Previous reports documented differential gene expression in A. marginale-infected tick guts and salivary glands and cultured tick cells . The expression of GST, SelM, vATPase and subolesin was upregulated in D. variabilis and/or IDE8 tick cells in response to infection with A. marginale [7, 9]. Conversely, functional analysis of these genes by RNAi demonstrated that A. marginale infection levels in D. variabilis guts and/or salivary glands were reduced after gene knockdown . However, these experiments did not provide evidence of how these genes affected the developmental cycle of A. marginale in ticks, which was the objective of the experiments reported herein.
The results reported in this study further confirm that GST, SelM, vATPase and subolesin are overexpressed in response to infection of ticks with A. marginale to increase infection/multiplication rate . In general, the number of A. marginale colonies was lower in most tissues in gene knockdown ticks when compared to controls. Notably, colonies were not seen by light microscopy in salivary glands of the gene silenced ticks, suggesting that transmission may be diminished or prevented. The results of the light microscopy analysis further suggested that the proteins encoded by these genes have different impacts on the development of A. marginale in ticks. GST may be important for the development of DF in guts of AF ticks. Subolesin was also essential for the multiplication of the pathogen in gut cells both after AF and TF as gene knockdown resulted in significantly lower RF-containing colonies. The increase in the densities of RFs in SelM dsRNA-injected ticks after AF and TF and DFs in the gut of vATPase dsRNA-injected ticks after TF provided interesting results suggesting that gene silencing affected the development of the pathogen. SelM knockdown in tick guts after AF and TF resulted in higher densities of colonies containing RFs, and thus appeared to inhibit development of A. marginale to the dense or infective forms. However, densities of colonies containing DFs were not significantly different from the controls in guts after AF or TF.
In most cases, the results of A. marginale infection levels determined by msp4 PCR were similar to light microscopy findings of RF- and DF-containing colonies in guts and salivary glands. However, some incongruence was observed between both types of analysis. In all cases except for the number of RF-containing colonies in the gut of SelM knockdown ticks after AF and TF, the msp4 PCR results showed higher infection levels than those predicted by light microscopy analysis when compared to controls. The detection of higher infection levels by PCR may be explained either by the PCR amplification of DNA from organisms not forming colonies or resulted from the sampling observed in a single cross section of the tick halves. Also, PCR did not differentiate between tissues that may be dissected together while light microscopy analysis allowed for examination of individual tick tissues. In ticks with SelM knockdown, light microscopy analysis showed an increase in A. marginale RF but not DF-containing colonies, which may have also influenced the results obtained by both methods.
The mechanism by which these proteins affect A. marginale developmental cycle in ticks is still unknown. However, information on the function of these proteins can be incorporated into discussion of their role in A. marginale infection/multiplication. Selenoproteins are selenocysteine (Sec)-containing proteins that are involved in a variety of cellular processes such as oxidant metabolism . In humans, SelM is expressed in many tissues and is localized in the endoplasmic reticulum . In ticks, Ribeiro et al.  identified selenoproteins in salivary glands of I. scapularis after blood feeding or B. burgdorferi infection. However, little is known about the function of these proteins in ticks. In other arthropods such as Drosophila, selenoproteins have been implicated in survival, salivary gland development and fertility [13, 14]. SelM was overexpressed in IDE8 tick cells infected with A. marginale and a selenoprotein gene was overexpressed in A. marginale-infected R. microplus ticks . SelM was also overexpressed in the gill of white shrimp (Litopenaeus vannamei) infected with the white spot syndrome virus . Taken together, these results suggest that selenoproteins may function to reduce the oxidative stress caused by pathogen infection in ticks. However, as shown herein, SelM may have other functions in ticks, perhaps related to salivary gland development, that explain why reduction in its expression prevents A. marginale from infection and/or multiplication in salivary glands after TF. The increase noted in the colony densities containing RFs in SelM silenced ticks both after AF and TF, suggests that expression of this gene directly impacts the A. marginale developmental cycle.
