INTRODUCTION
Marine microalgae are ubiquitous and major components of marine ecosystems (O’Halloran et al. 2006, Kudela and Gobler 2012, Jeong et al. 2021, Kang et al. 2023b, Ok et al. 2023a, Park et al. 2024a). They fuel marine food webs and interact with diverse groups, such as zooplankton, fish, and invertebrates, in marine ecosystems (Burkholder et al. 1995, LaJeunesse 2002, Rocha et al. 2008, Jeong et al. 2010, Kang et al. 2023a, Ok et al. 2023b, You et al. 2023, Park et al. 2024b). Some marine invertebrates form mutualistic symbioses with specific microalgae (Trench and Thinh 1995, Lee et al. 2001, LaJeunesse 2002, Burghardt et al. 2008). Through these symbioses, invertebrates obtain photosynthetic products or accumulate protective materials within their bodies while providing nutrients and habitats, to their symbiotic partners (Arillo et al. 1993, LaJeunesse 2002, Yellowlees et al. 2008, Rädecker et al. 2018). Moreover, invertebrates and microalgae can survive in various unfavorable environments because of their symbiotic partners (Berkelmans and Van Oppen 2006, LaJeunesse et al. 2018). For example, the adults of the hard coral Acropora millepora can acquire their increased thermal tolerance in stressful environments by shifting the dominant symbiotic dinoflagellate group in their tissues from Cladocopium spp. to Durusdinium spp. (Berkelmans and Van Oppen 2006). However, these symbiotic relationships can be disrupted by extreme environmental changes that can severely affect invertebrate survival, such as the bleaching of corals and giant clams (Glynn 1991, Brown 1997, Leggat et al. 2003).
Some mollusks form symbiotic relationships with microalgae, mainly with the dinoflagellates of the family Symbiodiniaceae (Kempf 1991, McFarland and Muller-Parker 1993, Farmer et al. 2001, LaJeunesse 2002, Burghardt et al. 2005, 2008). In addition, the dinoflagellate cells introduced into the host body may reside either intracellularly or extracellularly within the host mollusk. The nudibranchs Phyllodesmium spp., Pteraeolidia spp., and Melibe engeli in the suborder Cladobranchia are known to form intracellular symbioses with the dinoflagellate species in the genera Breviolum, Cladocopium, Durusdinium, and Symbiodinium (Loh et al. 2006, Burghardt et al. 2008, Ahmadian et al. 2016, LaJeunesse et al. 2018, Mizobata et al. 2023). Meanwhile, the giant clams Tridacna spp. and Corculum cardissa form extracellular symbioses with those in the genera Cladocopium and Symbiodinium (Farmer et al. 2001, Lee et al. 2020). Moreover, the sea snail Aliger gigas forms intracellular or extracellular symbioses with the species in the genera Breviolum, Cladocopium, and Symbiodinium (LaJeunesse 2002, Banaszak et al. 2013). Considering the symbiosis between microalgae and invertebrates, which includes both intracellular and extracellular forms, two critical questions arise: (1) do other microalgal species not engage in symbiosis with invertebrates?; and (2) do other invertebrate species not form symbioses with microalgae? You et al. (2024) answered the first question by reporting that the dinoflagellates Prorocentrum cordatum and Prorocentrum koreanum, the prasinophyte Tetraselmis suecica, the chlorophyte Dunaliella salina, and the raphidophyte Heterosigma akashiwo were able to survive inside the body of the medusa of the non-symbiotic jellyfish Aurelia aurita. Additionally, a free-living (FL) strain of Effrenium voratum survived within the medusa for 2 months, indicating the potential for artificially established symbiosis, although the precise localization (e.g., intracellular or extracellular) was not confirmed (You et al. 2024). To address the second question, it is necessary to investigate which invertebrates, whose symbioses with microalgae are not known, can support the survival of microalgae when transplanted into their bodies.
To investigate the symbioses of marine invertebrates in previous studies, either the infection or injection method has been used (Stevenson and South 1975, Colley and Trench 1983, McFarland and Muller-Parker 1993, Sachs and Wilcox 2006, You et al. 2024). The former, in which microalgae are naturally introduced into the body of invertebrates through the feeding of target animals, is widely used when the target animal is known to form a symbiotic relationship with certain microalgae (McFarland and Muller-Parker 1993, Sachs and Wilcox 2006). In contrast, the latter method, in which microalgae are artificially injected into the body of invertebrates using microinjectors, syringes, or pipettes, is relatively less commonly used and is usually used when the target animal feeds very slowly or when the symbiotic relationship of the target animal is unknown (Stevenson and South 1975, Colley and Trench 1983, You et al. 2024). Recently, You et al. (2024) used an injection method to artificially introduce microalgae or cyanobacteria into the medusa of the jellyfish A. aurita using syringes to investigate the potential of forming symbiotic relationships between diverse microalgae and the cnidarian. Thus, the injection method could be useful for identifying potential symbioses in marine invertebrates whose associations have not yet been discovered. However, it should be noted that the injection method does not necessarily allow for a precise determination of whether the resulting association is intracellular or extracellular.
