Sunday, 3 May 2020

'Stinging Water': How the Upside-down Jellyfish hunts prey beyond the reach of its tentacles.

Jellyfish, along with Corals, Anemones, Hydroids, and Myxozoans, belong to the phylum Cnidaria, the earliest diverging venomous animal lineage. These diploblastic Animals have two epithelial layers, the outer ectoderm and inner endoderm, separated by a gelatinous extracellular matrix called the mesoglea. Despite their seemingly simple morphology, Cnidarians have adapted globally to most saltwater habitats and some freshwater environments. As such, Cnidarians have evolved a remarkable envenomation mechanism that involves the deployment of subcellular stinging capsules called nematocysts from Cnidarian-specific cells called nematocytes, which vary in size, morphology, and bioactive contents. Sea Anemones possess unique nematocyte-rich structures (e.g., acrorhagi, acontia) and employ strategies such as tentacle and column contraction and expansion to enhance nematocyst deployment for prey capture and protection, while in Medusae (Jellyfish) the first line of defence is their extendable nematocyte-laden tentacles that envenomate prey and predators they encounter in the water column1, as well as Humans participating in marine recreation. In addition to direct stings caused by Jellyfish, indirect stings have also been reported. Some possible explanations for indirect Jellyfish stings are contact with tentacle fragments in the water (e.g., Jellyfish stings in offshore fishers), envenomation by juvenile venomous Jellyfish (e.g., Irukandji-like syndrome in United States Military combat divers), or Sea Bathers Eruption caused by microscopic jellyfish life forms (e.g., Linuche unguiculata).

Another indirect stinging mechanism is through mucus, such as in Medusae of the Upside-down Mangrove Jellyfish, Cassiopea xamachana, (Class Scyphozoa; Order Rhizostomeae), an emerging Cnidarian model for its relevance to the study of coevolution as well as symbiosis-driven development. The ubiquity of Cassiopea Medusae in healthy Mangroves has earned the Upside-down Jellyfish status as a potential bioindicator species for coastal management and conservation efforts. Cassiopea is known to release large amounts of mucus into the water column, which has been referred to as toxic mucus due to reports of nematocysts found freely suspended in the vicious substance. For instance, Cassiopea mucus is known to kill certain species of Fish on contact. Cassiopea is an exception to the iconic image of a Jellyfish in that it lacks marginal tentacles and, instead of swimming in the water column, lies apex-down on the substrate in Mangrove forests, Seagrass beds or other coastal waters with its relatively short oral arms facing upward. Despite this benthic lifestyle, warnings have been published alerting sea bathers of the stinging water or toxic mucus phenomena blamed on unidentified potent little grenades in the water column surrounding Cassiopea Medusae. In general, Cassiopea stings are categorised as mild to moderate in Humans, but crude venom extracted from the nematocysts displays hemolytic, cardiotoxic and dermonecrotic properties, suggesting that excessive exposure may be detrimental for Humans.

In a paper published in the journal Communications Biology on 13 February 2020, a team of scientists led by Cheryl Ames of the US Naval Research Laboratory in Washington DC, the Graduate School of Agricultural Science at Tohoku University, and the Department of Invertebrate Zoology at the Smithsonian National Museum of Natural History, and Anna Klompen, also of the Department of Invertebrate Zoology at the Smithsonian National Museum of Natural History, and of the Department of Ecology and Evolutionary Biology at the University of Kansas, present the results of a study that used a combination of histology, microscopy, microfluidics, videography, molecular biology, and mass spectrometry-based proteomics, to describe Cassiopea xamachana stinging-cell structures that they term cassiosomes, which are are released within the Jellyfish's mucus and are capable of killing prey.

During the course of this study, a review of the old literature on Cassiopea revealed a probable explanation for the grenades reported in stinging water. In 1908 zoologist Henry Farnham Perkins of the University of Vermont, discovered in the mucus of Cassiopea undeployed nematocysts and ciliated innumerable minute spherical bodies, the latter of which were dismissed as non-Coelenterate (i.e., non-Cnidarian) in nature. Then in 1936 marine biologist Horace Smith of the  Tortugas Laboratory of the Carnegie Institution published a brief description of peculiar structures found in the oral vesicles (i.e., vesicular appendages) of the oral arms of Cassiopea xamachana and  Cassiopea frondosa Medusae that were ‘shot’ at prey, which he dubbed small bags of mesoglea and nematocysts and suggested might play a role in predation. Funaly in 1997 Ronald Larson of the University of Puerto Rico reported polygonal-shaped bodies on the flatted sides of the oral vesicles (i.e., vesicular appendages) of the oral arms of Cassiopea xamachana and  Cassiopea frondosa corresponding to nematocyst clusters that released upon contact, to which he attributed a role in defense. The sum of these reports suggests that an investigation of the contents of Cassiopea mucus is needed to test the hypothesis that undeployed nematocysts and/or another nematocyst-bearing structure(s) present within the mucus of the Upside-down Jellyfish together are responsible for the phenomenon of stinging water experienced by Humans in the vicinity of Cassiopea Medusae.

