Potential for artificial symbiosis between marine microalgae and invertebrates: I. survival of marine microalgae injected into the medusa of the moon jellyfish Aurelia aurita

Article information

Algae. 2024;39(3):163-176

Publication date (electronic) : 2024 September 15

doi : https://doi.org/10.4490/algae.2024.39.9.6

Ji Hyun You ¹, Hae Jin Jeong ¹^,²^,, Sang Ah Park ¹, Se Hee Eom ¹, Hee Chang Kang ³, Min Ji Kwon ¹

¹School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 08826, Korea

²Research Institute of Oceanography, Seoul National University, Seoul 08826, Korea

³Department of Ocean Sciences, College of Natural Sciences, Inha University, Incheon 22212, Korea

^*Corresponding Author: E-mail: hjjeong@snu.ac.kr, Tel: +82-2-880-6746, Fax: +82-2-874-9695

Received 2024 April 12; Accepted 2024 September 6.

Abstract

Some marine microalgae and cyanobacteria form mutualistic symbioses with diverse invertebrates, particularly cnidarians. Among microalgae, dinoflagellates in the family Symbiodiniaceae are the most well-known symbiotic partners of jellyfish and corals. However, the symbioses involving other dinoflagellate families, nano- and micro-flagellates, diatoms, and cyanobacteria with cnidarians are not well understood. As an initial step, it is essential to explore the survival of these microorganisms inside cnidarians. We monitored the survival of eight microalgal species (nine strains) and one cyanobacterium species every day for seven days after injecting each into the medusa of the moon jellyfish Aurelia aurita. The dinoflagellates Effrenium voratum (free-living [FL] and living-in-coral strains), Cladocopium infistulum, Prorocentrum cordatum, Prorocentrum koreanum, Symbiodinium microadriaticum, the prasinophyte Tetraselmis suecica, the chlorophyte Dunaliella salina, and the raphidophyte Heterosigma akashiwo survived inside the medusa, while the cyanobacterium Synechococcus sp. was not detected. Additionally, E. voratum (FL) survived within the medusa for 60 days and gradually spread to adjacent areas, indicating potential for artificially established symbiosis. The results of this study provide a basis for artificial symbiosis between microalgae and invertebrates.

Keywords: cnidaria; cyanobacteria; dinoflagellate; flagellate; scyphozoa; Symbiodiniaceae

INTRODUCTION

Marine microalgae and cyanobacteria are crucial components of marine ecosystems (Sellner 1997, Ok et al. 2021, 2023a, Kang et al. 2023b, You et al. 2023). They play diverse roles as primary producers, prey, and symbionts (Thacker and Starnes 2003, Jeong et al. 2012, Hamilton et al. 2016, Lee et al. 2020, You et al. 2020, Lim and Jeong 2022, Kang et al. 2023a, Ok et al. 2023b, 2024). Some microalgae and cyanobacteria form symbiotic relationships with various marine organisms (Freudenthal 1962, LaJeunesse 2002, Lewis and Muller-Parker 2004, Di Camillo et al. 2005, Mutalipassi et al. 2021). In these symbioses, microalgae and cyanobacteria provide photosynthetic products to the host animals, which in turn supply nutrients to the microalgae and cyanobacteria through metabolic recycling (Arillo et al. 1993, Yellowlees et al. 2008, Rädecker et al. 2018). Symbiosis with microalgae grants invertebrates mixotrophic capabilities, aiding them in coping with environmental changes (Apprill 2020). These relationships are vital for the survival of certain hosts, such as corals; the disruption of these symbioses results in coral bleaching (Douglas 2003).

Among microalgae, dinoflagellates in the family Symbiodiniaceae are the most recognized symbiotic partners of corals and jellyfish (Freudenthal 1962, Trench and Thinh 1995, LaJeunesse 2002, Lee et al. 2016, LaJeunesse et al. 2018). Many species from different genera within this family engage in symbiosis with corals (LaJeunesse et al. 2018). The symbiotic dinoflagellates in the genera Cladocopium and Symbiodinium are sometimes crucial for the development of the jellyfish Cassiopea spp., as most polyps cannot metamorphose into ephyrae and complete their life cycles without symbionts (Rahat and Adar 1980, Colley and Trench 1983, Djeghri et al. 2019). Some cyanobacteria form symbioses with corals, the diatoms Amphora spp., Cocconeis spp., and Licmophora spp. with hydroids, and unidentified chlorophytes with sea anemones (Lewis and Muller-Parker 2004, Di Camillo et al. 2005, Mutalipassi et al. 2021). Considering the symbiosis between microalgae and invertebrates, critical questions arise. Do other microalgal species not engage in symbiosis with invertebrates? Do other invertebrate species not form symbioses with microalgae? To address the first question, it is essential to investigate whether dinoflagellates, flagellates, or cyanobacteria can survive inside invertebrates.

