​First described as Clade E (Toller et al., 2001a), and later reclassified as Clade D, finally in 2018 this clade of symbionts was designated as its own genus: Durusdinium of the family Symbiodiniaceae (LaJeunesse et al., 2018). Durusdinium has recently garnered lots of attention because of its ‘superior’ adaptations in the face of adversity. The name Durusdinium means “tough” and “whirling” (LaJeunesse et al., 2018), and, unlike other genera of Symbiodiniaceae commonly associated with corals (e.g., Breviolum and Cladocopium), members of Durusdinium are generalist extremophiles and are exceptionally tolerant to environmental stressors, including fluctuations in temperature, salinity, nutrients, sediments, turbidity, air exposure, rainfall, and light intensity (LaJeunesse et al., 2018; Muller-Parker et al., 2015; Toller et al., 2001​a; Toller et al., 2001b). ​Because of its exceptional ability to tolerate a multitude of environmental stressors, Durusdinium is commonly found in symbioses with hosts that are settled in less than favorable environments, like nearshore coastal reefs, coastal lagoons, and tidal pools where conditions are harsher and bleaching events are more common (Mashini et al., 2015; Muller-Parker et al., 2015; Toller et al., 2001a; Toller et al., 2001b).

​A noteworthy study by Baker et al. (2004) analyzed symbiont populations in coral hosts following a major bleaching event in the Indo-Pacific caused by the 1997-98 El Niño—Southern Oscillation (ENSO). Interestingly, they found that the coral colonies that survived the bleaching event were predominated by members of Durusdinium, lending credit to the theory that the tenacious adaptations of Durusdinium not only help the symbiont tolerate hostile conditions, but in turn bolster their host’s immunity to environmental stressors (Baker et al., 2004; LaJeunesse et al., 2009; LaJeunesse et al., 2018).
 
LaJeunesse et al. (2009) found similar results when they analyzed the symbiont populations of coral hosts that survived the 2005 mass bleaching event in the Caribbean; upon analysis, LaJeunesse et al. (2009) found that many of the coral colonies that survived the bleaching event harbored amplified populations of Durusdinium. Like the corals in Baker et al.’s study (2004), these corals’ susceptibility to environmental stressors appears to have been mitigated by the presence of Durusdinium, with corals disproportionately predominated by Durusdinium having an increased ability to withstand thermal stress and hence resist dissociation (i.e., bleaching) (LaJeunesse et al., 2009; LaJeunesse et al., 2018).
 
In the same vein, Manzello et al. (2018) came to analogous conclusions upon analysis of symbiont populations in coral colonies located in the Florida Keys following record-high ocean temperature spikes in sequential years (in 2014 and 2015). Manzello et al. (2018) discovered that during the 2014-15 bleaching event, corals in the Florida Keys had shifted away from hosting specialist symbionts (e.g., Breviolum), instead being in favor of hosting more resilient symbionts, specifically members of Durusdinium (Manzello et al., 2018). Manzello et al. (2018) found a significant relationship between Durusdinium presence and resistance to coral bleaching, with greater than 90% of the corals that survived the record bleaching event being primarily colonized by Durusdinium.
 
Likewise, Toller et al. (2001a) found Durusdinium to be the predominate symbiont in Montastraea (e.g., star corals) located in Panamanian regions known for their harsh environmental conditions. At Río Cartí, San Blas (a site that suffers from heavy freshwater runoff and increased sediments caused by its nearshore location in close proximity to the mouth of the major river: Río Cartí Grande), for the corals sampled, 35 of 43 corals were predominated by Durusdinium, and Durusdinium was the sole zooxanthella present for 18 of these corals (Toller et al., 2001a). Similarly, at Bocas del Toro (a large coastal lagoon known for exceptionally high rainfall and less than favorable water quality), Durusdinium was inordinately common in the sampled corals (Toller et al., 2001a). Based off their results, Toller et al. (2001a) concluded that Durusdinium’s stress-tolerant nature enables this symbiont to endure these environmental stressors, enabling its host coral to survive despite the hostile environmental conditions (Toller et al., 2001a).
 
In recent years, Huang et al. (2020) conducted a field experiment in which they transplanted Leptoria phrygia (e.g., smooth brain coral) between two sites with significant temperature differences and monitored the corals and their symbiont populations for 12 months. As theorized, in an effort to combat bleaching, the corals acclimatized to the thermal stress by ‘shuffling’ or ‘switching’ to hardier symbionts; specifically, the corals formed stable and strong associations with members of Durusdinium as a means of preventing bleaching (Huang et al., 2020).

