Endosymbiotic algal photosynthesis shapes diel transcriptome architecture in its ciliate host Paramecium bursaria

This study demonstrates that photosynthetic algal endosymbionts act as primary organizers of the host *Paramecium bursaria*'s diel transcriptome by driving rhythmic gene expression across diverse biological processes through photosynthesis-dependent mechanisms, a phenomenon also observed in the independently evolved symbiont *Tetrahymena utriculariae*.

The Gist

Imagine a tiny, single-celled organism called Paramecium bursaria. It's like a microscopic swimmer in a pond. But this isn't just any swimmer; it's a "host" that has invited thousands of tiny green algae (its roommates) to live inside its body. These algae are like tiny solar panels, using sunlight to make food for the host.

For a long time, scientists knew these roommates helped the host survive, but they didn't know how the host's internal "daily schedule" (its biological clock) changed because of them. Does the host just follow the sun, or does the algae's work inside it create a whole new rhythm?

This paper is like a detective story that solves that mystery. Here is the breakdown in simple terms:

1. The Two Lives: With and Without Roommates

The researchers did a clever experiment. They took two groups of these microscopic swimmers:

• The Roommates (Symbiotic): The normal ones with algae inside.
• The Solo Swimmers (Aposymbiotic): They removed the algae, leaving the host alone.

They then watched what genes (the instructions inside the cell) were turned on and off every 3 hours over a full 24-hour day.

The Discovery:

• The Solo Swimmers had a very quiet, simple daily schedule. Only a few hundred genes changed their activity between day and night. It was like a house with the lights dimmed and only a few people moving around.
• The Roommates had a massive, complex, and highly organized daily schedule. Thousands of genes were dancing to a specific rhythm. It was like a bustling city with traffic lights, construction crews, and factories all switching on and off at precise times.

The Analogy: Think of the host cell as a factory. Without the algae, the factory runs on a basic, slow loop. But with the algae (the solar power plant), the factory suddenly has enough energy to run a complex, high-speed assembly line with different shifts for different tasks.

2. The "No Alarm Clock" Surprise

Usually, when we think of biological clocks (like our own sleep-wake cycles), we think of a specific set of "clock genes" that act like the gears in a watch. These gears tick away even if you turn off the lights.

The Twist: The researchers looked for these "clock gears" in the Paramecium and couldn't find them. The host doesn't have the standard biological clock genes that animals and plants have.

So, how does it keep time?
The paper suggests the algae act as the master conductor. Because the algae are photosynthesizing (making sugar and oxygen) only when the sun is out, they create a wave of energy and chemical signals that sweeps through the host.

• Morning: The algae start working, sending a signal: "Wake up! Time to swim and eat!"
• Mid-day: The energy is high, so the host focuses on building new parts and digesting food.
• Night: The algae stop working. The host switches to "maintenance mode," cleaning up and preparing for tomorrow.

The host doesn't have a watch; it just listens to the rhythm of its roommate's work.

3. The "Blackout" Test

To prove that the algae were really the ones setting the schedule, the researchers used a chemical (paraquat) to temporarily "break" the algae's solar panels. The algae were still there, but they couldn't make energy.

The Result: The moment the algae stopped working, the host's complex daily schedule collapsed. The thousands of rhythmic genes went silent and reverted to the simple, "solo" pattern. It was like cutting the power to the city; the traffic lights stopped, and the bustling activity vanished.

This proved that the host's daily rhythm isn't just a habit; it is directly powered and organized by the algae's photosynthesis.

4. The "Twin" Discovery

Finally, the researchers looked at a different, unrelated microscopic swimmer (Tetrahymena) that also lives with algae. Even though these two creatures evolved separately millions of years ago, they both showed the exact same pattern: when they have algae, they get a complex daily schedule; when they don't, they go back to a simple one.

The Takeaway: This isn't a fluke. It seems that whenever a host invites a photosynthetic roommate, the host's entire daily life gets reorganized around the roommate's work.

Summary in One Sentence

This paper reveals that for these tiny swimmers, their internal clock isn't a built-in watch, but rather a dance routine choreographed entirely by their solar-powered roommates, creating a complex daily life that vanishes the moment the roommates stop working.

