Using ESTs derived from the cDNA libraries utilized in the sialotranscriptome analysis (Ribeiro et al., 2006
), we developed a first-generation microarray to observe I. scapularis
salivary gland transcript expression changes associated with tick feeding or with flavivirus infection.
Comparison of the salivary gland transcript expression profile between unfed and fed Ixodes scapularis nymphs
Blood feeding by ticks represents an enormous change from a conserving to a robust metabolism, which involves intense salivary biosynthetic capacity as well as increased water and ion transport. Microarray analysis comparing salivary transcripts from fed (days 1, 2, and 3) to unfed ticks confirmed this increase in metabolism showing 578 transcripts to be up-regulated and only 151 down-regulated gene products in feeding ticks (; specific contigs can be seen in Supplemental Table 2
). Further analysis of demonstrates 128 transcripts assigned to the secreted class are up-regulated, while only 3 secreted transcripts are down-regulated upon feeding. The down-regulated transcripts code for a threonine-rich protein (possibly a mucin or a protein needed for water vapor capture), for a Gly-rich peptide that is similar to antimicrobial peptides (AMP) of worms (Couillault et al., 2004
) and for a member of the 8.9-kDa protein family in ticks, of unknown function, but that could be an AMP (Francischetti et al., 2009
). Because transcripts containing a signal peptide encode proteins that are likely secreted into the saliva where they may function to modulate host responses during feeding, the secreted class of differentially regulated transcripts is of particular interest.
Differentially expressed family of transcripts when the comparison was made between fed (1, 2 and 3 days) or unfed ticks.
Nine transcripts coding for transporters or for storage proteins were up-regulated upon feeding, including 2 components of the V-ATPase complex, important for water and ion movement across membranes (Nelson, 2003
), and 2 ion transporters.
Two transcripts coding for putative transcription factors (TF) were up-regulated while 4 were down-regulated upon feeding. Transcripts coding for the TF e(y)2, which is highly conserved in eukaryotes (Georgieva et al., 2001
), were up-regulated in fed ticks. In contrast, the transcriptional repressor EED/ESC/FIE was down-regulated in fed ticks. Proteins of this family are important suppressors of homeotic genes (Ohad et al., 1999
), possibly functioning to prevent differentiation of the salivary glands while the tick is in the unfed state. In the category of nuclear regulation, 6 transcripts were up-regulated during blood feeding including 4 histones, a product homologous to the Anapc13 protein, which is important for cell cycle control and a protein potentially involved in replication/repair.
In unfed ticks, a small protein associated with negative regulation of transcription from the Pol II promoter was up-regulated, as was another transcriptional regulator similar to human HCNGP, which is associated with proteins that may inhibit cell survival genes. Products coding for a CRE-binding protein as well as a homolog of the homeodomain only protein were up-regulated in unfed ticks. Up-regulation of these transcripts classified as TF is consistent with a state of transcriptional paralysis in unfed ticks. Also associated with transcriptional control, 4 transcripts involved with nuclear regulation were up-regulated in unfed ticks, including products associated with transcriptional control and histone acetylation, with histone acetyltransferase, and with the nucleosome assembly protein NAP-1, which in human and yeast facilitates the association of histones with DNA (Ishimi and Kikuchi, 1991
). These transcripts suggest a role of histone modification in the DNA regulation of unfed ticks. Transcriptional up-regulation of a gene coding for the mitochondrial single-stranded DNA-binding protein was observed in unfed ticks and is important in mitochondrial genome maintenance and replication.
Somewhat puzzling was the up-regulation of 21 products associated with protein synthesis in unfed ticks, which consisted of elongation factors and ribosomal proteins, while 24 other products in the same category were found up-regulated in fed ticks, as expected. The same situation occurs with products associated with the transcription machinery. It is possible that some vital protein synthetic pathways are observed in unfed ticks (such as production of hygroscopic saliva), which use a unique set of proteins for transcriptional and protein synthesizing machinery, perhaps due to the vast biotic differences seen in unfed (dehydrated, metabolic conserving) and fed (overhydrated, high metabolism) ticks.
Many products classified as genes coding for metabolic enzymes, cytoskeletal and extracellular matrix components, as well as for protein modification and protein export machinery were up-regulated upon feeding, as expected.
Four transposable element-associated products were up-regulated in unfed ticks, all representing Class I elements. This finding is consistent with the idea that these elements transcribe more in organisms that are stressed, such as dehydrated, unfed ticks (Capy et al., 2000
A number of unknown transcripts were found in this analysis, representing a large portion of the transcripts identified as significantly altered in their expression levels. Specifically, 175 unknown or unknown, but conserved transcripts were up-regulated, whereas 69 were down-regulated in fed ticks. Some of the transcripts of the unknown class could represent probes that aim solely to untranslated regions of genes as was shown to occur in an analysis of the sialotranscriptome of Anopheles gambiae
(Arca et al., 2005
). In addition, it is possible that some of the proteins encoded by transcripts with unknown function may be secreted into the saliva, but their secretory signal peptide was not present because the transcript lacked the 5’ end of the sequence. For example, a histamine release factor (HRF) detected in Dermacentor variabilis
saliva did not contain a putative signal peptide (Mulenga et al., 2003
). In addition, transcripts from Ixodes ricinus
(Chmelar et al., 2008
) and Ixodes scapularis
(Dai et al., 2010
) were shown to have homology to the HRF and also lack a secretion signal. For this reason and the obvious possibility that some of the unknown transcripts may have novel functions, the unknown transcripts that were differentially regulated in fed ticks are interesting targets for future study.
