Chemical Stabilization Preserves Mosquito Immune Cell Transcriptomes for High-Yield Sequencing

Researchers have developed an optimized stabilization and sequencing protocol for hemocytes collected from adult female Anopheles gambiae mosquitoes, addressing longstanding technical obstacles that have impeded insect immune cell research. Hemocytes constitute the primary cellular arm of mosquito innate immunity, playing vital defensive roles against pathogens, including malaria-causing Plasmodium parasites. Investigating these immune cells at single-cell and bulk transcriptomic resolution has historically proven exceptionally difficult because mosquito hemocytes are scarce, diminutive, low in ribonucleic acid content, and highly reactive to handling. During classical hemocoel perfusion, thoracic tissue injury frequently introduces contaminating ambient RNA and debris from fragile tissues like fat body trophocytes, introducing confounding technical artifacts. The newly standardized workflow couples immediate chemical stabilization with multi-stage size filtration, enabling reproducible single-cell RNA sequencing and bulk messenger RNA analysis. By safeguarding cellular and transcriptomic integrity directly upon collection, the protocol minimizes batch variability and eliminates transcriptional changes during extended sample processing.

The experimental framework establishes precise mosquito rearing, blood-feeding, and micro-perfusion conditions to standardize biological inputs. Four- to six-day-old female Anopheles gambiae mosquitoes of the G3 strain were reared at 27 degrees Celsius and 80 percent humidity under a 12-hour light-dark cycle. Cohorts of 60 to 80 mosquitoes were starved for 12 to 16 hours before blood-feeding on female BALB/c mice sedated with 6.66 milligrams per kilogram xylazine hydrochloride and 66.6 milligrams per kilogram ketamine hydrochloride. For hemolymph perfusion, researchers constructed borosilicate glass capillary needles pulled at 68 degrees Celsius to a 6- to 8-millimeter taper with a 0.3- to 0.4-millimeter tip diameter. Operated via a micromanipulator and mineral-oil-filled dispenser, the needle injects five to six microliters of neutral collection buffer—comprising 95 percent Schneider Drosophila medium and five percent citrate buffer at pH 7.0 to 7.4—into the hemocoel. This perfusion yielded 5,000 to 10,000 hemocytes per sugar-fed mosquito, far exceeding historical recovery rates of 500 to 4,000 cells.

Immediate sample stabilization for single-cell assays is achieved by collecting perfused hemolymph into vivoPHIX, a specialized deep eutectic solvent that halts cellular RNA degradation and transcriptional drift at room temperature. Ten microliters of perfused hemolymph per mosquito are added directly into 250 microliters of vivoPHIX using siliconized tips, pooling ten mosquitoes per tube to obtain roughly 350 to 400 microliters of mixture. Following a one-hour room-temperature incubation with periodic inversion, the stabilized suspensions can be safely stored at 4 degrees Celsius for up to four weeks without transcriptomic deterioration. This prolonged stability removes the operational urgency inherent in processing live hemocytes immediately via plate-based single-cell platforms such as SMART-seq2. Live-cell fluorescence-activated sorting requires fluorescent dyes that compromise cellular viability and introduce transcriptomic perturbations. In contrast, eutectic stabilization preserves native molecular states immediately upon isolation, facilitating sample transportation from remote insectaries and allowing researchers to multiplex independent experimental cohorts to suppress technical batch artifacts.

Hemocyte recovery from vivoPHIX involves an ethanol dilution and dual-filtration protocol designed to enrich immune cells while discarding contaminating tissues. Stabilized samples are mixed with 500 microliters of 75 percent ethanol, piped through a 70-micrometer strainer, and passed through a 40-micrometer cell strainer to eliminate cuticle and muscle fragments. Centrifugation at 650 g for 15 minutes at room temperature produces a white cell pellet, which is washed with 75 percent ethanol to remove residual solvent, followed by a wash with 0.1 percent bovine serum albumin. Because the final cell pellet is completely transparent, technicians rely on rotor geometry to aspirate supernatants safely. Pellets from 50 to 60 mosquitoes are pooled into 40 to 50 microliters of albumin solution, generating working densities of 700 to 1,000 cells per microliter. Light microscopy and 7-amino-actinomycin D fluorescent nuclear staining confirmed that recovered cells maintain intact membranes and nuclei, although granulocytes contract from their spread diameter of 15 to 25 micrometers into rounded shapes under 10 micrometers.

Single-cell library preparation was executed by loading 10,000 recovered hemocytes into the Chromium Controller using Chromium Next GEM Single Cell 3 Prime Reagent Kits version 3.1. The microfluidic system encapsulated individual cells within nanoliter-scale gel beads in emulsion to capture polyadenylated messenger RNA. Reverse transcription inside droplets generated complementary DNA, which underwent 13 cycles of amplification to yield fragments between 400 and 2,000 base pairs at concentrations of 30 to 100 nanograms per microliter. Libraries were tagged with Dual Index Kit TT Set A and cleaned twice with solid-phase reversible immobilization magnetic beads to exclude sub-150-base-pair primer adapter artifacts. The resulting libraries exhibited average fragment lengths of 450 to 550 base pairs and DNA concentrations between 20 and 25 nanograms per microliter. Pooled sequencing on an Illumina NovaSeq 6000 instrument targeted 150 million reads per library across 14 samples, applying run parameters of 28 cycles for read one, 10 cycles for each index, and 90 cycles for read two alongside a one percent PhiX control.

