material 18 (PPTX 2108 kb)Supplementary Figure S4: Bar graphs showing the 35 most common hits to InterProScan Domains for the Unfed, 3 Hr Fed, 3 Hr Frustrated, and Adult Female Pooled transcriptomes

10493_2019_420_MOESM19_ESM.pptx

Supplementary material 19 (PPTX 1513 kb) Supplementary Figure S5: Bar graphs showing the 35 most common hits to InterProScan Sites for the Unfed, 3 Hr Fed, 3 Hr Frustrated, and Adult Female Pooled transcriptomes

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Bendele, K.G., Guerrero, F.D., Cameron, C. et al. Gene expression during the early stages of host perception and attachment in adult female Rhipicephalus microplus ticks. Exp Appl Acarol 79, 107–124 (2019). https://doi.org/10.1007/s10493-019-00420-1

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