In this study, we artificially injected the FL strain of the dinoflagellate E. voratum into seven mollusk species: the cephalopod Octopus minor and gastropods Chromodoris orientalis, Dendrodoris fumata, Nassarius sp., Tectus fenestratus, Babylonia spirata, and Haliotis discus hannai. The survival of the experimental mollusks was monitored every day for 7 d after the injections, and that of E. voratum (FL) was monitored after 7 d. Furthermore, the survival of the transplanted E. voratum (FL) and the octopus O. minor and nudibranch C. orientalis was monitored for 17–25 d until the mollusks died. The results in this study provide insights into the potential artificial symbioses of mollusks with microalgae, based on the observed persistence of algal cells within the host bodies. In the present study, endosymbiosis was not claimed or demonstrated; rather, the initial survival and retention of microalgae within the host body were investigated as a preliminary indicator of physiological compatibility.
MATERIALS AND METHODS
Preparation of experimental organisms
The adults of the octopus O. minor and abalone H. discus hannai were obtained from the Noryangjin Fisheries Wholesale Market (Seoul, Korea) and Mokpo Fish Market (Mokpo, Korea), respectively (Table 1). They were transported with oxygenation to a laboratory at Seoul National University and then maintained in two 27-L acrylic water tanks with a filtration system (BioPlus 100; Oase Living Water Co. Ltd., Hörstel, Germany) containing 0.5-μm-filtered seawater (FSW). The tanks were illuminated with a light intensity of 20 μmol photons m−2 s−1 from light-emitting diode (LED) lamps with a 14 : 10 h light : dark cycle and maintained at 10°C in a walled chamber. The adults of the nudibranch C. orientalis were obtained from the Cheonghae Industry aquarium (Jeju Island, Korea); those of the nudibranch D. fumata were obtained from the Parangbada aquarium (Tongyoung, Korea); and those of the sea snails Nassarius sp., T. fenestratus, and B. spirata were obtained from the Dongmulnara Aquarium (Anyang, Korea). They were transported to the laboratory and maintained in six 15-L glass water tanks, each equipped with a mechanical filter (Tetra, Melle, Germany) containing 0.5-μm-FSW (Table 1). These tanks were maintained under the same light intensity and photoperiod conditions, as mentioned above, at 25°C in a walled chamber. These animals were used in the experiments 1–2 d after they were brought to the laboratory.
A clonal culture of the dinoflagellate E. voratum (FL) (SvFL1) was isolated from waters off Jeju Island (Jeong et al. 2012). The cells of E. voratum (FL) were transferred monthly into 250-mL flat culture flasks (Corning Inc., Corning, NY, USA) containing freshly prepared f/2-Si medium (Guillard and Ryther 1962) and maintained under 20–50 μmol photons m−2 s−1 illumination using an LED lamp under a 14 : 10 h light : dark cycle at 20°C (Table 1).
Injection of Effrenium voratum (FL) into seven mollusk species
Through Experiment 1 (control n = 1, treatment n = 1), we aimed to determine whether E. voratum (FL) can survive inside the body of each mollusk species after being injected into the mollusk bodies (Table 2). The injection of E. voratum (FL) into mollusks was performed using the method described by You et al. (2024).
