An Insight Into the Microbial Diversity and Expression of Cysteine Protease Inhibitors (Cystatin) in \u3ci\u3eRickettsia parkeri\u3c/i\u3e Infected \u3ci\u3eAmblyomma maculatum\u3c/i\u3e

Amblyomma maculatum (Gulf Coast tick) is an emerging tick species of public health significance in United States. It is a competent vector of Rickettsia parkeri, an etiological agent of a human rickettsiosis. In this study, we investigated the spotted fever group of rickettsial diversity in A. maculatum based on rickettsial ompA gene PCR. Our results showed A. maculatum harbors R. parkeri, R. amblyommii, and R. endosymbiont of A. maculatum. While only R. parkeri was detected in female salivary glands which suggest its ability to traffic from midgut to salivary glands via hemocoel. The presence of R. parkeri was further confirmed by probe based qPCR assay. We found R. parkeri infection rate ranged 12-40% in field collected ticks. We also provided evidence of R. parkeri infection transovarially and transstadially transmitted in A. maculatum. We used a pyrosequencing approach to further study all possible bacterial diversity residing in field collected A. maculatum. The huge bacterial profiling in A. maculatum provided the basis of Amblyomma-bacterial interactions particularly in relation to R. parkeri. On the other side, we observed cystatins temporal transcriptional expression in A. maculatumacross the blood meal cycle and our finding suggested their importance during blood feeding. Further, we saw R. parkeri differentially regulates gene expressions of cystatins in A. maculatum, suggesting a possible role of cystatins in R. parkeri infection in ticks. This study encourages further study to assess the exact relationship of R. parkeri with bacterial diversity in A. maculatum and cystatins role during tick blood feeding and R. parkeri transmission. Link to doctoral dissertation - http://aquila.usm.edu/dissertations/1381

Study of \u3ci\u3eRickettsia parkeri\u3c/i\u3e Colonization and Proliferation in the Tick Host \u3ci\u3eAmblyomma maculatum\u3c/i\u3e (Acari: Ixodidae)

Amblyomma maculatum (Gulf coast tick) ticks are prevalent across the Atlantic to Gulf Coast region of United States. These ticks are recognized vectors of Rickettsia parkeri, a spotted fever group of Rickettsia (SFGR) known to cause American boutonneuse fever associated with fever and eschar rashes localized to the site of bites. We hypothesized that Rickettsia parkeri colonization and proliferation in the tick vector involve pathogen-symbiont dynamics and tick-pathogen interactions, which influence rickettsial transmission to the victims after tick bites. The rickettsial infection is maintained across the tick life cycle for many generations due to transovarial and transstadial transmission of the pathogen. In the first part, we hypothesized that dynamic interaction among pathogenic R. parkeri and other tick symbionts inside the tick favors the rickettsial pathogen to survive, which multiply and infect vertebrates host upon infestation. We maintained R. parkeri infected and uninfected tick colonies in our lab to study their colonization with I tick. The bacterial loads for R. parkeri, Francisella like endosymbionts and “Candidatus Midichloria mitochondrii” were estimated in both tick colonies during different life stages and within various tick organs that are vital for blood-feeding, reproduction, and disease transmission by using specific qRT-PCR primers. Our results showed that R. parkeri thrives when CMM is present but displaces FLE along the tick life-cycle. In the second part, we hypothesized that tick SECIS binding protein (SBP2) and selenoprotein P (SELENOP) are essential in selenoprotein biosynthesis, and thereby play a role in overall tick redox balance and rickettsial colonization. RNAi assays were employed to specifically silence tick SBP2 and SELENOP. The silencing of SBP2 and SELENOP impaired synthesis of many known selenoproteins except selenophosphate synthetase (SEPHS2) and selenoprotein O (SELENOO). Neither of the genes impaired tick feeding, but SBP2 silencing significantly impacted tick oviposition success and egg hatching. The silencing of SBP2 further impaired rickettsial colonization and reduced transovarial transmission, whereas the SELENOP did not show role in tick feeding success or ovipositioning, but it did impair rickettsial colonization and transovarial transmission. This study provided new avenues of pathogen-symbiont dynamics and tick-pathogen interactions within vectors. Link to thesis - http://aquila.usm.edu/masters_theses/272