Ames & Klompen et al. used a combination of microscopy, microfluidic devices, molecular biology techniques, mass spectrometry-based proteomics, and other experimental assays to provide the first detailed description, to thier knowledge, of the contents of the mucus liberated from lab-reared Cassiopea xamachana Medusae. Released within the mucus, they discovered three types of undeployed nematocysts, as well as microscopic, motile, cellular masses composed of nematocytes that Ames & Klompen et al. formally call cassiosomes. While cassiosomes bear some resemblance to another Cnidarian structure originating in mesenteries of the Starlet Sea Anemone, Nematostella, called nematosomes, the unique traits of cassiosomes in Cassiopea xamachana include their release into the water column within mucus, the ability to trap and kill prey as mobile grenades outside of the Medusa, their organization as an outer epithelial layer surrounding a mostly empty core (rather than a solid ball of cells), and the presence of centrally-located endosymbiotic Symbiodinium Dinoflagellates. Ames & Klompen et al. document the presence of cassiosomes in five species spanning four families of the order Rhizostomeae, while also confirming their absence in the Moon Jellyfish, Aurelia, (Semaeostomeae), a representative of the sister lineage, and discuss the possibility of a single evolutionary event behind this envenomation strategy which, to their knowledge, is unique. Despite the growing body of work on Cassiopea xamachana from an organismal biology perspective, Ames and Klompen et al.'s study is the first to directly investigate stinging properties of the mucus of this Jellyfish and the potential ecological and evolutionary relevance.

Observations were made on lab-reared Cassiopea xamachana and on Medusae in their natural habitat in waters of Florida Keys Mangrove forests. In both cases, Medusae were observed releasing copious amounts of mucus into the water when the surrounding water was disturbed (by Jellyfish aquarists and/or snorkelers), or when prey items were provided (e.g., Artemia nauplii in aquarium-reared Medusae). The stinging water phenomenon was experienced by Ames & Klompen et al. while handling lab-reared and/or wild Cassiopea xamachana and other Rhizostome Jellyfish examined during the study.

Medusae of the Upside-down Mangrove Jellyfish Cassiopea xamachana. (a), (b) Medusae (5–12 cm diameter) resting on umbrella apex (white arrow) with oral arms (cyan arrows) facing up, observed in the natural mangrove habitat in Key Largo, Florida (USA). (c)–(f) Cassiosome nests (pink arrows) observed as white bulging spots at the termini of vesicular appendages (green arrows) off-branching from areas of frilly digitate cirri (black arrows) on Medusa oral arms (cyan arrows). Some cassiosome nests appear less full than others. Scale bars: (a) 2 cm; (b) 5 cm; (c), (d) 1 mm; (e), (f 0.5 mm. Ames & Klompen et al. (2020).

Cassiopea xamachana Medusae start out like most Scyphozoan Jellyfish, as an asexual microscopic Polyp that metamorphosizes into a sexually reproducing Medusa via a process known as strobilation. However, they differ from most Jellyfish in that they host endosymbiotic Dinoflagellates (also called zooxanthellae). Colonisation of Polyps by Algal endosymbionts is the most common type of intracellular mutualism among Cnidarians of the class Anthozoa (e.g., Corals and Anemones), and although it is less common in Jellyfish species, endosymbiosis triggers the start of Cassiopea xamachana polyp strobilation. During the sessile life stage, these Polyps engulf Dinoflagellates (unicellular Algae called Symbiodinium) via the manubrium (feeding tube), which are then phagocytosed by endodermal cells. Bound by a membrane complex that combines host and infecting cell membranes (called a symbiosome), Symbiodinium spp. migrate to the Polyp mesoglea and remain there housed in endodermal cells, transformed into amoebocytes. Shortly after infection with endosymbionts, Cassiopea xamachana Polyps undergo strobilation, and the apical portion metamorphosis into an Ephyra (juvenile Medusa) which then develops into a sexually mature male or female Medusa, with multiple colour variants based on endosymbionts. Symbiodinium-generated photosynthates support the Jellyfish host metabolism, growth, reproduction and survival. This promotes conservation and recycling of essential nutrients, given their strategic presence amidst downwelling light, which is of unrivaled ecological importance for Coral reefs and Cassiopea populations alike.

Observations of mucus and cassiosome release in Cassiopea xamachana Medusae. (a) Cassiopea xamachana releasing mucus (yellow arrows) following collection in the field (Bonaire, The Netherlands Antilles). Cassiosome nests (pink arrows) appear as light bulging spots at the termini of vesicular appendages (cyan arrow). (b) Mucus (yellow arrow) released into the water by Cassiopea xamachana in the lab, small white flecks correspond to live cassiosomes (green arrows). (c) Live cassiosomes (green arrows) suspended in mucus (yellow arrow) harvested after release from Cassiopea xamachana. (d) Multiple motile cassiosomes isolated from Cassiopea xamachana mucus. (e) Live cassiosome close-up (green arrows), showing irregular shape and centralized Symbiodinium Dinoflagellates as amber spheres (red arrows). (f) Confocal image of highly motile cassiosome immobilized on glass bottom dishes coated with adhesive; image collected with × 60 objective (oil) reveals organization of the peripheral cell layer: nuclei (blue stained) of nematocytes with peripheral nematocytes bearing O-isorhiza nematocysts (blue arrows, differential interference contrast microscope image) and non-nematocyte ectodermal cells (purple). Differential interference contrast microscopy shows centrally Symbiodinium (dark spheres, red arrows) occupy presumptive Cassiopea xamachana amoebocytes in an otherwise acellular core. Scale bars: (a) 3 cm; (b) 3 mm, (c) 1 mm;, (d) 5 mm; (e) 300 μm, (f) 50 μm. Ames & Klompen et al. (2020).

