- Open Access
The tick endosymbiont Candidatus Midichloria mitochondrii and selenoproteins are essential for the growth of Rickettsia parkeri in the Gulf Coast tick vector
© The Author(s). 2018
- Received: 9 February 2018
- Accepted: 30 July 2018
- Published: 13 August 2018
Pathogen colonization inside tick tissues is a significant aspect of the overall competence of a vector. Amblyomma maculatum is a competent vector of the spotted fever group rickettsiae, Rickettsia parkeri. When R. parkeri colonizes its tick host, it has the opportunity to dynamically interact with not just its host but with the endosymbionts living within it, and this enables it to modulate the tick’s defenses by regulating tick gene expression. The microbiome in A. maculatum is dominated by two endosymbiont microbes: a Francisella-like endosymbiont (FLE) and Candidatus Midichloria mitochondrii (CMM). A range of selenium-containing proteins (selenoproteins) in A. maculatum ticks protects them from oxidative stress during blood feeding and pathogen infections. Here, we investigated rickettsial multiplication in the presence of tick endosymbionts and characterized the functional significance of selenoproteins during R. parkeri replication in the tick.
FLE and CMM were quantified throughout the tick life stages by quantitative PCR in R. parkeri-infected and uninfected ticks. R. parkeri infection was found to decrease the FLE numbers but CMM thrived across the tick life cycle. Our qRT-PCR analysis indicated that the transcripts of genes with functions related to redox (selenogenes) were upregulated in ticks infected with R. parkeri. Three differentially expressed proteins, selenoprotein M, selenoprotein O, and selenoprotein S were silenced to examine their functional significance during rickettsial replication within the tick tissues. Gene silencing of the target genes was found to impair R. parkeri colonization in the tick vector. Knockdown of the selenogenes triggered a compensatory response from other selenogenes, as observed by changes in gene expression, but oxidative stress levels and endoplasmic reticulum stress inside the ticks were also found to have heightened.
This study illustrates the potential of this new research model for augmenting our understanding of the pathogen interactions occurring within tick hosts and the important roles that symbionts and various tick factors play in regulating pathogen growth.
- Rickettsia parkeri
Ticks are blood-feeding ectoparasites of both humans and animals and are important from a public health perspective because they serve as competent vectors of various disease-causing infectious agents. Many tick-borne pathogens are equipped to infect various tick organs where they can multiply. Infection of the salivary glands enables tick pathogens to readily infect vertebrate hosts upon tick feeding. The spotted fever group rickettsial (SFGR) agent, Rickettsia parkeri, is maintained in tick populations through transstadial (between life stage molts) and transovarial or vertical transmission (deposition into eggs for next-generation pathogen development) . A. maculatum, the Gulf Coast tick, is an arthropod vector with increasing public health significance because of its role as the primary vector of R. parkeri in the USA . Rickettsial diseases are caused by obligate intracellular Gram-negative bacteria, and these organisms infect humans on all continents except Antarctica [3–5]. In modern times, the rate and ease of global movement has increased the risk of transporting ticks and tick-borne diseases that may have previously been restricted to one region.
The microorganisms that occupy a tick vector are collectively called the tick microbiome; however, the collection of commensal, symbiotic, and pathogenic microbes associated with ticks are more specifically termed the “pathobiome.” Although the inclusion of all three microbial types appears to be counterintuitive at first sight, it is possible that microbes living in association with pathogens within ticks can positively influence pathogen transmission. For instance, rickettsial endosymbionts are thought to alter the transmission of other rickettsial pathogens, as shown by the inverse relationship between the infection prevalence of Rickettsia rickettsii (pathogen) and R. peacockii (symbiont) in the Rocky Mountain wood tick Dermacentor andersoni [6, 7]. Likewise, the presence of Coxiella-related symbionts in the salivary glands of A. americanum ticks has been proposed to impair the transmission of Ehrlichia chaffeensis . In addition to symbionts, ticks maintain a natural bacterial flora predominantly composed of Proteobacteria, Firmicutes, and Bacteroides phyla [9–14], which have also been implicated in pathogen maintenance interference in the tick. For example, when Ixodes scapularis ticks are hatched and raised in a sterile environment, their microbiota is altered such that they experience impaired gut integrity and reduced colonization ability towards Borrelia burgdorferi . As seen with other arthropod vectors, altering the tick microbiome may also result in a modulated type of immune response that can interfere with pathogen survival and infection .
