Deep Sequencing Analysis of the Ixodes ricinus Haemocytome

Abstract

Background: Ixodes ricinus is the main tick vector of the microbes that cause Lyme disease and tick-borne encephalitis in Europe. Pathogens transmitted by ticks have to overcome innate immunity barriers present in tick tissues, including midgut, salivary glands epithelia and the hemocoel. Molecularly, invertebrate immunity is initiated when pathogen recognition molecules trigger serum or cellular signalling cascades leading to the production of antimicrobials, pathogen opsonization and phagocytosis. We presently aimed at identifying hemocyte transcripts from semi-engorged female I. ricinus ticks by mass sequencing a hemocyte cDNA library and annotating immune-related transcripts based on their hemocyte abundance as well as their ubiquitous distribution.

Methodology/principal findings: De novo assembly of 926,596 pyrosequence reads plus 49,328,982 Illumina reads (148 nt length) from a hemocyte library, together with over 189 million Illumina reads from salivary gland and midgut libraries, generated 15,716 extracted coding sequences (CDS); these are displayed in an annotated hyperlinked spreadsheet format. Read mapping allowed the identification and annotation of tissue-enriched transcripts. A total of 327 transcripts were found significantly over expressed in the hemocyte libraries, including those coding for scavenger receptors, antimicrobial peptides, pathogen recognition proteins, proteases and protease inhibitors. Vitellogenin and lipid metabolism transcription enrichment suggests fat body components. We additionally annotated ubiquitously distributed transcripts associated with immune function, including immune-associated signal transduction proteins and transcription factors, including the STAT transcription factor.

Conclusions/significance: This is the first systems biology approach to describe the genes expressed in the haemocytes of this neglected disease vector. A total of 2,860 coding sequences were deposited to GenBank, increasing to 27,547 the number so far deposited by our previous transcriptome studies that serves as a discovery platform for studies with I. ricinus biochemistry and physiology.

Fig 1. The tick microplusin family of antimicrobial peptides.

A) ClustalW alignment. The symbols at the top of the figure represent (*) identity, (:) similarity, and (.) lesser similarity. B) The neighbour-joining phylogenetic tree from the alignment in (A) following 1,000 bootstraps. Sequence names are represented by the first three letters of the genus name, followed by the first three letters of the species name, followed by their GenBank gene identifier (gi) accession number. Sequences from this work start with IrHem and include IrHemSgMg-674, which is 140-times overexpressed in haemocytes. Ixodes ricinus sequences are indicated by a red circle. The bar at the bottom represents 50% amino acid diversity. The numbers at the nodes represent the per cent bootstrap support. Values below 50 are not shown.

Fig 2. The tick defensin family of antimicrobial peptides.

A) ClustalW alignment. The symbols at the top of the figure represent (*) identity and (.) lesser similarity. B) The neighbour-joining phylogenetic tree from the alignment in (A) following 1,000 bootstraps. Sequence names are represented by the first three letters of the genus name, followed by the first three letters of the species name, followed by their GenBank gene identifier (gi) accession number. Ixodes scapularis scapularisin and scasins are indicated. Sequences from this work start with IrHem or IrSigp. The bar at the bottom represents 20% amino acid diversity. The numbers at the nodes represent the percent bootstrap support. Values below 50 are not shown.

Fig 3. Tick ML domain proteins.

A) ClustalW alignment. The symbols at the top of the figure represent (*) identity, (:) similarity, and (.) lesser similarity. B) The neighbour-joining phylogenetic tree from the alignment in (A) following 1,000 bootstraps. Sequence names are represented by the first three letters of the genus name, followed by the first three letters of the species name, followed by their GenBank gene identifier (gi) accession number. Sequences from this work start with IrHem. Ixodes ricinus sequences are identified with a red symbol. The bar at the bottom represents 20% amino acid diversity. The numbers at the nodes indicate the percentage bootstrap support.

Fig 4. Phylogeny of invertebrate peptidoglycan recognition proteins.

The A) sequence alignments and B) neighbour-joining phylogenetic tree following 1,000 bootstraps are shown. Sequence names are represented by the first three letters of the genus name, followed by the first three letters of the species name, followed by their GenBank gene identifier (gi) accession number. Sequences from this work start with IrHem or IrSigp. The bar at the bottom represents 10% amino acid diversity. The numbers at the nodes indicate the percentage bootstrap support.

Fig 5. The haemocyte-enriched cystatins of I. ricinus compared to other tick cystatins.

The neighbour-joining phylogenetic tree following 1,000 bootstraps is shown. Clades with significant bootstrap support are shown in colour. Sequence names are represented by the first three letters of the genus name, followed by the first three letters of the species name, followed by their GenBank gene identifier (gi) accession number. Sequences from this work start with IrHem or IrSigp and include peptide sequences highly expressed in haemocytes, identified by a red symbol. The bar at the top indicates 50% amino acid diversity. The numbers at the nodes indicate the percentage bootstrap support. Clades with strong bootstrap support are shown in colour.

Fig 6. The insect defence reeler protein family of arthropods.

A) ClustalW alignment. The symbols at the top of the figure represent (*) identity, (:) similarity, and (.) lesser similarity. B) The neighbour-joining phylogenetic tree from the alignment in A following 1,000 bootstraps. Sequence names are represented by the first three letters of the genus name, followed by the first three letters of the species name, followed by their GenBank gene identifier (gi) accession number. Sequences from this work start with IrHem or IrSigP. Tick sequences are identified with a red symbol. The bar at the bottom represents 50% amino acid diversity. The numbers at the nodes indicate the percentage bootstrap support.

Fig 7. The leucine-rich repeat family of Ixodes ricinus compared to other tick proteins.

The neighbour-joining phylogenetic tree following 1,000 bootstraps is shown. Sequence names are represented by the first three letters of the genus name, followed by the first three letters of the species name, followed by their GenBank gene identifier (gi) accession number. Sequences from this work start with IrHem or IrSigp and include peptide sequences highly expressed in haemocytes, identified by a red symbol. The bar at the top indicates 100% amino acid diversity. The numbers at the nodes indicate the percentage bootstrap support.