Feeding studies on Cassiopea Medusae show that prey capture occurs as a result of perpetual Medusa pulsation that carries the prey into the subumbrellar space and then onto the oral arms where they are held by nematocyst-rich digitate fringed lips and vesicular appendages (i.e., small oral vesicles), eventually being reduced to fragments. Finally, food particles are then forced into the oral ostia of secondary mouths, and ingested via ciliary action. Cassiopea are opportunistic predators, feeding on a broad range of prey items (e.g., Crustaceans, Mematodes, eggs) in the field, while in the lab polyps and medusae are fed 1–3 day old, lab reared Brine Shrimps, Artemia salina.

Numerous, motile cellular structures, which Ames & Klompen et al. call cassiosomes, were observed suspended within mucus released by Cassiopea xamachana Medusae (3.0–8.8 cm umbrella diameter) in response to feeding or mild disruption with short bursts of seawater from a pipette. Ames and Klompen et al. describe cassiosomes in Cassiopea xamachana as microscopic (100–550 μm in diameter), irregularly-shaped cellular masses whose peripheral cell layer is primarily composed of nematocytes and other irregular ectodermal cells that surrounds a space containing amoebocytes, some hosting Symbiodinium and others lacking them, among presumptive mesoglea.

When multiple Cassiopea xamachana Medusae were placed together and agitated by directing water at their oral arms using a glass pipette, they consistently released cassiosome laden mucus within 5–10 minutes for periods lasting several hours. When collected mucus was transferred to a small glass dish, cassiosomes moved around within the mucus for about 15 minutes and then descended to the bottom of the dish, leaving the neutrally buoyant mucus. This permitted efficient isolation of numerous cassiosomes which remained in constant motion by rotating and displacing in various directions along the bottom, but never elevating from the bottom of the dish. Isolated cassiosomes remained motile for up to 10 days, gradually losing their corrugated appearance after 5 or 6 days and shrinking in size to a smooth spherical shape until movement ceased and cassiosomes disintegrated. Additionally, custom-designed microfluidic devices with channels equal to or slightly bigger than the cassiosomes were used to observe cassiosomes of Cassiopea to gain a better understanding of their motility, and three-dimensional irregular, popcorn-shaped structure. These microscopic observations revealed motile cilia extending from the periphery that propel cassiosomes.

Cassiopea xamachana cassiosomes - various modes of motility. Random movement, central axis rotation, circular, and backing up (not shown here). Ames & Klompen et al. (2020).

Ames & Klompen et al. conducted assays to determine if cassiosomes were capable of trapping Artemia nauplii provided as food in aquarium-reared Medusae since so-called non-penetrant O-isorhiza nematocysts are the only type found in cassiosomes of Cassiopea. For this purpose, microfluidic chambers provided an arena in which to document immobilization and rapid trapping of Artemia, which were killed almost immediately upon substantial contact with cassiosomes. When isolated cassiosomes were added to dishes (150ml) containing abundant Artemia nauplii, cassiosomes that encountered the underside of the nauplii carapace immediately immobilized and killed the prey items. In cases where Artemia nauplii came into contact only briefly with cassiosomes, prey were able to escape rapid immobilization and death. Furthermore, during discharge assays when either Filtered Artificial Seawater or mucus containing no cassiosomes, following manual removal, was added Artemia were not affected and continued to swim around in the dish.

Cassiopea xamachana discharge assay; cassiosomes. Isolated cassiosomes concentrated in a petri dish, killing numerous 1-day old Artemia nauplii within 60 seconds. Ames & Klompen et al. (2020).

In order to better understand their organization, live cassiosomes were isolated from Cassiopea xamachana mucus. After fixing, dehydrated specimens were examined using scanning electron microscopy. The cassiosome perimeter was found to be lined with nematocyst capsules and numerous long, spiny tubules extruded from abundant O-isorhiza nematocysts in the periphery following spontaneous deployment (likely during the dehydration process). More abundant on the surface, however, were much thinner filaments corresponding to abundant cilia connected to ectodermal cells. Close observation of several cassiosomes via scanning electron microscopy revealed along the outer layer emptied out regions appearing as collapsed cell membrane remnants of deployed nematocysts, underneath which could be seen an amorphous thick central extracellular matrix-like substance. Confocal microscopy on fixed cassiosomes with labeled nuclei, nematocysts and cilia corroborated these findings of an organized cell mass. Cassiosomes are composed of a peripheral layer of nematocytes bearing O-isorhiza nematocysts patterned with presumptive ectoderm cells that lack nematocysts, from which numerous cilia protrude. This outer layer surrounds centralized clusters of Symbiodinium endosymbionts (i.e., hosted by amoebocytes) within an otherwise apparently acellular region.

