What is our ultimate plan to prevent coral bleaching?
The symbiotic algae inhabitants of reef-building corals get expelled from their host when their environment becomes stressful due to varying abiotic events including the rise of global ocean temperature, ocean acidification, pollution, and a myriad more (Mcleod et al, 2013). This reaction to environmental stress leaves the coral as a white skeleton and vulnerable to disease and malnutrition. However, this process is reversible if the conditions become favorable again before the corals die. These combined issues regarding the change of the corals’ habitat have threatened not only the mutualistic relationship between them and their algal symbiont, Symbiodinium, but also the entire ecosystem the coral reefs have established. The ultimate objective within our project design is to create the first stable transgenic procedure for Symbiodinium to allow for advancements in altering their genetic make up in hopes of introducing coral bleaching resistant genes.
To organize the project, we split up into five collaborative groups that included culturing algae, extensive researching of resistance genes in Symbiodinium, culturing coral, and research in the transformation of plasmid reporter genes.
This bleaching resistance part of our project entailed extensive research on what gene is in Symbiodinium that we can up or down-regulate to protect them from environmental stressors like heat and ocean acidification. We found that Symbiodinium has a gene that codes for heat shock protein (HSP) type A. Type A HSPs are regulated by a gene that codes for heat shock factor (HSF). Because HSF also regulates other protein chaperones such as dnaJ, up-regulating this gene will have a magnified effect on heat resistance.
Detection of DNA Integration?
Initially, the fluorescent proteins, GFP and RFP, were intended for the detection of gene expression to showcase successful foreign DNA integration. However, GFP that naturally emits from the corals have been speculated to hinder Symbiodinium light absorption, thus hindering the algae’s photosynthetic pathway. Additionally, Symbiodinium autoflruoresces RFP within their chloroplast, which would influence the results and make it harder to verify transformation. Hence, the reporter gene, uidA gene (commonly referred to as the GUS gene), encoding for β-glucuronidase was selected for accessible identification. The production of the enzyme will produce a blue pigment that can be detected by the human eye without additional tools.
What are our constructs?
Due to the nuclear complexity of not only the Symbiodinium genus but the dinoflagellate group itself, successful transformation has been unattainable for the algal symbionts. Not only would we have to design a feasible transformation protocol, but also DNA constructs that can successfully integrate into the nuclear chromosomal DNA, which is permanently condensed. Numerous articles have speculated that delivery of DNA into the nucleus may not even be the problem, but attempting to integrate the foreign DNA into the nuclear DNA and adhering to the divergent Symbiodinium regulatory elements is the most challenging aspect of all.
The DinoIII construct was selected due to being the only published dinoflagellate-optimized plasmid. This plasmid was constructed by the University of Connecticut by integrating sequences of different dinoflagellates and utilizing the TTTT-box. Although their chassis was the heterologous Oxyrrhis marina, the plasmid was constructed in hopes for a standard vector for all dinoflagellates.
pCB302 is a plasmid optimized for Agrobacterium tumefaciens-mediated transformation into plants. The plasmid was regulated by the nos promoter and terminator sequences and was previously used in a published A. tumefaciens transformation co-incubated with S. kawaguti and S. microadriaticum.
Another Agrobacterium tumefaciens mediated plant vector, pBI121, was obtained for its regulatory elements that were conveniently sought specifically for Symbiodinium gene expression. The vector contains the CAMV 35S promoter and terminator, nos promoter, neomycin phosphotransferase II (NPTII) selection marker (kanamycin and neomycin resistance), and GUS reporter gene. pBI121 was important in providing the GUS gene for the other two vectors as well. The plant cauliflower mosaic virus 35S (CAMV 35S) promoter conveniently incorporates the described Symbiodinium promoter elements. In addition, the CAMV 35S and nopaline synthase (nos) terminators both contain the dinoflagellate polyadenylation signal, AAAAG/C, which may explain the transgenic protein expression recorded in Symbiodinium produced in silicon-carbide whiskeys and Agrobacterium tumefaciens-mediated transformation.
Unlike pBI121, DinoIII and pCB302 had to be modified by replacing their reporter genes with the β-glucuronidase gene. All 3 plasmids are going to be utilized and observed to see which one was the most efficient in gene expression.
Culturing of the Algae
Symbiodinium microadriaticum was the appointed chassis to use as a model organism for the coral symbionts. The species’ stock was obtained from Bigelow labs and grown in vitro to be used for future intended transformation attempts. In order to determine the most effective growth conditions, varying factors such as temperature, media, and lights were tested. Quantitative and qualitative measurements were recorded.
