Single-cell RNA sequencing, or scRNA-seq, does something deceptively simple: instead of measuring the collective gene activity of an entire tissue, it profiles the transcriptome of individual cells. The transcriptome, the complete set of RNA molecules in a cell, acts as the bridge between the static genome and dynamic biological function. Bulk transcriptomics blurs this picture, masking the heterogeneity of the many cell types that make up even a small organ such as a mosquito midgut. By deconstructing tissues into their constituent cells, scRNA-seq allows researchers to discover rare cell populations, reconstruct developmental trajectories and map communication networks between cells. The technology has already driven breakthroughs in oncology, neuroscience and virology, and the review’s authors, led by Li-Bo Liu and Jia-Hong Wu of Guizhou Medical University, contend that mosquitoes are its next great frontier.

The technical pipeline is demanding at every step. Researchers must dissect tissues or collect hemolymph, isolate single cells or nuclei, capture them, prepare sequencing libraries and run high-throughput sequencing before any computational analysis begins. Each choice carries biological consequences. Enzymatic dissociation can stress cells and induce artificial transcriptional responses, and fragile or large cell types may be selectively lost. For tissues that resist dissociation, such as the brain or fat body, single-nucleus RNA sequencing offers an alternative that preserves cell-type proportions, though at the cost of losing cytoplasmic transcripts. The incorporation of unique molecular identifiers allows digital counting of original transcripts and mitigates amplification bias, while the choice between plate-based methods such as Smart-seq2, which deliver near-complete transcript coverage of relatively few cells, and droplet-based systems such as 10x Genomics Chromium, which profile tens of thousands of cells at shallower depth, fundamentally shapes what can be observed.

These trade-offs are not abstract. In studies of the malaria mosquito Anopheles gambiae, the high throughput of 10x Chromium succeeded in capturing megacytes, a rare hemocyte subpopulation making up only about 0.5 percent of immune cells, whereas Smart-seq2’s superior sensitivity identified phagocytic granulocytes that the droplet platform missed. Both findings were validated by in situ hybridization, confirming that the discrepancies stemmed from technical characteristics rather than false positives. The review’s authors emphasize that such variation demands rigorous reporting standards, including adherence to emerging minimum-information guidelines for single-cell experiments, explicit documentation of platforms and computational parameters, and independent validation of novel cell types before they are accepted as biological reality rather than computational artifacts.

What has the technology actually revealed? Perhaps the most striking lesson is that classical cell classifications were far too coarse. Mosquito hemocytes, the insect immune cells, were traditionally divided into granulocytes, oenocytoids and prohemocytes. Single-cell profiling has exploded this scheme into a multilevel system, showing that granulocytes comprise conventional, proliferative, antimicrobial and phagocytic subtypes, alongside megacytes that appear to coordinate immune responses mediated by hemocyte differentiation factor. Intriguingly, megacytes were not detected in Aedes aegypti, suggesting genuinely distinct immune lineages among vector species rather than a technical artifact, and challenging the assumption of a universal insect immune cell blueprint. The midgut epithelium has undergone a similar refinement: enterocytes once treated as a uniform class now resolve into five subtypes enriched for distinct digestive enzymes or immune factors, while enteroendocrine cells split into three subtypes specializing in different neuropeptide signals.

ScRNA-seq has also uncovered cell populations that fit no existing category. In the fat body of blood-fed Aedes aegypti, researchers identified fat-body-yolk cells, which coexpress markers of metabolic fat body tissue, ovarian reproductive tissue, stem cell genes and even eggshell formation genes. Their persistence after a blood meal, even when no oviposition sites are available, hints at a poised state dedicated to maintaining reproductive readiness, a possible adaptation to unpredictable breeding opportunities. The authors caution, however, that transcriptional identity does not equal validated function; the hypothesized roles of these cells in lipid transport, resource reallocation and defense remain compelling but untested. Cell atlases have now been extended to salivary glands, testes and the nervous system, laying groundwork for a comprehensive reference that could inform next-generation insecticides, gene-drive strategies and transmission-blocking interventions.