​In 2001, Toller et al. conducted a series of field experiments in which they simulated bleaching events in Montastraea by either transplanting them to shallow water (high light exposure) or shading them (low light exposure), and then they monitored their symbiont composition over the next several months. As predicted, the simulated bleaching event encouraged corals to associate with new zooxanthellae (Toller et al, 2001b). Many of these corals that bleached in the experiment subsequently formed new relationships with members of Durusdinium and remained repopulated by Durusdinium even 9 months post-recovery (Toller et al, 2001b). Intriguingly, even when a bleached coral still had a small reservoir of Breviolum or Cladocopium within its tissues post-bleaching, these remaining symbionts were not sufficient to prevent the establishment of new symbiont associations with members of Durusdinium in the coral’s bleached tissues (Toller et al., 2001b). Toller et al. (2001b) concluded that despite conditions of stress, Durusdinium remains a robust and thriving opportunist, and, in fact, seems to excel under these hostile environmental conditions. As such, this symbiont is adept at repopulating bleached corals and helping them recover post-bleaching (Toller et al., 2001b).
 
More recently, Wang et al. (2022) conducted a laboratory experiment on the host coral Pocillopora damicornis (e.g., cauliflower coral) in which they manipulated applied heat and cold stress and analyzed the symbiont-host relationship during both the period of applied stress and recovery. Results of their experiment concluded that when temperature was manipulated, corals lost most of their original symbionts (e.g., Cladocopium) and became predominated by Durusdinium. Their study found that Durusdinium has enhanced photochemical efficiency while its host coral is under thermal stress; this in turn improves growth via stimulating algal photosynthesis and carbon translocation to the host coral, making Durusdinium the preferential symbiont during conditions of thermal stress and recovery (Wang et al., 2022).

​Research has demonstrated that Durusdinium is both adept at bolstering its host’s invulnerability to bleaching, as well as repopulating a host and aiding in its recovery post-bleaching (Baker et al., 2004; Huang et al., 2020, LaJeunesse et al., 2009; LaJeunesse et al., 2018; Manzello et al., 2018, Toller et al., 2001a; Toller et al., 2001b; Wang et al., 2022). The evidence suggests that we can expect wild reefs to make adaptive shifts to Durusdinium (via ‘shuffling’ and/or ‘switching’) as both a means of recovery in the unfortunate event that the host has already succumbed to bleaching, as well as a preventative mechanism for resisting future bleaching events (Baker et al., 2004). Especially as oceans continue to get warmer, we may find that Durusdinium becomes increasingly prevalent among wild reefs; especially for hosts with greater sensitivity to thermal stress, it is likely that Durusdinium will play a pivotal role in their continued survival (La Jeunesse et al., 2009).

Whether in the wild or in captivity, hosts in symbioses primarily with members of Durusdinium are more likely to resist bleaching, survive through bleaching events, and recover from bleaching (Baker et al., 2004; Huang et al., 2020, LaJeunesse et al., 2009; LaJeunesse et al., 2018; Manzello et al., 2018, Toller et al., 2001a; Toller et al., 2001b; Wang et al., 2022). When searching for strains of symbionts for our REVIVE™: Live Zooxanthellae blend, Durusdinium’s aptitude at swiftly repopulating and aiding in the recovery of bleached corals made it an ideal candidate. Furthermore, the ability of this robust extremophile to withstand and thrive in a wide array of environmental stressors makes it the perfect addition to reef aquariums where it may act as a preventative against future bleaching episodes.

Two of the four new strains that we are currently having success with are from the genus Cladocopium (formerly Clade C). One of the new strains is C. goreaui and was isolated from a Rhodactis osculifera (formerly Discosoma sanctithomaei; commonly known as the “St. Thomas Mushroom” in the aquarium hobby) in Jamaica. The other strain of Cladocopium was isolated from a Mastigias papua (i.e., lagoon jellyfish) in Palau. While we have had some prior success commercially producing strains from this genus, Cladocopium is notoriously difficult to culture, so we are thrilled that these two new strains are currently doing so well at our facility.
​
Strains of zooxanthellae from Cladocopium are of great interest for use in a product like REVIVE™, as members of this genus are native symbionts of many of the symbiotic host invertebrates found in the aquarium trade. Cladocopium is the most ecologically abundant and broadly distributed genius of Symbiodiniaceae, and is commonly found in symbiosis with a plethora of corals, other cnidarians, clams, etc. (LaJeunesse et al., 2018; Riddle, 2016). This specialist genus of zooxanthellae is adapted for thriving in conditions that mimic a natural coral reef ecosystem, and symbionts of this genus are likely to be selected for uptake by many of the host species found in a typical reef aquarium.

