Introduction
Marine benthic invertebrates are frequently subjected to injury, affecting approximately 33–47% of them (Lindsay Reference Lindsay2010). Several authors have reviewed various aspects of regeneration at multiple levels of biological organization in different life stages of marine invertebrates (Allen et al. Reference Allen, Ross and Zayas2016; Bely and Nyberg Reference Bely and Nyberg2010; Fujita et al. Reference Fujita, Kuranaga and Nakajima2021; Lanna Reference Lanna2015; Leclère et al. Reference Leclère, Copley, Momose and Houliston2016; Lindsay Reference Lindsay2010), laying the groundwork for broader research on animal regeneration. In metazoans, self-repair and regeneration are essential for tissue maintenance and wound healing (Alvarado Reference Alvarado2000; Sinigaglia et al. Reference Sinigaglia, Peron, Eichelbrenner, Chevalier, Steger, Barreau, Houliston and Leclère2020). Among the animal phyla, planarians and cnidarian polyps have been the most studied for regeneration because of their remarkable regeneration capacities (Gold and Jacobs Reference Gold and Jacobs2013; Laurie-Lesh and Corriel Reference Laurie-Lesh and Corriel1973). The phylum Cnidaria exhibits remarkable regenerative capabilities at various stages of its life cycle, particularly during polyp and medusa stages (Agata and Inoue Reference Agata and Inoue2012; Fujita et al. Reference Fujita, Kuranaga and Nakajima2021; Galliot and Schmid Reference Galliot and Schmid2002). In general, polyps have demonstrated the ability to regenerate radial symmetry and functional structures from fragments (Curtis and Cowden Reference Curtis and Cowden1974). Researchers have extensively studied Hydra (Hydrozoa), a cnidarian known for its ability to regenerate an entire polyp from a small piece of tissue, or even a few cells (Bode Reference Bode2003; Holstein et al. Reference Holstein, Hobmayer and Technau2003). This regenerative capacity is attributed to the organized regulation of pluripotency and progressive cell differentiation observed in cnidarians (Alvarado Reference Alvarado2000). Because of this exceptional regenerative capability, Hydra has become a valuable model organism for studying hydrozoan regeneration, particularly in stem cell research, to better understand tissue organization and differentiation during medusa regeneration (Bode Reference Bode2003; Gold and Jacobs Reference Gold and Jacobs2013; Mills Reference Mills1993; Young Reference Young1974). The anthozoan Nematostella is a captivating example because of its extraordinary ability to regenerate its entire body from amputated polyp tissue (Bossert et al. Reference Bossert, Dunn and Thomsen2013; Darling et al. Reference Darling, Reitzel, Burton, Mazza, Ryan, Sullivan and Finnerty2005). Notable cnidarian model organisms include Hydra, Hydractinia, and Nematostella (Fujita et al. Reference Fujita, Kuranaga and Nakajima2021).
Furthermore, jellyfish species offer a unique model for exploring common principles and diverse mechanisms underlying complex organ regeneration. The clades within Hydrozoa, Scyphozoa, and Cubozoa exhibit notable regenerative ability (Gamero-Mora et al. Reference Gamero-Mora, Halbauer, Bartsch, Stampar and Morandini2019; Stamatis et al. Reference Stamatis, Worsaae and Garm2018). Cubozoan polyps regrow from small clusters of cells following metamorphosis (Stamatis et al. Reference Stamatis, Worsaae and Garm2018). Although Hydrozoa and Scyphozoa share the same regeneration morphogenesis, they differ in their stem cell dynamics (Straehler-Pohl and Jarms Reference Straehler-Pohl and Jarms2005). Notably, in Aurelia, the epithelial tissues of isolated polyps return to their original form and function as medusa (Fuchs et al. Reference Fuchs, Wang, Graspeuntner, Li, Insua, Herbst, Dirksen, Böhm, Hemmrich, Sommer and Domazet-Lošo2014; Loveridge et al. Reference Loveridge, Lucas and Ford2024). Compared with polyps, medusa, which have a complex morphology, are capable of regenerating organs and reconstructing their entire body from fragments (Sinigaglia et al. Reference Sinigaglia, Peron, Eichelbrenner, Chevalier, Steger, Barreau, Houliston and Leclère2020). A remarkable example is the immortal hydrozoan jellyfish Turritopsis dohrnii, which exhibits reverse development, wherein adult medusa can transform into cysts and revert to polyps (Piraino et al. Reference Piraino, Boero, Aeschbach and Schmid1996). In vitro studies have shown that differentiated jellyfish cells can alter their cellular commitment, thereby enabling the production of new cell types (Piraino et al. Reference Piraino, De Vito, Schmich, Bouillon and Boero2004; Schmid Reference Schmid1992). However, studies on scyphozoan jellyfish regeneration are scarce and there is a lack of evidence regarding wound healing (He et al. Reference He, Zheng, Zhang and Lin2015). Some hydrozoan species from the genera Aequorea, Clytia, and Gastroblasta have been reported to regenerate the medusa (Russell Reference Russell1953; Stretch and King Reference Stretch and King1980). Scyphozoa species of the genera Chrysaora, Cassiopea, and Aurelia also exhibit regeneration and return to their original shapes (Black and Riley Reference Black and Riley1985; Curtis and Cowden Reference Curtis and Cowden1974; Kroiher et al. Reference Kroiher, Siefker and Berking2000; Steinberg Reference Steinberg1963). Researchers have documented structural reorganization during the regeneration and self-repair stages, particularly when cell differentiation is more pronounced, as observed in ephyra (Abrams et al. Reference Abrams, Basinger, Yuan, Guo and Goentoro2015; Silveira et al. Reference Silveira, Jarms and Morandini2002; Stockard and Beebe Reference Stockard and Beebe1910). Reports on medusa-stage regeneration include the replacement of body structures such as oral arms and appendages, with regeneration accelerated in the presence of sensory organs (Bossert et al. Reference Bossert, Dunn and Thomsen2013).