A dynamic interaction happens between tick vectors and their associated disease-causing agents, and this has been referred to as a continuous “bellum omnium contra omes” or war of all against all . An unavoidable interaction between a pathogen and the obligate symbiont(s) in a vector occurs during colonization and transmission. However, understanding about the interactions between rickettsial endosymbionts and pathogenic bacteria in ticks and how they influence each other is limited. There are few published reports on the roles played by symbionts in ticks and whether these bacteria have an impact on tick proliferation or transmission [18, 19]. The symbionts commonly associated with hard ticks belong to Rickettsia, Francisella, Coxiella, Wolbachia, and Candidatus Midichloria genera . Francisella-like endosymbionts (FLE), which have been detected in many ticks [21–23], are γ-proteobacterial symbionts and are related to the bacterium that causes tularemia, Francisella tularensis . Genetically distinct FLEs have been reported in D. variabilis and D. andersoni  and across the tick’s developmental stages . Gerhart et al.  hypothesized that pathogenic F. tularensis was capable of transforming into symbiotic FLE in ticks. Candidatus Midichloria mitochondrii (CMM), an α-proteobacterial symbiont first detected in I. ricinus, has a unique intramitochondrial lifestyle . Based on phylogenetic and statistical studies of the 16S rRNA sequences from Midichloria and “similar organisms,” CMM is proposed to belong to a novel family known as “Candidatus Midichloriaceae”  and is widespread in various ixodid ticks . However, our understanding of the interactions between endosymbionts (FLE, CMM) and pathogenic bacteria (R. parkeri) in tick tissues and how they influence each other remains limited.
In the absence of preventive measures, the increasing number of tick-borne diseases poses a significant threat to public health. To survive, ticks must maintain homeostasis (stable equilibrium maintained by physiological processes) and obtain gigantic blood meals of up to 100 times their unfed weight. Selenium (Se) is an essential trace element that is incorporated as selenocysteine (Sec) into selenoproteins (SELENO), many of which form an essential line of defense against oxidative stress damage . These proteins are also responsible for myriad other functions including Se transport, protein folding, and endoplasmic reticulum-associated degradation (ERAD). The endoplasmic reticulum (ER) is involved in intracellular signaling, protein synthesis and protein folding, glycosylation, and secretion of saliva via the exocytotic pathway. Tick saliva composition, as revealed by our sialotranscriptome (from the Greek, sialo means saliva), indicated the presence of over 5000 putative secreted peptides representative of dozens of protein families [32, 33]. Protein folding is dependent on the oxidation of disulfide bridges via reactive oxygen species (ROS). A heightened oxidative environment can impair protein folding, leading to the accumulation of unfolded or misfolded proteins and ultimately ER stress. ER homeostasis can be disrupted by a variety of insults such as the accumulation of misfolded proteins, elevated levels of ROS, pathogen infections, and abnormalities in Ca+ 2 signaling. These disturbances are able to trigger the unfolded-protein response (UPR), a protective counter-measure that acts to reestablish homeostatic balance and promote survival by increasing the production of the chaperones involved in protein folding or by inhibiting global translation and eliminating chronically misfolded proteins. Proteins that fail to properly fold are eliminated via ERAD. ER-resident selenoproteins play a critical role in modulating oxidative and ER stress during prolonged tick feeding on the host. We have discovered multiple factors involved in the synthesis of the tick selenoproteome (a full set of novel selenoproteins in ticks), including a novel eukaryotic elongation factor (eEFSec), a novel SECIS-binding protein (SBP2), and Sec-tRNASec [31–34]. The serendipitous RNA-Seq findings from our experimental gene silencing of eEFSec indicated that dramatic changes in the expression patterns of the transcripts encoding secreted salivary proteins had occurred . Our further studies revealed that selenoproteins and antioxidants participate in SFGR colonization within the tick vector and in their vertical transmission to the next generation [31, 34–38]. Interestingly, I. scapularis Salp25D (a glutathione peroxidase) and D. variabilis SELENOM confer a survival advantage on B. burgdorferi  and Anaplasma marginale .