Technical Summary

1. Problem Statement

While time-of-day transcriptional programs are well-documented in unicellular eukaryotes, the mechanism by which photosynthetic endosymbionts reorganize the host's daily gene regulation remains unclear. Specifically, it is unknown whether the diel (daily) rhythms observed in the facultative photosymbiotic ciliate Paramecium bursaria are driven by:

• Canonical circadian clock genes (Transcription-Translation Feedback Loops, TTFL).
• Metabolic inputs from the algal symbiont (Chlorella spp.).
• Direct light entrainment of the host.

Previous studies in coral-algal systems suggest symbionts alter host rhythms, but disentangling the effects of light from photosynthesis is difficult because constant darkness suppresses both. This study aims to determine if algal photosynthesis acts as a primary organizer of the host's diel transcriptome and to identify the regulatory mechanisms involved, particularly in the absence of recognizable canonical clock genes.

2. Methodology

The researchers employed a multi-faceted approach combining comparative transcriptomics, chemical perturbation, and cross-species analysis:

• Organism & Strains: The study utilized the P. bursaria DK2 strain, which contains both symbiotic (with Chlorella) and aposymbiotic (algae-free) states derived from the same genetic background.
• Experimental Design:
  • Time-Series RNA-seq: Samples were collected every 3 hours over a 24-hour Light-Dark (LD) cycle (8 time points) for both symbiotic and aposymbiotic cells.
  • Photosynthesis Perturbation: Symbiotic cells were treated with Paraquat (PQ), a redox-active herbicide that diverts electrons from Photosystem I, generating reactive oxygen species and suppressing photosynthetic electron transport without immediately killing the host or removing the symbionts.
  • Cross-Species Comparison: Temporal profiles were compared with the distantly related ciliate Tetrahymena utriculariae, which has independently evolved a similar endosymbiosis with green algae.
• Bioinformatics & Analysis:
  • Genome: A high-quality, haplotype-resolved non-redundant pangenome (18,325 loci) was generated for P. bursaria DK2.
  • Rhythmicity Detection: The RAIN algorithm (Rhythmic Analysis Incorporating Nonparametric methods) was used to detect 24-hour periodicity without assuming sinusoidal waveforms.
  • Clustering: Gaussian Mixture Models (GMM) were used to group genes into temporal clusters based on expression profiles.
  • Orthology Screening: The genome was screened for canonical TTFL clock components (e.g., CLOCK, BMAL1, PER, CRY) using BLASTp and DIAMOND against 42 reference proteins.
  • Functional Enrichment: GO and KEGG pathway analyses were performed to characterize the biological functions of rhythmic gene clusters.
  • Perturbation Analysis: PQ-treated samples were correlated against untreated symbiotic and aposymbiotic references to classify genes as "disrupted," "converged," or "maintained."

3. Key Contributions

• Discovery of Symbiont-Driven Transcriptome Expansion: Demonstrated that the presence of photosynthetic endosymbionts expands the host's rhythmic transcriptome by approximately 7-fold (3,300 symbiotic-specific rhythmic genes vs. 467 aposymbiotic-specific).
• Absence of Canonical Clocks: Provided strong evidence that P. bursaria lacks recognizable orthologs of canonical TTFL clock genes, suggesting its diel organization relies on alternative mechanisms.
• Role of Post-Translational Regulation: Identified that the expanded rhythmic program is enriched for genes encoding post-translational regulatory domains (kinases, ubiquitin ligases, calcium-binding proteins, WD40 scaffolds), pointing to a non-transcriptional timing mechanism.
• Photosynthesis as the Organizing Cue: Showed that disrupting photosynthetic electron transport (via Paraquat) shifts the host's temporal identity toward an aposymbiotic-like state, proving that active photosynthesis, not just symbiont presence, drives the rhythmic architecture.
• Evolutionary Convergence: Demonstrated that two independently evolved ciliate-algal symbioses (P. bursaria and T. utriculariae) share convergent diel expression programs specifically in symbiotic-specific genes, implying a universal organizing principle of photosymbiosis.