The upside-down jellyfish Cassiopea (Family: Cassiopeidae) occurs in both tropical and subtropical coastal ecosystems and is known for its unique benthic lifestyle, symbiotic relationship with photosynthetic dinoflagellates (Gamero-Mora et al. Reference Gamero-Mora, Collins, Boco, Geson, Morandini and Wilson2022; Maggio et al. Reference Maggio, Allegra, Bosch-Belmar, Cillari, Cuttitta, Falautano, Milisenda, Nicosia, Perzia, Sinopoli and Castriota2019; Ohdera et al. Reference Ohdera, Abrams, Ames, Baker, Suescún-Bolívar, Collins, Freeman, Gamero-Mora, Goulet, Hofmann and Jaimes-Becerra2018), and phenotypic plasticity (Anthony et al. Reference Anthony, Heagy and Bentlage2022; Salas et al. Reference Salas, Anthony and Bentlage2024). Along the Indian coast, medusa of Cassiopea species have been recorded mainly in shallow and calm environments (Browne Reference Browne1916; CMFRI 2018; Gopalakrishnan Reference Gopalakrishnan1970; Haldar and Chowdhury Reference Haldar and Chowdhury1995; Menon Reference Menon1930, Reference Menon1936; Prasade et al. Reference Prasade, Nagale and Apte2016; Ramakrishna and Sarkar Reference Ramakrishna and Sarkar2003; Ranjith et al. Reference Ranjith, Saravanan, Ramkumar and Behera2014; Rao Reference Rao1931; Venkataraman et al. Reference Venkataraman, Raghunathan, Raghuraman and Sreeraj2012). All earlier reports in the study region involved Cassiopea andromeda, except Rao (Reference Rao1931), who reported C. frondosa in the backwater muddy tidal pool of Visakhapatnam. However, the occurrence of this species in the region remains unclear. Consequently, there is a lack of clarity regarding the identity of Cassiopea; confounded, unreproducible, or less comparable experimental results may occur because of incorrect species documentation (Muffett and Miglietta Reference Muffett and Miglietta2023).
Although various researchers have observed the processes of self-repair and regeneration in scyphozoans, research on tissue regeneration in Cassiopea remains limited in scope and not as extensive as in other genera. Cassiopea exhibits high regenerative capacity, with documented cases of complete body structure regeneration in polyps after strobilation or injury (Neumann Reference Neumann1977; Zeleny Reference Zeleny1907). Although Cassiopea species exhibit reduced regenerative potential during the medusa stage, they can still regenerate various missing structures (Hofmann et al. Reference Hofmann, Fitt and Fleck1996; Stockard Reference Stockard1908). Studies have demonstrated that marginal sense organs play a key role in the rate of regeneration in C. xamachana (Cary Reference Cary1916). Although many studies have highlighted the remarkable regenerative ability of Cassiopea species (Cary Reference Cary1916; Stockard Reference Stockard1908; Zeleny Reference Zeleny1907), only a few studies have suggested that umbrella tissue can fully regenerate into medusae (Bossert et al. Reference Bossert, Dunn and Thomsen2013; Fujita et al. Reference Fujita, Kuranaga and Nakajima2021). Following injury or amputation, medusa umbrella tissue has the potential to regenerate entire body structures (Ostendarp et al. Reference Ostendarp, Plewka, Flathmann, Tilstra, El-Khaled and Wild2022). This study aimed to elucidate the self-repair and regenerative potential of the upside-down jellyfish C. xamachana under captive conditions, with a specific focus on the regeneration of umbrella tissue and amputated medusa with umbrella tissue and oral arms.
