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Introduction: Ticks rely on robust cellular and humoral responses to control microbial infection. However, several aspects of the tick's innate immune system remain uncharacterized, most notably that of the immune cells (called hemocytes), which are known to play a significant role in cellular and humoral responses. Despite the importance of hemocytes in regulating microbial infection, our understanding of their basic biology and molecular mechanisms remains limited. Therefore, we believe that a more detailed understanding of the role of hemocytes in the interactions between ticks and tick-borne microbes is crucial to illuminating their function in vector competence and to help identify novel targets for developing new strategies to block tick-borne pathogen transmission. Methods: This study examined hemocytes from the lone star tick (Amblyomma americanum) at the transcriptomic level using the 10X genomics single-cell RNA sequencing platform to analyze hemocyte populations from unfed, partially blood-fed, and Ehrlichia chaffeensis-infected ticks. The functional role of differentially expressed hemocyte markers in hemocyte proliferation and Ehrlichia dissemination was determined using an RNA interference approach. Results and discussion: Our data exhibit the identification of fourteen distinct hemocyte populations. Our results uncover seven distinct lineages present in uninfected and Ehrlichia-infected hemocyte clusters. The functional characterization of hemocytin, cystatin, fibronectin, and lipocalin demonstrate their role in hemocyte population changes, proliferation, and Ehrlichia dissemination. Conclusion: Our results uncover the tick immune responses to Ehrlichia infection and hematophagy at a single-cell resolution. This work opens a new field of tick innate immunobiology to understand the role of hemocytes, particularly in response to prolonged blood-feeding (hematophagy), and tickmicrobial interactions. [ABSTRACT FROM AUTHOR]

Introduction: Blood-feeding arthropods rely on robust cellular and humoral immunity to control pathogen invasion and replication. Tick hemocytes produce factors that can facilitate or suppress microbial infection and pathogenesis. Despite the importance of hemocytes in regulating microbial infection, understanding of their basic biology and molecular mechanisms remains limited. Methods: Here we combined histomorphology and functional analysis to identify five distinct phagocytic and non-phagocytic hemocyte populations circulating within the Gulf Coast tick Amblyomma maculatum. Results and discussion: Depletion of phagocytic hemocytes using clodronate liposomes revealed their function in eliminating bacterial infection. We provide the first direct evidence that an intracellular tick-borne pathogen, Rickettsia parkeri, infects phagocytic hemocytes in Am. maculatum to modify tick cellular immune responses. A hemocyte-specific RNA-seq dataset generated from hemocytes isolated from uninfected and R. parkeri-infected partially blood-fed ticks generated ~40,000 differentially regulated transcripts, >11,000 of which were immune genes. Silencing two differentially regulated phagocytic immune marker genes (nimrod B2 and eater-two Drosophila homologs), significantly reduced hemocyte phagocytosis. Conclusion: Together, these findings represent a significant step forward in understanding how hemocytes regulate microbial homeostasis and vector competence. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In arthropods, hemolymph carries immune cells and solubilizes and transports nutrients, hormones, and other molecules that are involved in diverse physiological processes including immunity, metabolism, and reproduction. However, despite such physiological importance, little is known about its composition. We applied mass spectrometry-based label-free quantification approaches to study the proteome of hemolymph perfused from sugar-fed female and male Aedes aegypti mosquitoes. A total of 1403 proteins were identified, out of which 447 of them were predicted to be extracellular. In both sexes, almost half of these extracellular proteins were predicted to be involved in defense/immune response, and their relative abundances (based on their intensity-based absolute quantification, iBAQ) were 37.9 and 33.2%, respectively. Interestingly, among them, 102 serine proteases/serine protease-homologues were identified, with almost half of them containing CLIP regulatory domains. Moreover, proteins belonging to families classically described as chemoreceptors, such as odorant-binding proteins (OBPs) and chemosensory proteins (CSPs), were also highly abundant in the hemolymph of both sexes. Our data provide a comprehensive catalogue of A. aegypti hemolymph basal protein content, revealing numerous unexplored targets for future research on mosquito physiology and disease transmission. It also provides a reference for future studies on the effect of blood meal and infection on hemolymph composition.