The technology’s power to dissect dynamic physiology is vividly illustrated by the response to a blood meal, the event that makes mosquitoes dangerous. Within one day of feeding, three nascent enterocyte clusters surged from 0.4 percent to 52.2 percent of midgut cells, an expansion too rapid for new cell generation and one that points to transdifferentiation of existing cells, though the origin remains unresolved. In the fat body, trophocyte proportions shifted between two and seven days post-feeding, with upregulation of trehalose synthesis genes and immune regulators coupling metabolic reprogramming to immune activation. Hemocyte numbers doubled by day seven, and even the brain participated: single-nucleus sequencing revealed dramatic transcriptional changes in glial cells within three hours of feeding, including upregulation of steroid hormone receptors and circadian clock genes, implicating glia as regulators of the post-blood-meal behavioral switch.

Sexual dimorphism, too, has acquired a cellular basis. Female Aedes aegypti brains are enriched for specific Kenyon cells, dopaminergic neurons and projection neurons, while male brains contain higher proportions of particular glial cells and other neuronal classes. Sex-biased gene expression follows, with males showing higher expression of the sex determination factor Nix and females expressing dipeptidyl peptidases at elevated levels. Notably, the sex determination gene doublesex is more highly expressed in glial cells than in neurons, suggesting an underappreciated role for glia in establishing sexual identity. In sensory organs, male-specific epithelial-like cells in antennae and female-specific neurons in the proboscis provide cellular correlates for sex-specific behaviors such as host-seeking. Single-nucleus data have likewise challenged the one neuron–one receptor dogma of sensory coding, revealing frequent coexpression of odorant and ionotropic receptors, and coexpression of taste and temperature sensors in gustatory neurons, expanding the theoretical coding capacity of the mosquito nervous system.

The most consequential applications may lie in host–pathogen interactions. Conventional scRNA-seq pipelines discard non-host reads as contamination, but adapted protocols, notably 5′ capture methods compatible with microfluidic platforms, can capture viral RNAs that lack polyadenylated tails. Using such approaches, researchers found that Zika virus preferentially targets enteroendocrine cells and enterocytes in the Aedes aegypti midgut, while West Nile virus reaches its highest loads in enteroendocrine cells of Culex tarsalis, hinting at a conserved flaviviral susceptibility tied to these cells’ signaling and secretory functions. In Anopheles gambiae, dual host–parasite sequencing showed that Plasmodium ookinetes preferentially interact with midgut progenitor cells during epithelial traversal, an interaction conserved across Anopheles species and parasite isolates. Immune responses are equally illuminating: enterocytes infected with Zika express antimicrobial peptides, whereas West Nile virus infection in Culex tarsalis produced no broad tissue-level immune activation, a finding that challenges the midgut’s reputation as a key site of innate immune defense and may partly explain that species’ vector competence.

These discoveries are already generating testable intervention targets. The enrichment of apolipophorin III in Zika-infected enteroendocrine cells led to RNA interference experiments confirming that this lipid transport protein supports viral replication, marking it as a candidate target. Similarly, single-cell analysis identified the LL3 gene as highly expressed in megacytes and essential for the hemocyte differentiation factor-mediated anti-Plasmodium response, establishing a rare cell type as a critical regulatory node in malaria vector immunity. The review’s authors are candid about remaining obstacles: cell annotation still leans heavily on Drosophila orthology, atlases remain incomplete for organs such as ovaries and compound eyes, and most findings await functional validation. The path forward, they argue, lies in integrating spatial transcriptomics, single-cell multiomics and perturbation-based experiments to build predictive models of mosquito–pathogen interactions. If that integration succeeds, single-cell insights accumulated over the past several years could mature from a discovery engine into the foundation of a new generation of targeted vector control strategies.