Materials and methods
Experimental animals and maintenance
The young experimental medusae, 75 ± 15 days old, were developed from polyp cultures and ephyra were maintained in the Cnidarian Laboratory of the ICAR-Tuticorin Regional Station of the Central Marine Fisheries Research Institute (CMFRI), India. The original polyps were collected during field surveys along the Gulf of Mannar, southeast coast of India, and maintained in the laboratory to produce ephyra and young medusa (Figure 1a). A total of nine experimental jellyfish (5.8 ± 0.4 cm), comprising the control group (n = 6), an injured medusa that was accidentally torn into four unequal fragments when it was unintentionally sucked into the suction pump housed in the aquarium tank (Figure 1b); a single medusa that was radially amputated into four equal fragments, were maintained individually in different glass aquaria (20 × 20 × 20 cm) under optimum seawater conditions (salinity, 33 ± 2 ppt; water temperature, 30 ± 2°C; light intensity, 60 ± 20 photosynthetically active radiation (PAR); photoperiod, 18 L/6 D) without any substrate for one week during June 2021 and another medusa was amputated similarly during July 2021 and maintained for four weeks. Initial observations focused on a single accidentally injured medusa, which was divided into four parts: an umbrella with four oral arms (UTWA), an umbrella tissue without oral arms (UT), and two separate oral arms without umbrella tissue (OAs). The injured portions were monitored for 3 months, from January to April 2021, and morphological changes were recorded. In the amputation experiment, a scalpel blade was used to cut the medusa radially through the umbrella, without damaging the oral arms, resulting in four equal fragments from each medusa. The first four amputated jellyfish fragments (FAF 1, FAF 2, FAF 3, and FAF 4) were classified as radially cut medusa fragments, each possessing oral arms and umbrella tissue, and the second amputated fragments from the repeated experiments were designated as SAF 1, SAF 2, SAF 3, and SAF 4. Table 1 details the Cassiopea medusa, fragments used for the experiments, broader characteristics, and their respective monitoring periods as follow-up experiments. The medusa/fragments were maintained in glass aquaria with optimum seawater parameters and adequate water exchange (25–50% daily). The medusa was fed freshly hatched Artemia nauplii and mucus, and any uneaten nauplii were siphoned out daily.
(a) Cnidarian laboratory of ICAR-Tuticorin Regional Station of CMFRI; (b) recirculatory system having glass aquarium tank used for the study.
Details of Cassiopea xamachana medusae/fragments used for experimental studies and monitoring period
Morphological and microscopic observation
Morphological regeneration and growth of the umbrella tissue were studied using a stereo-zoom microscope (Leica S8 APD Stereo Zoom, Switzerland). Tissue samples were carefully transferred into a 50-ml beaker for examination under a microscope. Growth measurements and corrected image parameters were performed using GNU Image Manipulation Program (GIMP, ver. 3; https://www.gimp.org ), an open-source image-editing software. Measurements were taken using a calibrated ocular micrometre attached to the microscope before the images were analysed and processed using Adobe Photoshop image editing software.
Pulsation
A minute video recording was used to monitor and document the pulsing of injured or amputated medusa/fragments. Pulsation was recorded before and after feeding Artemia nauplii. The average pulse rate per minute was compared with that of uninjured control medusa.