To inject the dense cells of E. voratum (FL), 100-mL aliquots of an E. voratum (FL) culture were transferred into two 50-mL Falcon tubes (Corning Inc.) and centrifuged at 2,063 ×g for 10 min (Labogene 1696R; Gyrozen Co., Gimpo, Korea). The pellets obtained through centrifugation were resuspended in 0.5 or 1 mL of 0.2-μm-FSW to facilitate effective injection into the organisms. A 30-μL aliquot of the re-suspended solutions of E. voratum (FL) was transferred to 1-mL (U-100) insulin syringes (31G, 8 mm; Angel Syringe; Yong Chang Co. Ltd., Gimpo, Korea), and 30 μL from the syringe was injected into an individual of each of the sea snails Nassarius sp., T. fenestratus, and B. spirata, while a 40-μL aliquot of the solutions was injected into each nudibranch, C. orientalis, and D. fumata, and the abalone H. discus hannai, and 90 μL into the octopus O. minor (Table 2). One individual from each mollusk species, not injected with E. voratum (FL), was used as a control. Each experimental and control mollusk was individually transferred to fourteen 15-L glass water tanks and maintained for over 7 d under the same conditions, as described above. The survival of the experimental mollusks and the changes in the morphology of the site where E. voratum (FL) cells were present within their bodies were monitored daily with the naked eye. Seven days after E. voratum (FL) was injected into each experimental mollusk, or when the experimental mollusks died, E. voratum (FL) cells were retrieved from the presence sites. The viability of the transplanted E. voratum (FL) cells was determined by fluorescence analysis using an epifluorescence microscope (EVOS M5000; Thermo Fisher Scientific, Waltham, MA, USA).
Experiment 2 (control n = 1, treatment n = 2) was designed to investigate whether the injected E. voratum (FL) cells were alive or dead when the nudibranch C. orientalis and octopus O. minor injected with E. voratum (FL) died naturally (Table 2). The preparation and injection of E. voratum (FL) into the nudibranch and octopus were conducted as described above. Two experimental individuals of the nudibranchs (or octopuses) and one individual as a control were set up in several 15-L glass water tanks. Survival of the experimental nudibranch and octopus and changes in the shape of the presence site of E. voratum (FL) within their bodies were monitored with the naked eye until the experimental mollusks died naturally. After the death of the experimental organisms, E. voratum (FL) cells were retrieved from the presence sites, and whether the transplanted E. voratum (FL) cells were alive or dead was determined by fluorescence analysis using an epifluorescence microscope.
To determine the abundance of E. voratum (FL) cells before the centrifugation step, a 5-mL aliquot was taken from the E. voratum (FL) culture, and the cells were fixed with a 5% acidic Lugol’s solution. The fixed E. voratum (FL) cells were enumerated using three 1-mL Sedgewick–Rafter chambers under an inverted microscope (CX21; Olympus Corporation, Tokyo, Japan) at 100× magnification. During the experimental period, 40–50% of the total water volume in the water tanks was replaced with fresh FSW twice a week, and no prey items were added.
RESULTS
Survival of the transplanted Effrenium voratum cells and host mollusks for 7 d after transplantation
All mollusks, except H. discus hannai, survived for at least 7 d after the transplantation; however, H. discus hannai died on day 3. Furthermore, the transplanted E. voratum (FL) cells survived for at least 7 d inside the bodies of all mollusks, except the abalone H. discus hannai.
The nudibranchs C. orientalis and D. fumata survived the 7-d observation period after the injection of E. voratum (FL) cells and were subsequently sacrificed and dissected for further analysis (Figs 1 & 2). Moreover, the transplanted E. voratum (FL) cells retrieved from inside the bodies of C. orientalis and D. fumata emitted red fluorescence even 7 d after their artificial injection into the bodies of the nudibranchs, indicating that the microalgal cells remained detectable/alive within the nudibranch body during the study period (Table 3, Figs 1 & 2).
In addition, the sea snails Nassarius sp., T. fenestratus, and B. spirata survived the 7-d observation period after the injection of E. voratum (FL) cells and were subsequently sacrificed and dissected for further analysis (Figs 3–5). The transplanted E. voratum (FL) cells retrieved from inside the bodies of Nassarius sp., T. fenestratus, and B. spirata also emitted red fluorescence even 7 d after their artificial injection into the bodies of the sea snails, indicating that the microalgal cells remained detectable/alive within the body of the sea snail during the study period (Table 3, Figs 3–5).
Similarly, the octopus O. minor survived the 7-d observation period after the injection of E. voratum (FL) cells; subsequently, it was also sacrificed and dissected for further analysis (Fig. 6). As observed with nudibranchs and sea snails, in this case also, the transplanted E. voratum (FL) cells retrieved from inside the body of O. minor emitted red fluorescence even 7 d after their artificial injection into the body of the octopus, indicating that the microalgal cells remained detectable/alive within the octopus body during the study period (Table 3, Fig. 6).
However, the abalone H. discus hannai died 3 d after E. voratum (FL) cells were injected into its body. Moreover, the transplanted E. voratum (FL) cells also did not survive inside the body of H. discus hannai, as red fluorescence from these cells was not detected on day 3 after their transplantation, indicating that they died within 3 d after their artificial injection into the body of the abalone (Table 3).