The other two promising new symbionts are unique strains of Durusdinium (formerly Clade D). One of the new strains of D. trenchii that we are culturing was isolated from an Acropora sp. in Okinawa, and the other was isolated from an Orbicella faveolata (i.e., Mountainous Star Coral) in the Florida Keys.
 
We have hoped for a while to bring this particular genus of Symbiodiniaceae to the aquarium industry, as research has shown that members of Durusdinium tend to be highly robust and stress-tolerant, bolstering their host’s immunity to environmental stressors (LaJeunesse et al., 2009; LaJeunesse et al., 2018). In the last twenty years, numerous studies have illustrated that Durusdinium is remarkably resilient, with these extremophiles being specially adapted to tolerate harsh conditions including: significant fluctuations in temperature, salinity, nutrients, sediments, turbidity, air exposure, rainfall, and light intensity (LaJeunesse et al., 2018; Mashini et al., 2015; Muller-Parker et al., 2015; Toller et al., 2001a). Research has shown that corals in symbioses primarily with members of Durusdinium are more likely to resist bleaching, survive through bleaching events, and recover from bleaching (Baker, 1999; Baker et al., 2004; LaJeunesse et al., 2009; LaJeunesse et al., 2018; Manzello et al., 2018, Toller et al., 2001a; Toller et al., 2001b; Wang et al., 2022). Theoretically, including strains of Durusdinium in a product like REVIVE™ could be revolutionary, significantly increasing aquarists’ ability to ‘revive’ bleached inhabitants.

​Currently we are in the process of further refining our culture protocols for each of these four new strains and gradually ramping up their production. Our goal is to continue to optimize our culture methods and soon be able to reliably produce these new strains in commercial volumes. Once we have achieved consistent commercial-scale volumes, we will begin the extensive ‘Testing Phase’ that each of our strains currently utilized in REVIVE™ has undergone. During the ‘Testing Phase’ we will ensure that these new strains have the potential to be beneficial to reef aquarium inhabitants (and in no way harmful to them). We will also analyze each strain’s ability to withstand periods of prolonged refrigerated storage, as every strain included in REVIVE™ must be able to achieve a minimum four-month shelf life in order to be viable for commercial distribution.

In sum, while we are excited that these four new strains are the most promising zooxanthella cultures that we have worked with in a very long time and we are eager to add them to REVIVE™, it will still be a while before any of these new strains will be ready for commercial distribution. However, we feel confident that the wait will be worth it! With every new zooxanthella strain added to REVIVE™, we enhance its overall efficacy and increase the capacity for aquarists’ success maintaining their reef aquariums.
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Stay tuned as we continue to work to bring these new strains to you and your aquarium. Cheers!

​The ovoid morphotype is either oval or round in appearance and possesses a raphe that enables motility (Tesson et al., 2009). This shape is preferentially benthic, with ovoid cells tending to clump together and sink to the bottom (Lewin et al., 1958). In laboratory cultures the ovoid morph usually predominates cultures grown on solid media (e.g., agar plates) or is found adhering to the walls of its culture vessel (Barker, 1935; Lewin et al., 1958; Tesson et al., 2009). Research has shown that the elongated versus rounded shape of the ovoid morphotype is dependent upon culture conditions, with a tendency for ovoid cells to take on a more circular shape when stressed (e.g., nutrient depleted) and an elongated shape when healthy and thriving (Tesson et al., 2009).

​The fusiform morphotype is best described as spindle or peapod shaped. In laboratory cultures grown in liquid media, the fusiform morph usually predominates (Barker, 1935; DeMartino, 2007; Lewin et al., 1958). The large peripheral vacuoles of the fusiform morph make this cellular shape significantly more buoyant than ovoid cells (Lewin et al., 1958; Tesson et al., 2009). Therefore, in an actively growing laboratory culture, fusiform cells will be found floating throughout the liquid media (Lewin et al., 1958).