Molecular identification
Tissue samples taken from the control medusa before amputation were fixed in 96% ethyl alcohol and stored at −20°C. DNA extraction, amplification, and sequencing were performed using the ammonium acetate salt precipitation protocol (Cunningham and Buss Reference Cunningham and Buss1993; Fetzner Reference Fetzner1999). A 647-bp fragment of the ribosomal gene 16S rRNA was amplified from the specimen. The DNA samples were checked and amplified using forward C&B1 (5′-TCGACTGTTTACCAAAAACATAGC-3′) and reverse C&B2 (5′-ACGGAATGAACTCAAATCATGTAAG-3′) primers (Cunningham and Buss Reference Cunningham and Buss1993). PCR conditions used by Gamero-Mora et al. (Reference Gamero-Mora, Halbauer, Bartsch, Stampar and Morandini2019) included DNA denaturation at 94°C for 3 minutes. Amplification was performed for 35 cycles, each consisting of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The final extension step was performed at 72°C for 10 minutes (Jiang et al. Reference Jiang, Wang, Zhao, Yan, Han, Shang, Du and Wu2019). PCR products were visualized by electrophoresis on a 2% TAE-buffer agarose gel stained with ethidium bromide. Sequencing was performed using a BigDye Terminator Cycle Sequencing Kit (ver. 3.1; Applied Biosystems, USA), and the primers were subjected to Sanger bidirectional sequencing (Sanger et al. Reference Sanger, Nicklen and Coulson1977) via PCR using an ABI 3730xl DNA analyzer (Applied Biosystems, USA). The cycled products were purified using 3 M sodium acetate ethyl alcohol, followed by Sephadex G-50 (Sigma Aldrich, Switzerland). Chromatograms of sequence reads were checked, edited, and assembled using the BioEdit software (Hall et al. Reference Hall, Biosciences and Carlsbad2011) to produce a consensus sequence. The generated mitochondrial 16S rRNA gene sequence was deposited in the GenBank database of the National Center for Biotechnology Information (NCBI). The genera and species were tentatively identified based on the lowest E value and highest similarity in the BLAST program. For subsequent phylogenetic analysis and species identification, the 16S rRNA reference sequence database (Table 2) was obtained from the GenBank NCBI database for Cassiopea species such as C. xamachana (MT709260.1), C. andromeda (MT709258.1, KY610612.1), C. culionensis (MW164879.1, MW164885.1), C. mayeri (MW164866.1, MW164864.1), C. ornata (OL721669.1, KY610616.1), and C. frondosa (KY610617.1, MT709259.1), and outgroup species comprising Mastigias papua (KY610621.1), Versuriga anadyomene (KX904852.1), and Aurelia sp. (MT709255.1). Multiple-sequence alignment of the consensus and reference sequences was performed using ClustalX (Thompson et al. Reference Thompson, Gibson, Plewniak, Jeanmougin and Higgins1997). Evolutionary genetic distance was calculated using Kimura’s two-parameter model (Kimura Reference Kimura1980) in MEGA 11.0 (Tamura et al. Reference Tamura, Stecher and Kumar2021). The Maximum Likelihood (ML) dendrogram was constructed using Kimura’s two-parameter model (Kimura Reference Kimura1980) with 1000 bootstrap replications in MEGA 11.0 (Liang et al. Reference Liang, Jiang, Wang, Zhang, Liu, Xu, Zhang and Xiao2019; Tamura et al. Reference Tamura, Stecher and Kumar2021). The ML tree was rooted with the outgroup sequences M. papua (KY610621.1), V. anadyomene (KX904852.1), and Aurelia sp. (MT709255.1).
Details of 16S rRNA sequences of jellyfish species used for phylogenetic analysis
Results
Genus and species identity
The specimens were classified under the genus Cassiopea based on the general morphological features and key identification characteristics of the medusae: absence of tentacles, presence of vesicles, upside-down orientation of the jellyfish, presence of eight oral arms, and presence of 16 rhopalia. Further, molecular sequence of the ribosomal gene (16S rRNA), confirmed the species identity of the specimen Cassiopea xamachana (Bigelow Reference Bigelow1892) (Class: Scyphozoa; Order: Rhizostomeae; Family: Cassiopeidae). Sequencing and contig assembly of the 16S rRNA gene produced a 647 bp sequence. Together with the reference sequences from NCBI, multiple sequence alignment and trimming yielded a final alignment length of 508 bp which was used for further analysis. The generated gene sequence was submitted to the NCBI GenBank (Accession No. ON545804.1). The BLAST search result of the query sequence showed 98.91% similarity (99% query coverage) with that of the Cassiopea xamachana isolate from the USA (GenBank Accession no. MT709260.1). The maximum likelihood dendrogram (Figure 2) also confirmed the species identity of sequence ON545804.1 as C. xamachana because of its strong phylogenetic affinity to the C. xamachana reference sequence MT709260.1, supported by a high bootstrap value. The genetic distance value (K2P) between C. xamachana species was 0.003, whereas that of the other Cassiopea species ranged from 0.029 to 0.138, with the outgroup ranging from 0.173 to 0.315 (Table 3).
Maximum likelihood (ML) tree based on mitochondrial 16S rRNA sequences showing phylogenetic relationship of Cassiopea spp.
Genetic distance values (Kimura 2 Parameter) of 16S rRNA gene sequences between 15 jellyfish taxa comprising Cassiopea, Mastigias, Versuriga and Aurelia species
* Sl. = Number 1 to 15 correspond to species and sequences listed in Table 2.
Pulsation rate
The control medusa, C (Figure 3a and b), exhibited an initial pulse rate of 16 ± 2 pulses per minute, and a higher pulsation rate was observed in the accident injury, and the amputation medusa ranged from 22 ± 8 to 40 ± 5 pulse/min. The umbrella tissue, with and without the oral arms, continued to pulsate and remained viable and the OAs did not display any pulsations (Figure 4). The pulse rate increased after Artemia nauplii was fed which is a comparatively lower rate than that observed before Artemia nauplii feeding. Initially, the UT had a pulse rate of 39 ± 3 pulses/min, and the UTWA had a pulse rate of 23 ± 6 pulses/min. The first and second amputated medusa exhibited initial pulse rates of 33 ± 6 pulses/min and 29 ± 7 pulses/min, respectively. Variations in the pulsation levels of the different medusa used in the experimental rearing period and pulse rate during the Artemia nauplii feeding experiment are presented in Table 4.