Temporal analysis of the salivary gland transcript expression profile in unfed and fed I. scapularis nymphs
Because the acquisition of a blood meal by ticks is a highly dynamic process, we analyzed the transcript expression profile as ticks fed over a period of 3 days. To graphically depict expression patterns over the course of feeding, a heat map of the microarray data was generated showing relative expression of differentially regulated transcripts over the 3-day feeding period (). These data demonstrate the temporal pattern of salivary gland gene transcript expression over time.
Fig. 1 Temporal pattern of salivary gland gene expression in nymphs. A heat map showing expression ratios (fed vs unfed) of 911 contigs that had statistically significant* changes during feeding or between any of the feeding timepoints. Red encodes positive (more ...)
The different times post attachment (24 h, 48 h, and 72 h) were compared to each other to ascertain if there were transcript expression changes that could be attributed to the different stages of feeding. The first day of feeding involves attachment to the host and transformation from a metabolically inactive to a metabolically active tick. Comparison of differentially expressed transcripts between day 1 and subsequent days post attachment may indicate genes that are associated with the attachment process, when up-regulated. Down-regulated transcripts in this comparison set may indicate genes that are significantly expressed in the mid and final feeding stages. Notably when comparing day 1 to days 2 and 3 post attachment, 56 transcripts coding for putative secreted proteins, as well as transcripts associated with energy metabolism, protein export machinery and cytoskeleton, which may represent the growth of the tissues as feeding progresses were found to be down-regulated during the earliest phase of blood meal acquisition (). Transcripts of the unknown class are the most abundant up-regulated gene products on day 1 (). Only 3 transcripts classified as putative secreted salivary products were up-regulated at 24 h post attachment, including a transcript coding for a Pro-Gly-rich peptide, which may be part of the attachment cement, and members of the 8.9-kDa and 18.7-kDa families, which have no known function (). Specific contigs that were differentially expressed on day 1 of feeding can be seen in Supplemental Table 3
Differentially expressed family of transcripts when the comparison was made between day 1 of feeding with days 2 and 3.
The comparison in shows differentially expressed transcripts unique to the second day of feeding (48 h post attachment) with relationship to both the previous and the following days. It provides for a relatively small number of differentially expressed transcripts, including very few related to metabolism, and protein synthesis, modification, and export as expected. Notably, 17 transcripts coding for secreted proteins are up-regulated, including several coding for Gly-Tyr-rich peptides that are similar to worm AMP, 2 members of the 9.4-kDa secreted protein family (164, 165) and a peptide containing a trypsin inhibitor like (TIL) domain that suggests it functions as a protease inhibitor and/or as an AMP (Otvos, 2000
). None of these putative secreted proteins have been functionally characterized. Specific contigs that were differentially expressed on day 2 of feeding can be seen in Supplemental Table 4
Differentially expressed family of transcripts when the comparison was made between day 2 of feeding with days 1 and 3.
The third day of nymphal feeding (72 h post attachment) is characterized by a relatively high number of up-regulated transcripts when compared to the previous 2 days of feeding (). Fifty-four transcripts coding for putative secreted proteins, possibly involved with salivary function were up-regulated on day 3 of feeding. This category includes members of TIL domain containing peptides as well as transcripts coding for putative protease inhibitors, including those coding for 2 different serpins (1070, 2940), 2 members of the Kunitz family (469, 470), and one cystatin. A transcript coding for a lipocalin was also differentially up-regulated on day 3. A member of the AMP defensin family as well as several members of the Gly-Tyr rich protein family, possibly an AMP as per their similarity to nematode peptides (Couillault et al., 2004
), were up-regulated. Transcripts coding for a secreted ribonuclease were also up-regulated and could be related to AMP function (Deshpande and Shankar, 2002
). Mucins, a Gly-rich peptide and a peritrophin were also up-regulated. Except for the cystatin, which has anti-inflammatory and immunosuppressive properties (Kotsyfakis et al., 2006
), the remaining proteins have not been functionally characterized. Notably, many transcripts coding for proteins or enzymes associated with oxidant metabolism were up-regulated, including several coding for alkyl hydroperoxide reductases, selenoproteins, and superoxide dismutase. These proteins, if secreted, could help to counteract oxygen radicals released by activated leukocytes, or intracellularly to prevent oxidative damage that could result from the very active prostaglandin synthetic activity during feeding that leads to high amounts of PGE2
in tick saliva (Ribeiro et al., 1992
; Sa-Nunes et al., 2007
). Dopamine is a salivary secretagogue in ticks, and possibly associated with its catabolism is the increased expression of transcripts homologous to cytosolic sulfotransferases that are known detoxicants of catecholamines (Negishi et al., 2001
). Enzymes associated with proline hydroxylation were also up-regulated, suggesting, as previously stated (Ribeiro et al., 2006
), that many of the proline-rich salivary proteins could be modified to look more like collagen, which has many hydroxyproline and hydroxylysine post-translation modifications. Transcripts coding for homologs of the vertebrate cytokine Macrophage Inhibition Factor were also elevated (Orita et al., 2002
), but it is not clear whether this ubiquitous protein is secreted in saliva, as it does not have a signal peptide. As in the previous comparisons, many transcripts of unknown function and putative secreted proteins were differentially transcribed. Specific contigs that were differentially expressed on day 3 of feeding can be seen in Supplemental Table 5
Differentially expressed family of transcripts when the comparison was made between day 3 of feeding with days 1 and 2.