​The triradiate morphotype is three-rayed in appearance and resembles the shape of a three-pointed star. Like the fusiform morphotype, the triradiate morphotype is very buoyant and therefore best suited for a planktonic lifestyle (DeMartino et al., 2007; Tesson et al., 2009). This morph usually thrives in standard laboratory cultures, and occasionally even outcompetes fusiform cells, especially if the culture is static. It is theorized in these instances that the triradiate cells were favored by selection due to their increased buoyancy (and decreased rate of sinking) enabling them to outcompete other cellular shapes (Lewin et al., 1958).

Aquaholic Aquaculture was founded ten years ago on April 22, 2015. ​Our company was established on Earth Day as a symbolic representation of our mission to foster marine conservation and sustainability within the aquarium industry through marine ornamental aquaculture. Aquaholic Aquaculture's mission is grounded in a love for the aquarium hobby and a devotion to marine conservation. At Aquaholic Aquaculture, all of our live products (from live feeds to marine fish and coral) are 100% aquacultured.

In 2017, we released a very unique plankton product: REVIVE™: Live Zooxanthellae. We had worked for years behind the scenes trying to acquire and mass culture various species of zooxanthellae (i.e., members of the family Symbiodiniaceae), and finally had success with some cultures of Symbiodinium, Breviolum, and Cladocopium. We are very proud of this product, as it is the only of its kind available to the aquarium industry and since its release has helped numerous aquarists save their bleached corals (see our Testimonials).  ​

While many members of the family Symbiodiniaceae can live independently of an animal host, it is challenging to find species that can thrive asymbiotically for long-term culture in a laboratory. As phototrophs, laboratory cultures require adequate light and nutrients in order to grow. While zooxanthellae living within an animal host are primarily found in the coccoid (non-motile) stage, in laboratory cultures zooxanthellae tend to alternate between both the coccoid and dinomastigote (motile) stages (Muller-Parker et al., 2015). Like other species of microalgae grown for the aquarium industry, species of Symbiodiniaceae reproduce asexually, increasing their population by creating identical clones of the parent cell. Different genera of Symbiodiniaceae have varied growth rates (Toller et al., 2001). In our experience, cultures of Symbiodinium or Breviolum generally multiply faster than Cladocopium cultures.

(1) New Starter Cultures
New starter cultures are difficult and often very expensive to obtain. There are very few resources for obtaining symbiont starter cultures, and these resources usually only have a select few strains available at a given time. Moreover, our specific interest in only acquiring symbionts that have the potential to host aquarium inhabitants further amplifies the challenge of finding an appropriate starter culture.

​(2) Commercial-Scale Volumes
Zooxanthellae are notoriously difficult to grow in a laboratory setting separate from their hosts, and it is very challenging to reliably produce these zooxanthellae at commercial-scale volumes. Most obtainable starter cultures are only available in extremely small quantities (e.g., test tube starters), and it is a slow and arduous process growing out these starter cultures to commercial volumes.

​Acanthophyllia deshayesiana are large, solitary-polyp stony corals that are known for their fluffy flesh and array of vibrant colors. They are eye-catching corals, and a species that many aquarists aspire to own.
 
For most corals, asexual propagation (i.e., fragmentation) is the easiest and fastest method of farming. However, for solitary-polyp corals like A. deshayesiana, asexual propagation is more challenging, theoretically making them better candidates to attempt to farm via sexual propagation.
 
Sexual propagation is significantly more difficult than asexual propagation, which is why many aquaculturists do not attempt this coral farming method. Everything needs to be perfect to encourage broodstock corals to spawn and larvae to settle. Factors such as specific lunar cycles, light intensity, water motion, etc. (usually mimicking the coral’s native environment) need to be refined in order to successfully illicit coral spawning.

​The A. deshayesiana broodstock at ACI Aquaculture spawned twice in January 2024 (on the 14th and 17th days following the December 27th full moon), and I was honored to be able to assist Amanda and Chris with collecting the eggs from the second spawn. On January 10th around 7:30 in the evening, Chris excitedly called saying to come down to ACI because there were “more babies!”. I hurriedly jumped in the car and headed to ACI.
 