(a) Cassiopea xamachana used for the experimental studies; (b) control specimens used for the experiment.
Accidentally injured C. xamachana fragments: umbrella tissue without arms, UT (black circle); umbrella tissue with oral arms, UTWA (yellow circle), oral arms, OAs without umbrella tissue (black and yellow squares).
Pulsation variation of the injured and amputation medusae of Cassiopea xamachana and pulsation rate (pulse/minute) before and after feeding Artemia nauplii
General description and growth of control animals
Most control individuals had flat bell and disc shapes, with a variable number of short, blunt marginal lappets. The colours of the medusa exumbrella and oral arm ranged from clear to golden brown. Rhopalia varied from 16 to 22 in different ways, with 20 being the most common. Lappets between rhopalia varied from four to five. Eight oral arms, lateral branches, and short side branches arose from each arm in a tree-like manner and were turned to provide offside branchlets in an alternating pattern. Vesicles are large or small ribbon-like filaments found in central discs and arms.
Regeneration in an accidentally injured medusa
The medusa with UTWA exhibited pulsation and regained symmetry through tissue formation (Figure 5a and b). The UT did not develop any rounded or dome-like structures and lacked visible features such as mouth, oral arms, vesicles, and symmetry; however, it still exhibited pulsation. The OAs possessing secondary branches and vesicles, but without any tissue portion of the umbrella, did not show any growth toward complete structural restoration. Over time, the accidentally cut portions of C. xamachana umbrella tissue (UTWA and UT) remained alive and continued to actively pulse. The first signs of regeneration were visible within one day. The UTWA and UT fragments exhibited pulsation and regained symmetry, whereas the oral arms failed to restore their body structure (Figure 6a and b). New tissue developed at the site of injury; however, the UTWAs and UTs gradually decreased in size to 0.5 cm and 4.0 cm, respectively. After 15 days, the UTWA fragments regenerated and regained symmetry, forming a bell-shaped tissue from the injured region (Figure 7a and b). Although the size of UTWAs initially decreased, they regained tissue and symmetry within 7 days. After 30 days, a gastrovascular system with radial canals and anastomoses was observed. The oral anastomosing vessels and radial canals extended from the mouth to the bell margin, forming nearly spherical structures (Figure 7c and d). The presence of marginal lappets and the development of oral arms extending from the central disc, along with the presence of yellow-orange zooxanthellae in the umbrella region, were visualized under a microscope. The oral arms also developed vesicles (Figure 7e and f). After 60 days, three oral arms emerged from the mouth, along with rhopalia sensory organs and marginal lappets (Figure 7g and h). By the third month, the oral arms had enlarged, and oral vesicles had started to develop from the centre of the disc (Figure 7i and j). At this point, the medusa had more oral vesicles and central disc vesicles, and the zooxanthellae spread out more clearly (Figure 7k and l). Figure 7a–l shows the complete sequence of self-repair and regeneration of injured bell tissues in C. xamachana.
(a) Injured umbrella tissue with oral arms (UTWA); (b) regenerated UTWA.
(a) Oral arm without umbrella tissue (OA) immediately after injury and (b) OA showing no growth even after 2 weeks post-injury.
Regeneration process in accidentally injured C. xamachana with only umbrella tissue (UT). (a) UT soon after injury (day one); (b) initiation of bell tissue regeneration (7 days); (c) formation of gastrovascular system with radial canals and anastomoses (30 days); (d) development of radial canals extending from mouth to bell margins (30 days); (e) formation of complete bell tissue, oral arms, rhopaliar niche and marginal lappets (30 days); (f) regenerated oral arms and vesicles (30 days); (g) 3 oral arms regenerated from the mouth region, rhophalia sensory organs and marginal lappets (60 days); (h) aboral side showing marginal lappet (60 days); (i) 3 well-developed oral arms from the mouth region (75 days); (j) regenerated oral arms with well-developed vesicles (75 days); (k) regenerated medusa with more number of oral vesicles (90 days); (l) regenerated UT showing oral vesicles with zooxanthellae (90 days).
