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.

BackgroundSeveral 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.ResultsHere, 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0581-6) contains supplementary material, which is available to authorized users.

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.

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. Graphic abstract

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 genetic and non-genetic 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 their relative contribution 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. Infection with parasites and viruses only contributed very marginally and the main driver of the bacterial diversity was the location where 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 sites where the mosquitoes are collected is the main driver of the bacterial diversity of wild-caught mosquitoes, although further studies will be needed to determine how the specific components of the local environment affecting the bacterial composition.

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 Maryland. 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 novel 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.

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 the high costs, vector-borne disease surveillance is often limited to current threats, and the investigation of emerging pathogens typically occur 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 Maryland. 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, insect Flaviviruses, and novel DNA sequences from previously uncharacterized organisms. Our results also revealed that analysis of the content of an entire trap is an efficient approach to monitor and identify potential vector-borne pathogens in large surveillance studies, and that analyses of RNA extracted from mosquitoes is preferable, when possible, over DNA-based analyses. Overall, we describe a flexible and easy-to-customize assay that can provide important information for vector-borne disease surveillance and research studies to efficiently complement current approaches.