In the present study, we used two approaches to gain a better understanding of the replication and physiology of A. maculatum R. parkeri infected (Rp+) and R. parkeri-free (Rp−) colonies isolated from field collection and continuously propagated at The University of Southern Mississippi, USA. First, we examined the potential interplay between pathogenic Rickettsia (R. parkeri) and the dominant non-pathogenic tick symbionts by quantifying the FLE and CMM symbiont loads with or without rickettsial infections. Second, we investigated the differential gene expression of specific tick selenogenes in R. parkeri-infected tissues. Third, we utilized an RNA interference approach to deplete the expression of differentially regulated SELENOM, SELENOO, and SELENOS selenogenes and assess their functional significance in pathobiome maintenance in the tick vector. Overall, we have shown that R. parkeri replication success is correlated with the quantity of CMM present in the tick at the expense of FLE in the tick, and that selenogenes play important roles in tick–pathogen interactions.
Quantitation of R. parkeri, FLE, and CMM across the tick life cycle
R. parkeri-infected (Rp+) and R. parkeri-free (Rp−) A. maculatum tick colonies were established and maintained in the laboratory for studying the dynamics of symbiont–Rickettsia interactions. Our previous microbiome analysis of A. maculatum identified FLE and R. parkeri in A. maculatum ticks . Recently, our Illumina-sequenced sialotranscriptome work has also detected significant numbers of reads from CMM in A. maculatum (Shahid Karim, unpublished results).
The relative concentrations of FLE and CMM in the total bacterial load were estimated in the Rp+ and Rp− ticks to assess the potential interplay that may occur during bacterial replication among R. parkeri, FLE, and CMM (Figs. 2 and 3) across the immature and mature developmental stages of the ticks. The total bacterial concentration was significantly reduced in the Rp− eggs compared with the Rp+ eggs (Fig. 2a–c). In the unfed Rp+ larvae, the total bacterial concentration was lower than in the unfed Rp− ticks, but a significant effect was not observed for FLE or CMM (Fig. 2d–f). However, the larval blood meal in the Rp+ ticks greatly enhanced the total bacterial load and the FLE and CMM concentrations relative to those in the Rp− ticks (Fig. 3d–f). The outcome of this experiment in the nymphs did not parallel those for the larval stage (Fig. 3g–i). Indeed, the Rp+ ticks had reduced FLE loads in the fed and unfed nymphs as compared with the Rp− ticks. Similarly, the total bacterial and CMM loads were seen to increase in the Rp+ nymphs compared with the Rp− nymphs regardless of feeding status (Fig. 3g–i).
The presence of an R. parkeri infection increased the total bacterial load in the Rp+-infected female gut tissues, salivary glands, and ovarian tissues (Fig. 2). The total bacterial load  in naïve ticks (from the Oklahoma State Tick Rearing Facility), along with the FLE levels, were found to decrease rapidly in both tick midgut and salivary gland tissues after the blood meal in the Rp− female adults (Additional file 1: Figure S1), whereas the CMM level remained fairly constant in the midgut tissues over the course of the blood meal but decreased rapidly in the salivary glands after the blood meal (Additional file 1: Figure S1).