Regeneration in amputated medusa
The first four radially amputated jellyfish fragments (FAF 1, FAF 2, FAF 3, and FAF 4), which initially had oral arms and umbrella tissue, exhibited wound healing, specifically regeneration of umbrella tissue to restore symmetry. The canal system plays a crucial role in this process by forming whitish tissues at both ends of the amputated tissue. This process restored symmetry within 3–4 days, depending on the severity of the injury, and the fragments regained complete symmetry within a week (Figure 8). Repeated experiments on additional medusa (SAF 1, SAF 2, SAF 3, and SAF 4) also showed similar results, regaining symmetry within a week (Figure 9). All the amputated fragments exhibited signs of self-repair and regeneration. After four weeks, microscopic observations revealed the presence of marginal lappets in the regenerated tissue, emergence of additional oral arms from the central disc, and expansion of zooxanthellae in the umbrella region. In the oral region, the gastrovascular cavities, canal structures, and lappet systems are fully developed, and more oral arms emerge from the mouth. Oral vesicles were also formed on the arms and in the centre of the mouth, resulting in a complete structure that resembled a normal adult medusa in appearance and function (Figure 9).
Regeneration process in first amputated C. xamachana medusae fragment (FAF); a) immedately after amputation, b) oral and c) aboral view of FAF 1, FAF 2, FAF 3, and FAF 4 depicting the restoration of symmetry after one week.
Regeneration process in second amputated C. xamachana medusae fragment (SAF); a, b, c and d depicts the regenerated oral view whereas e, f, g and h depicts the regenerated aboral view of SAF 1, SAF 2, SAF 3, and SAF 4 after 1st, 2nd, 3rd week and 4th week, respectively.
Discussion
The morphological features of these specimens are comparable to those of the original species described in Jamaica (Bigelow Reference Bigelow1892, Reference Bigelow1900; Bossert et al. Reference Bossert, Dunn and Thomsen2013). In addition, BLAST and phylogenetic analyses of the 16S rRNA gene sequence confirmed the identity of the jellyfish as C. xamachana and validated the presence of this species in the Gulf of Mannar, Indian waters. The sequence of the isolate (GenBank Accession No. ON545804.1) showed 98.91% similarity (99% query coverage) with an isolate from the USA (GenBank Accession No. MT709260.1) in the NCBI GenBank database, indicating that the samples obtained in this study were C. xamachana. The phylogenetic tree based on the maximum likelihood method also supported the above findings by revealing distinct clades where similar species were clustered under the same nodes, that is, the sequences derived from the present study and other available resources for C. xamachana, but dissimilar to different species (Figure 2). The genetic distance between our retrieved sequence and C. xamachana was lower (0.003) than that of other species, such as C. andromeda, C. culionesis, C. mayeri, C. frondosa, and C. ornata (0.029–0.138), whereas the outgroup species, such as M. papua, V. anadyomene, and Aurelia sp. had the highest distance values (0.173–0.315).
Comparative data on internationally reported species have provided valuable insights into the expanded distribution of this species. Specifically, this species extends to the Gulf of Mannar, India, originating from its nearest known region, the tropical South American Brazilian coast, which is located in the Western Atlantic Ocean (Morandini et al. Reference Morandini, Stampar, Maronna and Da Silveira2017; Stampar et al. Reference Stampar, Gamero-Mora, Maronna, Fritscher, Oliveira, Sampaio and Morandini2020). However, C. xamachana has not been reported in Indian waters, and the present study reports its distribution range in the Gulf of Mannar, Bay of Bengal Large Marine Ecosystem, necessitating the implementation of a comprehensive methodology other than conventional DNA sequencing to evaluate the spread of Cassiopea species in Indian waters. A portable field-able environmental DNA sequencing (FeDS) kit is useful for identifying the presence of this species in Florida waters (Ames et al. Reference Ames, Ohdera, Colston, Collins, Fitt, Morandini, Erickson and Vora2021). A scientometric study revealed that C. xamachana is the most researched upside-down jellyfish globally, mostly from Florida, the Caribbean, and the Indo-Pacific geographic areas (López-Figueroa et al. Reference López-Figueroa, Stoner and Hallock2024). Future field surveys of Cassiopea spp. in Indian waters will uncover new studies on species diversity, ecological spread, and bioindicators. Cassiopea xamachana can be used as a model organism to understand cnidarian photosymbiotic structure, which has implications for the conservation of coral reef ecosystems from mass bleaching (Medina et al. Reference Medina, Sharp, Ohdera, Bellantuono, Dalrymple, Gamero-Mora, Steinworth, Hofmann, Martindale, Morandini, DeGennaro and Fitt2021). Cassiosome structure in Cassiopea is believed to be an adaptation for ecological success, and is responsible for the symbiotic nutrient exchange and increased lifespan of this species (Toullec et al. Reference Toullec, Lyndby, Banc-Prandi, Pogoreutz, Martin Olmos, Meibom and Rädecker2024). Moreover, the success of C. xamachana larval settlement and metamorphosis is induced by bacterial induction, which also plays a role in ecological succession (Ohdera et al. Reference Ohdera, Attarwala, Wu, Henry, Laird, Hofmann, Fitt and Medina2023).
