Unbiased classification of mosquito blood cells by single-cell genomics and high-content imaging.

Mosquito blood cells are immune cells that help control infection by vector-borne pathogens. Despite their importance, little is known about mosquito blood cell biology beyond morphological and functional criteria used for their classification. Here, we combined the power of single-cell RNA sequencing, high-content imaging flow cytometry, and single-molecule RNA hybridization to analyze a subset of blood cells of the malaria mosquito Anopheles gambiae By demonstrating that blood cells express nearly half of the mosquito transcriptome, our dataset represents an unprecedented view into their transcriptional program. Analyses of differentially expressed genes identified transcriptional signatures of two cell types and provide insights into the current classification of these cells. We further demonstrate the active transfer of a cellular marker between blood cells that may confound their identification. We propose that cell-to-cell exchange may contribute to cellular diversity and functional plasticity seen across biological systems

Genomic Insights Into The Ixodes scapularis Tick Vector Of Lyme Disease

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retrotransposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing B57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick–host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host ‘questing’, prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent

Genomic Insights Into The Ixodes scapularis Tick Vector Of Lyme Disease

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retrotransposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing B57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick–host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host ‘questing’, prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent

Decoding the ubiquitin-mediated pathway of arthropod disease vectors.

Protein regulation by ubiquitin has been extensively described in model organisms. However, characterization of the ubiquitin machinery in disease vectors remains mostly unknown. This fundamental gap in knowledge presents a concern because new therapeutics are needed to control vector-borne diseases, and targeting the ubiquitin machinery as a means for disease intervention has been already adopted in the clinic. In this study, we employed a bioinformatics approach to uncover the ubiquitin-mediated pathway in the genomes of Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus, Ixodes scapularis, Pediculus humanus and Rhodnius prolixus. We observed that (1) disease vectors encode a lower percentage of ubiquitin-related genes when compared to Drosophila melanogaster, Mus musculus and Homo sapiens but not Saccharomyces cerevisiae; (2) overall, there are more proteins categorized as E3 ubiquitin ligases when compared to E2-conjugating or E1-activating enzymes; (3) the ubiquitin machinery within the three mosquito genomes is highly similar; (4) ubiquitin genes are more than doubled in the Chagas disease vector (R. prolixus) when compared to other arthropod vectors; (5) the deer tick I. scapularis and the body louse (P. humanus) genomes carry low numbers of E1-activating enzymes and HECT-type E3 ubiquitin ligases; (6) R. prolixus have low numbers of RING-type E3 ubiquitin ligases; and (7) C. quinquefasciatus present elevated numbers of predicted F-box E3 ubiquitin ligases, JAB and UCH deubiquitinases. Taken together, these findings provide novel opportunities to study the interaction between a pathogen and an arthropod vector

<it>Ixodes scapularis</it> saliva mitigates inflammatory cytokine secretion during <it>Anaplasma phagocytophilum</it> stimulation of immune cells

<p>Abstract</p> <p>Background</p> <p><it>Ixodes scapularis</it> saliva enables the transmission of infectious agents to the mammalian host due to its immunomodulatory, anesthetic and anti-coagulant properties. However, how <it>I. scapularis</it> saliva influences host cytokine secretion in the presence of the obligate intracellular rickettsial pathogen <it>Anaplasma phagocytophilum</it> remains elusive.</p> <p>Methods</p> <p>Bone marrow derived macrophages (BMDMs) were stimulated with pathogen associated molecular patterns (PAMPs) and <it>A. phagocytophilum.</it> Cytokine secretion was measured in the presence and absence of <it>I. scapularis</it> saliva. Human peripheral blood mononuclear cells (PBMCs) were also stimulated with Tumor Necrosis Factor (TNF)-α in the presence and absence of <it>I. scapularis</it> saliva and interleukin (IL)-8 was measured.</p> <p>Results</p> <p><it>I. scapularis</it> saliva inhibits inflammatory cytokine secretion by macrophages during stimulation of Toll-like (TLR) and Nod-like receptor (NLR) signaling pathways. The effect of <it>I. scapularis</it> saliva on immune cells is not restricted to murine macrophages because decreasing levels of interleukin (IL)-8 were observed after TNF-α stimulation of human peripheral blood mononuclear cells. <it>I. scapularis</it> saliva also mitigates pro-inflammatory cytokine response by murine macrophages during challenge with <it>A. phagocytophilum</it>.</p> <p>Conclusions</p> <p>These findings suggest that <it>I. scapularis</it> may inhibit inflammatory cytokine secretion during rickettsial transmission at the vector-host interface.</p

The E3 Ubiquitin Ligase XIAP Restricts Anaplasma phagocytophilum Colonization of Ixodes scapularis Ticks

Ubiquitination is a posttranslational modification that regulates protein degradation and signaling in eukaryotes. Although it is acknowledged that pathogens exploit ubiquitination to infect mammalian cells, it remains unknown how microbes interact with the ubiquitination machinery in medically relevant arthropods. Here, we show that the ubiquitination machinery is present in the tick Ixodes scapularis and demonstrate that the E3 ubiquitin ligase named x-linked inhibitor of apoptosis protein (XIAP) restricts bacterial colonization of this arthropod vector. We provide evidence that xiap silencing significantly increases tick colonization by the bacterium Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis. We also demonstrate that (i) XIAP polyubiquitination is dependent on the really interesting new gene (RING) catalytic domain, (ii) XIAP polyubiquitination occurs via lysine (K)-63 but not K-48 residues, and (iii) XIAP-dependent K-63 polyubiquitination requires zinc for catalysis. Taken together, our data define a role for ubiquitination during bacterial colonization of disease vectors