Rickettsia parkeri infection differentially regulates tick selenogene expression
Temporal- and tissue-specific SELENOO and SELENOS transcript levels were assessed in the unfed and partially-fed midguts and salivary glands in from the Rp− female adults (Additional file 2: Figure S2). Interestingly, the gene expression level of SELENOO in the midgut tissues was upregulated 2–2.5-fold, while the salivary glands showed decreased transcriptional expression from 2 to 8 dpi (Additional file 2: Figure S2a). In contrast, the SELENOS transcript level was fourfold upregulated during the early phase of tick feeding compared with the tissue levels in the unfed ticks (Additional file 2: Figure S2). SELENOS expression remained unchanged in the midgut tissues from 2 to 8 dpi (Additional file 2: Figure S2b). The transcriptional activity of SELENOM has been reported to gradually increase up to 2 dpi but diminish thereafter in both tissue types .
Impact of selenoprotein gene silencing on tick physiology
Summary of results
Rickettsia parkeri (Rp-) ticks
Rickettsia parkeri (Rp+) ticks
Rickettsia parkeri and symbionts dynamics
R. parkeri load (Rp)
No. R. parkeri
Increases load with blood meal in immature ticks.
Decreases load along the blood meal in adult (female) tissues.
Candidatus Midichloria mitochondrii (CMM)
Present across the tick life cycle.
Blood meal reduces the abundances.
Proliferates with Rp infections.
Francisella-like endosymbiont (FLE)
Present across the tick life cycle.
Blood meal reduces the abundances.
May get displaced with Rp infections.
Total bacterial load (BL)
Blood meal reduces the abundances.
Increase with Rp infections.
Transcriptional expressions of selenoproteins
SelO and SelS constantly express while SelM is cyclical (gradually peaking up and then decreases).
SELENOK, SELENOO, SELENOS, SELENON, and SELENOX upregulated
Spiked at unfed (SelO) and spiked at early feeding and later diminishes (SelS) while SelM cyclical.
eEFSec, SELENOM, SELENOK, SELENOS, TrxR, and GST upregulated
SELENOM and SELENOO upregulated
Knockdown of selenoproteins
SG: depleted Rp, BL, and FLE
MG: depleted Rp, BL, and CMM
MG: depleted BL, FLE
SG: depleted CMM
Impact of selenoprotein silencing on total bacterial load and R. parkeri replication
Knocking-down SELENOM resulted in a significant decrease in the R. parkeri concentration and the total bacterial load in the salivary gland tissues, but not in the midgut tissues after 5 dpi in the female ticks (Fig. 6a, b). The total bacterial load decreased in the midguts upon SELENOO knockdown, but the result was not statistically significant (p = 0.07), and the bacterial load in the salivary glands remained unaffected (Fig. 6c). The SELENOO knockdown depleted R. parkeri (p = 0.0062) in the midgut tissues, but the R. parkeri levels remained unchanged in the salivary glands (p = 0.06) (Fig. 6d). Finally, the SELENOS gene silencing depleted the total bacterial load in the midgut but not in the salivary gland tissues (Fig. 6e). Similarly, the SELENOS knockdown did not alter the R. parkeri load in the midgut tissues (p = 0.97), unlike in the salivary gland tissues where it was reduced, but not significantly so (p = 0.06) (Fig. 6f).
Quantification of tick symbionts upon selenogene silencing
We further investigated the dynamic interplay occurring among R. parkeri, CMM, and FLE microbes in ticks depleted of R. parkeri via selenoprotein silencing (Figs. 6 and 7). We found that infection with R. parkeri differentially regulated a battery of tick selenoproteins in the midgut, salivary glands, and ovarian tissues (Fig. 4). We showed the differential expression of selenogenes within and among the tick organs with respect to the pathogen infection. The selenoproteins in the salivary glands were highly expressed during infection with R. parkeri compared with those in the midguts and ovarian tissues (Fig. 4). Pathogen development and the secretory functions of the tick salivary glands might have resulted in the higher levels of selenoprotein expression in tick salivary glands that were observed. Furthermore, during tick feeding, the tick salivary glands probably remained under stress because these glands participate in the constant supply of anti-hemostatic, anti-inflammatory, and analgesic compounds during the continuous flow of the blood meal [57, 58]. ROS generation is one of the first lines of defense against invading microbes [59, 60]. However, despite minimal investigation, evidence is now accumulating that the tick selenoproteome and antioxidant enzymes may play critical roles in detoxifying ROS and in maintaining both vector microbiota and R. parkeri colonization [10, 31, 34, 36–38].