An Insight Into the Functional Role of Thioredoxin Reductase, a Selenoprotein, in Maintaining Normal Native Microbiota in the Gult Coast tick (\u3ci\u3eAmbylomma maculatum\u3c/i\u3e)

Tick selenoproteins have been associated with antioxidant activity in ticks. Thioredoxin reductase (TrxR), also a selenoprotein, belongs to the pyridine nucleotide‐disulphide oxidoreductase family of proteins and is an important antioxidant. Molecular interactions between native microbiota and tick hosts have barely been investigated to date. In this study, we determined the functional role of TrxR in tick feeding and in maintenance of the native microbial community. TrxR transcript levels remained high and microbial load was reduced throughout tick attachment to the vertebrate host. RNA interference (RNAi) showed that depletion of TrxR activity did not interfere with tick haematophagy or phenotype but did reduce the viability of the microbiome within the tick tissues, presumably by perturbing redox homeostasis. The transcriptional activity of various antioxidant genes remained unaffected whereas the antioxidant genes Manganese superoxide dismutase (MnSOD), copper/zinc superoxide dismutase (Cu/Zn SOD) and selenoprotein M (SelM) were significantly down‐regulated in salivary glands of the ticks subjected to RNAi. The perturbed TrxR enzymatic activity in the knocked‐down tick tissues negatively affected the bacterial load as well. Furthermore, we observed the altered bacterial profiles in TrxR‐silenced tick tissues. Taken together, these results indicate an essential functional role for TrxR in maintaining the bacterial community associated with ticks

\u3ci\u3eAmblyomma maculatum\u3c/i\u3e SECIS Binding Protein 2 and Putative Selenoprotein P are Indispensable for Pathogen Replication and Tick Fecundity

Selenium, a vital trace element, is incorporated into selenoproteins to produce selenocysteine. Our previous studies have revealed an adaptive co-evolutionary process that has enabled the spotted fever-causing tick-borne pathogen Rickettsia parkeri to survive by manipulating an antioxidant defense system associated with selenium, which includes a full set of selenoproteins and other antioxidants in ticks. Here, we conducted a systemic investigation of SECIS binding protein 2 (SBP2) and putative selenoprotein P (SELENOP) by transcript silencing in adult female Gulf-coast ticks (Amblyomma maculatum). Knockdown of the SBP2 and SELENOP genes depleted the respective transcript levels of these tick selenogenes, and caused differential regulation of other antioxidants. Importantly, the selenium level in the immature and mature tick stages increased significantly after a blood meal, but the selenium level decreased in ticks after the SBP2 and SELENOP knockdowns. Moreover, the SBP2 knockdown significantly impaired both transovarial transmission of R. parkeri to tick eggs and egg hatching. Overall, our data offer new insight into the relationship between the SBP2 selenoprotein synthesis gene and the putative tick SELENOP gene. It also augments our understanding of selenoprotein synthesis, selenium maintenance and utilization, and bacterial colonization of a tick vector

Catalase is a Determinant of the Colonization and Transovarial Transmission of \u3ci\u3eRickettsia parkeri\u3c/i\u3e in the Gulf Coast Tick \u3ci\u3eAmblyomma maculatum\u3c/i\u3e

The Gulf Coast tick (Amblyomma maculatum) has evolved as a competent vector of the spotted‐fever group rickettsia, Rickettsia parkeri. In this study, the functional role of catalase, an enzyme responsible for the degradation of toxic hydrogen peroxide, in the colonization of the tick vector by R. parkeri and transovarial transmission of this pathogen to the next tick generation, was investigated. Catalase gene (CAT) expression in midgut, salivary glands and ovarian tissues exhibited a 2–11‐fold increase in transcription level upon R. parkeri infection. Depletion of CAT transcripts using an RNA‐interference approach significantly reduced R. parkeri infection levels in midgut and salivary gland tissues by 53–63%. The role of CAT in transovarial transmission of R. parkeri was confirmed by simultaneously blocking the transcript and the enzyme by injecting double‐stranded RNA for CAT and a catalase inhibitor (3‐amino‐1,2,4‐triazole) into gravid females. Simultaneous inhibition of the CAT transcript and the enzyme significantly reduced the egg conversion ratio with a 44% reduction of R. parkeri transovarial transmission. These data suggest that catalase is required for rickettsial colonization of the tick vector and transovarial transmission to the next generation

