Shinichiro Maruyama

Oophila spp. are the only green algae known to establish symbiotic associations with the eggs of amphibians, including salamanders and frogs. They comprise two phylogenetic lineages, Clades A and B, each exhibiting a distinct degree of host specificity. In the North American spotted salamander Ambystoma maculatum, a symbiotic green alga Oophila amblystomatis has been shown to colonize not only the egg capsules but also the embryonic tissues and cells. Consequently, this system has attracted considerable attention as the only known algal-vertebrate symbiosis in nature. While previous studies have demonstrated the geographic distribution, phylogenetic diversity, and symbiotic lifestyle of Oophila, many fundamental questions remain unanswered: Where does Oophila originate? How does it establish symbiosis within amphibian eggs? How has this symbiosis evolved? Here we summarize the current knowledge and remaining challenges at each stage of the algal-amphibian symbiosis, including the life history and ecology of Oophila, host–symbiont interactions, and the origin and evolution of this unique symbiotic lifestyle.

Medakamo hakoo is an ultrasmall green alga with a simplified cellular structure, offering potential as a new model organism. To explore the genetic basis of cell morphology and its physiological implications, we applied carbon-ion beam irradiation to induce mutations in M. hakoo and successfully isolated two mutants: LRG (Large) and TTR (Tetra). LRG exhibited significantly enlarged cell size and increased chlorophyll content, but lower photosynthetic efficiency compared to the wild-type. Proteomic analysis of LRG revealed upregulation of photosynthesis-related proteins, stress response proteins, and ribosomal proteins, suggesting a link between increased cell size and disrupted photosynthetic homeostasis. TTR showed a higher proportion of dividing cells throughout the light–dark cycle and exhibited cell aggregation. Proteomic profiling revealed increased abundance of dynein and cell wall–modifying enzymes such as expansin and mannosidases, indicating that TTR may undergo cell cycle delay or dysregulation of cytoplasmic and cell wall dynamics. TTR also displayed decreased levels of photosynthetic proteins and reduced photosynthetic activity. Both mutants demonstrated slower growth compared to the wild-type. These findings highlight the close relationship between cell size and cell cycle/cell wall dynamics with photosynthetic activity, providing new insights into the cellular regulatory mechanisms of photosynthetic microalgae. Our study also demonstrates the utility of heavy-ion mutagenesis in dissecting microalgal physiology in species for which genetic transformation systems have not yet been developed.

Photosymbiosis is a symbiotic relationship between microalgae and non-photosynthetic eukaryotes. Such associations have independently evolved multiple times across a wide range of eukaryotic lineages. In some photosymbiotic relationships, photosymbionts supply photosynthetic products to the host, and, on the other hand, host organisms supply some nutrients. Considering these trophic interactions, photosymbiosis appears to be mutualistic, but the balance between benefits and costs is highly dependent on environmental conditions. In this article, we focus on photosymbioses in green algae, which show particularly high diversity in both symbiont and host species, and discuss how these symbiotic relationships affect the fitness of symbionts and their hosts.

Symbiosis is a basis for species diversification through interactions between organisms. In tropical and subtropical oceans, dinoflagellate symbionts belonging to the family Symbiodiniaceae, including the genus Symbiodinium, support the flourishment of cnidarian hosts, including corals, and thereby the ecology of oligotrophic oceans through their photosynthate carbon transfers. Although the genus Symbiodinium includes both free-living and symbiotic species, the detailed genetic background of their lifestyle differences remains unclear. In this study, we identified candidate genes involved in the evolutionary acquisition or maintenance of symbiosis in Symbiodinium spp. by detecting genes that have undergone positive selection during symbiotic and free-living lifestyle diversification. Using multiple Symbiodinium genomes to detect positive selection, 35 genes were identified, including a gene encoding soluble starch synthase SSY1 and genes related to metabolite secretion, which may be preferred for symbiotic lifestyles. In particular, our in silico analyses revealed that the SSY1 gene family has undergone extensive gene duplications in an ancestral dinoflagellate, and that the mutations detected as positive selection have occurred in the intrinsically disordered region of one of the homologs. Consistent with molecular evolution, the phenotypes of intracellular starch synthesis/accumulation were distinct between the symbiotic and free-living species of Symbiodinium when cultured under different pH and nitrogen conditions. These results provide molecular and phenotypic insights into symbiotic Symbiodinium-coral relationships.