Subject of Research: Application of single-cell RNA sequencing to mosquito biology and mosquito–pathogen interactions

Article Title: Single-cell RNA sequencing unravels mosquito biology and host–pathogen interactions

Article References: Liu, L.-B., Ye, H.-B., Tian, Z.-H., Zeng, X.-H., & Wu, J.-H. (2026). Single-cell RNA sequencing unravels mosquito biology and host–pathogen interactions. Parasites & Vectors, 19(1), Article 426. https://doi.org/10.1186/s13071-026-07460-8

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07460-8

Keywords: single-cell RNA sequencing, mosquito, vector biology, host–pathogen interactions, cell atlas, Zika virus, West Nile virus, Plasmodium, malaria, dengue, vector control, transcriptomics

The study, published in Virology Journal, focused on Culex pipiens sensu lato and Culiseta annulata, two species that dominate the UK mosquito fauna and are considered among the most plausible vectors should arboviruses such as West Nile or Usutu virus establish local transmission cycles. Between 2021 and 2022, the team collected adult mosquitoes across the grounds of the two zoological collections, sites chosen deliberately because zoos concentrate large numbers of animals in outdoor enclosures where mosquitoes feed readily. That combination of abundant vectors, diverse vertebrate hosts and regular human visitors makes zoological collections potential interfaces for virus exchange between mosquitoes, animals and people, and therefore valuable sentinel locations for surveillance.

In total, 4,042 mosquitoes were processed and sequenced across 44 pools. The researchers extracted total nucleic acids and applied a metagenomic sequencing strategy designed to capture both RNA and DNA viruses, allowing them to recover viral genomes without relying on prior assumptions about which viruses might be present. Bioinformatic assembly and taxonomic classification of the resulting reads yielded 26 viral genomes, a striking haul for a country whose mosquito viromes have remained comparatively understudied. Nine of these represented entirely novel species, distributed across both RNA and DNA virus families, underscoring how much basic viral diversity in northern European mosquitoes still awaits description.

A consistent pattern emerged across the sampling sites: several viruses were detected in every pool analysed, indicating substantial overlap in virome composition between the two zoos. This shared core of viruses suggests that certain mosquito-associated viruses are widespread and stable components of the local mosquito fauna rather than isolated curiosities confined to a single habitat. Such ubiquity matters for risk assessment, because a virus that maintains itself efficiently across geographically separated mosquito populations has, by definition, the ecological flexibility that emergence requires, even if most of these viruses ultimately prove harmless to vertebrates.

The headline findings are two novel orthobunyaviruses, named Atherstone virus and Deva virus, belonging to the family Peribunyaviridae. Orthobunyaviruses are a genus with a serious pedigree: it includes recognised human and animal pathogens such as Schmallenberg virus, which devastated European ruminant farming after its emergence in 2011, and Oropouche virus, a growing public health concern in the Americas. The tripartite RNA genomes of orthobunyaviruses encode a viral RNA-dependent RNA polymerase on the large segment, surface glycoproteins on the medium segment, and nucleoprotein plus accessory proteins on the small segment. Finding two previously unknown members of this genus in British mosquitoes immediately elevates them to candidates for closer scrutiny.

Not all orthobunyaviruses pose equal risk, and the study went beyond simple detection to ask which of the two new viruses resembles a genuine arbovirus, capable of replicating in both mosquitoes and vertebrate hosts. The team employed MosViR, a machine-learning classification tool trained on known mosquito viruses to predict whether a given virus is likely to be mosquito-restricted or arthropod-borne. For Atherstone virus, detected at both zoos, the prediction pointed towards arbovirus status, a result that alone would justify follow-up. More compelling still was the genomic evidence: Atherstone virus carries an open reading frame encoding a non-structural protein S, or NSs, a well-characterised virulence factor in orthobunyaviruses that antagonises host antiviral responses, including the interferon system, and is a hallmark of pathogenic members of the genus.