Artificial injection of microalgae and cyanobacteria into the eggs and larvae of zebrafish, frog larvae (tadpoles), mice and rats using devices has been conducted to explore the potential for oxygen supply and the survival of microalgae and cyanobacteria within animal bodies (Agapakis et al. 2011, Alvarez et al. 2015, Chávez et al. 2016, Cohen et al. 2017, Özugur et al. 2021, Ehrenfeld et al. 2023). The microalgal and cyanobacterial species used in these studies included the nanoflagellate Chlamydomonas reinhardtii and the cyanobacteria Synechococcus elongatus and Synechocystis sp. These microalgal and cyanobacterial species supplied oxygen to the animals (Cohen et al. 2017, Özugur et al. 2021).

The symbiosis of dinoflagellates from other families, nano- and micro-flagellates, and cyanobacteria with cnidarians is not well understood. In this study, we monitored the survival of eight microalgal species (Effrenium voratum, with two strains—free-living [FL] and living-in-coral [IC], Cladocopium infistulum, Prorocentrum cordatum, Prorocentrum koreanum, and Symbiodinium microadriaticum) as well as the prasinophyte Tetraselmis suecica, the chlorophyte Dunaliella salina, the raphidophyte Heterosigma akashiwo, and one cyanobacterium species (Synechococcus sp.) every day over seven days after injecting each species into the medusa of the moon jellyfish Aurelia aurita. Additionally, we examined the survival of E. voratum (FL) inside the medusa of the jellyfish for 60 days. The results of this study provide a foundation for artificial symbiosis between microalgae and invertebrates.

MATERIALS AND METHODS

Preparation of experimental organisms

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.

Table 1

Information on the isolation of microalgal and cyanobacterial species used in this study

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.

Injection of microalgae into the moon jellyfish

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).

Table 2

Experimental conditions in this study

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.

Fig. 1

Preparation (A) and process (B & C) for the injection (yellow circle) of microalgal and cyanobacterial cells into the target jellyfish Aurelia aurita (yellow arrows) and the actual experimental setup (D & E).

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).

RESULTS

Survival of microalgal and cyanobacterial species inside the medusa of the moon jellyfish after injection

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).

Table 3

Results of Experiments 1 and 2

Fig. 2

Survival of cells of Effrenium voratum free-living strain (EVFL) after the cells were injected into the mesoglea of the medusa of the moon jellyfish Aurelia aurita. A non-injected cell of EVFL (A). The package containing injected cells of EVFL inside the jellyfish body (yellow arrow) for 0 (B–D), 3 (E & F), and 7 days (G & H) after artificial injection. The red fluorescence in (D, F & H) indicates the survival of the microalgal species. Micrographs in (C, E & G) were taken under a light microscope and those in (D, F & H) under an epifluorescence microscope. Scale bars represent: A, 5 μm; B, 5 mm; C–H, 300 μm.

Fig. 3

Survival of cells of Effrenium voratum living-in-coral strain (EVIC) after the cells were injected into the mesoglea of the medusa of the moon jellyfish Aurelia aurita. A non-injected cell of EVIC (A). The package containing injected cells of EVIC inside the jellyfish body (yellow arrow) for 0 (B–D), 3 (E & F), and 7 days (G & H) after artificial injection. The red fluorescence in (D, F & H) indicates the survival of the microalgal species. Micrographs in (C, E & G) were taken under a light microscope and those in (D, F & H) under an epifluorescence microscope. Scale bars represent: A, 5 μm; B, 5 mm; C & D, 300 μm; E–H, 150 μm.

Fig. 4

Survival of cells of Cladocopium infistulum (CI) after the cells were injected into the mesoglea of the medusa of the moon jellyfish Aurelia aurita. A non-injected cell of Cl (A). The package containing injected cells of CI inside the jellyfish body (yellow arrow) for 0 (B–D), 3 (E & F), and 7 days (G & H) after artificial injection. The red fluorescence in (D, F & H) indicates the survival of the microalgal species. Micrographs in (C, E & G) were taken under a light microscope and those in (D, F & H) under an epifluorescence microscope. Scale bars represent: A, 5 μm; B, 5 mm; C–H, 300 μm.