Photosymbioses, the symbiotic relationships between microalgae and non‐photosynthetic eukaryotes, are sporadically found in many eukaryotic lineages. Only a few taxa, such as cnidarians and ciliates hosting algal endosymbionts, have been actively studied, which has hindered understanding the universal mechanisms of photosymbiosis establishment. In Amoebozoa, few species are reported as photosymbiotic, and how the photosymbioses are established is still unclear. To investigate the extent to which one of the photosymbiotic amoebae, Mayorella viridis, depends on their symbionts, the amoebae were treated with reagents known to induce the collapsing of photosymbioses in other species. We succeeded in removing algal symbionts from the hosts with 2‐amino‐3‐chloro‐1,4‐naphthoquinone. While the apo‐symbiotic amoebae grew to the same extent as the symbiotic state when they fed on prey, their survival rates were lower than those of the symbiotic ones during starvation, suggesting that the impact of the photosymbiosis on fitness is condition‐dependent. Furthermore, we showed that the photosymbiotic state was reversible by feeding two strains of the green alga Chlorella to the apo‐symbiotic amoebae. The efficiencies of ingesting algal cells significantly differed between algal strains. These results suggest that the photosymbiotic relationship in the amoeba is facultative and that different algal strains have discrete symbiotic abilities to the amoeba.

Symbiosis is a basis for species diversification through interactions between organisms. In tropical and subtropical oceans, dinoflagellate symbionts belonging to the family Symbiodiniaceae, including the genus Symbiodinium , support the flourishment of cnidarian hosts, including corals, and thereby the ecology of oligotrophic oceans through their photosynthate carbon transfers. Although the genus Symbiodinium includes both free-living and symbiotic species, the detailed genetic background of their lifestyle differences remains unclear. In this study, we identified candidate genes involved in the evolutionary acquisition or maintenance of symbiosis in Symbiodinium spp. by detecting genes that have undergone positive selection during symbiotic and free-living lifestyle diversification. Using multiple Symbiodinium genomes to detect positive selection, 35 genes were identified, including a gene encoding soluble starch synthase SSY1 and genes related to metabolite secretion, which may be preferred for symbiotic lifestyles. In particular, our in silico analyses revealed that the SSY1 gene family has undergone extensive gene duplications in an ancestral dinoflagellate, and that the mutations detected as positive selection have occurred in the intrinsically disordered region of one of the homologs. Consistent with molecular evolution, the phenotypes of intracellular starch synthesis/accumulation were distinct between the symbiotic and free-living species of Symbiodinium when cultured under different pH and nitrogen conditions. These results provide molecular and phenotypic insights into symbiotic Symbiodinium -coral relationships. Significance statement: Clarifying how coral symbiont algae of the family Symbiodiniaceae are functionally diversified is crucial for understanding the molecular mechanisms of symbiotic relationships and interactions in animal-algal symbioses. However, gene-level molecular evolution that contributes to symbiotic relationships remains unclear. Our genome-wide single-gene analyses of the genus Symbiodinium , comprising both symbiotic and free-living species, successfully detected several dozen protein-coding genes that underwent symbiont-specific positive selection after divergence from the free-living species in the genus. Furthermore, our cultivation experiments demonstrated differences in photosynthate-related phenotypes between the symbiotic and free-living species. Our findings provide a key to illuminating the molecular evolution involved in phenotypic changes that potentially contribute to symbiotic lifestyles in Symbiodinium .