Brine Shrimp in microfluidic device. Isolated Cassiopea xamachana cassiosomes within a single chamber of a microfluidic device subdue, and then kill two-day old Artemia when introduced into the chamber. Ames & Klompen et al. (2020).

The term cnidome refers to the dynamic repertoire of nematocyst types in a Cnidarian species. The cnidome, a species-specific trait, often changes throughout the life cycle of the Jellyfish as it undergoes metamorphosis from a sessile Polyp, to strobila, and then to juvenile and sexually mature Medusa. Given reports implicating nematocysts, or tiny little grenades, within Cassiopea mucus as the cause of stinging water, Ames & Klompen et al. sought to characterize the cnidome of this species at several life stages, and within the cassiosomes and contents of the mucus.

Cassiopea xamachana discharge assay; mucus only. Mucus concentrated in a petri dish, following manual removal of cassiosomes, subduing and trapping, but not killing, numerous one-day old Artemia nauplii within 60 seconds. Ames & Klompen et al. (2020).

Nematocyst measurements were plotted for the following life stages of Cassiopea: Polyps (3); strobilating/released Ephyrae (3) and Medusae, 2.4–8.8 cm in diameter (3); mucus; and cassiosomes (isolated from mucus). Measurements of undischarged nematocysts of each type in the corresponding subsample revealed that O-isorhizas nematocysts are absent in Polyps, but appear in Medusae from the onset of Ephyra development during strobilation. Ames & Klompen et al. also observed penetrant nematocytes, birhopaloids and euryteles, which cannot be distinguished in the undeployed state (intact) within Cassiopea tissue using light microscopy. Therefore in this study, these two nematocyst types were analysed together as rhopaloids; hence, rhopaloids account for a larger proportion of the cnidome in the Medusa and mucus than distinct isorhiza types. An assessment of the inventory of nematocysts freely suspended within the mucus yielded a similar nematocyst profile to that of the Medusa, albeit with a proportionately higher number of rhopaloids, which are implicated in envenomation. Conversely, isolated cassiosomes of Cassiopea contain exclusively O-isorhiza nematocysts which are a ubiquitous type in Jellyfish tentacles, functioning in prey capture and predation.


Nematocyte type proportion (cnidome) varies within different life stages and structures of Cassiopea xamachana. (a) Figure displaying life cycle stages of Cassiopea xamachana and associated cassiosome-laden mucus release by the Medusae. Pie charts indicate proportion of nematocyte types for Polyps (3), Strobila/Ephyrae (3), Medusa (3), mucus (from 4 Medusae), and cassiosomes (from 4 Medusae), based on measurements of multiples of each nematocyst type per life stage. (b), (c) Different nematocyst types isolated from Cassiopea xamachana Medusae oral-arm filaments corresponding to colours in pie charts in (a): a-isorhiza intact (light blue arrow), O-isorhiza intact (green arrows) and deployed (dashed green arrow), and rhopaloid intact (lavender arrow) and deployed (dashed lavender arrow); and Symbidinium (brown arrows). (d) Mucus contents of Cassiopea xamachana containing a triplet of rhopaloid nematocysts (lavender arrows) intact within nematocytes, and Symbiodinium (brown arrow) disassociated from Jellyfish tissue but still within amoebocytes (pink arrows). Scale bars: (b), (d) 10 μm; (c) 20 μm. Ames & Klompen et al. (2020).

Over a century ago, Henry Farnham Perkins documented that disturbed Cassiopea xamachana Medusae produced mucus containing ciliated structures as innumerable minute spherical bodies containing unicellular zooxanthellae within the interior, which he considered to be parasitic larvae, and claimed it was 'impossible to regard [these structures] as of Coelenterate [Cnidaria] affinities'. These details suggest that Perkins observed what Ames & Klompen et al. have identified as cassiosomes, but mistook them for entirely unique, non-Cnidarian organisms. In order to test this theory, and properly classify cassiosomes as belonging to Cassiopea xamachana, rather than being unknown organism, Ames & Klompen et al. used real-time quantitative polymerase chain reaction assays to target species specific Cnidarian toxins, employing three custom-designed primer pairs that they designed from the publicly available Cassiopea xamachana genome.


Cassiosomes are capable of killing Brine Shrimp. (a) Dead 2-day old Artemia nauplii (orange arrow) with cassiosome (green arrow) attached to carapace, imaged within a microfluidic chamber. (b) Cassiosomes (green arrow) lodged into two different 1-day old Artemia nauplii (orange arrow). (c) 1-day old Artemia nauplii (orange arrow), immobilized following cassiosome (green arrow) attachment to the carapace with visibly discharged nematocysts (fuchsia arrows). Scale bars are 200 μm. Ames & Klompen et al. (2020).