The compensatory actions of the redox genes following selenogene transcript depletion via RNAi differed between the Rp− and Rp+ ticks (Fig. 5, Additional file 3: Figure S3). SELENOM depletion in the Rp+ ticks showed evidence of compensation by overexpression of TrxR, Mn-SOD, and Salp25D in the midgut and ovary tissues, whereas CAT, SELENOK, and SELENOS were only upregulated in the salivary glands (Fig. 5a). Cu/Zn-SOD and Duox were significantly upregulated after SELENOO and SELENOS silencing; these are involved in known defense mechanisms against invading pathogens and are also involved in repairing the tissue damage from Rickettsia-dependent superoxide generation . Superoxide generation is associated with rickettsial infections , and tick extracellular Cu/Zn-SOD is the main quencher for dismutation of the superoxides generated during rickettsial infections . Upregulated Cu/Zn-SOD probably provides the redox balance required to offset the superoxide radicals generated by the rickettsial infections in the ticks after SELENOO and SELENOS were knocked down, but Cu/Zn-SOD was not upregulated in the SELENOM knockdowns (Fig. 5). Further investigation of the unfolded protein response sensor genes (ATF6 and IRE1) provided evidence of the altered protein folding homeostasis inside the ER, the organelle necessary for the proper folding of all secretory and transmembrane proteins (Additional file 4: Figure S4). The sensor genes for the unfolded protein response (ATF6 and IRE1) and tick selenoprotein silencing potentially cause ER stress . Knockdown of the mitochondrial resident selenogene, SELENOO, likely induced high oxidative stress in the gut tissues (Fig. 5, Additional file 4: Figure S4), which in turn induced ER stress. Studies have shown that there is a physical and biochemical interaction between the ER and mitochondria , and mitochondrial ROS can induce ER stress . We proposed a model to summarize the important points arising from our study (Fig. 8). In this model, we suggest that successful R. parkeri replication within the tick vector is enhanced by the presence of CMM, probably by displacing FLE. The selenogenes responding to R. parkeri infection by transcriptional upregulation favor R. parkeri replication, and this in turn enhances the overall vectorial competence of A. maculatum for R. parkeri.
The successful growth of a human pathogenic spotted fever group rickettsia, R. parkeri, inside its competent vector, A. maculatum, offers it a chance to dynamically interact with tick symbionts and modulate its host’s defenses by upregulating tick selenoproteins. This study illustrates the potential of a new research model aimed at providing better understanding of tick–pathogen interactions and the important roles played by symbionts and various tick factors in regulating pathogen growth.
All the animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, USA. The protocols for tick blood feeding were approved by the Institutional Animal Care and Use Committee of the University of Southern Mississippi (protocol#15101501 and 15011402).