Deva virus tells a different story. Its small segment lacks an NSs open reading frame altogether, and MosViR did not predict it to be an arbovirus. The absence of NSs, combined with the absence of a vertebrate-infecting signature, is consistent with Deva virus being an insect-specific virus, locked into replication within mosquito cells and their close relatives. Insect-specific viruses of this kind are increasingly recognised as common and sometimes abundant residents of mosquito viromes. Although they do not infect vertebrates directly, they are far from biologically irrelevant: they can modulate mosquito immunity, compete with arboviruses for cellular resources, and in some cases reduce the ability of mosquitoes to transmit pathogenic viruses, making them subjects of interest both for basic virology and for potential interference-based control strategies.

The broader viral inventory recovered in the study reinforces this picture of a layered virome. Alongside the orthobunyaviruses, the sequencing identified viruses spanning multiple families, including flavivirus-like and other RNA virus groups as well as DNA viruses, with several classified as novel species. Distinguishing arboviruses from insect-specific viruses is one of the central analytical challenges in mosquito metagenomics, because both can be present in the same sample and their genomes can be difficult to interpret from sequence data alone. Tools such as MosViR, combined with comparative genomics against reference databases and catalogues of known arboviruses, provide a probabilistic framework for triage, flagging which discoveries warrant experimental characterisation in cell culture and vertebrate systems and which can be provisionally set aside as likely insect-restricted.

Why does this matter for the United Kingdom specifically? The country sits at the northern edge of the range of many mosquito species, and climate warming, changes in land use and the movement of animals and goods are all expected to alter the dynamics of mosquito-borne disease in temperate Europe. Usutu virus has already established itself in British birds and mosquitoes, and West Nile virus circulates periodically in neighbouring countries. Risk frameworks built around these known threats are essential, but the new study argues that they should be complemented by surveillance for the unknown: viruses already resident in local vectors whose properties have never been tested. A virus that today appears to be insect-specific could, through reassortment with a related pathogen or through evolutionary change, acquire new capabilities. Orthobunyaviruses are particularly notable in this respect because their segmented genomes can reassort when mosquitoes are co-infected with related viruses, generating novel combinations with unpredictable host ranges.

The authors are careful not to overstate the immediate danger. Detection of a viral genome in a mosquito does not demonstrate that the virus infects or harms vertebrates, and neither Atherstone nor Deva virus has been shown to cause disease. What the study provides is a prioritised list of candidates and a demonstration of method: that large-scale metagenomic surveillance of UK mosquitoes is feasible, productive and capable of revealing both the diversity of local viromes and the specific viruses that merit laboratory follow-up. Isolation of Atherstone virus, characterisation of its host range, and serological surveys of animals and humans near the sampling sites would be the logical next steps. In the meantime, the work adds the United Kingdom to the growing map of regions where the viruses closest to home, rather than those arriving from abroad, are proving to be the first order of business for emerging infection surveillance.

Subject of Research: Metagenomic surveillance of mosquito viromes at two UK zoos reveals novel mosquito-associated orthobunyaviruses

Article Title: Emergence risks from within? Metagenomic analysis of mosquito viromes from two zoos reveals mosquito-associated orthobunyaviruses in the UK

Article References: Pilgrim, J., Seechurn, N., Cunningham-Oakes, E., Gillespie, L., Dobbs, P., Lopez, J., Kohl, A., Hughes, G. L., Blagrove, M. S. C., Baylis, M., & Darby, A. C. (2026). Emergence risks from within? Metagenomic analysis of mosquito viromes from two zoos reveals mosquito-associated orthobunyaviruses in the UK. Virology Journal. https://doi.org/10.1186/s12985-026-03305-z

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03305-z

Keywords: orthobunyavirus, mosquito, virome, metagenomics, arbovirus, Culex pipiens, Culiseta annulata, surveillance, zoonotic risk, insect-specific virus, United Kingdom, Peribunyaviridae