Ames & Klompen et al. targeted a cnidarian-restricted CrTX/CaTX family toxin gene in DNA extracted separately from Cassiopea xamachana Medusa tissue and isolated cassiosomes, and also from tissue of the Moon Jellyfish, Aurelia sp. (Class Scyphozoa) and a more divergent Jellyfish species, the Sea Wasp, Alatina alata (Class Cubozoa) for comparison. Amplification of the quantitative polymerase chain reaction gene target was observed for both Cassiopea xamachana tissue and cassiosome samples using primers for the CrTX/CaTX gene. Conversely, failure to amplify the target in non-Cassiopea Medusozoans used in this study (despite reports of CrTX/CaTX genes documented in both Aurelia sp and Alatina alata validates the specificity of Ames & Klompen et al.'s primers to a Cassiopea xamachana -derived gene target, indicating that cassiosomes originate in Cassiopea Medusae.

Cassiosome organization revealed via scanning electron microscopy. (a) An individual cassiosome poised in a 100-μm mesh opening, revealing the irregular ‘popcorn’ shape of the cassiosome bearing numerous cilia (pink arrows) protruding from the peripheral layer. White rectangles correspond to magnified region shown in (b) and (c). (b), (c) Close up of the cassiosome reveals cilia (pink arrows), and thicker tubules (white arrows) of discharged O-isorhiza nematocyst capsules (blue arrows) in the periphery which is lined with collapsed nematocytes (cyan arrows). Spiny nematocyst tubules (white arrows) are outnumbered by the abundant cilia (thinner filaments) (pink arrows). (d) Depressions fringed by deflated cell membranes outlining nematocytes (cyan arrows in (c) and (d)) following deployment of O-isorhiza nematocysts (blue arrows in (c)) along the cassiosome surface. (e) A different cassiosome from that in (a)–(d). All cellular components were lost (possibly in dehydration stage of preparation) but for discharged O-isorhiza nematocyst capsules (blue arrows) and corresponding spiny tubules (white arrows) in the cassiosome periphery lined with collapsed nematocytes (cyan arrows). White rectangle corresponds to magnified region shown in (f) revealing the dense central, fibrous extra-cellular matrix (green arrow). Unidentified microscopic particles also present (orange arrows). Scale bars as indicated. Ames & Klompen et al. (2020).

To validate the potential for envenomation by cassiosomes, rather than solely by suspended intact nematocysts in the mucus released by medusae, Ames & Klompen et al. used LC-MS/MS analyses to confirm the presence of the same CrTX/CaTX toxin proteins in two Cassiopea xamachana sample types: cassiosomes isolated from mucus released from about 20 Medusae over a 7 hour period, and several vesicular appendages containing cassiosome nests, dissected from multiple Medusae. A shotgun proteomic analysis identified three isoforms of the target toxin family encoded in the Cassiopea xamachana genome, which Ames & Klompen et al. call CassTX-A, CassTX-B and CassTX-C. Each toxin protein was identified with multiple unique peptides and a minimum of 17.0% protein coverage, with the exception of CassTX-C, which in the cassiosomes was not assigned with sufficient confidence.


Characterization of cassiosome ultrastructure. (a)–(f) Individual cassiosome fixed and labeled with Tubulin Antibody, ActinGreen™ and NucBlue™, and mounted in 80% glycerol in phosphate buffered saline on glass slides for imaging. Imaging was performed with both differential interference contrast and confocal laser scanning with lines at 405, 488, 561, and 640 nm, and collected with a Plan Apo × 100 objective. (a) Differential interference contrast reveals peripheral layer of nematocytes bearing spherical O-isorhiza nematocysts (lavender arrows). Tubulin (red) reveals cnidocils (short filaments marked by white arrows) extending  from apex of nematocytes, and motile cilia (long filaments) originating from non-nematocytes ectoderm cells organized in patches along the peripheral layer among nematocyte-rich areas. NucBlue (blue) reveals nuclei (yellow arrows) of peripheral epithelial layer; nematocytes and other ciliated ectoderm cells. Actin (green) reveals actin basket (pink arrows) formed around the apex of nematocysts. (b) 3-D construction of Z-stack magnified confocal images corresponding to (a) and (c)–(f) shows tubulin (red) of cnidocils (short filaments marked by white arrows) extending from around apex of nematocytes and motile cilia (long filaments) originating from non-nematocytes putative ectoderm cells organized in patches along the peripheral layer among nematocyte-rich areas. NucBlue (blue) reveals nuclei of peripheral epithelial layer; nematocytes and other ciliated ectoderm cells. Actin (green) reveals actin basket forming around the apex of nematocysts. Scale bars are 10 μm. Ames & Klompen et al. (2020).

