Photosymbiotic algae acquisition and their interactions with the acoel Convolutriloba macropyga

This study characterizes the unique symbiotic relationship between the acoel *Convolutriloba macropyga* and *Tetraselmis* algae, revealing a complex lifecycle involving both vertical and horizontal transmission, distinct spatial localization of symbionts (both extracellular and intracellular), and specific metabolic adaptations in the host.

The Gist

Imagine a tiny, flatworm-like creature called Convolutriloba macropyga. It's no bigger than a grain of sand, but it has a superpower: it can turn itself into a solar-powered machine by hosting tiny green algae inside its body.

This new study is like a behind-the-scenes documentary showing exactly how this partnership is formed, how the two partners live together, and what happens inside their tiny bodies to make it work.

Here is the story of their relationship, broken down into simple parts:

1. The "Blank Slate" Start

Think of these flatworms like a newborn baby who hasn't learned to cook yet. When they hatch from their eggs, they are completely empty of algae. They are "aposymbiotic" (meaning "without partners").

• The Twist: Unlike some animals that inherit their algae from their parents (vertical transmission), these babies have to go out and find their own roommates. They must swim around, find the right kind of green algae (Tetraselmis), and invite them in.
• The Process: The baby worm swims in circles, creating a little whirlpool that traps algae. It then swallows them through its mouth, just like eating food.

2. The "Makeover" and the Move

Once the algae are inside the baby worm, things get interesting. It's not a simple "eat and keep" situation.

• The Shedding: Imagine the algae are like swimmers wearing wetsuits and carrying flippers. Once they enter the worm, they realize they don't need the gear anymore. They shed their outer shell (the "theca") and their flippers (the "flagella"). They become rounder and simpler, ready to settle down.
• The Migration: At first, the algae are scattered all over the worm's body. But over a few days, they start moving. Think of them like tenants moving from the basement to the top floor of a house to get better sunlight. They migrate toward the worm's outer skin (the body wall), specifically the top side (dorsal side), so they can soak up the sun.

3. The "Two-Mode" Living Arrangement

This is where the study found something really surprising. In many other animals, algae are locked inside a specific "room" (a cell) or a specific "building" (an organ). But in this worm, the algae are more like free spirits.

• The Sunbathers (Extracellular): Most of the algae live outside the worm's cells, just under the skin. They are like sunbathers lying on a beach towel (the worm's skin), soaking up light to make energy. They aren't trapped in a cage; they are free-floating in the space between cells.
• The Travelers (Intracellular): Some algae are found inside the worm's cells, deep in the body. The researchers think these might be "in transit." Maybe they are being moved from one place to another, or perhaps the worm is temporarily digesting them to get a quick snack of nutrients. It's a bit like a hotel where some guests are sleeping in the rooms, while others are just passing through the lobby.

4. The "Energy Exchange" (The Deal)

Why do they do this? It's a classic trade deal.

• What the Algae get: A safe home, protection from predators, and a steady supply of carbon dioxide (which they need to breathe).
• What the Worm gets: The algae act as solar panels. They take sunlight and turn it into food (sugars and fats) and oxygen. The worm then "harvests" this food.
• The Molecular Magic: The study looked at the worm's "instruction manual" (its genes) and found that when the algae are present, the worm turns on specific switches. It starts making more amino acids (building blocks for proteins), managing its water balance better, and building up defenses against stress (like too much sunlight or salt). It's like the worm's body is saying, "Okay, we have a power plant now; let's upgrade the whole factory!"

5. The Big Picture

This research is important because it shows that nature is more flexible than we thought. We used to think that for an animal to host algae, the algae must be locked inside a cell. But here, the algae are mostly living outside the cells, right under the skin, acting like a living, breathing solar panel.

In a nutshell:
This tiny worm is a master of adaptation. It starts life alone, goes out to find a roommate, helps the roommate shed its heavy gear, and then lets the roommate set up a solar farm on its skin. In exchange, the worm gets a free lunch and a supercharged metabolism. It's a perfect example of how two very different life forms can team up to survive and thrive in the ocean.

Technical Summary

1. Problem Statement

Photosymbiosis (the association between heterotrophic hosts and photoautotrophic microbes) is a widespread phenomenon in marine animals, yet the mechanisms of symbiont acquisition, transmission, and spatial organization vary significantly across taxa. While many acoel flatworms are known to host photosymbionts, the specific dynamics of how the species Convolutriloba macropyga acquires its symbionts, the ultrastructural changes the algae undergo, and the molecular mechanisms maintaining the symbiosis remain poorly understood. Specifically, there is a lack of clarity regarding:

• Whether symbionts are transmitted vertically (to asexual offspring) or horizontally (acquired by sexual offspring).
• The precise localization of symbionts within the host (intracellular vs. extracellular) and whether they reside in specific tissues.
• The morphological and physiological changes algae undergo upon entering the host.
• The host's molecular response to establishing and maintaining this symbiosis.