Photosymbioses, the symbiotic relationships between photosynthetic algal symbionts and non-photosynthetic eukaryotic hosts, are sporadically found in a lot of eukaryotic lineages, but only a few taxa, such as cnidarians and ciliates hosting algal endosymbionts, have been actively studied for a long time. That has hindered understanding the universal mechanisms of the photosymbiosis establishment. Especially in Amoebozoa, only two species, Mayorella viridis and Parachaos zoochlorella , are reported as photosymbiotic in nature, and their mechanisms of establishing symbiotic relationships are still unclear. To investigate the extent to which and how photosymbiotic amoebae depend on the symbiotic relationships, M. viridis were treated with reagents that are known to induce the collapsing of photosymbiotic relationships, or bleaching, in other photosymbiotic species. As a result, we succeeded in artificially removing algal symbionts from host M. viridis cells with an herbicide, 2-amino-3-chloro-1,4-naphthoquinone. The apo-symbiotic state amoeba cells were able to survive and grow to the same extent as the symbiotic state cells when they fed microbial prey, indicating that the algal symbionts are not essential for the host growth under certain conditions. Furthermore, to see whether the photosymbiotic state is reversible, we fed two strains of algal symbionts to the apo-symbiotic amoeba host. The result showed that the apo-symbiotic hosts were able to ingest symbiont cells and re-establish the symbiotic state. The efficiencies of ingesting algal cells were significantly different depending on algal symbiont strains, indicating that different algal strains possess discrete symbiotic abilities to M. viridis . To our knowledge, we provide first insights on the establishment and collapse of photosymbiosis in Amoebozoa, which pave the way to understand the universal mechanism of photosymbiosis utilizing M. viridis as a model system.

Aligning spawning timing with seasonal environmental changes is critical for both terrestrial and aquatic organisms. However, mechanisms to regulate reproductive activity in response to environmental factors are not well understood, partly owing to the technical difficulty in maintaining detailed long-term observational data of the reproductive activities in the same population across years. In this study, we present an application of the aquarium system to examine the long-term spawning properties of corals. Spawning records over a 15-year period at the Okinawa Churaumi Aquarium revealed that the spawning timing of Acropora corals in aquarium tanks aligned well with that of wild corals from a neighbouring reef. Using the aquarium population as a model, we investigated the relationship between key environmental factors and the timing of the first and peak spawning dates of Acropora corals during a 15-year period. The results suggest that the spawning window of each spawning season is largely influenced by the water temperature and that the timing of peak spawning can be fine-tuned in response to environmental fluctuations. This behavioural feature can prevent synchronous spawning events during unfavourable environmental conditions and increase long-term reproductive reliability.

Chloroplasts are photosynthetic organelles that evolved through the endosymbiosis between cyanobacteria-like symbionts and hosts. Many studies have attempted to isolate intact chloroplasts to analyze their morphological characteristics and photosynthetic activity. Although several studies introduced isolated chloroplasts into the cells of different species, their photosynthetic activities have not been confirmed. In this study, we isolated photosynthetically active chloroplasts from the primitive red alga Cyanidioschyzon merolae and incorporated them in cultured mammalian cells via co-cultivation. The incorporated chloroplasts retained their thylakoid structure in intracellular vesicles and were maintained in the cytoplasm, surrounded by the mitochondria near the nucleus. Moreover, the incorporated chloroplasts maintained electron transport activity of photosystem II in cultured mammalian cells for at least 2 days after the incorporation. Our top-down synthetic biology-based approach may serve as a foundation for creating artificially photosynthetic animal cells.

Reef-building corals thrive in oligotrophic environments due to their possession of endosymbiotic algae. Confined to the low pH interior of the symbiosome within the cell, the algal symbiont provides the coral host with photosynthetically fixed carbon. However, it remains unknown how carbon is released from the algal symbiont for uptake by the host. Here we show, using cultured symbiotic dinoflagellate, Breviolum sp., that decreases in pH directly accelerates the release of monosaccharides, that is, glucose and galactose, into the ambient environment. Under low pH conditions, the cell surface structures were deformed and genes related to cellulase were significantly upregulated in Breviolum. Importantly, the release of monosaccharides was suppressed by the cellulase inhibitor, glucopyranoside, linking the release of carbon to degradation of the agal cell wall. Our results suggest that the low pH signals the cellulase-mediated release of monosaccharides from the algal cell wall as an environmental response in coral reef ecosystems.