Cell-Free Ribosomal RNA Offers a New Molecular Yardstick for Viral Lysis of Marine Algae

Every day, viruses kill an estimated 10 to 40 percent of the microbes in the ocean, a process known as the viral shunt. When a virus bursts its host cell, the cell’s contents spill into the surrounding seawater as dissolved organic matter that bacteria rapidly consume, rerouting carbon and nutrients away from the classic food web and into the microbial loop. For marine phytoplankton, the single-celled primary producers that fix carbon dioxide at rates comparable to land plants, viral lysis is one of the dominant causes of mortality. Yet despite its ecological importance, scientists have long lacked a reliable molecular marker that directly indicates when and how much plankton lysis is occurring in a water sample.

A new laboratory study, published in MicrobiologyOpen, suggests that a solution may lie in one of the most abundant molecules inside every living cell: ribosomal RNA. Previous work on prokaryotes demonstrated that when viruses lyse bacterial cells, substantial amounts of rRNA are released into the extracellular medium, where it can be detected and quantified. Building on that observation, researchers developed an approach called Mortality by Ribosomal Sequencing, or MoRS, which compares ribosomal RNA sequences in cellular and dissolved fractions of seawater to estimate taxon-specific cell lysis. What remained untested was whether the concept could be extended to eukaryotic microalgae, whose complex internal organization and diverse viral enemies make such an extrapolation far from trivial.

To test the idea, a research team led by investigators working with marine algal virus systems conducted controlled infection experiments with two contrasting host-virus pairs. The first was the diatom Chaetoceros tenuissimus, a small coastal phytoplankton species less than ten micrometers across that is susceptible to Chaetoceros tenuissimus RNA virus type II, a small single-stranded RNA virus with particles roughly 22 to 38 nanometers in diameter. The second was the raphidophyte Heterosigma akashiwo, a globally distributed, flagellated species notorious for forming harmful algal blooms, which was challenged with HaV strain 120, a large double-stranded DNA virus with strain-specific infectivity. Together, these systems span different host physiologies and viral genome types, providing a rigorous framework for testing whether cell-free rRNA behaves as a general lysis marker in eukaryotes.

The experimental design was carefully controlled. Host cultures were grown at 22 degrees Celsius under a 12-hour light and 12-hour dark cycle in sterile artificial seawater media and acclimated for multiple generations before infection. For each host-virus system, triplicate flasks received virus at a multiplicity of infection of ten, while triplicate control flasks received none. The cultures were incubated for six days and sampled daily. To track viral proliferation, the team used the most probable number method to determine infectious virus titers at the beginning and end of the incubation. Critically, they also added purified Escherichia coli ribosomes to every flask as spike-in controls, at a concentration of approximately 2.9 times ten to the seventh copies per milliliter, allowing them to measure how quickly free rRNA degrades in the medium.

Separating the cellular from the dissolved fraction was achieved by gentle filtration through 0.22-micrometer polycarbonate filters under low vacuum. RNA retained on the filter was operationally defined as the cellular fraction, while RNA passing through was defined as cell-free. Cellular RNA was extracted with a silica-column kit enhanced by bead-beating, whereas cell-free RNA was concentrated from twenty-milliliter filtrates using a column-based vacuum system optimized for large water volumes. After genomic DNA removal and complementary DNA synthesis, absolute quantification was performed with a digital PCR system, using newly designed primers targeting the V4 region of the algal 18S rRNA gene and primers specific to the major capsid protein genes of each virus. Extraction efficiencies of 21.9 percent for the cellular fraction and 83.1 percent for the cell-free fraction were used to correct the measured concentrations.

The growth dynamics of the two hosts diverged sharply. In the Chaetoceros experiment, cells in the virus-added treatment grew at rates comparable to controls until day three but then grew significantly more slowly, while cell density, chlorophyll a, and cellular rRNA all continued to rise until day three or four. In striking contrast, Heterosigma cultures declined immediately after viral addition, with most cells losing motility within 24 hours, echoing earlier reports on this host-virus system. Infectious virus particles increased dramatically in both systems, from 5.8 times ten to the fifth to 1.9 times ten to the eighth most probable number units per milliliter for the diatom RNA virus, and from 7.6 times ten to the fifth to 1.4 times ten to the seventh for the Heterosigma DNA virus, confirming active viral replication.