To confirm the validity of the microarray analysis, 15 transcripts shown to be differentially expressed in the microarray analysis were analyzed using quantitative real-time PCR (qPCR). I. scapularis 16s rRNA was used for normalization and fold changes were calculated using the standard curve method. Linear regression analysis was performed and correlation values calculated by plotting the fold changes observed in the microarray against the fold changes observed in qPCR. This analysis gave a good degree of correlation (R2=0.70, data not shown), typical for this kind of cross comparison, validating the results of the microarray analysis.
Comparison of the salivary gland transcription profile between Langat virus-infected and mock-infected I. scapularis nymphs
Validation of the microarray comparing fed to unfed ticks provided us with the background to observe the salivary gland transcript expression changes associated with flavivirus infection in ticks. LGTV-infected or mock-infected nymphs were fed on mice and subsequently harvested at 1, 2, and 3 days post attachment. Small, but significant groups of salivary gland transcript expression changes were observed at all 3 time points of feeding when LGTV-infected and mock-infected ticks were compared (). The small number of differentially expressed salivary gland transcripts observed in this study is similar to other studies involving infection of ticks with Borrelia burgdorferi
and Theileria parva
(Alarcon-Chaidez et al., 2006
; Nene et al., 2004
Differentially expressed transcripts in LGTV infected compared to mock infected nymphs over the average of the three feeding timepoints (A) or at 1 day (B), 2 days (C) or 3 days (D) post attachment.
When all 3 time points post attachment were averaged, 3 transcripts were statistically differentially regulated in LGTV-infected ticks compared to uninfected ticks (). One of these is classified into the 25-kDa salivary gland protein family and has homology to the lipocalins. The other 2 transcripts (213, 2446) have no known function.
On day 1 of feeding (24 h post attachment), transcripts that were up-regulated in LGTV-infected nymphs include several putative secreted proteins, which fall into the 5.3-kDa, 8.4-kDa, 10.2-kDa, and 22.5-kDa (1368, 2827) protein families (). Up-regulated transcripts also include a serpin, a glutathione S-transferase homolog, a metalloprotease, and a lipocalin-like protein. Only 5 transcripts were down-regulated on day 1 in LGTV-infected ticks and include rRNA, a 16-kDa salivary gland protein, 2 putative secreted proteins (156, 2520), and an unknown transcript.
Only 5 of 16 differentially regulated transcripts were up-regulated in LGTV-infected ticks 2 days post attachment (). The up-regulated transcripts include a Kunitz domain-containing protein, an NADH dehydrogenase subunit, a member of the 25-kDa salivary gland protein family with homology to the lipocalins, and 2 unknown proteins (1117, 2607). The majority of differentially regulated transcripts on day 2 is down-regulated and includes 2 microplusin-related proteins, several housekeeping genes, a serpin, a lipocalin, and a Kunitz protease inhibitor.
The smallest number of differentially regulated transcripts in LGTV-infected ticks was observed 3 days post attachment (). Up-regulated transcripts include rRNA, an ATP synthase subunit, an NADH dehydrogenase subunit, and a 26-kDa salivary gland protein family member with homology to lipocalins. One putative secreted protein had a fold change of 5.1 (log2), suggesting this transcript to be highly up-regulated in LGTV-infected ticks. Another interesting transcript is classified into the 25-kDa salivary gland protein family and also has homology to the lipocalins. This protein had a fold change of 3.5 (log2) compared to uninfected ticks on day 3. Down-regulated transcripts include a defensin precursor, an AMP, a putative 8.9 secreted protein family member, and a putative 18.7-kDa secreted protein family member with homology to the lipocalins. At all 3 time points of feeding, several differentially regulated transcripts were classified as unknown.
Sixteen transcripts were analyzed by qPCR to confirm the validity of the microarray results. All of the transcripts tested were validated in the direction of their change in the microarray analysis, but not necessarily in the magnitude of the change. For example, if the transcript was up-regulated in the microarray analysis, it was also found to be up-regulated by qPCR. However, the actual fold change values for each transcript varied leading to a low correlation when linear regression analysis was performed (R2=0.47, data not shown). The low correlation may be further explained because of differences in normalization methods or differences in the location of the primers for real-time PCR compared to hybridization location of the microarray probes.