\u3ci\u3eRickettsia parkeri\u3c/i\u3e Colonization in \u3ci\u3eAmbylomma maculatum\u3c/i\u3e: The Role of Superoxide Dismutases

Background The Gulf Coast tick (Amblyomma maculatum) is an arthropod vector of Rickettsia parkeri, the causative agent of American boutonneuse fever and an infectious agent of public health significance. In this study, we evaluated the biological significance of the superoxide dismutases (SODs) of A. maculatum in hematophagy and R. parkeri colonization within the tick host. Methods An RNA interference approach was used to measure the functional roles of tick SODs (Cu/Zn-SOD and Mn-SOD) in R. parkeri colonization of the tick vector. Total microbial load, R. parkeri infection rate, and compensatory mechanisms by tick genes were examined using quantitative polymerase chain reaction (PCR) and quantitative reverse-transcriptase PCR assays. SOD enzymatic activity assays and malondialdehyde (MDA) lipid peroxidation were employed to determine the redox states in the tick tissues. Results Knockdown of the Cu/Zn-SOD gene caused the upregulation of Mn-SOD in transcript levels. Single and dual knockdowns of the SOD genes caused an increase in MDA lipid peroxidation while SOD enzymatic activities did not show a significant change. Mn-SOD knockdown resulted in a substantial increase in the microbial load; however, Cu/Zn-SOD transcript depletion prompted an upsurge in the midgut bacterial load, and significantly decreased the bacterial load in salivary gland tissues. Additionally, Cu/Zn-SOD transcript silencing led to significantly fewer R. parkeri DNA copy numbers in both tick tissues (midguts and salivary glands). Conclusions SOD enzymes play an important function in the regulation of bacterial communities associated with tick vectors and also in the defense mechanisms against the damage caused by reactive oxygen species within the tick. Knockdown experiments increased the levels of total oxidative stress in ticks, revealing the interplay between SOD isozymes that results in the transcriptional regulation of tick antioxidants. Moreover, the tick\u27s Cu/Zn-SOD aids in the colonization of R. parkeri in tick tissues providing evidence of A. maculatum\u27s vectorial success for a spotted fever group rickettsial pathogen

Hematophagy and Tick-Borne Rickettsial Pathogen Shape the Microbial Community Structure and Predicted Functions Within the Tick Vector, \u3ci\u3eAmblyomma maculatum\u3c/i\u3e.

Background: Ticks are the primary vectors of emerging and resurging pathogens of public health significance worldwide. Analyzing tick bacterial composition, diversity, and functionality across developmental stages and tissues is crucial for designing new strategies to control ticks and prevent tick-borne diseases. Materials and methods: Here, we explored the microbial communities across the developmental timeline and in different tissues of the Gulf-Coast ticks (Amblyomma maculatum). Using a high-throughput sequencing approach, the influence of blood meal and Rickettsia parkeri, a spotted fever group rickettsiae infection in driving changes in microbiome composition, diversity, and functionality was determined. Results: This study shows that the core microbiome of Am. maculatum comprises ten core bacterial genera. The genus Rickettsia, Francisella, and Candidatus_Midichloria are the key players, with positive interactions within each developmental stage and adult tick organ tested. Blood meal and Rickettsia parkeri led to an increase in the bacterial abundance in the tissues. According to functional analysis, the increase in bacterial numbers is positively correlated to highly abundant energy metabolism orthologs with blood meal. Correlation analysis identified an increase in OTUs identified as Candidatus Midichloria and a subsequent decrease in Francisella OTUs in Rickettsia parkeri infected tick stages and tissues. Results demonstrate the abundance of Rickettsia and Francisella predominate in the core microbiome of Am. maculatum, whereas Candidatus_Midichloria and Cutibacterium prevalence increase with R. parkeri-infection. Network analysis and functional annotation suggest that R. parkeri interacts positively with Candidatus_Midichloria and negatively with Francisella. Conclusion: We conclude that tick-transmitted pathogens, such as R. parkeri establishes infection by interacting with the core microbiome of the tick vector

