Additional file 1: of Rickettsia parkeri colonization in Amblyomma maculatum: the role of superoxide dismutases

Figure S1.  Evolutionary relationships of taxa based on the SOD amino acid sequence using maximum likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [15]. The tree is drawn to scale, with the branch lengths measured by the number of substitutions per site. Evolutionary analyses were conducted using MEGA6 software [14]. The sequences were obtained from Amblyomma maculatum, Amblyomma variegatum, Ixodes scapularis, Anopheles gambiae, Mus musculus, Sus scrofa, and Homo sapiens. GenBank accession numbers followed by species names are shown in the tree. Figure S2A. Multiple sequence alignments of Cu/Zn-SOD amino acid sequences from different taxa. Regions outlined by red boxes indicate metal-binding sites that are conserved between all of the listed species. The sequences for Cu/Zn-SODs were obtained from Drosophila melanogaster, Apis mellifera, Amblyomma maculatum, Thermostable mutant of Human Cu/Zn SOD, Mus musculus, Equus caballus, Danio rerio, Anopheles gambiae and Culex quinquefasciatus. Figure S2B. Multiple sequence alignments of Mn-SOD amino acid sequences from different taxa. Regions outlined by red boxes indicate metal-binding sites that are conserved between all the listed species. The sequences were obtained from Haliotis discus, Lottia gigantea, Perinereis nuntia, Stegastes partitus, Takifugu rubripes, Struthio camelus, Tauraco erythrolophus, Thamnophis elegans, Sus scrofa, Macaca mulatta, Mus musculus, Xenopus laevis, Amblyomma variegatum, Amblyomma maculatum and Ixodes scapularis. Figure S3. The engorged tick weight in dsRNA-SODs injected ticks. The engorged weights of ticks injected with dsLacZ, dsCu/Zn-SOD, dsMn-SOD and dual SODs (dsCu/Zn-SOD and dsMn-SOD) observed at the detachment of ticks from Sheep. There were no significant effects in tick engorged weights with dsRNA-SODs (ANOVA, F (3,52) = 0.6274, P = 0.6006). (DOCX 473 kb

Table_1_Hematophagy and tick-borne Rickettsial pathogen shape the microbial community structure and predicted functions within the tick vector, Amblyomma maculatum.xlsx

BackgroundTicks 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 methodsHere, 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.ResultsThis 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.ConclusionWe conclude that tick-transmitted pathogens, such as R. parkeri establishes infection by interacting with the core microbiome of the tick vector.</p

DataSheet_1_Hematophagy and tick-borne Rickettsial pathogen shape the microbial community structure and predicted functions within the tick vector, Amblyomma maculatum.docx

BackgroundTicks 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 methodsHere, 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.ResultsThis 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.ConclusionWe conclude that tick-transmitted pathogens, such as R. parkeri establishes infection by interacting with the core microbiome of the tick vector.</p

Evidence of transcriptional knockdown of SDS3 components in control and SEF-knockdowns.

<p>Transcriptional gene expression of two SDS3 components of the Sin3 histone deacetylase corepressor complexes in LacZ- and SEF-dsRNA knockdowns in pathogen-free and <i>R. parkeri-</i>infected <i>A. maculatum</i>.</p

Evolutionary relationships of taxa based on the SEF amino acid sequence using the Neighbor-joining method.

<p>Bootstrap values <70 and branch lengths shorter than 0.10 were not displayed.</p

Effects of SEF-knockdown on selenogenes and antioxidant genes.

<p>Normalized fold change in the transcriptional activity of selected selenogenes and associated antioxidant genes in the salivary glands of pathogen-free <i>A. maculatum</i> blood fed seven days and preceded by SEF-dsRNA injection. The transcriptional level of each candidate gene in tissues injected with LacZ-dsRNA was set to 1.0 as a reference point. </p

Knockdown of Selenocysteine-Specific Elongation Factor in <i>Amblyomma maculatum</i> Alters the Pathogen Burden of <i>Rickettsia parkeri</i> with Epigenetic Control by the Sin3 Histone Deacetylase Corepressor Complex

<div><p>Selenocysteine is the 21st naturally-occurring amino acid. Selenoproteins have diverse functions and many remain uncharacterized, but they are typically associated with antioxidant activity. The incorporation of selenocysteine into the nascent polypeptide chain recodes the TGA stop codon and this process depends upon a number of essential factors including the selenocysteine elongation factor (SEF). The transcriptional expression of SEF did not change significantly in tick midguts throughout the blood meal, but decreased in salivary glands to 20% at the end of the fast feeding phase. Since selenoprotein translation requires this specialized elongation factor, we targeted this gene for knockdown by RNAi to gain a global view of the role selenoproteins play in tick physiology. We found no significant differences in tick engorgement and embryogenesis but detected no antioxidant capacity in tick saliva. The transcriptional profile of selenoproteins in <i>R. parkeri</i>-infected <i>Amblyomma maculatum</i> revealed declined activity of selenoprotein M and catalase and increased activity of selenoprotein O, selenoprotein S, and selenoprotein T. Furthermore, the pathogen burden was significantly altered in SEF-knockdowns. We then determined the global impact of SEF-knockdown by RNA-seq, and mapped huge shifts in secretory gene expression that could be the result of downregulation of the Sin3 histone deacetylase corepressor complex.</p> </div

Transcriptional activity of selenogenes in pathogen-free and <i>R. parkeri-</i>infected tick midgut tissues.

<p>Normalized fold change in the transcriptional activity of selected selenoprotein and associated antioxidant genes in pathogen-free or <i>R. parkeri-</i>infected <i>A. maculatum</i> midguts at three and five days post-infestation. Gene expression was normalized to pathogen-free levels in three and five days to highlight differences observed in <i>R. parkeri-</i>infected tick midguts. (*<i>p</i><0.05).</p

Evidence of transcriptional knockdown of SEF in dsRNA-injected ticks.

<p>Normalized fold change in the salivary gland transcriptional activity of SEF in pathogen-free and <i>R. parkeri</i>-infected <i>A. maculatum</i> injected with LacZ-dsRNA or SEF-dsRNA.</p

<i>R. parkeri</i> pathogen burden in RNAi tissues. (n.d.: not detected).

<p><i>R. parkeri</i> pathogen burden in RNAi tissues. (n.d.: not detected).</p