Tick saliva helps blood feeding by its antihemostatic and immunomodulatory activities. Tick salivary gland transcriptomes (sialotranscriptomes) revealed thousands of transcripts coding for putative secreted polypeptides. Hundreds of these transcripts code for groups of similar proteins, constituting protein families, such as the lipocalins and metalloproteases. However, while many of these transcriptome-derived protein sequences matches sequences predicted by tick genome assemblies, the majority are not represented in these proteomes. The diversity of these transcriptome-derived transcripts could derive from artifacts generated during assembly of short Illumina reads or derive from polymorphisms of the genes coding for these proteins. To investigate this discrepancy, we collected salivary glands from blood-feeding ticks and, from the same homogenate, made and sequenced libraries following Illumina and PacBio protocols, with the assumption that the longer PacBio reads would reveal the sequences generated by the assembly of Illumina reads. Using both Rhipicephalus zambeziensis and Ixodes scapularis ticks, we have obtained more lipocalin transcripts from the Illumina library than the PacBio library. To verify whether these unique Illumina transcripts were real, we selected 9 uniquely Illumina-derived lipocalin transcripts from I. scapularis and attempted to obtain PCR products. These were obtained and their sequences confirmed the presence of these transcripts in the I. scapularis salivary homogenate. We further compared the predicted salivary lipocalins and metalloproteases from I. scapularis sialotranscriptomes with those found in the predicted proteomes of 3 publicly available genomes of I. scapularis. Results indicate that the discrepancy between the genome and transcriptome sequences for these salivary protein families is due to a high degree of polymorphism within these genes., (Copyright © 2023. Published by Elsevier GmbH.)

The Gulf Coast tick, Amblyomma maculatum, inhabits the Southeastern states of the USA bordering the Gulf of Mexico, Mexico, and other Central and South American countries. More recently, its U.S. range has extended West to Arizona and Northeast to New York state and Connecticut. It is a vector of Rickettsia parkeri and Hepatozoon americanum. This tick species has become a model to study tick/Rickettsia interactions. To increase our knowledge of the basic biology of A. maculatum we report here a draft genome of this tick and an extensive functional classification of its proteome. The DNA from a single male tick was used as a genomic source, and a 10X genomics protocol determined 28,460 scaffolds having equal or more than 10 Kb, totaling 1.98 Gb. The N50 scaffold size was 19,849 Kb. The BRAKER pipeline was used to find the protein-coding gene boundaries on the assembled A. maculatum genome, discovering 237,921 CDS. After trimming and classifying the transposable elements, bacterial contaminants, and truncated genes, a set of 25,702 were annotated and classified as the core gene products. A BUSCO analysis revealed 83.4% complete BUSCOs. A hyperlinked spreadsheet is provided, allowing browsing of the individual gene products and their matches to several databases., (Copyright © 2022. Published by Elsevier GmbH.)

Mosquitoes are responsible for the death and debilitation of millions of people every year due to the pathogens they can transmit while blood feeding. While a handful of mosquitoes, namely those in the Aedes, Anopheles, and Culex genus, are the dominant vectors, many other species belonging to different genus are also involved in various pathogen cycles. Sabethes cyaneus is one of the many poorly understood mosquito species involved in the sylvatic cycle of Yellow Fever Virus. Here, we report the expression profile differences between male and female of Sa.cyaneus salivary glands (SGs). We find that female Sa.cyaneus SGs have 165 up-regulated and 18 down-regulated genes compared to male SGs. Most of the up-regulated genes have unknown functions, however, odorant binding proteins, such as those in the D7 protein family, and mucins were among the top 30 genes. We also performed various in vitro activity assays of female SGs. In the activity analysis we found that female SG extracts inhibit coagulation by blocking factor Xa and has endonuclease activity. Knowledge about mosquitoes and their physiology are important for understanding how different species differ in their ability to feed on and transmits pathogens to humans. These results provide us with an insight into the Sabethes SG activity and gene expression that expands our understanding of mosquito salivary glands., (Copyright © 2022. Published by Elsevier Ltd.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Guizzo, Budachetri, Adegoke, Ribeiro and Karim.)