Fig. 5

Survival of cells of Symbiodinium microadriaticum (SM) after the cells were injected into the mesoglea of the medusa of the moon jellyfish Aurelia aurita. A non-injected cell of SM (A). The package containing injected cells of SM inside the jellyfish body (yellow arrow) for 0 (B–D), 3 (E & F), and 7 days (G & H) after artificial injection. The red fluorescence in (D, F & H) indicates the survival of the microalgal species. Micrographs in (C, E & G) were taken under a light microscope and those in (D, F & H) under an epifluorescence microscope. Scale bars represent: A, 5 μm; B, 5 mm; C–H, 300 μm.

Fig. 6

Survival of cells of Prorocentrum cordatum (PC) after the cells were injected into the mesoglea of the medusa of the moon jellyfish Aurelia aurita. A non-injected cell of PC (A). The package containing injected cells of PC inside the jellyfish body (yellow arrow) for 0 (B–D), 3 (E & F), and 7 days (G & H) after artificial injection. The red fluorescence in (D, F & H) indicates the survival of the microalgal species. Micrographs in (C, E & G) were taken under a light microscope and those in (D, F & H) under an epifluorescence microscope. Scale bars represent: A, 5 μm; B, 5 mm; C–F, 300 μm; G & H, 150 μm.

Fig. 7

Survival of cells of Prorocentrum koreanum (PK) after the cells were injected into the mesoglea of the medusa of the moon jellyfish Aurelia aurita. A non-injected cell of PK (A). The package containing injected cells of PK inside the jellyfish body (yellow arrow) for 0 (B–D), 3 (E & F), and 7 days (G & H) after artificial injection. The red fluorescence in (D, F & H) indicates the survival of the microalgal species. Micrographs in (C, E & G) were taken under a light microscope and those in (D, F & H) under an epifluorescence microscope. Scale bars represent: A, 5 μm; B, 5 mm; C & D, 300 μm; E–H, 150 μm.

Fig. 8

Survival of cells of Tetraselmis suecica (TS) after the cells were injected into the mesoglea of the medusa of the moon jellyfish Aurelia aurita. Non-injected cells of TS (A). The package containing injected cells of TS inside the jellyfish body (yellow arrow) for 0 (B–D), 3 (E & F), and 7 days (G & H) after artificial injection. The red fluorescence in (D, F & H) indicates the survival of the microalgal species. Micrographs in (C, E & G) were taken under a light microscope and those in (D, F & H) under an epifluorescence microscope. Scale bars represent: A, 5 μm; B, 5 mm; C–H, 300 μm.

Fig. 9

Survival of cells of Dunaliella salina (DS) after the cells were injected into the mesoglea of the medusa of the moon jellyfish Aurelia aurita. A non-injected cell of DS (A). The package containing injected cells of DS inside the jellyfish body (yellow arrow) for 0 (B–D), 3 (E & F), and 7 days (G & H) after artificial injection. The red fluorescence in (D, F & H) indicates the survival of the microalgal species. Micrographs in (C, E & G) were taken under a light microscope and those in (D, F & H) under an epifluorescence microscope. Scale bars represent: A, 5 μm; B, 5 mm; C & D, 300 μm; E–H, 150 μm.

Fig. 10

Survival of cells of Heterosigma akashiwo (HA) after the cells were injected into the mesoglea of the medusa of the moon jellyfish Aurelia aurita. A non-injected cell of HA (A). The package containing injected cells of HA inside the jellyfish body (yellow arrow) for 0 (B–D), 3 (E & F), and 7 days (G & H) after artificial injection. The red fluorescence in (D, F & H) indicates the survival of the microalgal species. Micrographs in (C, E & G) were taken under a light microscope and those in (D, F & H) under an epifluorescence microscope. Scale bars represent: A, 5 μm; B, 5 mm; C & D, 300 μm; E–H, 150 μm.

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.

