RNA-seq analyses of the midgut from blood- and serum-fed Ixodes ricinus ticks

Abstract

Adult females of the genus Ixodes imbibe blood meals exceeding about 100 times their own weight within 7‒9 days. During this period, ticks internalise components of host blood by endocytic digest cells that line the tick midgut epithelium. Using RNA-seq, we aimed to characterise the midgut transcriptome composition in adult Ixodes ricinus females during early and late phase of engorgement. To address specific adaptations to the haemoglobin-rich diet, we compared the midgut transcriptomes of genetically homogenous female siblings fed either bovine blood or haemoglobin-depleted serum. We noted that tick gut transcriptomes are subject to substantial temporal-dependent expression changes between day 3 and day 8 of feeding. In contrast, the number of transcripts significantly affected by the presence or absence of host red blood cells was low. Transcripts relevant to the processes associated with blood-meal digestion were analysed and involvement of selected encoded proteins in the tick midgut physiology discussed. A total of 7215 novel sequences from I. ricinus were deposited in public databases as an additional outcome of this study. Our results broaden the current knowledge of tick digestive system and may lead to the discovery of potential molecular targets for efficient tick control.

Figure 1. Blood- and serum-fed adult Ixodes ricinus females used in this study.

First-generation siblings I. ricinus females were membrane-fed for 3 days (partial engorgement) or 8 days (full engorgement) with either reconstituted bovine blood or bovine serum. At particular time points, ticks were dissected and individual midgut caeca were used for RNA extractions. Resulting RNA extracts from individual ticks were used for RNA-seq analyses.

Figure 2. Scanning electron microscopy of tick gut caecum and digest cells.

(A) Illustration of tick gut caecum dissected from a partially-fed adult I. ricinus female. Such caeca were used for RNA-seq analyses. Scale bars indicate 100 μm. (B) Manually disrupted digest cells maturing along tick midgut epithelium from blood- (left) and serum-fed (right) fully engorged adult I. ricinus females. Note that digest cells from either tick contain both small and large digestive vesicles. Scale bars indicate 10 μm.

Figure 3. Transcriptome analysis.

The transcriptomes from individual blood-fed (B1‒B8) and serum-fed (S1‒S8) ticks were characterised using an EdgeR package. (A) MDS plot of 16 libraries indicating the related nature of transcriptomes from a given time-point of feeding. (B) Heat map of differentially expressed intestinal contigs in individual libraries. The gene expression (RPKM) values for each gene were normalised to the standard normal distribution in order to generate Z-scores. (C) Volcano plot visualising the number of differentially expressed genes (2676) with respect to tick feeding stage. (D) Volcano plot visualising the number of differentially expressed genes (15) with respect to tick RBCs (+/−) diet composition.

Figure 4. Overview of average RKPM values for contigs differentially expressed between serum- and blood-fed ticks.

Encoded proteins and their respective average RPKM values in particular libraries are listed. ¹Abbreviations: GST-glutathione S-transferase; TNF-tumor necrosis factor; DRAM-damage-regulated autophagy modulator; PHGPx-Phospholipid hydroperoxide glutathione peroxidase; ²For contig names, sequences and other and RKPM data, see the Source data 1 at http://exon.niaid.nih.gov/transcriptome/Ixric-MG/Ir-web.xlsx; ³BvsS logFC represents the base 2 logarithm of the fold change in transcript expression when the Blood treatment was compared with the Serum treatment. 3D, FE-indicate time periods of feeding 3 days and 8 days (fully engorged), respectively. Background color coding: No colour-low expression; Grey colour-insignificant differences; Red vs. blue-significant differences.

Figure 5. Biological (independent) RT-qPCR validation of RNA-seq data.

(A) Correlation between fold change (up-regulation) of RT-qPCR (independent cDNA sets from unrelated ticks collected in the wild) and RNA-seq data on selected transcripts. The table of selected differentially expressed genes for blood-fed ticks and serum-fed ticks is shown below the graph. (B) RT-qPCR analyses of differentially expressed genes in the tick midgut dissected from I. ricinus females during and after feeding. Data were obtained from three independent cDNA sets, and normalised to elongation factor 1 (ef1α). UF-unfed; B-blood-fed; S-serum-fed; 3D, 5D, FF-indicate time periods of feeding 3 days, 5 days, 8 days (fully fed), respectively; 2 ABM and 6 ABM indicate time periods after blood meal, 2 days and 6 days, respectively. Mean and SEM are shown, n = 3 (biological replicates).