Ticks and tissue preparations
A. maculatum ticks were maintained at the University of Southern Mississippi according to established methods . The Rp+ tick colonies and Rp− tick colonies from Mississippi field collections were established and maintained in the laboratory. A. maculatum colonies containing individual Rp+ and Rp− ticks were established in our laboratory in 2013. Questing unfed adult ticks were collected from Mississippi Sandhill Crane, National Wildlife Refuge, Gautier, Mississippi (www.fws.gov/refuge/mississippi_sandhill_crane/) using the drag cloth method during July and August 2013. The hundreds of ticks collected from the field were blood-fed on sheep and allowed to fully engorge and drop off. Fully engorged female adult ticks were kept in snap vials for egg laying. Typically, a single fully engorged female A. maculatum lays on average a 450–950 mg egg mass containing 15,000–18,000 eggs. To determine the presence of R. parkeri in an egg mass, 10–20 mg of it from each gravid female was subjected to genomic DNA extraction using the DNeasy extraction kit as described by the manufacturers (Qiagen, CA) followed by PCR amplification of the extracted DNA to identify SFGR species . Individual Rp+ and Rp− eggs were selected from individual gravid females and allowed to hatch into unfed larva. The unfed larval ticks were blood-fed by allowing them to infest golden Syrian hamsters until they reached repletion. Fully engorged larvae were allowed to molt into nymphs and then blood-fed on hamsters. Fully engorged nymphs molted as male or female ticks. Each developmental stage was routinely tested for the presence of R. parkeri infection. Closed colonies from the third and fourth generation of the original wild-caught ticks were used in this study after confirming the presence or absence of infection in the colonies. Freshly laid eggs, freshly molted larvae and blood-fed larvae, freshly hatched nymphs, and blood-fed nymphs were collected from Rp+ and Rp− colonies separately. Eggs, unfed larvae (from three individual ticks, 20 mg each), three fed and pooled larval batches, unfed nymphs (20 mg), and fed nymphs were stored in RNAlater (Invitrogen, Carlsbad, CA). At least three biological replicates were used in all the experiments. Tick tissues from the unfed and partially blood-fed female adult ticks were dissected and stored immediately in RNAlater (Invitrogen) prior to extracting the mRNA using our previously described laboratory method .
RNA preparation, cDNA synthesis, and qRT-PCR
Total RNA extraction, cDNA synthesis, and qRT-PCR were conducted as previously described . The gene-specific primer sequences, which were designed to amplify specific cDNA fragments from A. maculatum tissues, are listed in Additional file 5: Table S1. Transcriptional gene expression of the tick genes in Rp− ticks was normalized against β-actin gene expression, while GAPDH gene expression was used to normalize tick gene expression in the Rp+ tick tissues because it is stably expressed irrespective of the infection status . The synthesized cDNA was used to measure mRNA levels by qRT-PCR using the CFX96 Real Time System (Bio-Rad Inc., Hercules, CA) as described previously [10, 38].
Double-stranded RNA (dsRNA) synthesis, tick injections, and hematophagy
dsRNA was synthesized to allow for the in vivo analysis of SELENOM, SELENOO, and SELENOS in the ticks. Tick manipulations were performed according to the methods described previously [36, 68]. The dsRNAs for each selenoprotein gene (dsSELENOM, dsSELENOO, dsSELENOS) were diluted to working concentrations of 1 μg/μL in nuclease-free water. The same protocol was used to synthesize dsLacZ, which was used as an irrelevant dsRNA control. Twenty-five unfed adult female ticks were each microinjected with 1 μl of dsRNA or dsLacZ using a 27-gauge needle, then kept overnight at 37 °C to alleviate needle trauma and promote their survival, after which they were blood-fed using routine laboratory procedures .
Quantification of total bacterial loads
The bacterial load in each tick tissue was estimated as described previously [10, 15]. The bacterial copy numbers were normalized against A. maculatum actin expression in the uninfected ticks and GAPDH expression in the Rp+ ticks.
Quantification of FLE in tick tissues
The FLE from A. maculatum tick tissues was quantified using the primers described elsewhere . The serially diluted copies (108 to 101) of each gene were PCR-amplified using predetermined thermal cycling conditions, and the Ct values for known dilutions were used to construct a standard curve from which the copy number of each gene was calculated. The 25 μL qRT-PCRs comprised 125 nM of each primer, SYBR Green Master Mix (Bio-Rad, Inc. USA), and the serially diluted PCR products prepared for each standard curve. The reaction mixtures were subjected to the thermal cycle parameters of 95 °C for 5 min followed by 29 cycles of 95 °C for 30s, 52 °C for 30s, and 72 °C for 30s with a final extension of 72 °C for 5 min in a CFX96 Real Time System (Bio-Rad Inc.). The FLE copy numbers were normalized against the A. maculatum GAPDH gene. As with the other qRT-PCRs, all the samples were run in triplicate.