Survival of the transplanted Effrenium voratum (FL) cells inside the bodies of the nudibranch Chromodoris orientalis and octopus Octopus minor for a long time after their injection
Two individuals of the experimental nudibranch C. orientalis injected with E. voratum (FL) cells survived for up to 23 and 24 d but died on days 24 and 25, respectively, after the injections (Table 4). The control nudibranch survived for up to 25 d but died on day 26. Based on visual observations during the experimental period, the morphology of the site where E. voratum (FL) cells were injected into the bodies of the experimental nudibranchs gradually changed from a thinly spread form to a condensed circular form (Fig. 7A–D). The E. voratum (FL) cells retrieved from inside the bodies of the two C. orientalis individuals after the natural death of the experimental nudibranchs on days 24 and 25 emitted red fluorescence, indicating that the microalgal cells remained detectable/alive within the nudibranch body (Fig. 7E & F).
In addition, two individuals of the experimental octopus O. minor injected with E. voratum (FL) cells survived for up to 13 and 16 d but died on days 14 and 17, respectively, after the injections (Table 4). The control octopus survived for up to 15 d but died on day 16. Based on visual observations during the experimental period, the morphology of the site where E. voratum (FL) cells were injected into the bodies of the experimental octopuses gradually changed from a thin spread to a condensed form (Fig. 8A–C). The cells of E. voratum (FL) retrieved from the body of O. minor after the natural death of the experimental octopuses on days 14 and 17 emitted red fluorescence, indicating that the microalgal cells remained detectable/alive within the octopus body (Fig. 8D & E).
DISCUSSION
Survival of the transplanted Effrenium voratum cells and host mollusks for 7 d after transplantation
Prior to the present study, the potential symbiosis between marine mollusks and microalgae had rarely been investigated using the artificial symbiosis method. The results of the present study revealed that six mollusk species of diverse taxonomic orders and the microalga E. voratum transplanted within the bodies of these mollusks survived for at least 7 d after the injection. This suggests physiological compatibility between these mollusks and the microalga, although the present study does not provide histological evidence to determine whether the association is extracellular or intracellular. However, in the case of the abalone H. discus hannai, both the mollusk host and the microalgal cells transplanted within its body died 3 d after the transplantation. The symbioses of these seven mollusk species with microalgae have not yet been reported. Therefore, this method used in the present study could be useful for determining potential symbiotic compatibility between microalgae and marine mollusks, whose symbioses have not yet been determined. In non-symbiotic hosts, injection of microorganisms into the digestive tract may lead to rapid digestion and elimination of the algae, thereby precluding the opportunity to observe physiological compatibility. In the present study, E. voratum cells were injected into the muscular layer under the epidermis of mollusks to avoid the digestive tract as much as possible, however, precise localization was not confirmed. This experimental design was intended to assess whether microalgae could survive and persist in mollusk species not previously known to form algal symbioses. An exploratory approach was therefore adopted to evaluate algal survival within the general internal environment of the host.
The sea slugs of the order Nudibranchia are classified into the suborders Doridina and Cladobranchia, and intracellular symbiotic relationships with microalgae have only been reported for species belonging to the suborder Cladobranchia (Kempf 1991, McFarland and Muller-Parker 1993, Burghardt et al. 2005, 2008). The two nudibranchs C. orientalis and D. fumata used in this study belong to the suborder Doridina. To our knowledge, no symbiotic relationships with microalgae have been reported for C. orientalis or D. fumata. Furthermore, no symbiotic relationships with microalgae have been reported for the three sea snails Nassarius sp., T. fenestratus, or B. spirata used in the present study. In addition, no symbiotic relationships are known to exist between microalgae and the octopus O. minor used in the present study, although O. minor has been reported to engage in symbiotic relationships with a few mesozoans and ciliates (Furuya et al. 2004). The observed persistence of the injected alga within these mollusk species suggests a physiological compatibility between these mollusks and microalgae. However, further evidence is needed to confirm true symbiosis.
The observed persistence of algal cells in multiple mollusk species may not simply result from passive entrapment caused by anatomical constraints, because mollusks possess diverse immune responses―including phagocytosis for small particles and encapsulation, melanization, and oxidative burst for larger ones―in both open and closed circulatory systems (Gliński and Jarosz 1997, Reiber and McGaw 2009). However, further studies are needed to determine whether such persistence of algal cells in mollusks represents a true symbiosis.
In the present study, the sample sizes were relatively small. It may limit the statistical power and generalization of the findings. In addition, survival of algae was inferred based on the detection of chlorophyll fluorescence, which may persist even after algal cell death; thus, the possibility of false positives cannot be fully excluded.