Figure 6. Gene ontology analysis.

Bar graphs show the enriched GO terms of molecular function (A,E) and biological process (B,F) for 3D ticks (A,B) and FE ticks (E,F); n indicates the number of contigs concerned. Results are shown only for E value match of <1e-15 and n ≥5. Pie charts show absolute representation of GO terms of molecular function (C,G) and biological process (D,H) for 3D ticks (C,D) and FE ticks (G,H); sum of RPKM values are shown below the charts. The three most represented categories are highlighted.

Figure 7. Overview of average RKPM of putative digestive peptidases.

Encoded proteins and their respective average RPKM values in particular libraries are listed. ¹Abbreviations and GenBank Accession Nos: IrCD–I. ricinus cathepsin D, IrCD1 (EF428204); IrCD2 (HQ615697), IrCD3 (HQ615698); IrAE–I. ricinus asparaginyl endopeptidase (legumain), IrAE1 (AY584752), IrAE2 (ortholog of I. scapularis XM_002402043); IrAE3 (unpublished); IrCB–I. ricinus cathepsin B, IrCB1 (EF428206); IrCB2 (unpublished); Ir-putative longipain (orthologue of I. scapularis XM_002433755); IrCL–I. ricinus catheppsin L, IrCL1 (EF428205); IrCL3 (ortholog of I. scapularis XM_002405329 ); IrCC–I. ricinus cathepsin C (EU128750); ²For contig names, sequences and other and RKPM data, see the Source data 1 at http://exon.niaid.nih.gov/transcriptome/Ixric-MG/Ir-web.xlsx. Background color coding: No colour-low expression; Grey colour-insignificant differences; Red vs. blue-substantial differences. *-non homogenous expression over four biological replicates.

Figure 8. Overview of average RKPM of selected protease inhibitors.

Encoded proteins and their respective average RPKM values in particular libraries are listed. ¹Abbreviations: BPTI-bovine pancreatic trypsin inhibitor, TIL-trypsin inhibitor-like domain (cysteine rich); ²contig names, sequences and other and RKPM data, see the Source data 1 at http://exon.niaid.nih.gov/transcriptome/Ixric-MG/Ir-web.xlsx. Background color coding: Grey colour-insignificant differences; Red vs. blue-substantial differences. *-non homogenous expression over four biological replicates.

Figure 9. Overview of average RKPM values for contigs encoding redox, antioxidant, and detoxification proteins.

Encoded proteins and their respective average RPKM values in particular libraries are listed. ¹Abbreviations: Glu-6-P DH-Glucose-6-phosphate dehydrogenase; 6-PG DH-6-phosphogluconate dehydrogenase; Glu-Cys ligase-Glutamate-cysteine ligase; Trx-GSH Reductase-Thioredoxin Glutathione Reductase; PHGPx-Phospholipid hydroperoxide glutathione peroxidase; SOD-Superoxide dismutase; CYP450-Cytochrome P450; GST-Glutathione S-transferase; ²For contig names, sequences and other and RKPM data, see the Source data 1 at http://exon.niaid.nih.gov/transcriptome/Ixric-MG/Ir-web.xlsx. Background colour coding: Grey colour-insignificant differences; Red vs. blue-substantial differences.

Figure 10. Overview of average RKPM values for contigs encoding proteins involved in iron and haem meatbolism.

Encoded proteins and their respective average RPKM values in particular libraries are listed. ¹Abbreviations and GenBank Accession Nos: IrFer1-I. ricinus ferritin 1 (AF068224); IrFer2-I. ricinus ferritin 2 (EU885951), IrIRP-I. ricinus iron regulatory protein/cytoplasmic aconitase (EU885952); DMT-divalent metal transporter; m-Aconitase-mitochondrial aconitase; Tf-transferrin; HCP-haem carrier protein; PCFT-proton-coupled folate transporter; MRP-multidrug resistance protein; Hrg-haem responsive gene; FLVCR-group C feline leukemia virus receptor; IrCPOX-I. ricinus coproporphyrinogen oxidase; IrPPOX-I. ricinus protoporphyrinogen oxidase; ²For contig names, sequences and other and RKPM data, see the Source data 1 at http://exon.niaid.nih.gov/transcriptome/Ixric-MG/Ir-web.xlsx. Background color coding: No colour-low expression; Grey colour-insignificant differences; Red vs. blue-substantial differences.