Bioinformatic analysis coupled ambient noise decontamination with dimensionality reduction to resolve hemocyte lineages cleanly. Reads were aligned to the Anopheles gambiae PEST P4.14 genome using CellRanger version 7.0.0 and a custom hemocyte annotation. To isolate true cells from background, analysts treated the top 20,000 droplets by unique molecular identifier counts as candidate cells and eliminated droplets exceeding a 0.5 decontX ambient RNA threshold. In Seurat version 4.3.0, quality filters selected cells possessing 250 to 2,500 detected genes and under 15 percent mitochondrial transcripts. For a Toll-activated dsCactus cohort enriched in giant granulocytes termed megacytes, the gene threshold was raised to 4,000 to accommodate larger RNA volumes. Following principal component analysis and UMAP projection, preliminary clustering at resolution 0.1 removed fat body trophocyte contaminants. Final clustering at resolution 0.3 recovered an average of 2,600 high-quality hemocytes per sample, with mitochondrial transcript levels below three percent demonstrating intact cellular viability.

For bulk transcriptomic sequencing, hemolymph collection was adapted to utilize RNAprotect Cell Reagent, arresting enzymatic degradation immediately during sampling. The protocol harvests eight microliters of hemolymph from each of 20 mosquitoes into 800 microliters of reagent, followed by one hour of room-temperature stabilization and storage at 4 degrees Celsius for up to four weeks. To eliminate ambient tissue debris and large cells, the mixture is filtered through sequential 70-micrometer and 40-micrometer strainers and centrifuged at 650 g for 15 minutes at 4 degrees Celsius. The resulting cell pellet is lysed in one milliliter of Trizol LS. Organic phase extraction in prespun Phasemaker tubes with 200 microliters of chloroform, followed by precipitation with linear acrylamide carrier and isopropanol, yields intact total RNA. After duplicate washes in ice-cold 70 percent ethanol, the RNA pellet is resuspended in warm nuclease-free water and stored stably at -80 degrees Celsius for up to four months.

Quality assessments performed on Agilent TapeStation instruments demonstrated exceptional RNA preservation, yielding 70 to 200 nanograms of total RNA per sample with RNA Integrity Numbers between 8.8 and 9.0 across 34 sugar-fed mosquito samples. Electrophoretic profiles displayed a characteristic single band because insect 28S ribosomal RNA contains two fragments joined by hydrogen bonds that separate under denaturing conditions to migrate alongside 18S ribosomal RNA, distinguishing insect profiles from the two-band pattern seen in vertebrates. Quantitative PCR measuring normalized expression of the hemocyte-specific leucine-rich repeat ficolin marker LRR8 (AGAP004017) against ribosomal protein S7 verified significant cell enrichment. Relative LRR8 expression was 5.6 times higher in filtered hemolymph from blood-fed mosquitoes and 2.5 times higher in sugar-fed mosquitoes compared to unfiltered perfusates (unpaired t-tests, p < 0.0001 across three biological replicates), confirming that filtration after stabilization effectively strips away contaminating fat body trophocytes and ambient tissue transcripts.

Bulk sequencing libraries were generated from stabilized hemocytes using NEBNext Poly(A) mRNA magnetic isolation modules and NEBNext Ultra II RNA library preparation kits. Libraries underwent paired-end 150-base-pair sequencing on the Illumina NovaSeq 6000 platform, generating an average of 49.72 million total reads per library with high accuracy, exceeding 97 percent Q20 and 93 percent Q30 Phred scores. Bioinformatic processing evaluated read quality via FastQC and trimmed adapters and low-quality bases below Phred 20 using TrimGalore. Filtered reads were aligned to the custom hemocyte transcriptome reference using RSEM version 1.3.3 with Bowtie 2. For differential expression analysis, raw transcript counts were analyzed in edgeR, applying trimmed mean of M-values normalization to adjust for library compositional variations. Model fitting with quasi-likelihood F-tests via glmQLFit identified differentially expressed transcripts, establishing significance at an absolute log2 fold-change of 1 or greater with a false discovery rate below 0.05.

Despite these technical advancements, the authors highlight specific procedural limitations that warrant careful consideration in downstream experimental interpretations. The recovery of diminutive prohemocytes measuring under five micrometers in diameter remains lower, between 20 and 30 percent, compared to larger oenocytoids and granulocytes, introducing potential representation bias during single-cell cell capture. Additionally, generating adequate cell numbers for microfluidic encapsulation requires pooling hemocytes from 50 to 60 mosquitoes, which obscures individual biological variation. Nevertheless, the stabilized extraction methodology successfully captures rare immune subsets and provides an adaptable template for emerging multi-omic assays combining single-cell transcriptomics with open chromatin profiling. The authors conclude that these stabilized workflows can be readily extended to other mosquito and insect vectors, providing vital molecular toolsets to study cellular immunity, pathogen tropism, and novel vector-borne disease control strategies.

Subject of Research: Transcriptomic profiling protocols for stabilized Anopheles gambiae mosquito hemocytes

Article Title: Optimized protocol for single-cell and bulk transcriptomic profiling of stabilized hemocytes from Anopheles gambiae mosquitoes

Article References: Saha, B., McNinch, C. M., & Barillas-Mury, C. (2026). Optimized protocol for single-cell and bulk transcriptomic profiling of stabilized hemocytes from Anopheles gambiae mosquitoes. BMC Methods, 3(1), Article 39. https://doi.org/10.1186/s44330-026-00092-5

Image Credits: AI Generated

DOI: 10.1186/s44330-026-00092-5

Keywords: Anopheles gambiae, hemocyte transcriptomics, single-cell RNA sequencing, bulk RNA sequencing, vivoPHIX, RNAprotect Cell Reagent, hemolymph perfusion, prohemocytes, granulocytes, oenocytoids, 10X Genomics Chromium, NovaSeq 6000, decontX, Seurat analysis, LRR8 gene expression, insect innate immunity, malaria vector biology