Amanda’s and Chris’ pure excitement from finally having successfully spawned A. deshayesiana was infectious. The spawn that I helped collect eggs from was the larger of the two spawns at ACI, and it took a while to carefully collect all of the eggs and transfer them to holding bins. Afterwards, we looked at some of the eggs under a microscope and confirmed that many were fertilized. In fact, several were already developing into motile planula larvae before our eyes.

Zooxanthellae are special microalgae that form a symbiotic relationship with corals (as well as certain other invertebrates, including: anemones, clams, jellyfish, sea fans, etc.). On a taxonomic level, the colloquial term "zooxanthellae" is synonymous to members of the family Symbiodiniaceae. These zooxanthellae live within the host’s tissue and provide the host with oxygen, food, and its iridescent colors. When a host is stressed, it expels its zooxanthellae and ‘bleaches’, losing its color. The symbiotic relationship that these hosts have with their zooxanthellae is necessary for the host’s growth and survival; without their zooxanthellae, hosts lose their color and eventually starve to death.

Corals expel their zooxanthellae when exposed to intolerable environmental stressors (e.g., extreme temperatures, sudden changes in water quality, etc.).
 
In the ocean when corals bleach and expel their zooxanthellae, the zooxanthellae remain in the water column. If the stressor that caused the coral to originally bleach is removed, the coral can uptake zooxanthellae from the water column and recover.
 
In contrast, when corals bleach in an aquarium setting, the expelled zooxanthellae are removed from the water column via mechanical filtration (e.g., protein skimmers). As such, when a coral bleaches in an aquarium, it cannot uptake the zooxanthellae that it expelled because those zooxanthellae have been effectively removed. This is where REVIVE™ comes in! REVIVE™ adds zooxanthellae back into the aquarium that the coral can uptake once the stressor that caused the bleaching event has been corrected.

Corals can be selective as to which zooxanthellae they will form a symbiotic relationship with. Because of this, we try to incorporate a variety of zooxanthellae species into REVIVE™ in order to increase the chances that target corals will be receptive to zooxanthellae uptake.
 
There are seven genera (formerly known as "clades") of Symbiodiniaceae, and numerous species (formerly referred to as "sub-clades") of zooxanthellae within each genus (LaJeunesse et al., 2018). 
 
Of the seven genera of Symbiodiniaceae, the genera Symbiodinium, Breviolum, Cladocopium, and Durusdinium (formerly clades A-D) are the main genera associated with coral symbiosis. As such, we have focused our efforts at Aquaholic Aquaculture on culturing zooxanthellae from these genera.
 
Typically REVIVE™ contains multiple species from either Symbiodinium, Breviolum, and/or Cladocopium. However, the specific zooxanthella composition of REVIVE™ does vary, as it is dependent on which species are thriving at the time that we bottle the product.

All of the species that we have selected thus far for REVIVE™ are from either the genus Symbiodinium, Breviolum, or Cladocopium. The species that we culture were selected because of their relative hardiness and because they are known symbionts of corals kept in marine aquaria.
 
Most of our zooxanthella cultures have been isolated from motile hosts (e.g., jellyfish), and, as such, are able to adapt to various environments as well as rapid shifts in environmental conditions. These species can acclimate to both low and high light levels and are tolerant of both low and high temperatures, with some of them (e.g., Symbiodinium microadriaticum) being able to tolerate temperatures as high as almost 90F (Robinson & Warner, 2006).
 
All of our zooxanthella cultures are known symbionts of corals found in marine aquaria. Most of our Breviolum cultures are from the Caribbean, whereas our Symbiodinium and Cladocopium cultures are Indo-Pacific based. While some of our cultures were isolated from corals, most of our cultures were isolated from jellyfish (Cassiopeia), anemones (Aiptasia), and clams (Tridacna). However, despite being isolated from non-coral hosts, these zooxanthellae are also symbionts of corals in the wild and thrive in corals common in the aquarium industry, including but not limited to: Acropora sp., Capnella sp., Favia sp., Pocillopora sp., Porites sp., Sinularia sp., Stylophora sp., and Zoanthus sp.

Recovery time for most corals fed REVIVE™ is at least one month, so do not expect a miracle overnight. It takes time for these corals to heal after being stressed. Removal of the initial stressor, consistent dosage of REVIVE™, and patience is key to successful coral recovery.
 
To read testimonials from customers who swear by our REVIVE™, please visit our Testimonials page.

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