Tick hemocytes have a pleiotropic role in microbial infection and arthropod fitness.

Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods.

Single-cell analyses of polyclonal Plasmodium vivax infections and their consequences on parasite transmission.

Most Plasmodium vivax infections contain genetically distinct parasites, but the consequences of this polyclonality on the development of asexual parasites, their sexual differentiation, and their transmission remain unknown. We describe infections of Saimiri monkeys with two strains of P. vivax and the analyses of 117,350 parasites characterized by single cell RNA sequencing and individually genotyped. In our model, consecutive inoculations fail to establish polyclonal infections. By contrast, simultaneous inoculations of two strains lead to sustained polyclonal infections, although without detectable differences in parasite regulation or sexual commitment. Analyses of sporozoites dissected from mosquitoes fed on coinfected monkeys show that all genotypes are successfully transmitted to mosquitoes. However, after sporozoite inoculation, not all genotypes contribute to the subsequent blood infections, highlighting an important bottleneck during pre-erythrocytic development. Overall, these studies provide new insights on the mechanisms regulating the establishment of polyclonal P. vivax infections and their consequences for disease transmission.

Tick extracellular vesicles impair epidermal homeostasis through immune-epithelial networks during hematophagy.

Hematophagous ectoparasites, such as ticks, rely on impaired wound healing for skin attachment and blood feeding. Wound healing has been extensively studied through the lens of inflammatory disorders and cancer, but limited attention has been given to arthropod-borne diseases. Here, we used orthogonal approaches combining single-cell RNA sequencing (scRNAseq), flow cytometry, murine genetics, and intravital microscopy to demonstrate how tick extracellular vesicles (EVs) disrupt networks involved in tissue repair. Impairment of EVs through silencing of the SNARE protein vamp33 negatively impacted ectoparasite feeding and survival in three medically relevant tick species, including Ixodes scapularis. Furthermore, I. scapularis EVs affected epidermal γδ T cell frequencies and co-receptor expression, which are essential for keratinocyte function. ScRNAseq analysis of the skin epidermis in wildtype animals exposed to vamp33-deficient ticks revealed a unique cluster of keratinocytes with an overrepresentation of pathways connected to wound healing. This biological circuit was further implicated in arthropod fitness when tick EVs inhibited epithelial proliferation through the disruption of phosphoinositide 3-kinase activity and keratinocyte growth factor levels. Collectively, we uncovered a tick-targeted impairment of tissue repair via the resident γδ T cell-keratinocyte axis, which contributes to ectoparasite feeding.

Tick hemocytes have pleiotropic roles in microbial infection and arthropod fitness.

Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here, we describe immune cells or hemocytes from the clinically relevant tick Ixodes scapularis using bulk and single cell RNA sequencing combined with depletion via clodronate liposomes, RNA interference, Clustered Regularly Interspaced Short Palindromic Repeats activation (CRISPRa) and RNA-fluorescence in situ hybridization (FISH). We observe molecular alterations in hemocytes upon tick infestation of mammals and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We predict distinct hemocyte lineages and reveal clusters exhibiting defined signatures for immunity, metabolism, and proliferation during hematophagy. Furthermore, we perform a mechanistic characterization of two I. scapularis hemocyte markers: hemocytin and astakine. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, which impacts blood feeding and molting behavior of ticks. Hemocytin specifically affects the c-Jun N-terminal kinase (JNK) signaling pathway, whereas astakine alters hemocyte proliferation in I. scapularis. Altogether, we uncover the heterogeneity and pleiotropic roles of hemocytes in ticks and provide a valuable resource for comparative biology in arthropods.

High-throughput detection of eukaryotic parasites and arboviruses in mosquitoes.

Vector-borne pathogens cause many human infectious diseases and are responsible for high mortality and morbidity throughout the world. They can also cause livestock epidemics with dramatic social and economic consequences. Due to its high costs, vector-borne disease surveillance is often limited to current threats, and the investigation of emerging pathogens typically occurs after the reports of clinical cases. Here, we use high-throughput sequencing to detect and identify a wide range of parasites and viruses carried by mosquitoes from Cambodia, Guinea, Mali and the USA. We apply this approach to individual Anopheles mosquitoes as well as pools of mosquitoes captured in traps; and compare the outcomes of this assay when applied to DNA or RNA. We identified known human and animal pathogens and mosquito parasites belonging to a wide range of taxa, as well as DNA sequences from previously uncharacterized organisms. Our results also revealed that analysis of the content of an entire trap could be an efficient approach to monitor and identify rare vector-borne pathogens in large surveillance studies. Overall, we describe a high-throughput and easy-to-customize assay to screen for a wide range of pathogens and efficiently complement current vector-borne disease surveillance approaches.

Transcriptional heterogeneity and tightly regulated changes in gene expression during Plasmodium berghei sporozoite development.