GST belongs to a gene family that functions in the detoxification of xenobiotic compounds and metabolites produced by cell oxidative stress [16–18]. GSTs have been found to be overexpressed in both infected [19, 20] and uninfected ticks . In human cells infected with A. phagocytophilum or R. rickettsii, GST genes were down-regulated [22, 23]. GST was overexpressed both in IDE8 tick cells and D. variabilis salivary glands in response to infection with A. marginale . However, congruent with proteomics results, real-time RT-PCR analysis of GST expression in D. variabilis guts and R. microplus ticks revealed that mRNA levels were higher in uninfected ticks . These results suggest that ticks have multiple GST genes with different tissue-specific expression patterns that could play different roles during A. marginale infection . As in other arthropods [24–27], GSTs may be involved in tick innate immunity by protecting cells from oxidative stress as a result of bacterial infection . Additionally, GST may function as a stress response protein during blood feeding in ticks [19–21]. As determined by RNAi combined with PCR and light microscopy analysis of A. marginale, GST appears to be required for pathogen infection of D. variabilis guts and salivary glands and IDE8 cells, thus suggesting that the pathogen benefits from GST function, perhaps by diminishing the deleterious effect that cell oxidative stress metabolites may have on bacterial multiplication and development [7, 28]. Most interesting in this study was the notable increase of A. marginale infection in fat body cells in the GST silenced ticks, which represents a change in the A. marginale developmental cycle.
vATPase is a multisubunit enzyme that mediates acidification of eukaryotic intracellular organelles which has been associated with the cytoskeleton and clathrin-coated vesicles that facilitate receptor-mediated endocytosis required for rickettsial infection [6, 29, 30]. Functional vATPase was shown to be required for the normal function of the Golgi complex, endoplasmic reticulum, vacuoles and endocytotic and exocytotic vesicles . vATPase was also implicated in immunity . Genetic knockout of vATPase subunits resulted in lethal phenotypes in yeast, Neurospora, Drosophila and mice . The vATPase knockdown in Drosophila and human cells reduced influenza virus replication . In ticks, vATPase has been implicated in salivary fluid secretion in Amblyomma americanum . The results in A. marginale-infected tick cells were similar to those in D. variabilis ticks infected with R. montanensis in which vATPase mRNA levels were increased [7, 34], as well as studies in which human HL-60 cells were infected with A. phagocytophilum . Furthermore, RNAi of vATPase expression reduced A. marginale infection of D. variabilis gut cells but not pathogen multiplication in IDE8 cells . These results together with those reported herein suggest that vATPase may be functionally important for A. marginale development in ticks by affecting pathogen infection of guts and salivary glands. Additionally, vATPase knockdown resulted in testis and salivary gland degeneration, suggesting a role for this molecule in the function of these organs.
The tick subolesin was recently discovered as a tick protective antigen in Ixodes scapularis . Subolesin was shown by both RNAi gene knockdown and immunization trials using the recombinant protein to protect hosts against tick infestations, reduce tick survival and reproduction, and cause degeneration of gut, salivary gland, reproductive tissues and embryos [36–41]. Subolesin was shown to function in the control of gene expression in ticks through the interaction with other regulatory proteins [7, 42, 43]. These studies demonstrated a role of subolesin in the control of multiple cellular pathways by exerting a regulatory function on global gene expression in ticks. Subolesin was also shown to be differentially expressed in Anaplasma-infected ticks and cultured tick cells [7, 42]. The targeting of tick subolesin by RNAi or immunization was also resulted in decreased vector capacity of ticks for A. marginale and A. phagocytophilum, respectively . Consistent with these results, in the experiments reported herein subolesin knockdown resulted in gut and salivary gland degeneration and affected the development of both DFs and RFs in the gut and the movement to and infection of salivary glands. These results provide additional evidence of the role of subolesin during A. marginale developmental cycle in ticks. RNAi has become an important tool for the study of gene expression and function in ticks . However, little is known about the process of RNAi in ticks . In a recent study, we analyzed the possible off-target effects after tick subolesin RNAi and found that it is a highly specific process . However, these studies have not been performed for other tick genes. Therefore, the possibility of off-target effects may exist particularly for multigene families such as those including SelM and GST. Nevertheless, it is likely that off-target effects, if present would affect the expression of other members of the gene family that are relevant for the results presented and discussed herein.