Fig. 11

Survival of cells of Synechococcus sp. (SYN) after the cells were injected into the mesoglea of the medusa of the moon jellyfish Aurelia aurita. Non-injected cells of SYN (A & B). The package containing injected cells of SYN inside the jellyfish body (yellow arrow) for 0 (C–E), 1 (F & G), and 4 days (H & I) after artificial injection. The red fluorescence in (B, E, G & I) indicates the survival of the cyanobacterial species. Micrographs in (A, D, F & H) were taken under a light microscope and those in (B, E, G & I) under an epifluorescence microscope. Scale bars represent: A & B, 5 μm; C, 5 mm; D–I, 300 μm.

Survival of Effrenium voratum FL strain inside the medusa of the jellyfish after injection

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.

Fig. 12

Survival of cells of Effrenium voratum free-living strain for 60 days after the cells were injected into the mesoglea of the medusa of the moon jellyfish Aurelia aurita. The package containing injected cells inside the jellyfish body for 0 (A & B), 3 (C & D), 6 (E & F), 9 (G & H), 15 (I & J), 30 (K & L), 45 (M & N), and 60 days (O & P) after artificial injection. The red fluorescence indicates the survival of the microalgal species. Micrographs in (A, C, E, G, I, K, M & O) were taken under a light microscope and those in (B, D, F, H, J, L, N & P) under an epifluorescence microscope. Scale bars represent: A–P, 300 μm.

DISCUSSION

To the best of our knowledge, this study is the first to report the artificial injection of microalgae into the medusa of jellyfish using devices such as syringes or microinjectors. Previously, symbiotic algae were injected into the scyphistoma of jellyfish (Rahat and Adar 1980, Trench et al. 1981, Colley and Trench 1983, Fitt and Trench 1983). In many previous studies, microalgal cells were ingested by invertebrates to establish symbiosis, known as the infection method (Sachs and Wilcox 2006, Thornhill et al. 2006, Newkirk et al. 2018, Jinkerson et al. 2022, Mammone et al. 2023, Sharp et al. 2024).

In this infection method, only identified symbiotic microalgal and invertebrate species have been investigated. This study tested the survival of diverse microalgal, cyanobacterial, and invertebrate species without identified symbiosis, and all tested microalgal species survived during the study period. Furthermore, all experimental jellyfish survived. Therefore, the artificial microalgal injection method can be used to explore potential symbiosis between diverse microalgae, cyanobacteria, and invertebrate species.

Microalgal and cyanobacterial species survived inside cnidarians

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).

Table 4

Marine symbiotic microalgae and cyanobacteria with invertebrates in the phylum Cnidaria

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.

ACKNOWLEDGEMENTS

This research was supported by Korea Institute of Marine Science & Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries (MOF) (20230018) and the National Research Foundation (NRF) funded by the Ministry of Science and ICT (NRF-2021M3I6A1091272; 2021R1A2C1093379; RS-2023-00291696) award to HJJ.

Abbreviations

free-living strain

living-in-coral strain

NCMA

National Center for Marine Algae and Microbiota

Notes

The authors declare that they have no potential conflicts of interest.

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Type	Organism	Strain name	ESD (μm)	Isolation

				Location	Date	T (°C)	S
Bacterium	Synechococcus sp.	N54-2	1	East China Sea	Jul 2005	25.5	33.2
Prasinophyte	Tetraselmis suecica	CCMP904	10	Bembridge, UK	Jan 1959	-	-
Chlorophyte	Dunaliella salina	DSJH1710	10.3	Jinhae, Korea	Oct 2017	21.6	32.5
Raphidophyte	Heterosigma akashiwo	HAKS9905	11.5	Kunsan, Korea	May 1999	16.0	27.7
Dinoflagellate	Cladocopium infistulum	rt-203	8.6	Palau	-	-	-
Dinoflagellate	Symbiodinium microadriaticum	CCMP2464/rt-061	9.5	Florida Keys, USA	1977	-	-
Dinoflagellate	Prorocentrum cordatum	PMKS9906	12.1	Kunsan, Korea	Jun 1999	21.1	30.1
Dinoflagellate	Effrenium voratum (FL)	SvFL1	12.8	Jeju Island, Korea	May 2008	-	-
Dinoflagellate	Effrenium voratum (IC)	SvIC2	12.8	Jeju Island, Korea	Aug 2011	-	-
Dinoflagellate	Prorocentrum koreanum	PKMS1807	30	Masan, Korea	Jul 2018	31.3	28.7