Culturing of the Coral
Culturing and growing coral entailed a 4 step process that essentially included mimicking oceanic conditions to successfully grow coral and proceed with aggressive bleaching protocols. The overall goal for this group was to design a protocol that enables us to bleach corals of their existing, bleaching susceptible algae while keeping the corals alive before introducing modified Symbiodinium. If transformation is successful, the coral can be exposed to extreme thermal or pH conditions while still retaining their symbionts. This portion of the project wished to attempt the reconstruction of a bleaching event using at-home aquariums and using Acropora species and one highly resistant coral species, such as Goniopora from the Poritidae family. We used Acropora species of coral because they are the key source of surface reef structure, supporting a high diversity of reef species characteristic of the Indo-Pacific. We also used two other genus of coral species: Seriatopora spp and Stylophora spp of coral species are generally sensitive to thermal stress (Marshall & Baird, 2000; Loya et al., 2001) and ocean acidification (Anthony et al., 2008), and thus are an important indicator group for estimating climate change and ocean acidification effects on coral reefs. They are also relatively easy to culture.
Removing Natural Algae and Replacing with Modified Algae
The first step for the coral group was attempting to successfully culture and grow healthy Acropora spp, Seriatopora spp and Stylophora spp and the resistant coral in the at-home aquariums for at least 3 weeks by mimicking natural, oceanic saltwater conditions including wildlife such as fish, herbivores, bacteria, etc. After successfully growing the coral, the next step is to perform aggressive bleaching of the coral by increasing the water temperature. We did a trial run of a potential at-home bleaching system, which was ultimately unsuccessful, but it gave us more ideas to try in the future. The third step is to perform bleaching protocols by mimicking an ocean acidification event by increasing dissolved CO2 levels in the tanks, thus decreasing the pH levels of the tank. The rationale for this protocol is that CO2 reacts with water by decreasing calcium carbonate levels while raising carbonic acid levels. This leaves the corals with less calcium to build their calcium carbonate shells needed for survival. We wanted to create this phenomenon by manually filtering CO2 into our aquarian systems. The final step after this is to duplicate the previous acclamation and bleaching protocols with Acropora spp, Seriatopora spp, Stylophora spp, and another coral of a more resistant species (such as Goniopora from the Poritidae coral family) introduced into the same aquarian environment. After another 3 week acclimation period where growth differences will be recorded, all of the corals will experience temperature and acidity extremes at varying levels to test resistance to stressors based on the type of stressor. We have also explored using symbiodinium and coral fluorescence in our experiments via confocal imaging. This is a potential method to assess the extent of bleaching and its effects on coral health in future experiments.
We have found several papers that have guided our attempts at transforming our algae. Some labs have been able to transform Symbiodinium but only temporarily. The algae are left either unable to reproduce or they only transiently express the DNA. We’re testing out these protocols and slightly modifying them to produce better results.
One of the methods utilizes the lonza nucleofector electroporation device. This allowed us to electroporate the cells while maintaining them in a salty solution. A regular electroporation device requires that all salts are removed from solution before shocking cells. Symbiodinium are cultured in a salt-based media, so removing salts adds yet another stress on top of the shock. This device should help prevent some of the cells from dying simply due to stress.
The second method we attempted involved agrobacterium tumefaciens, which is known for infecting plant cells and inserting its DNA into them. We had to first transform the agrobacterium with the pcb302 plasmid. We then mixed glass beads and Symbiodinium into an overnight culture of this transformed bacteria and shook the solution. The glass beads poke holes into the algae’s tough cell wall, which should make it easier for DNA to enter the cell and integrate into the genome.
This is very similar to the agrobacterium- mediated transformation except it doesn’t use agrobacterium. The protocol calls for simply adding DNA into the solution
This method uses a standard biorad electroporation device on the yeast setting.
Silicon Carbide Whiskers
This technique was one of the first used for transforming algae. This technique is less common now because of safety concerns. We attempted this method under a fume hood to prevent any respiratory injuries.
Biolistics Gene Particle Delivery System
This system shoots DNA into the cell chloroplasts using a gene gun.
1. Brittany N. Sprecher, Huan Zhang, Senjie Lin (2019, April 9). Nuclear gene transformation in a dinoflagellate.doi: 10.1101/602821
2. Mario Fernando Ortiz-Matamoros, Tania Islas-Flores, Boris Voigt, Diedrik Menzel, František Baluška, Marco A. Villanueva (2015, July 13). Heterologous DNA Uptake in Cultured Symbiodinium spp. Aided by Agrobacterium tumefaciens. doi:10.1371/journal.Pone.0132693
3. Ten Lohuis, M. R., and Miller, D. J. (1998). Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs. Plant J. 13, 427–435. doi: 10.1046/j.1365-313X.1998.00040.x