Clonal cultures of the dinoflagellates Effrenium voratum (FL) (SvFL1), Prorocentrum cordatum (PMKS9906), and Prorocentrum koreanum (PKMS1807) were isolated from waters off Jeju Island, Kunsan, and Masan, Korea, respectively (Table 1). The dinoflagellates E. voratum (IC) (SvIC2), Cladocopium infistulum (rt-203), and Symbiodinium microadriaticum (CCMP2464 or rt-061) were obtained from the tissues of the stony coral Alveopora japonica from Jeju Island, the giant clam Hippopus hippopus from Palau, and the jellyfish Cassiopea xamachana from Florida Keys, USA, respectively (LaJeunesse 2001, Jeong et al. 2014, Lee et al. 2020) (Table 1). The cyanobacterium Synechococcus sp. (N54-2), chlorophyte Dunaliella salina (DSJH1710), and raphidophyte Heterosigma akashiwo (HAKS9905) were isolated from the East China Sea, Jinhae, and Kunsan, Korea, respectively. The prasinophyte Tetraselmis suecica (CCMP904), originally isolated from Bembridge, UK, was obtained from the National Center for Marine Algae and Microbiota (NCMA) (Boothbay Harbor, ME, USA) (Table 1). All microalgal cultures were transferred monthly into 250-mL flat culture flasks (Corning Inc., New York, NY, USA) containing freshly prepared f/2-Si medium (Guillard and Ryther 1962) and maintained at 20°C under 50 μmol photons m⁻² s⁻¹ illumination using a light-emitting diode lamp under a 14 : 10 h light/dark cycle. The cyanobacterium Synechococcus sp. was maintained under 5–10 μmol photons m⁻² s⁻¹ from cool-white fluorescent light with a 14 : 10 h light/dark cycle at 20°C.

Farmed medusae of the moon jellyfish A. aurita (Cnidaria, Scyphozoa) were obtained from the Korea Jellyfish Lab (Seoul, Korea). They were transported with fresh seawater to the laboratory and maintained in a 50-L acrylic round jellyfish culture tank equipped with an internal filter using 0.5-μm filtered seawater (FSW) under 20 μmol photons m⁻² s⁻¹ from cool-white fluorescent light with a 14 : 10 h light/dark cycle at 20°C in a walk-in chamber. Each jellyfish was fed daily with approximately 4,000 Artemia nauplii (SEP-Art Magnetic Artemia; Great Salt Lake Artemia, Ogden, UT, USA), and the seawater was changed weekly with 0.5-μm FSW at the same temperature. The salinity of the seawater used in the present study was 29.3. Jellyfish were not fed for one day prior to the experiment.

Experiment 1 aimed to investigate the survival of each microalgal and cyanobacterial species inside the medusa of the moon jellyfish, A. aurita, after injecting microalgae or cyanobacteria into the mesoglea of the medusa of the jellyfish (Table 2).

For Experiment 1, 50- or 100-mL aliquots from each culture of the microalgal and cyanobacterial species were transferred into several 50-mL Falcon tubes (Corning Inc.) and centrifuged at 3,000 rpm for 10 min (microalgae) or 30 min (Synechococcus sp.) (Labogene 1696R; Gyrozen Co., Gimpo, Korea) (Fig. 1). The pellets obtained through centrifugation were resuspended in 1 mL of 0.2-μm FSW. Forty microliters of the resuspended solutions of each microalgal and cyanobacterial species were transferred to 1-mL (U-100) insulin syringes (30 G, 8 mm; Angel Syringe, Yong Chang Co., Ltd., Gimpo, Korea) and injected into 10 jellyfish (experimental jellyfish) (Table 2, Fig. 1). One jellyfish without injected microalgae or cyanobacteria served as a control. The experimental and control jellyfish were transferred to eleven 8-L round jellyfish-water tanks and maintained for 7 days under 50 μmol photons m⁻² s⁻¹ from cool-white fluorescent light with a 14 : 10 h light/dark cycle at 20°C. The jellyfish and injected microalgal and cyanobacterial cells were observed daily using an epi-fluorescent microscope (EVOS M5000; Thermo Fisher Scientific, Waltham, MA, USA) and photographed until the injected cells were no longer detected inside the experimental jellyfish.

Experiment 2 aimed to investigate the survival of E. voratum (FL) inside jellyfish for up to 60 days after artificial injection into A. aurita (Table 2, Fig. 1). The preparation and injection process of E. voratum (FL) into the jellyfish and the monitoring of E. voratum (FL) inside the jellyfish followed the same procedures as in Experiment 1 (Fig. 1). Two experimental jellyfish and two control jellyfish were set up in four 8-L round jellyfish-water tanks. The jellyfish and injected E. voratum (FL) cells were observed daily using the epi-fluorescent microscope (EVOS M5000; Thermo Fisher Scientific) and photographed until the injected cells were no longer detected inside the experimental jellyfish.

Before centrifugation, the abundance of microalga and cyanobacterium cultures was determined by enumerating the cells of each strain in three 1-mL Sedgewick-Rafter chambers using an inverted microscope (CX21; Olympus Corporation, Shinjuku, Tokyo, Japan) at 100- or 200-fold magnification. Throughout all experiments, 30–40% of the total water volume in the tanks containing A. aurita was replaced with fresh FSW every 3–4 days. During the experiments, Artemia sp. nauplii were provided daily to the jellyfish as prey (approximately 4,000 Artemia nauplii for each jellyfish).

Cells of the dinoflagellates E. voratum (FL), E. voratum (IC), C. infistulum, S. microadriaticum, P. cordatum, and P. koreanum inside the mesoglea of the medusa of the jellyfish A. aurita produced red fluorescence, indicating the survival of the microalgal species during the study period, 7 days after artificial injection into the jellyfish (Table 3, Figs 2–7). Furthermore, the nanoflagellates T. suecica, D. salina, and H. akashiwo inside the mesoglea of the jellyfish also produced red fluorescence during the study period (Table 3, Figs 8–10).