A visual inspection of Cassiopea xamachana oral arms during mucus release revealed that cassiosomes occur as warty clusters within a shallow pocket on the vesicular appendages which are formed of ectoderm, endoderm and mesoglea; the cavity of these appendages communicates with the canals of the oral arms. Vesicular appendages are capable of independent movement, and during feeding of lab-reared  Cassiopeaxamachana Medusae, when Artemia nauplii approach the oral arms, the vesicular appendage bends to cover the Shrimp, thereby trapping the prey item; this trapping method was also reported in the conspecific Cassiopea frondosa. Clusters of cassiosomes (i.e., 30–100 individuals) line the surface of the numerous, variably sized vesicular appendages present in  Cassiopea xamachana. In 1900 Henry Bryant Bigelow called these appendages nettle batteries, referring to their functional role in subduing prey, and possibly also in defense.


Characterisation of the ultrastructure of the vesicular appendages during cassiosome production and development in Cassiopea xamachana. Line drawing of vesicular appendage demonstrates how early developing cassiosome protrusions (pro) are connected peripherally to the pocket surface of the vesicular appendage by their shared epithelium (epi vap), whereas fully developed cassiosomes (cass) awaiting deployment are only loosely attached to the pocket and neighboring cassiosomes. (a)–(e) Semithin sections (about 1 μm) of resin-embedded vesicular appendages corresponding to arrows labeled (a)–(e) in the line drawing of the vesicular appendages (va) extending from the oral arms (arm) of the medusae. Clusters of cassiosomes (pink arrows) developing from protrusions (pro) in the epithelium of the concave vesicular appendage pocket (epi vap) give rise to the cassiosome peripheral layer comprising nematocytes bearing O-isorhiza nematocysts (dark spheres stained with 1% toluidine blue) interspersed with other ectodermal cells. Clusters of amoebocytes hosting Symbiodinium (green arrows) move into the cassiosome core at protrusions points. Cassiosome core containing presumptive mesoglea indicated by difference in diffractive index with differential interference contrast. (f) Partial 3-D reconstruction showing protrusions developing from epithelium of the vesicular appendage pocket (epi vap) into popcorn-shaped cassiosomes. Reconstruction based on sections from a different vesicular appendage than seen above but corresponds to the region between sections (a)–(d), revealing the empty core (core) of cassiosomes (cass) (3-D image orientation is vertical with respect to cross sections in the line drawing). Abbreviations: arm, medusa oral arm; cass, cassiosomes(s); core, presumptive mesoglea; pro, protrusion(s); va, vesicular appendage(s); epi vap, epithelial layer of the vesicular appendage pocket. Scale bar is 250 μm. Ames & Klompen et al. (2020).

Images of semithin sections of five separate vesicular appendages revealed that cassiosomes develop within a depression externally on one side of a vesicular appendage, but occasionally on both sides. During development, cassiosomes originate proximally as protrusions of the epithelium (ectoderm) of the vesicular appendage, and then spread out distally as they develop, incorporating presumptive amoebocytes (endoderm cells that have migrated into the mesoglea), some of which host Symbiodinium. Early developing cassiosome protrusions are connected peripherally to the pocket surface of the vesicular appendage by their shared ectoderm epithelial layer, whereas fully developed cassiosomes awaiting deployment are only loosely attached to the pocket and neighboring cassiosomes. This development process results in irregular popcornshaped cassiosomes, as shown in the 3-D reconstruction of their organisation within the vesicular appendages, based on semithin images.


Early report of cassiosome nests and detailed documentation in Ames & Klompen et al.'s study. (a), (b) Line drawing of putative cassiosomes being released by Cassiopea frondosa, identified as 'grey bodies' or a 'nematocyst mass' through an apparent opening in the vesicular appendage, modified from Smith, H. G. (1936) Contribution to the anatomy and physiology of Cassiopea frondosa. Tortugas Laboratory of Carnegie Institution of Washington.Volume  XXIX. pp 18–52. Abbreviations: g.b.,  nematocyte mass and op., opening at tip of 'oral vesicles' (i.e., vesicular appendage). (c), (d) Vesicular appendages (green arrows) of Cassiopeaxamachana photographed study lacking aperture (only a groove with no opening occurs at the tip, white arrow). In Cassiopeaxamachana, cassiosomes are shed from loosely organized nests (pink arrows) and released within mucus. Ames & Klompen et al. (2020).

The peripheral layer (nematocytes and ectoderm) surrounds a central space containing clusters of amoebocytes often hosting Symbiodinium, randomly interspersed among clear empty patches that exhibit substantially different refractive index properties (as seen in differential interference contrast microscopy) reminiscent of the small bags of mesoglea and nematocysts witnessed being released in the colosely related Cassiopea frondosa by HG Smith. These findings corroborate those of Ames & Klompen et al.'s scanning electron microscopy and confocal analyses, and suggest the central region of cassiosomes is amorphous, containing only some loose cells, likely amoebocytes, many of which host Symbiodinium.