2. Methodology

The authors employed a multidisciplinary approach combining developmental biology, microscopy, phylogenetics, and transcriptomics:

• Organism & Culturing: C. macropyga adults and juveniles were cultured in artificial seawater. Juveniles were isolated from egg clusters to ensure they were aposymbiotic (symbiont-free) at hatching.
• Symbiont Identification: The symbionts were identified as Tetraselmis sp. (Chlorophyta) using phylogenetic analysis of the rbcL gene (Rubisco) from both the host holobiont and free-living algae (strain SAG 35.93).
• Experimental Exposure: Aposymbiotic juveniles were exposed to Tetraselmis algae for varying durations (5 hours to 3 days) and monitored over time (Post-Exposure Days, PED) to track uptake and proliferation.
• Microscopy:
  • Light & Confocal Microscopy: Used to visualize algal autofluorescence, host nuclei (DAPI), and muscle fibers (phalloidin) to quantify algal numbers and spatial distribution (body wall vs. parenchyma).
  • Transmission Electron Microscopy (TEM): Used to examine ultrastructural changes in algae (loss of flagella/theca) and determine the intracellular vs. extracellular status of symbionts in both juveniles and adults.
• Transcriptomics: Bulk RNA sequencing was performed on aposymbiotic juveniles and symbiotic adults. Differential gene expression analysis (DESeq2) and Gene Set Enrichment Analysis (GSEA) using GO and KEGG databases were conducted to identify metabolic and physiological pathways activated during symbiosis.

3. Key Results

A. Transmission and Acquisition

• Dual Transmission Modes: Symbionts are transmitted vertically during asexual reproduction (budding), where offspring inherit algae directly. However, sexually produced offspring hatch as aposymbiotic juveniles.
• Horizontal Acquisition: These aposymbiotic juveniles must acquire Tetraselmis algae horizontally from the environment by ingesting them through the mouth.
• Uptake Dynamics: Upon exposure, juveniles ingest algae immediately. The number of algae within the juvenile increases significantly over time, indicating that the algae proliferate within the host tissues rather than just accumulating passively.

B. Morphological and Ultrastructural Changes

• Loss of Motility Structures: Upon uptake, Tetraselmis algae lose their flagella and theca (cell wall). However, they retain eyespots and Golgi apparatus.
• Spatial Distribution:
  • Juveniles: Algae migrate from the central parenchyma toward the body periphery (body wall), specifically accumulating on the dorsal side (optimal for light).
  • Adults: Symbionts are found in two distinct states:
    1. Extracellular: Located at the body wall (between epidermis and muscle layers), where they are likely performing photosynthesis.
    2. Intracellular: Found within the parenchyma, often inside vacuolated, multinucleated cells. These intracellular algae are rounder and may be undergoing digestion or transport.

C. Transcriptomic Insights

Comparative analysis between symbiotic adults and aposymbiotic juveniles revealed significant upregulation of specific pathways in adults:

• Nutrient Exchange: Enrichment in pathways for amino acid synthesis, lipid metabolism (beta-oxidation), and carbohydrate metabolism (including trehalose biosynthesis), suggesting the host receives organic compounds from the algae.
• Stress Response: Upregulation of genes related to oxidative stress (ROS detoxification) and osmoregulation (e.g., betaine synthesis), likely compensating for the metabolic byproducts of photosynthesis and the marine environment.
• Immunity: Enrichment in immune-related pathways, potentially managing the recognition and tolerance of the symbiont.
• Growth: Upregulation of cell cycle and protein synthesis genes, correlating with the growth and asexual reproduction of the symbiotic adults.

4. Key Contributions

1. Clarification of Transmission: The study confirms that C. macropyga utilizes a mixed transmission strategy: vertical for asexual clones and horizontal for sexual offspring, a pattern not fully characterized in this species previously.
2. Spatial Complexity of Symbiosis: The authors provide the first detailed evidence that C. macropyga hosts algae in both extracellular and intracellular compartments simultaneously. This challenges the binary view of endosymbiosis (strictly intracellular) and suggests a dynamic system where algae may be "farmed" extracellularly for photosynthesis and internalized for nutrient processing or transport.
3. Ultrastructural Characterization: Detailed TEM evidence showing the specific loss of theca and flagella while retaining eyespots, distinguishing the symbiotic state from free-living Tetraselmis.
4. Molecular Mechanism: The transcriptomic data offers a molecular blueprint for how acoels manage the metabolic costs and benefits of photosymbiosis, highlighting the role of osmoregulation and oxidative stress management.

5. Significance

This research significantly advances the understanding of animal-microbe interactions in basal bilaterians. By demonstrating that acoels can maintain symbionts both inside and outside cells, the paper suggests a high degree of plasticity in host-symbiont relationships. The findings imply that the host may actively regulate the symbiont population through non-physical mechanisms (e.g., chemical signaling) rather than strict physical containment (symbiosomes).

Furthermore, the identification of specific metabolic pathways (lipid transfer, amino acid synthesis, stress response) provides a foundation for comparative studies across diverse photosymbiotic systems (e.g., corals, giant clams). The study also highlights the potential of using extracellular photosymbionts in animal tissues as a model for understanding allorecognition, transplant biology, and even targeted therapies using microalgae.