The central result concerned the redistribution of rRNA between particulate and dissolved pools. In the Chaetoceros experiment, cell-free 18S rRNA remained low in controls, below about 4 times ten to the eighth copies per milliliter, but rose sharply in infected flasks to a maximum of 3.7 times ten to the ninth copies per milliliter on day four. Per-cell rRNA content varied up to 2.8-fold within each condition but showed no significant treatment effect, and cellular rRNA correlated strongly with cell density in both species, with Pearson correlation coefficients of 0.72 and 0.94. These findings established that rRNA abundance tracks plankton biomass at the population scale, while its release into the dissolved phase signals cell rupture.

To convert rRNA dynamics into lysis rates, the team built a rate-based model describing daily changes in cell-free rRNA concentration. The model rests on first-order degradation kinetics, an assumption validated by the spike-in ribosomes, which decayed exponentially with rate constants of roughly 1.78 to 2.16 per day in the Chaetoceros experiment, corresponding to half-lives of 7.7 to 9.3 hours. By rearranging a simple mass-balance equation, the researchers solved for the cell-free rRNA production rate, a direct proxy for cell lysis. Normalizing this production rate by host cell abundance yielded a metric they call cell-free rRNA production per cell. In the Chaetoceros system, this metric was 46.1-fold higher in infected treatments than controls between days two and three, and in the Heterosigma system it reached 302.1-fold higher between days three and four, with maximum per-cell release rates up to 46.1- and 302.1-fold above controls across the experiments.

The two host-virus systems also revealed fundamentally different infection strategies. In Chaetoceros, viral marker genes accumulated progressively in the cellular fraction over days one to four, and lysis peaked between days two and three even as the population was still growing, demonstrating that active viral lysis can occur during the apparent growth phase of a bloom. In Heterosigma, elevated per-cell rRNA release appeared both early, during days zero to one, and late, during days three to five. The early mortality could not be explained by canonical viral lysis, since viral capsid gene expression peaked only on day two. The authors suggest several possible mechanisms, including abortive infection, membrane destabilization during viral entry, a phenomenon analogous to lysis from without described in bacteriophage systems, or damage caused by bacteria introduced with the nonaxenic viral lysate, which flow cytometry confirmed had proliferated to high densities in infected treatments.

The implications reach well beyond the laboratory. Unlike dilution assays, which are labor-intensive and lack taxonomic resolution, cell-free rRNA quantification combined with high-throughput sequencing could simultaneously assess lysis across diverse taxa in natural communities, offering a taxon-resolved measure of lytic mortality that plankton ecologists have long sought. Because viral lysis is estimated to contribute up to roughly 45 percent of the labile dissolved organic carbon pool in marine systems, better quantification of who is being lysed, and when, could sharpen models of carbon cycling and microbial loop dynamics. The authors caution that the approach has limits: ribosome content per cell varies several-fold, infected bacteria can actively reduce their ribosome pools, degradation constants differ among seawater samples and must be measured for each one, and nonviral processes such as sloppy feeding, parasitism, and algicidal bacteria can also release intracellular rRNA. They also note that the degradation kinetics of E. coli 16S rRNA may not perfectly match those of eukaryotic 18S rRNA, calling for spike-in standards derived from representative plankton. Even with these caveats, the study provides a mechanistic foundation for reading cell death directly from the RNA dissolved in seawater, turning a ubiquitous molecule into a quantitative witness of the viral shunt at work.

Subject of Research: Quantifying viral lysis of eukaryotic microalgae using cell-free ribosomal RNA as a molecular marker

Article Title: Quantifying Viral Lysis in Microalgae Using Cell‐Free rRNA

Article References: Kikuya, S., Tomaru, Y., Nagasaki, K., Morimoto, D., Yamagishi, Y., Ogata, H., & Endo, H. (2026). Quantifying Viral Lysis in Microalgae Using Cell‐Free rRNA. MicrobiologyOpen, 15(5), Article e70402. https://doi.org/10.1002/mbo3.70402

Image Credits: AI Generated

DOI: 10.1002/mbo3.70402

Keywords: viral lysis, microalgae, ribosomal RNA, phytoplankton, viral shunt, marine viruses, Chaetoceros tenuissimus, Heterosigma akashiwo, MoRS, digital PCR, plankton mortality, biogeochemistry