Characterization of the ultrastructure of mature cassiosomes in Cassiopea xamachana. (a) Line drawing, and (b), (c) thin sections of fully developed popcorn-shaped cassiosomes from semithin sections (about 1 μm) of resin-embedded vesicular appendage. Cassiosome peripheral layer comprising nematocytes (cyan arrows) bearing O-isorhiza nematocysts (as peripheral dark spheres stained blue with Richardson’s stain in (b) and (c)) interspersed with patches of oddly shaped ectoderm cells (red arrows), and motile cilia (pink arrows); blue-stained nuclei (yellow arrows) visible below the base of large nematocysts capsule in nematocytes, and also in non-nematocyte ectoderm cells. Cassiosome core containing presumptive mesoglea (gray central region in (a), gray arrow in (b) and (c)), speckled with amoebocytes (purple arrow), hosting Symbiodinium (green arrows) or empty. Rigid stereocillia/cnidocil complex (orange arrows) visible as a point at the nematocyst apical portion, and deployed tubules (black arrows in (a)) on surface present as long, thick spiny threads. Abbreviation: epi vap, epithelial layer of the vesicular appendage pocket. Scale bars are 50 μm. Ames & Klompen et al. (2020).

Jellyfish of the taxonomic order Rhizostomeae, including Cassiopea xamachana, all lack marginal tentacles, possessing instead oral arms covered with minute vesicular appendages. Although the main focus of Ames & Klompen et al.'s study is to provide a detailed description of cassiosomes in Cassiopea xamachana, in an effort to ascertain if cassiosome production is a possible apomorphy of the Rhizostome Jellyfish clade, they examined the mucus of additional Rhizostome Jellyfish taxa and documented cassiosomes in a total of four Rhizostome Jellyfish lineages (five different species). Cassiosomes from all six species are classified into two main types: motile, bearing cilia that propel them in the water column and nonmotile, bearing no apparent motile cilia. Mucus was directly examined (using light microscopy) from three additional Rhizostomes, the Spotted Jelly, Mastigias papua, the Floating Bell, Phyllorhiza punctata, and the Jelly Blubber, Catostylus mosaicus, as well as a single Semeastomeae (sister group) species, the Moon Jellyfish, Aurelia sp., all reared at the National Aquarium in Baltimore, USA. Additionally, Ames & Klompen et al. obtained a video from the author of the Jelly Club citizen scientist blog showing abundant motile particles reportedly released by another Rhizostome, the Crown Jellyfish, Netrostoma setouchianum, collected in Japan. Although Ames & Klompen et al. were unable to directly examine these cellular masses from  Netrostoma setouchianum, their motility, the irregular shape they possess when released from the oral arms, and the eventual loss of bumpiness and disappearance after several days matches the general description of cassiosomes Ames & Klompen et al. first discovered in Cassiopea xamachana. Cassiosomes of Mastigias papua and Phyllorhiza punctata Medusae are highly motile, and share the same fundamental structure, albeit exhibiting slight variations with respect to nematocyst types present within the peripheral nematocyte layer of each type. Superficially, Netrostoma setouchianum cassiosomes appear to match the morphology of the two aforementioned species, however, as Ames & Klompen et al. were not able to examine them directly using microscopy (solely via video), the presence of associated Dinoflagellates could not be confirmed. Conversely, cassiosomes in Catostylus mosaicus exhibit several differences in that neither motility nor centralised Symbiodinium were observed but, rather, unidentified Microalgae are distributed homogenously throughout the cell mass. No cassiosomes were found in the mucus of the Semaeostome Aurelia sp. which lacks both vesicular appendages and endosymbiotic Algae.

Cassiosomes observed in jellyfish species of the order Rhizostomeae: Cladogram of species examined in this study from two orders Rhizostomeae: (a)–(c) Cassiopea xamachana, (d)–(f) Mastigias papua, (g)–(i) Phyllorhiza punctata, (j)–(l) Netrostoma setouchianum, and (m)–(o) Catostylus mosaicus, and (p) Semaeostomeae: Aurelia sp., and their respective cassiosome structures, when present. Abbreviations: iso, isorhiza nematocysts; rhp, rhopaloid nematocysts; *, could not confirm type of nematocysts. Blue symbols: star, motile via cilary movement; hexagon, non-motile; circle, endosymbiotic Dinoflagellates within cassiosomes confirmed; oval, Microalgae on the surface of cassiosomes confirmed; asterix, could not confirm presence or absence of Algal symbioints; X, no cassiosomes witnessed within the mucus.Scale bar: (a) 1.5 cm; (d), (g), (j), (m), (p) 2.5 cm; (b), (e), (h), (k), (n), (o) 300 μm; (c), (i), (l) 200 μm; (f), (o) 100 μm. Ames & Klompen et al. (2020).

Jellyfish are remarkable aquatic animals that diverged over 600 million years ago and have in spite of, or possibly because of, their diploblastic nature, evolved a remarkable envenomation system in the form of stinging cells, nematocytes, for prey capture and defense. Ames & Klompen et al. report the findings of an extensive investigation into the provenance, development and ultrastructure of cassiosomes, a newly described cnidarian stinging-cell structure. Based on these findings, they hypothesise that cassiosomes evolved within a single lineage of Jellyfish, Rhizostomeae, to further weaponise the Jellyfish by sequestering nematocytes (and other cells) into grenade-like structures that are freely released into the water within exuded mucus. Ames & Klompen et al.'s findings strongly implicate cassiosomes as a major contributor to the stinging water phenomenon reported by sea bathers and aquarists when interacting with Rhizostome Jellyfish species.