The ability of upside-down jellyfish polyps to regenerate is well known (Bossert et al. Reference Bossert, Dunn and Thomsen2013). Gamero-Mora et al. (Reference Gamero-Mora, Halbauer, Bartsch, Stampar and Morandini2019) reported that C. xamachana medusa had similar regenerative abilities; the present experimental studies also demonstrated that umbrella tissues can grow and regenerate in all medusa without any mortality of the fragments. Similar regenerative abilities have been reported for Tripedalia cystophora (Stamatis et al. Reference Stamatis, Worsaae and Garm2018). However, the regeneration of disaggregated umbrella tissue in C. xamachana has not been previously reported, although studies have demonstrated that Cassiopea umbrella tissue can regenerate into a fully functional medusa (Gamero-Mora et al. Reference Gamero-Mora, Halbauer, Bartsch, Stampar and Morandini2019). In contrast, the moon jellyfish Aurelia aurita cannot regenerate new tissue; instead, the medusa rearranges their existing body structures during regeneration (Abrams et al. Reference Abrams, Basinger, Yuan, Guo and Goentoro2015). The present study also demonstrated that umbrella tissue with an arm can regain symmetry and regenerate body structures, as previously reported (Gamero-Mora et al. Reference Gamero-Mora, Halbauer, Bartsch, Stampar and Morandini2019; Ostendarp et al. Reference Ostendarp, Plewka, Flathmann, Tilstra, El-Khaled and Wild2022), which the oral arm alone cannot accomplish. This study revealed that the pulsation behaviour of injured medusa (UTWA and UT) had a higher pulsation rate in the initial stage of the experiment, whereas Ostendarp et al. (Reference Ostendarp, Plewka, Flathmann, Tilstra, El-Khaled and Wild2022) reported that OA failed to perform the same observation. However, both wounded and amputated medusa displayed noticeably increased pulsation activity due to stress, which increased further during feeding compared to the normal control medusa. The oral arms did not exhibit any pulsation similar to that of the umbrella part; hence, physiological activities were not active in the oral arms for the formation of biological components. However, the umbrella exhibits a series of morphological modifications, starting from symmetry to complete structures, including most of the vital body parts. This statement is supported by Ostendarp et al. (Reference Ostendarp, Plewka, Flathmann, Tilstra, El-Khaled and Wild2022). In the initial stages of the wound healing process, we observed a decrease in body size during regeneration, which was likely due to increased energy demand. A similar observation was made for A. aurita, where medusa can digest certain body parts and later regrow them (Hamner and Jenssen Reference Hamner and Jenssen1974). Mammone et al. (Reference Mammone, Sharp, Hewitt and Medina2023) revealed that the presence of symbionts increased the likelihood of regenerating C. xamachana polyps, whereas symbiont species did not affect the regeneration success. In addition to its greater potential for regeneration, Cassiopea is renowned for its ability to withstand heat stress, which is linked to the colour of its appendages, increasing the survival of this species by having blue appendages (Maloney et al. Reference Maloney, Buckley and Strader2024). The medusa of C. xamachana have a strong symbiotic relationship with the symbiotic alga Symbiodinium microadriaticum (Fitt et al. Reference Fitt, Hofmann, Kemp and Ohdera2021; Medina et al. Reference Medina, Sharp, Ohdera, Bellantuono, Dalrymple, Gamero-Mora, Steinworth, Hofmann, Martindale, Morandini, DeGennaro and Fitt2021).
