Antimicrobial gene expression is the result of an undoubtedly complex detection/signaling pathway. The study of antimicrobial gene expression may provide clues to mechanisms employed by the tick's immune system to prevent the overgrowth of resident or imbibed microbes. Additionally, fluctuations in gene expression profiles may suggest rickettsial modulation of the host immune response as an evasion mechanism used by rickettsiae to gain access to their host. Ultimately, defining the balance between vector and pathogen will aid in elucidating factors that determine symbiotic relationships as commensal or parasitic.
In this study, we report a new defensin isoform from D. variabilis
, defensin-2, which belongs to the arthropod family of defensins. An alignment of defensin-2 with other tick defensins highlighted the conserved cysteines common to all arthropod defensins. Similarity to the other defensins was moderate but high enough to implicate defensin-2 as an antimicrobial. Recent reviews describe defensin as a multifunctional antimicrobial with abilities to act as signaling molecules that promote immune responses; as inhibitors of microbial nucleic acid, cell wall, and protein synthesis; and as pore-forming peptides (2
). The variety in the functions of antimicrobials appears to be attributable, in part, to differences in primary structures and charges at physiological pHs (2
). The corresponding gene phylogeny (based on primary structures) and the predicted acidic pI of the mature defensin-2 peptide relative to those of other tick defensins, especially defensin-1, call into question the putative function of defensin-2. The observation that Ornithodoros moubata
and Amblyomma americanum
defensins group with one another and that defensin-1 groups with other Acari defensins may indicate that the gene phylogeny classifies similarly functioning peptides. These data argue for a difference in the antimicrobial specificity or the mechanism of antimicrobial action of defensin-2.
The potential for antimicrobial gene expression at the tissue level could feasibly determine the pervasiveness and intensity of microbial infection in the tick. Antimicrobial gene expression in ticks is localized to the hemolymph, hemocytes, midgut, and fat body (4
). In this study, we investigated the basal-level tissue distributions (those in fed, uninfected ticks) of defensin-1 and -2 and reexamined the lysozyme distribution using qRT-PCR. In the soft tick Ornithodoros moubata
, there is a contrast in the tissue distribution patterns of defensin isoforms. Ornithodoros moubata
isoforms A and B are expressed in the midgut exclusively; isoforms C and D exhibit a broader distribution, with expression of the corresponding genes occurring in the fat body and midgut (34
). In our study, defensin-2 exhibited widespread tissue distribution in comparison to defensin-1. The diffuse expression pattern of defensin-2, similar to that of isoform C from Ornithodoros moubata
, suggests tissue-specific roles for each of the defensins. Because R. montanensis
infects the ovary and the sylvatic cycle is perpetuated through transovarial transmission, the abundant defensin-2 expression in the ovary is interesting. Future studies will address antimicrobial gene expression in the context of ovary invasion, survival of rickettsiae, and transovarial transmission. It is also of interest that our method of dissection of the fat bodies did not provide fat body cells free of tracheolar tissue. Because of this contamination, we cannot rule out the possibility that tracheolar tissue could be a source of defensin. However, we feel that this possibility does not detract from our findings given that the fat body is integral to defensin production in other arthropods.
As hematophagous arthropods feed, there is the potential to imbibe microbes that the host harbors. It is well documented that defensin is up-regulated in response to feeding in insects. In the blood-sucking fly Stomoxys calcitrans
, the abundance of transcripts for defensins Smd 1 and 2 increases to peak levels at 24 and 36 h post-blood meal, respectively (25
). A recent review discusses the immune signaling capabilities of cell-derived hydrophobic portions (hyppos) of molecules (41
). It is possible that damage to blood cells, incurred as hemolysis progresses during blood meal digestion by the tick (30
), could stimulate an immune response. Thus, antimicrobial gene expression during feeding may have evolved for dealing with the threat of invading microbes or for responding to free hydrophobic molecules in the midgut lumen. Therefore, the increase in the expression of defensin-1 and -2 genes in response to feeding was expected. Defensin-1 transcript levels in the fat bodies of both the fed and unfed ticks may have remained at constitutively high levels in “anticipation” of impending hemocoelic microbial infiltration.
Studies involving differential gene expression in D. variabilis
in response to R. montanensis
infection are not without precedent. Subtractive hybridization to decipher expression patterns in R. montanensis
-infected and uninfected ovaries has revealed a number of ovary-specific, differentially expressed genes that were classified according to putative functions and include receptor and adhesion genes, genes for stress response proteins, and most relevant to this study, immune function genes (28
). Of equal interest was the use of a differential display to parse the same experimental design for ovary-specific differentially expressed genes. Macaluso et al. (26
) found that Ena/vasodilator-stimulated phosphoprotein-like protein, vacuolar ATPase, and α-catenin transcripts were all up-regulated in infected ovaries, reflecting the potential importance of these proteins for rickettsial entry and intra- and intercellular mobility.
To date, there are no studies describing the rate at which or the intensity with which rickettsiae become established in the tick or the immune system recognition and action by the tick host. We assume that the recognition of rickettsiae by immune system effectors can occur as rickettsiae are acquired through blood feeding and as they begin their migration through the tick. As rickettsiae migrate from the gut to the hemocoel, they are exposed to soluble factors known (and unknown) to be active in the D. variabilis
). From our observations, we can say that rickettsiae effectively infect their tick host within 72 h postfeeding, as 100% of the ticks we tested at 72 h post-experimental feeding were infected with R. montanensis
. One hundred percent detection of rickettsiae was not observed at earlier time points, possibly because the numbers of live bacteria were below the limits of detection for our assays. We chose to continue the analyses for the 18 (0%)-, 24 (50%)-, and 48 (50%)-h time points, as ticks were challenged with rickettsiae and visibly imbibed blood, which constitutes a bacterial challenge.
In general, we saw an increase in gene expression for all tested antimicrobials, as time increased, in both the midgut and fat body. In the midgut, we saw a positive difference between the challenged and control groups as early as 24 h and as late as 72 h post-experimental feeding. This finding may be correlated to the increase in the percentage of ticks with detectable levels of rickettsiae, i.e., the increase in the number of rickettsiae that had been imbibed at these time points. While the same general trend appeared to be true for the fat bodies, we noted an apparent delay in defensin-1 (18 to 48 h) and defensin-2 (24 to 48 h) gene expression at the time when rickettsiae may have been entering the hemocoel. Pathogen-directed modulation of antimicrobial gene expression is common. Down-regulation of β-defensin is observed when mice are infected with the obligate intracellular parasite Cryptosporidium parvum
). Similarly, an inverse correlation between the E. coli
multiplicity of infection and diptericin gene expression in the Drosophila
cell line mbn-2 has been observed (15
In summary, we have described a second defensin isoform, defensin-2, from the hard tick D. variabilis, a vector for the spotted fever group rickettsia R. rickettsii. The bioinformatic and phylogenetic analyses predict that defensin-2 has antimicrobial properties. The observation that defensin-2 is expressed in a number of tissues, especially the ovary, calls into question its specificity as an antimicrobial and its involvement in rickettsial infection of the ovary. Based on the gene expression data and statistical analysis, we accept our hypothesis that differential antimicrobial gene expression levels in the midgut and fat body occur in response to R. montanensis challenge. We are continuing our work with antimicrobials to answer questions regarding their functions as general antimicrobials and antirickettsial agents. Future work will address the importance of local versus systemic responses with regard to antimicrobial expression and defensin-2 gene expression in the ovary in response to rickettsial challenge. This is the first report of profiles of antimicrobial gene expression in a vector tick in response to a spotted fever group rickettsia acquired per os.