Ames & Klompen et al. used extensive microscopy techniques, video-documentation and microfluidics to describe these cnidarian innovations first in Cassiopea xamachana, and then in taxa belonging to four additional Rhizostomeae Jellyfish families. Their preliminary findings suggest there are motile and non-motile types of cassiosomes among the Rhizostomes they examined in this study, and that some host endosymbiotic Algae Symbiodinium while at least one bears Microalgae instead. However, the fundamental trait distinguishing cassiosomes from nematosomes, analogous cell masses deriving from the mesenteries of the Sea Anemone Nematostella, is that the structure of cassiosomes is organized into a distinct outer epithelial layer surrounding a central, mostly empty, core. Further studies are needed to elucidate the role of these photosynthetic endosymbionts in cassiosomes. The complete absence of cassiosomes in the mucus released by the Semeastome Aurelia sp. supports Ames & Klompen et al.'s theory that cassiosomes are a Rhizostome evolutionary novelty. However, a comparative examination of the mucus contents across all Rhizostome lineages, including the eight nominal Cassiopea species is needed to test this hypothesis.

Furthermore, Ames & Klompen et al. identified the provenance of cassiosome production and release from oral arm vesicular appendages, corroborating earlier works suggesting these vesicles (as oral vesicles) function in defense and predation. These previous works noted similar structures in the oral vesicles of Cassiopea species, dubbed either gray bodies, bags of nematocysts and mucous cells, minute spherical bodies, or grenades, that shot when contacted. Although those reports fell short of providing an adequate description,  Ames & Klompen et al. are confident that the structures mentioned therein correspond to what they described as cassiosomes. The mechanism of cassiosome deployment may vary across different Rhizostome taxa, or even between closely related species, as according to Horace Smith, when prey was provided to Cassiopea frondosa, an aperture opened at the tip of the vesicular appendages releasing gray bodies (putative cassiosomes). Conversely, in Ames & Klompen et al.'s study on Cassiopea xamachana, upon disturbance, cassiosomes spontaneously detached from the surface of vesicular appendage pockets, which lack a terminal aperture.

All Jellyfish have envenomation capabilities due to bioactive proteins comprising the venom cocktail of the cnidome (i.e., repertoire of nematocysts types). The Cnidarian-specific poreforming CrTX/CaTX toxin family is one of the most potent toxin groups, and represents the main proteinaceous component of the venom of Cubozoans (Box Jellyfish), a clade that includes species whose sting results in a deadly cardiovascular condition. In Ames & Klompen et al.'s study, the presence of Cassiopea xamachana-specific CrTX/CaTX toxin family homologs was confirmed in cassiosomes at the DNA and protein level (i.e., CassTX), validating their expression in cassiosomes and their contribution to the stinging water phenomenon.

Although Cassiopea xamachana in Florida waters is considered a mild stinger, reports exist of painful Human envenomation resulting in rash, vomiting, painful joints, swelling and irritation for this broadly distributed species, in addition to documentation of hemolytic and cytolytic activity in crude venom of Cassiopea xamachana and close relatives. Following a characterisation of the cnidome in Cassiopea xamachana in this study, Ames & Klompen et al. discovered a large proportion of undeployed penetrant rhopaloid nematocysts in released mucus. These findings suggest that the toxic mucus phenomenon is due to the combined effect of cassiosomes and free, undeployed nematocysts.

Overall, the topic of Jellyfish mucus is an understudied field, despite the ecological importance of mucus in antimicrobial and environmental stress protection and chemical defense, relevance of toxic bioactive compounds found within venom, and the potential importance to non-Cnidarian taxa. Given the recent publication of the first reference genome for Cassiopea xamachana, a thorough investigation into the molecular pathways underlying the development and release of cassiosomes and comparisons with nematocyst-enriched structures in other Cnidarians (e.g., nematosomes, acontia and acrorhagi) can now be undertaken.

See also...


https://sciencythoughts.blogspot.com/2020/04/large-numbers-of-cannonball-jellyfish.htmlhttps://sciencythoughts.blogspot.com/2020/03/crambione-cf-mastigophora-bloom-of.html
https://sciencythoughts.blogspot.com/2019/12/huge-swarms-of-moon-jellyfish-seen-in.htmlhttps://sciencythoughts.blogspot.com/2019/10/closure-of-nuclear-power-plant-allows.html
https://sciencythoughts.blogspot.com/2019/10/millions-of-moon-jellyfish-seen-in.htmlhttps://sciencythoughts.blogspot.com/2019/01/mercury-and-selenium-levels-in.html
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      MAKANYA SENGAJA NAMA BELIAU SAYA CANTUNKAN DI INTERNET !!!

      assalamualaikum wr, wb, saya IBU SUCHI saya Mengucapkan banyak2
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