4. Key Results

A. Transcriptome Reconfiguration

• Global Shift: Symbiotic cells exhibit a broad shift in gene expression compared to aposymbiotic cells, with a significant downregulation of biosynthetic genes in symbionts and upregulation of recycling/lysosomal genes in aposymbionts.
• Rhythmic Expansion: RAIN analysis identified 3,538 rhythmic genes in symbiotic cells versus 705 in aposymbiotic cells. The vast majority (3,300) are specific to the symbiotic state.
• Temporal Ordering: Symbiotic rhythmic genes show a highly ordered, phase-structured progression:
  • Early Light: Motility, ciliary movement, and sensory signaling.
  • Mid-Light: Oxidative phosphorylation, ribosome biogenesis, and vesicle trafficking (linked to increased bacterial ingestion).
  • Dark Phase: Translation machinery, ubiquitin-linked protein turnover, and checkpoint signaling.
• Aposymbiotic State: Lacks this complex phase ordering; rhythmic genes are fewer and show lower contrast, focusing on growth and lipid metabolism.

B. Mechanism of Timing

• No TTFL: No full-length orthologs of canonical clock genes were found in the P. bursaria genome.
• Post-Translational Machinery: The symbiotic-specific rhythmic genes are heavily enriched for timing-associated domains:
  • Casein kinases (17 rhythmic).
  • EF-hand Ca²⁺-binding proteins (86 rhythmic).
  • F-box ubiquitin ligases (4 rhythmic).
  • WD40 scaffolds (77 rhythmic).
  • This suggests a timing mechanism driven by phosphorylation, calcium signaling, and targeted protein degradation rather than transcriptional feedback loops.

C. Photosynthesis Perturbation (Paraquat)

• Shift to Aposymbiotic State: When photosynthesis was inhibited by Paraquat, the temporal expression profiles of symbiotic cells shifted significantly.
  • Symbiotic-specific rhythmic genes lost their rhythmicity or amplitude (31.7% abolished, 27.2% partially disrupted).
  • Core rhythmic genes and aposymbiotic-specific genes in PQ-treated cells aligned more closely with the aposymbiotic reference (median correlation $r=0.771$) than the symbiotic reference.
• Domain Sensitivity: Regulatory domains (F-box, PAS, WD40) were highly sensitive to PQ treatment, with >70% showing disruption, confirming that photosynthetic output is required to maintain the post-translational timing infrastructure.

D. Cross-Species Conservation

• Convergent Evolution: Comparing P. bursaria with T. utriculariae (diverged ~600 million years ago) revealed that symbiotic-specific rhythmic genes show significant positive cross-species correlation ($median \ r = 0.354$).
• Divergent Hosts: In contrast, aposymbiotic-specific rhythmic genes showed negative correlation ($median \ r = -0.305$), indicating that intrinsic host rhythms have diverged, while the symbiont-driven program is conserved.

5. Significance

This study fundamentally changes the understanding of how photosymbiotic relationships regulate host physiology:

1. Metabolic Entrainment: It establishes that photosynthetic metabolic outputs (likely redox signals or sugar flux) act as a potent "organizer" of the host's daily gene expression, capable of expanding and structuring the transcriptome even in the absence of a canonical circadian clock.
2. Alternative Timing Mechanisms: It provides a robust example of a eukaryotic system where diel rhythms are maintained via post-translational regulatory networks (kinases, ubiquitination, calcium) rather than TTFL, challenging the dogma that circadian rhythms require specific clock genes.
3. Ecological Implications: The conservation of these programs across independently evolved ciliate-algal systems suggests that photosymbiosis imposes a universal selective pressure that converges on specific host regulatory architectures to optimize resource allocation and behavior (e.g., motility, feeding) in sync with the symbiont's photosynthetic cycle.
4. Methodological Advance: The use of Paraquat to decouple photosynthesis from light presence offers a novel experimental paradigm for studying photosymbiosis, allowing researchers to distinguish between light-entrained and photosynthesis-dependent effects.

In summary, the paper concludes that in P. bursaria, the endosymbiont does not merely provide nutrients but actively reorganizes the host's temporal architecture, creating a complex, phase-ordered diel program driven by photosynthetic inputs and executed through post-translational regulation.