Despite the critical role of Plasmodium sporozoites in malaria transmission, we still know little about the mechanisms underlying their development in mosquitoes. Here, we use single-cell RNA sequencing to characterize the gene expression profiles of 16,038 Plasmodium berghei sporozoites isolated throughout their development from midgut oocysts to salivary glands, and from forced salivation experiments. Our results reveal a succession of tightly regulated changes in gene expression occurring during the maturation of sporozoites and highlight candidate genes that could play important roles in oocyst egress, sporozoite motility, and the mechanisms underlying the invasion of mosquito salivary glands and mammalian hepatocytes. In addition, the single-cell data reveal extensive transcriptional heterogeneity among parasites isolated from the same anatomical site, suggesting that Plasmodium development in mosquitoes is asynchronous and regulated by intrinsic as well as environmental factors. Finally, our analyses show a decrease in transcriptional activity preceding the translational repression observed in mature sporozoites and associated with their quiescent state in salivary glands, followed by a rapid reactivation of the transcriptional machinery immediately upon salivation.

Relative contributions of various endogenous and exogenous factors to the mosquito microbiota.

BACKGROUND: The commensal microbiota of mosquitoes impacts their development, immunity, and competency, and could provide a target for alternative entomological control approaches. However, despite the importance of the mosquito/microbiota interactions, little is known about the relative contribution of endogenous and exogenous factors in shaping the bacterial communities of mosquitoes. METHODS: We used a high-throughput sequencing-based assay to characterize the bacterial composition and diversity of 665 individual field-caught mosquitoes, as well as their species, genotype at an insecticide resistance locus, blood-meal composition, and the eukaryotic parasites and viruses they carry. We then used these data to rigorously estimate the individual effect of each parameter on the bacterial diversity as well as the relative contribution of each parameter to the microbial composition. RESULTS: Overall, multivariate analyses did not reveal any significant contribution of the mosquito species, insecticide resistance, or blood meal to the bacterial composition of the mosquitoes surveyed, and infection with parasites and viruses only contributed very marginally. The main driver of the bacterial diversity was the location at which each mosquito was collected, which explained roughly 20% of the variance observed. CONCLUSIONS: This analysis shows that when confounding factors are taken into account, the site at which the mosquitoes are collected is the main driver of the bacterial diversity of wild-caught mosquitoes, although further studies will be needed to determine which specific components of the local environment affect bacterial composition.

A high-throughput sequencing assay to comprehensively detect and characterize unicellular eukaryotes and helminths from biological and environmental samples.

BACKGROUND: Several of the most devastating human diseases are caused by eukaryotic parasites transmitted by arthropod vectors or through food and water contamination. These pathogens only represent a fraction of all unicellular eukaryotes and helminths that are present in the environment and many uncharacterized organisms might have subtle but pervasive effects on health, including by modifying the microbiome where they reside. Unfortunately, while we have modern molecular tools to characterize bacterial and, to a lesser extent, fungal communities, we lack suitable methods to comprehensively investigate and characterize most unicellular eukaryotes and helminths: the detection of these organisms often relies on microscopy that cannot differentiate related organisms, while molecular assays can only detect the pathogens specifically tested. RESULTS: Here, we describe a novel sequencing-based assay, akin to bacterial 16S rRNA sequencing, that enables high-throughput detection and characterization of a wide range of unicellular eukaryotes and helminths, including those from taxonomical groups containing all common human parasites. We designed and evaluated taxon-specific PCR primer pairs that selectively amplify all species from eight taxonomical groups (Apicomplexa, Amoeba, Diplomonadida, Kinetoplastida, Parabasalia, Nematoda, Platyhelminthes, and Microsporidia). We then used these primers to screen DNA extracted from clinical, biological, and environmental samples, and after next-generation sequencing, identified both known and previously undescribed organisms from most taxa targeted. CONCLUSIONS: This novel high-throughput assay enables comprehensive detection and identification of eukaryotic parasites and related organisms, from a wide range of complex biological and environmental samples. This approach can be easily deployed to many settings and will efficiently complement existing methods and provide a holistic perspective on the microbiome.

NagC represses N-acetyl-glucosamine utilization genes in Vibrio fischeri within the light organ of Euprymna scolopes.

Bacteria often use transcription factors to regulate the expression of metabolic genes in accordance to available nutrients. NagC is a repressor conserved among γ-proteobacteria that regulates expression of enzymes involved in the metabolism of N-acetyl-glucosamine (GlcNAc). The polymeric form of GlcNAc, known as chitin, has been shown to play roles in chemotactic signaling and nutrition within the light organ symbiosis established between the marine bacterium Vibrio fischeri and the Hawaiian squid Euprymna scolopes. Here, we investigate the impact of NagC regulation on the physiology of V. fischeri. We find that NagC repression contributes to the fitness of V. fischeri in the absence of GlcNAc. In addition, the inability to de-repress expression of NagC-regulated genes reduces the fitness of V. fischeri in the presence of GlcNAc. We find that chemotaxis toward GlcNAc or chitobiose, a dimeric form of GlcNAc, is independent of NagC regulation. Finally, we show that NagC represses gene expression during the early stages of symbiosis. Our data suggest that the ability to regulate gene expression with NagC contributes to the overall fitness of V. fischeri in environments that vary in levels of GlcNAc. Furthermore, our finding that NagC represses gene expression within the squid light organ during an early stage of symbiosis supports the notion that the ability of the squid to provide a source of GlcNAc emerges later in host development.