The injected cells of Synechococcus sp. inside the mesoglea of the medusa of the jellyfish produced red fluorescence for the first 4 days after artificial injection into the jellyfish but did not produce it after 5 days (Table 3, Fig. 11). All experimental and control jellyfish survived for 7 days.

The injected cells of E. voratum (FL) inside the mesoglea of the medusa of the jellyfish A. aurita produced red fluorescence, indicating the survival of the microalgal species for 60 days after artificial injection into the jellyfish (Fig. 12). Additionally, cells of E. voratum (FL) gradually spread toward adjacent areas 15 days after injection, suggesting potential for artificially established symbiosis (Fig. 12). The injected cells of E. voratum (FL) inside the mesoglea of another jellyfish A. aurita also produced red fluorescence, indicating the survival of the microalgal species for 22 days after injection. All experimental and control jellyfish survived during the study period.

Several species in the dinoflagellate genera Breviolum, Cladocopium, Durusdinium, and Symbiodinium in the family Symbiodiniaceae are known to survive inside invertebrates (Freudenthal 1962, Trench and Thinh 1995, LaJeunesse 2002) (Table 4). Additionally, the dinoflagellates Gloeodinium viscum and Scrippsiella velellae have survived inside the bodies of the fire coral Millepora dichotoma (Hydrozoa) and sea raft Velella velella (Hydrozoa), respectively (Banaszak et al. 1993) (Table 4). The present study added Prorocentrum cordatum and P. koreanum as dinoflagellates that survived inside a cnidarian. Unidentified green chlorophytes have been reported to survive in the sea anemones Anthopleura elegantissima and A. xanthogrammica (O’Brien and Wyttenbach 1980, Lewis and Muller-Parker 2004) (Table 4). The present study added T. suecica, H. akashiwo, and D. salina as nanoflagellates that also survived inside a cnidarian. Diverse cyanobacterial species are known to survive inside corals (Table 4). However, in the present study, cells of Synechococcus sp. were not detected inside the jellyfish 5 days after being artificially injected. Therefore, Synechococcus cells may be digested by phagocytosis of mesogleal amoebocytes or decomposed by the antimicrobial activity of peptides in the mesoglea (Chapman 1974, Ovchinnikova et al. 2006).

Although eight microalgal species survived inside the medusa of the moon jellyfish A. aurita in this study, it was unclear whether these microalgal species were symbiotic with the moon jellyfish. Based on comparative studies of their genomes and transcriptome protein sets, among the dinoflagellates Karenia brevis, Lingulodinium polyedra, Amphidinium carterae, Crypthecodinium cohnii, P. cordatum, S. microadriaticum, Breviolum minutum, and Fugacium kawagutii, only the Symbiodinium lineage (i.e., S. microadriaticum, B. minutum, and F. kawagutii) possesses a wide range of transporters associated with the delivery of carbon and nitrogen (Aranda et al. 2016). These molecular characteristics may allow the Symbiodinium lineage to form symbiotic relationships with a wide range of cnidarians in the natural environment. It is worth exploring whether each of the eight microalgal species has a wide range of transporters linked to the delivery of carbon and nitrogen.

The moon jellyfish A. aurita used in this study is a non-symbiotic jellyfish that develops into an adult without symbionts in its polyp or scyphistoma stage (Kakinuma 1975). However, when S. microadriaticum isolated from the jellyfish Cassiopea xamachana just before the experiments and the dinoflagellates Amphidinium carterae and Gymnodinium sp. were provided to the scyphistoma of A. aurita, only S. microadriaticum cells were phagocytosed by the endodermal cells and retained inside the body of the scyphistoma for 4 days (Colley and Trench 1983). Furthermore, S. microadriaticum cells disappeared after 7 days of ingestion. However, E. voratum (FL) survived for 60 days inside the medusa of A. aurita. This evidence suggests the potential to establish symbiotic relationships between E. voratum (FL) and A. aurita by artificial injection.

Sharp et al. (2024) reported that the polyps of the symbiotic jellyfish Ca. xamachana were not infected with E. voratum. However, Starzak et al. (2020) reported that the sea anemone Exaiptasia pallida could be infected by E. voratum (FL). Additionally, E. voratum survives in diverse corals (Jeong et al. 2014, Yang et al. 2020). Therefore, E. voratum can survive inside the body of jellyfish by artificial injection rather than by natural infection. The results of this study suggest that injecting E. voratum cells into the medusa of jellyfish can lead to temporary or long-term symbiosis.

Due to climate change, the global surface and seawater temperatures in 2011–2020 were 1.09°C and 0.88°C higher, respectively, compared to those in 1850–1900 (Intergovernmental Panel on Climate Change 2023). Additionally, global warming increases sea surface temperature, preventing vertical mixing and accelerating hypoxic conditions on the seafloor (Moffitt et al. 2015, Intergovernmental Panel on Climate Change 2023). Hypoxic conditions on the seafloor can lead to the mass mortality of benthic marine organisms, such as corals, clams, sea slugs, and octopuses. Artificially transplanting marine microalgae into benthic animals to induce mixotrophy could help the animals adapt to and overcome hypoxic environments by the microalgae inside the animals. Symbiosis between microalgae and invertebrates can increase the survival of invertebrates, and transplanting microalgae into vertebrates can supply oxygen to vertebrates (Cohen et al. 2017, Apprill 2020, Özugur et al. 2021). Therefore, biotechnology involving the transplantation of microalgae into invertebrates may increase invertebrate survival under unfavorable environmental conditions and restore invertebrate populations.