Quantification of CMM in tick tissues
We followed the protocol described previously for quantifying CMM . The CMM-specific GyrB gene and tick GAPDH gene were PCR-amplified from A. maculatum ticks using the primers shown in Table 1. The amplified PCR products serially diluted tenfold (108 to 101 copies) were used to generate a standard curve. The qRT-PCRs comprised 400 nM of each primer and 25 ng of the cDNA samples. The reaction mixture containing SYBR Green (Bio-Rad Inc. USA) was subjected to thermal cycling at 95 °C for 2 min, 40 cycles at 95 °C for 15 s, and at 60 °C for 30s, and a melting curve from 55 °C to 95 °C with increasing increments of 0.5 °C per cycle was prepared using the CFX96 Real Time System (Bio-Rad Inc.). The standard curves generated were used to calculate the copy numbers of the CMM GyrB gene and the tick GAPDH gene. The CMM copy numbers were normalized against the A. maculatum GAPDH gene. As with the other qRT-PCRs, all the samples were run in triplicate.
Quantification of the R. parkeri load in tick tissues
The level of infection with R. parkeri within the tick tissues across the developmental stages was quantified using a slightly modified version of a previously published method [9, 70]. The R. parkeri load was estimated as the ratio of R. parkeri-specific rompB gene copies to tick GAPDH copies. GAPDH and rompB genes were amplified using 250 nM of each specific primer (Table 1) in a reaction containing SYBR Green Master Mix (Bio-Rad Inc.) in the CFX96 Real Time System (Bio-Rad Inc.) with thermal cycling conditions of 95 °C for 10 min, followed by 35 cycles of 95 °C for 15 s, 60 °C for 30s, and 72 °C for 30s. The standard curves for tick GAPDH and rompB were prepared based on the amplification profiles of known concentrations of purified GAPDH and rompB PCR products. The standard curves generated were used to estimate the copy numbers of each gene in the tick samples.
Quantification of total oxidative stress levels
The malondialdehyde lipid peroxidation assay kit (Sigma-Aldrich, St. Louis, MO, USA) was used to quantify lipid degradation as a result of oxidative damage . All the procedures followed the manufacturer’s recommendations, and all the samples were balanced by weight.
All data are expressed as mean values ± SEM unless otherwise indicated. Statistical significance between two experimental groups or their respective controls was determined by a t test (p value, 0.05). Comparative differences among multiple experimental groups were determined by analysis of variance with statically significant p values of < 0.05 (GraphPad Prism 6.05, La Jolla, CA). Transcriptional expression levels were determined using Bio-Rad software (Bio-Rad CFX MANAGER v.3.1), and the gene expression values obtained were considered statistically significant if a p value of 0.05 was obtained when compared with the control.
This work was supported by grants from the National Institute of Allergy and Infectious Diseases (award #AI099919), United States Department of Agriculture, National Institute of Food and Agriculture (awards #2017-67016-26864), and the National Institutes of General Medical Sciences (award # P20RR016476). The funders played no role in the study design, data collection, analysis, decision to publish, or manuscript preparation.
SK conceived and designed the experiments . KBC, DK, GC, CB, and SK performed the experiments. KBC, DK, and SK analyzed the data. SK and GD contributed reagents/materials/analysis tools. KBC, DK, GD, and SK wrote the paper. All authors have read and approved the manuscript.
Ethics approval and consent to participate
Use of animals for tick blood-feeding was approved by the IACUC of the University of Southern Mississippi.
The authors declare that they have no competing interests.
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