Single-cell RNA sequencing reveals hidden cell types shaping mosquito disease transmission

Mosquitoes remain the deadliest animals on Earth, not through any venom of their own but because of the pathogens they ferry between hosts. Malaria, dengue fever, Zika, chikungunya and West Nile virus together account for an estimated 350 million cases and roughly 700,000 deaths every year, a burden that public health agencies warn is being aggravated as climate change pushes vector species into new territories. For decades, researchers probing how these insects host, tolerate and transmit such a menagerie of microbes have relied on bulk tissue analysis or candidate gene studies, approaches that average away the very cellular detail that matters most. A new review published in Parasites & Vectors argues that a transformative technology, single-cell RNA sequencing, is now rewriting the rules of mosquito biology, exposing cell types and host–pathogen interactions that older methods could never resolve.

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