Molecular functions indentified in <i>D. reticulatus</i> unfed larvae.

<p>(A) Transcripts identified in <i>D. reticulatus</i> unfed larvae were functionally annotated and grouped according to the molecular function of the encoded proteins after removing transcripts with unknown function. (B) Proteins identified in <i>D. reticulatus</i> unfed larvae after searching against Ixodida database were functionally annotated and grouped according to their molecular function. (C) Proteins identified in <i>D. reticulatus</i> unfed larvae after searching against transcripts database (PIT) were functionally annotated and grouped according to their molecular function. The number of proteins on each category is shown.</p

Five hundred most highly represented genes.

<p>(A) The 500 more represented unigenes (protein clusters) identified in <i>D. reticulatus</i> unfed larvae were functionally annotated and grouped according to the biological process of the encoded proteins. The number of proteins on each category is shown. (B) The 500 more represented unigenes (protein clusters) identified in <i>D. reticulatus</i> unfed larvae were functionally annotated and grouped according to the molecular function of the encoded proteins. The number of proteins on each category is shown.</p

Transcriptomics of <i>D. reticulatus</i> unfed larvae.

<p>(A) Transcripts identified in <i>D. reticulatus</i> unfed larvae were functionally annotated and grouped according to the biological process of the encoded proteins. The number of proteins on each category is shown. (B) Transcripts identified in <i>D. reticulatus</i> unfed larvae were functionally annotated and grouped according to the molecular function of the encoded proteins. The number of proteins on each category is shown.</p

Stress response in <i>D. reticulatus</i> unfed larvae.

<p>(A) Stress response transcripts identified in <i>D. reticulatus</i> unfed larvae were grouped according to the function of their encoded protein. The number of proteins and percent in each category is shown. (B) Number of counts per protein (Ave+S.E.) in stress response proteins identified by transcriptomics analysis in <i>D. reticulatus</i> unfed larvae. (C) Stress response proteins identified in <i>D. reticulatus</i> unfed larvae were grouped according to the function of their encoded protein. The number of proteins and percent in each category is shown. (D) Number of peptides per protein (Ave+S.D.) in stress response proteins identified by proteomics analysis in <i>D. reticulatus</i> unfed larvae.</p

Biological processes identified in <i>D. reticulatus</i> unfed larvae.

<p>(A) Transcripts identified in <i>D. reticulatus</i> unfed larvae were functionally annotated and grouped according to the biological process of the encoded proteins after removing transcripts with unknown function. (B) Proteins identified in <i>D. reticulatus</i> unfed larvae after searching against Ixodida database were functionally annotated and grouped according to their biological process. (C) Proteins identified in <i>D. reticulatus</i> unfed larvae after searching against transcripts database (PIT) were functionally annotated and grouped according to their biological process. The number of proteins on each category is shown.</p

mRNA levels for selected genes encoding for stress response proteins.

<p>(A) The mRNA levels were characterized by real-time RT-PCR in <i>D. reticulatus</i> unfed larvae and adult female and male guts and salivary glands (N = 3), normalized against tick ribosomal protein S4 and shown as Ave+S.D. in arbitrary units. Normalized Ct values were compared between larvae and adult samples by Student's t-test with unequal variance (*P≤0.05). (B–D) The mRNA levels were characterized by real-time RT-PCR in <i>D. reticulatus</i> guts and salivary glands from adult female and male ticks incubated at 4, 19 and 37°C for 4.5 h prior to RNA extraction (N = 3), normalized against tick ribosomal protein S4 and shown as Ave+S.D. in arbitrary units. Normalized Ct values were compared between samples from ticks incubated at 4 or 37°C and 19°C by Student's t-test with unequal variance (*P≤0.05). (E) For genes with significant differences between samples from ticks incubated at 4°C or 37°C and 19°C, the log2 4/19°C or 37/19°C normalized Ct values ratio was calculated to show differential expression in response to temperature. Abbreviations: FG, female gusts; FSG, female salivary glands; MG, male guts; MSG, male salivary glands.</p

Primer sequences used for real-time RT-PCR.

<p>PCR conditions: 40 cycles of 30 sec denaturation at 95°C, 30 sec annealing at (a) 55°C or (b) 60°C and 1 min extension at 72°C.</p

Tick stress response proteins identified in <i>D. reticulatus</i> unfed larvae after transcriptomics analysis.

<p>Tick stress response proteins identified in <i>D. reticulatus</i> unfed larvae after transcriptomics analysis.</p

A Systems Biology Approach to the Characterization of Stress Response in <i>Dermacentor reticulatus</i> Tick Unfed Larvae

<div><p>Background</p><p><i>Dermacentor reticulatus</i> (Fabricius, 1794) is distributed in Europe and Asia where it infests and transmits disease-causing pathogens to humans, pets and other domestic and wild animals. However, despite its role as a vector of emerging or re-emerging diseases, very little information is available on the genome, transcriptome and proteome of <i>D. reticulatus</i>. Tick larvae are the first developmental stage to infest hosts, acquire infection and transmit pathogens that are transovarially transmitted and are exposed to extremely stressing conditions. In this study, we used a systems biology approach to get an insight into the mechanisms active in <i>D. reticulatus</i> unfed larvae, with special emphasis on stress response.</p><p>Principal Findings</p><p>The results support the use of paired end RNA sequencing and proteomics informed by transcriptomics (PIT) for the analysis of transcriptomics and proteomics data, particularly for organisms such as <i>D. reticulatus</i> with little sequence information available. The results showed that metabolic and cellular processes involved in protein synthesis were the most active in <i>D. reticulatus</i> unfed larvae, suggesting that ticks are very active during this life stage. The stress response was activated in <i>D. reticulatus</i> unfed larvae and a <i>Rickettsia</i> sp. similar to <i>R. raoultii</i> was identified in these ticks.</p><p>Significance</p><p>The activation of stress responses in <i>D. reticulatus</i> unfed larvae likely counteracts the negative effect of temperature and other stress conditions such as <i>Rickettsia</i> infection and favors tick adaptation to environmental conditions to increase tick survival. These results show mechanisms that have evolved in <i>D. reticulatus</i> ticks to survive under stress conditions and suggest that these mechanisms are conserved across hard tick species. Targeting some of these proteins by vaccination may increase tick susceptibility to natural stress conditions, which in turn reduce tick survival and reproduction, thus reducing tick populations and vector capacity for tick-borne pathogens.</p></div

<i>D. reticulatus</i> 80S ribosome transcriptomics and proteomics data.

<p>Unigenes corresponding to 80S ribosomal proteins are shown. New names refer to current nomenclature for <i>D. melanogaster</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089564#pone.0089564-Anger1" target="_blank">[7]</a>. The 80S ribosomal proteins (new/old name) L2/L8, L8e/L7A, L10/LP0, L13e/L13, L18e/L18, L20e/L18A, L22e/L22, L24/L26, L27e/L27, L29e/L29, L41e/L41, L43e/L37A, P2/LP2, RACK1/RACK1, S3/S3, S6e/S6, S10e/S10, S19e/S19, S24e/S24, S25e/S25, S26e/S26, S28e/S28, and S30e/S30 were not identified.</p