Expt. No.	Class	Used marine animal	Used microorganism	Abundance (total of cells) of the microorganism injected to the animal
Expt 1	Scyphozoa	Aurelia aurita	Synechococcus sp.	20,000,000
			Tetraselmis suecica	1,121,600
			Dunaliella salina	1,016,000
			Heterosigma akashiwo	1,016,000
			Cladocopium infistulum	1,032,000
			Symbiodinium microadriaticum	1,240,000
			Prorocentrum cordatum	1,036,667
			Effrenium voratum (FL)	1,112,000
			Effrenium voratum (IC)	1,217,000
			Prorocentrum koreanum	302,000
Expt 2	Scyphozoa	Aurelia aurita	Effrenium voratum (FL)	1,112,000–1,500,000

Used animal			Microorganism injected into the animal	ESD (μm)	Microorganism surviving in the animal for ≥ 7 d	Animal surviving for ≥ 7 d

Phylum	Class	Species
Cnidaria	Scyphozoa	Aurelia aurita	Synechococcus sp. (Bac)	1	×	○
			Tetraselmis suecica (Pra)	10	○	○
			Dunaliella salina (Chl)	10.3	○	○
			Heterosigma akashiwo (Rap)	11.5	○	○
			Cladocopium infistulum (Din)	8.6	○	○
			Symbiodinium microadriaticum (Din)	9.5	○	○
			Prorocentrum cordatum (Din)	12.1	○	○
			Effrenium voratum (FL) (Din)	12.8	○	○
			Effrenium voratum (IC) (Din)	12.8	○	○
			Prorocentrum koreanum (Din)	30	○	○

Type	Microorganism	Symbiotic relationship		Reference

		Class	Animal
Cyanobacteria	The families Aphanothecaceae, Chroococcaceae, Chroococcidiopsidaceae, Calotrichaceae, Gloeotrichiaceae, Hapalosiphonaceae, Chlorogloeopsidaceae, Tolypothrichaceae, Nostocaceae, Cyanothecaceae, Microcoleaceae, Hyellaceae, Oscillatoriaceae, Prochloraceae, Synechococcaceae	-	Coral	Mutalipassi et al. (2021)
Dinoflagellate	Breviolum sp./spp.	Hydrozoa	Fire coral Millepora complanata	LaJeunesse (2002)
	Breviolum sp./spp., Cladocopium sp./spp.	Octocorallia	Sea fan order Gorgonacea
	Breviolum sp./spp., Cladocopium sp./spp., Durusdinium sp./spp., Symbiodinium sp./spp.	Hexacorallia	Coral order Scleractinia
	Breviolum sp./spp., Cladocopium sp./spp., Symbiodinium sp./spp.	Hexacorallia	Sea anemone order Actiniaria
		Hexacorallia	Coral order Zoantharia
	Breviolum sp./spp., Symbiodinium sp./spp.	Hydrozoa	Fire coral Millepora alcicornis
	Cladocopium sp./spp.	Hexacorallia	Coral order Corallimorpharia
	Cladocopium sp./spp., Symbiodinium linucheae	Scyphozoa	Jellyfish Linuche unguiculata	Trench and Thinh (1995), LaJeunesse (2002)
	Cladocopium sp./spp., Symbiodinium microadriaticum	Scyphozoa	Jellyfish Cassiopea sp., Ca. xamachana	Freudenthal (1962), LaJeunesse (2002)
	Gloeodinium viscum	Hydrozoa	Fire coral Millepora dichotoma	Banaszak et al. (1993)
	Scrippsiella velellae	Hydrozoa	Sea raft Velella velella	Banaszak et al. (1993)
	Symbiodinium sp./spp.	Hydrozoa	Fire coral Myrionema ambionense	LaJeunesse (2002)
Diatom	Amphora spp., Cocconeis neothumensis var. marina, Co. scutellum, Co. stauroneiformis, Licmophora flabellata, L. oedipus	Hydrozoa	Hydroid Eudendrium racemosum	Di Camillo et al. (2005)
	Cocconeis notata, Co. pseudonotata	Hydrozoa	Hydroid Campanularia hincksii
	Cocconeis dirupta, Co. pseudonotata, Cylindrotheca sp.	Hydrozoa	Hydroid Clytia linearis
	Cocconeis neothumensis var. marina, Co. lineata, Navicula sp.	Hydrozoa	Hydroid Synthecium evansi
Flagellate	Unidentified green chlorophytes	Hexacorallia	Sea anemones Anthopleura elegantissima, A. xanthogrammica	O’Brien and Wyttenbach (1980), Lewis and Muller-Parker (2004)