A Snapshot of the Microbiome of \u3ci\u3eAmbylomma tuberculatum\u3c/i\u3e Ticks Infesting the Gopher Tortoise, an Endangered Species

The gopher tortoise tick, Amblyomma tuberculatum, has a unique relationship with the gopher tortoise, Gopherus polyphemus, found in sandy habitats across the southeastern United States. We aimed to understand the overall bacterial community associated with A. tuberculatum while also focusing on spotted fever group Rickettsia. These tortoises in the Southern Mississippi region are a federally threatened species; therefore, we have carefully trapped the tortoises and removed the species-specific ticks attached to them. Genomic DNA was extracted from individual ticks and used to explore overall bacterial load using pyrosequencing of bacterial 16S rRNA on 454-sequencing platform. The spotted fever group of Rickettsia was explored by amplifying rickettsial outer membrane protein A (rompA) gene by nested PCR. Sequencing results revealed 330 bacterial operational taxonomic units (OTUs) after all the necessary curation of sequences. Four whole A. tuberculatum ticks showed Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes as the most dominant phyla with a total of 74 different bacterial genera detected. Together Rickettsiae and Francisella showed \u3e85% abundance, thus dominating the bacterial community structure. Partial sequences obtained from ompA amplicons revealed the presence of an uncharacterized Rickettsia similar to the Rickettsial endosymbiont of A. tuberculatum. This is the first preliminary profile of a complete bacterial community from gopher tortoise ticks and warrants further investigation regarding the functional role of Rickettsial and Francisella-like endosymbionts in tick physiology

The Microbiome of Neotropical Ticks Parasitizing On Passerine Migratory Birds

Seasonal migration of passerine birds between temperate North America and tropical Central and South America is an ecological phenomenon. Migration of birds has been associated with the introduction of ectoparasites like ticks or tick-borne pathogens across the avian migration routes. In this study, the microbial diversity was determined in the ticks and bird DNA samples using 454 pyrosequencing of bacterial 16S rRNA gene. Tick DNA samples showed the dominance of genera Lactococcus, Francisella, Raoultella, Wolbachiaand Rickettsia across all the ticks, but birds DNA did not share common microbial diversity with ticks. Furthermore, “Candidatus Rickettsia amblyommii” infection in the 91 ticks collected off the songbirds was also quantified by qPCR assay. Interestingly, “Candidatus R. amblyommii” was tested positive in 24 ticks (26% infection), and infection varied from as low as three copies to thousands of copies, but bird blood samples showed no amplification. Our results provide evidence that songbirds serve as transport carrier for immature ticks, and less likely to be a reservoir for “Candidatus R. amblyommii”

\u3ci\u3eRickettsia parkeri\u3c/i\u3e Infection Modulates the Sialome and Ovariome of the Gulf Coast Tick, \u3ci\u3eAmblyomma maculatum\u3c/i\u3e

The Gulf Coast tick, Amblyomma maculatum, is a vector of several tick-borne pathogens, including Rickettsia parkeri. The ability of R. parkeri to persist within the tick population through transovarial and transstadial transmission, without apparently harming the ticks, contributes to the pathogen’s perpetuation in the tick population. Previous studies have shown that the R. parkeri load in A. maculatum is regulated by the tick tissues’ oxidant/antioxidant balance and the non-pathogenic tick microbiome. To obtain further insights into the interaction between tick and pathogen, we performed a bulk RNA-Seq for differential transcriptomic analysis of ovaries and salivary glands from R. parkeri-infected and uninfected ticks over the feeding course on a host. The most differentially expressed functional category was of bacterial origin, exhibiting a massive overexpression of bacterial transcripts in response to the R. parkeri infection. Candidatus Midichloria mitochondrii and bacteria from the genus Rickettsia were mainly responsible for the overexpression of bacterial transcripts. Host genes were also modulated in R. parkeri-infected tick organs. A similar number of host transcripts from all analyzed functional categories was negatively and positively modulated, revealing a global alteration of the A. maculatum transcriptome in response to pathogen infection. R. parkeri infection led to an increase in salivary transcripts involved in blood feeding success as well as a decrease in ovarian immune transcripts. We hypothesize that these transcriptional alterations facilitate pathogen persistence and transmission within tick population