The present study is the first to report the self-repair and regeneration of C. xamachana from India, where the regeneration of umbrella tissue fragments was initiated within one day and the virtual medusa structure development was completed within 3 months; similar results have been documented in previous experiments, where virtual body structures began to form just one day after tissue fragmentation. The umbrella margin of C. xamachana exhibits signs of fission that precede the regeneration process (Ostendarp et al. Reference Ostendarp, Plewka, Flathmann, Tilstra, El-Khaled and Wild2022). Furthermore, this study suggests that wound healing in C. xamachana begins with tissue regeneration, but does not end with mere wound closure. The umbrella tissue of the medusa supports the growth of almost all body structures, including the canals, oral arms, and vesicles. In contrast to a previous report (Ostendarp et al. Reference Ostendarp, Plewka, Flathmann, Tilstra, El-Khaled and Wild2022), this study showed that C. xamachana could rebuild whole structures by splitting them apart from the umbrella tissue. This shows that this genus has hidden regenerative abilities, which have not been previously reported. This study contributes to existing knowledge on the regeneration of Aurelia sp. 1, Chrysaora sp., and C. xamachana (Abrams et al. Reference Abrams, Basinger, Yuan, Guo and Goentoro2015; Ostendarp et al. Reference Ostendarp, Plewka, Flathmann, Tilstra, El-Khaled and Wild2022), showing that C. xamachana shares similar healing and growth abilities. Scyphomedusae such as C. xamachana may possess tissue plasticity traits similar to those found in Hydromedusae, indicating the need for further research on this group (Piraino et al. Reference Piraino, Boero, Aeschbach and Schmid1996). Interstitial cells have been found in the endoderm of Cassiopea planula in previous studies and have also been observed in Hydrozoan stem cells (Curtis and Cowden Reference Curtis and Cowden1974; Martin and Chia Reference Martin and Chia1982). Previous reports have suggested that amoeboid cells play a similar regenerative role in Cassiopea scyphistoma (Curtis and Cowden Reference Curtis and Cowden1974). Researchers have made significant progress in understanding the genetic factors that control jellyfish regeneration (Fujita et al. Reference Fujita, Kuranaga and Nakajima2021). In terms of regenerative capabilities, both Hydrozoa and Scyphozoa exhibit morphogenesis (Galliot and Schmid Reference Galliot and Schmid2002), and exposing striated muscle cells in Podocoryne carnea umbrella tissue to different culture conditions can induce their differentiation into different cell types (Schmid and Reber-Muller Reference Schmid and Reber-Muller1995). Similarly, cells from the exumbrella, epidermis, and gastrovascular systems can reverse the life cycle of Turritopsis medusae (Piraino et al. Reference Piraino, Boero, Aeschbach and Schmid1996).
This study revealed that the umbrella tissue of C. xamachana possesses unique pluripotency. However, much remains to be understood regarding the regeneration mechanism in Cassiopea jellyfish, as well as the factors that promote pluripotency and subsequent regeneration. Understanding the regeneration characteristics of these non-bilaterian lineages may aid scientists in developing evolutionary hypotheses for this group (Allen et al. Reference Allen, Ross and Zayas2016; Bell Reference Bell2002). Amplatz et al. (Reference Amplatz, Zieger, Abed-Navandi, Weissenbacher and Wanninger2024) elucidated the implications of the evolution of cnidarian nervous systems, specifically focusing on the neuromuscular development of Cassiopea xamachana as an emerging scyphozoan model system for regeneration. Consequently, further research on regeneration in Scyphozoa will require well-designed laboratory experiments and manipulation techniques to accurately identify their self-repair and regeneration potential. Species, injury size, and tissue type are critical factors in determining wound healing and regeneration processes. Future studies should focus on investigating the molecular inductors or inhibitors and cellular cues that facilitate regeneration and organ formation.
Acknowledgements
The authors are grateful to the former and present Directors of ICAR-Central Marine Fisheries Research Institute (CMFRI), Kochi, for their kind help and constant encouragement, and the Heads of Marine Biodiversity and Environment Management Division, ICAR-CMFRI, Kochi, for their support and keen interest in conducting this study. The first author is grateful to the Scientist-in-charges (Dr. I. Jagadis and Dr. P. P. Manojkumar), ICAR-Tuticorin Regional Station of CMFRI, for the support and facilities provided during this study. Dr. Andre Carrara Morandini, University of São Paulo, Brazil and Dr. Edgar Gamero Mora, Department of Technology of Food of Animal Origin, Research Center for Food and Development, A. C. (CIAD), Mexico, for providing valuable input to the first author during the study of animal maintenance and for providing constructive suggestions on the molecular methods of the paper.
Author contributions
R. L. and S. R. developed the overall concept and designed the experiments. R. L., C. K., and M. A. performed the experiments and recorded the observations. R. L., S. K. S., D. L. P., R. S., and S. D. carried out the morphological and molecular analyses. R. L., M. A., and S. D. prepared the manuscript. R. L., S. K. S., S. R., A. P. S, and G. G. critically revised the manuscript for intellectual content. L. R., S. R., A. P. S., and G. G. contributed to project formulation, fund acquisition, and project management. R. L., S. R., S. K. S., R. S., C. K., D. L. P., R. S., M. A., S. D., A. P. S., and G. G approved the final version to be published.
Funding
This “Research was supported by the Indian Council of Agricultural Research (ICAR), Department of Agricultural Research and Education, Government of India” under the Institute Project of the Central Marine Fisheries Research Institute (CMFRI), Project code MBD/JBD/32.
Competing interests
The authors declare that they have no competing interests.
Ethical standards
The upside-down jellyfish Cassiopea xamachana (Bigelow Reference Bigelow1892) is not a protected species in the study area. All international, national, and/or institutional guidelines for the care and use of animals were followed.
Data and availability
The datasets generated in this study are available from the corresponding author upon request.