Survival of the transplanted Effrenium voratum (FL) cells inside the bodies of the nudibranch Chromodoris orientalis and octopus Octopus minor for a long time after their injection
The experimental individuals of the nudibranch C. orientalis and octopus O. minor injected with E. voratum (FL) survived for 23–24 and 13–16 d after injection, respectively, whereas their respective control individuals survived for 25 and 15 d, respectively. Therefore, starvation was partially responsible for the death of the experimental and control individuals of both mollusk species, and persistence of E. voratum (FL) within the host body may not support the survival of both mollusk species.
In the present study, the duration for which E. voratum (FL) remained (or survived) within C. orientalis was 24 d. In previous studies, the duration for which microalgae remained (or survived) within the host, referred to as the retention time, varied depending on the species (Kempf 1991, McFarland and Muller-Parker 1993, Burghardt et al. 2005, 2008, Burghardt and Wägele 2014, Monteiro et al. 2019, Silva et al. 2021). For example, it has been reported that the cladobranchs M. engeli and Pteraeolidia ianthina could retain one of the Symbiodinium spp. for over 200 d under starvation (Burghardt et al. 2005, 2008, Burghardt and Wägele 2014). Furthermore, the cladobranchs Phyllodesmium longicirrum, Ph. colemani, and Ph. briareum have been reported to stay in symbioses with one of the Symbiodinium spp. for over 70–100 d (Table 5) (Burghardt et al. 2005, 2008, Burghardt and Wägele 2014). In these studies, the symbiosis durations for these cladobranchs have been reported to be limited by experimental endpoints rather than the host death or the loss of the symbionts, suggesting that actual retention periods may be even longer under natural conditions. In contrast, the cladobranch Berghia stephanieae retained microalgae for up to 14 d, whereas the cladobranchs Be. verrucicornis and Aeolidia papillosa exhibited the maximum microalgal retention times of only 6 d (Table 5) (Kempf 1991, McFarland and Muller-Parker 1993, Monteiro et al. 2019, Silva et al. 2021). These relatively short retention durations result from either host death or the complete disappearance of symbionts, indicating that such limited retention durations may also be representative of natural conditions. Therefore, the retention time between E. voratum (FL) and C. orientalis was shorter than that between one of Symbiodinium spp. and M. engeli, Pt. ianthina, Ph. longicirrum, Ph. colemani, or Ph. briareum but longer than that between microalgae and Be. stephanieae, Be. verrucicornis, and A. papillosa. This result falls within the known range of retention durations in nudibranchs and may reflect physiological tolerance in C. orientalis, although the underlying symbiotic mechanism may differ (e.g., extracellular vs. intracellular localization).
These interspecific differences in retention times are likely influenced by differences in gene expression associated with symbiosis. For establishing stable symbiosis, the host must recognize the symbiont and suppress phagosome maturation and immune responses to enable their intracellular maintenance (Davy et al. 2012, Chan et al. 2018, Clavijo et al. 2020, 2022). For instance, transcriptomic analyses have revealed that despite recognizing algal symbionts, Be. stephanieae fails to sufficiently downregulate the immune response against the dinoflagellate Breviolum minutum or inhibit phagosome maturation, leading to an unstable symbiosis (Clavijo et al. 2022). Therefore, Clavijo et al. (2022) suggested that Be. stephanieae and Symbiodinium spp. may be in a transitional phase toward stable photosymbiosis. Furthermore, evolutionary perspectives suggest that symbiotic capabilities may be closely linked to the nudibranch phylogeny (Wollscheid-Lengeling et al. 2001, Moore and Gosliner 2011, Rola et al. 2022). However, Rola et al. (2022) suggested that it remains unclear whether stable symbiosis evolved gradually from unstable symbiosis or whether both states evolved independently through convergent pathways. Future research investigating the gene expression patterns in the dorid nudibranch C. orientalis injected with E. voratum (FL) is needed.
There have been no reports on the natural symbiotic relationships between octopuses and microalgae, but the present study indicated the persistence of the injected algae within O. minor until the death of the host. This finding suggests that short-term retention of microalgae by a complex cephalopod host is physiologically possible, even though the cephalopod host has a well-developed immune system and high metabolic activity (reviewed in Gestal and Castellanos-Martínez 2015).
In natural environments, symbiotic microalgae provide photosynthetic products and useful materials to their invertebrate partners. Therefore, artificial symbiosis by transplanting microalgae into invertebrates could provide the materials necessary for their survival and growth.
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