Sea urchins should not only be viewed as venomous terrors of the sea. These spikey seabed dwellers are critical to maintaining the health of coral reefs. Unfortunately dead long-spined sea urchins of the species Diadema antillarum are reported to be ‘rolling like tumbleweeds’ across the Caribbean sea floor in a catastrophic die-off of an unknown cause.[1] This die-off is reminiscent of the mass mortality of the same species that occurred in the 1980s, which spread from the coast of Central America northward across the Caribbean Islands to Bermuda.[2] The exact cause of the 1980’s die-off was never discovered though mass mortality followed the direction of currents, indicating a waterborne pathogen may have been responsible.[3] The current death rate of D. antillarum is especially alarming as their population densities have not recovered since the 1980s, and population currently stands at a mere 12% of their pre-die-off numbers.[4]
These sea urchins are integral components of marine ecosystems, acting as ocean lawn mowers that graze upon macroalgae on the ocean’s coral reefs.[5] The 1980s die-off emphasized the importance of D. antillarum in controlling algal abundance and community structure.[6] The diminished populations of D. antillarum resulted in a 75–95% decrease in coral cover over a six-year period as a result of corals being smothered to death by algal overgrowth.[7] The current mass mortality of D. antillarum is likely to inflict further harm on coral populations in the Caribbean. Rich in biodiversity, coral reefs are one of the most valuable ecosystems in the world providing habitats for thousands of marine organisms, protecting shorelines from erosion, and adding profit to local economies through tourism and fisheries.[8] As a result of their immense value, the dwindling numbers of coral reefs is extremely worrisome. Therefore, the study of algal grazers, organisms that maintain the health of coral reefs, is critical to making informed conservation decisions in marine ecosystems worldwide.
| Figure 1: Coral bommies in Sosoikula Reef, Suva, Fiji (Coppard). | Figure 2: Algal assemblage on Sosoikula Reef, Suva, Fiji (Coppard). |
Diadema antillarum is a member of the family Diadematidae. Another species belonging to the Diadematidae, Echinothrix calamaris, is distributed across the Indo-Pacific, including the Red Sea, and is an important grazer on coral reefs.[9] Since its discovery, E. calamaris was believed to be a single species. However, recent research by Coppard et al. determined that E. calamaris, actually contains three species, one of which inhabits the Red Sea and Gulf of Oman, and two others which have overlapping distributions across regions of the Indo-Pacific (see Figure 3).[10] These latter species lack spatial isolation as they occur side by side on coral reefs and temporal isolation as they both spawn on or around the new moon.[11] Spawning is a form of mating in which organisms release gametes (sperm or eggs) into the water at the same time in hopes they will bind with each other and produce a fertilized egg.
Figure 3: Phylogeography of the Echinothrix. Clade 2 and Clade 3 of E. calamaris represent discrete species with sympatric distributions across the Indo-Pacific Ocean (Coppard et al., 2021).
Although both species live in the same area and spawn at the same time, they do not exhibit hybridization or introgression. Hybridization occurs when two individuals, each of different species, interbreed to produce a hybrid offspring. Introgression occurs when genetic information is transferred from one species to another.[12] This genetic information is then incorporated into the latter species population through a process known as backcrossing, in which a hybrid offspring mates with a parent or an individual genetically similar to their parent.[13] Given the lack of spatial and temporal isolation between the two species in the Indo-Pacific, what keeps the species separate and prevents hybridization or genetic introgression? This is the question that I am trying to answer under the supervision of Dr. Simon Coppard. Dr. Coppard and I hope that by determining the mechanisms by which species maintain their integrity and avoid hybridization, we will further our understanding of evolution and speciation, and improve conservation decisions for Echinothrix calamaris.
| Figure 4: E. calamaris from Korolevu, Fiji (Coppard). | Figure 5: E. calamaris with porities in Sosoikula Reef, Suva, Fiji (Coppard) |
The aim of this research project is to examine sperm recognition genes from members of E. calamaris that reside in the Indo-Pacific to determine whether gametic isolation (incompatible sperm and eggs) is preventing interbreeding between the newly identified species. Genes are short sequences of DNA that carry the instructions to make proteins. These instructions are written by nucleotides, individual building blocks that are linked together to make a sequence. Nucleotides can be ordered in countless ways to create many different sequences and thus many different proteins. The proteins that genes encode dictate physical traits and bodily functions. Generally, members of the same species share genes with sequences that are nearly identical. It is genetic variation that accounts for divergent characteristics across different species. Gametes have genes that code for proteins which aid in the formation of a fertilized egg. The sperm of E. calamaris contains the protein speract which guides sperm to an egg and the protein bindin which fuses the gametes together.[14] As part of my research, I will be analyzing the sequences of the gene that codes for the protein bindin from E. calamaris obtained from the Red Sea and from the two species that occur in the Indo-Pacific. I will be looking for differences in the gene sequences across the individuals that could potentially result in the production of structurally different proteins. Diverging proteins could inhibit gamete attraction and fusion, thus preventing the formation of a hybrid species.
The first step of my research was to determine the DNA sequence of the gene that encodes the bindin protein. The sequence for the bindin gene was obtained by my supervisor, Dr. Coppard, from the transcriptome of an E. calamaris male gonad, a sperm producing organ (see Figure 6). A transcriptome is a long sequence of DNA that contains all the genes expressed by an organism. To study the bindin gene, millions of copies of the gene sequence will need to be produced in a process known as amplification.
Figure 6: The DNA sequence and primers for the gene encoding the protein bindin in E. calamaris
Amplification is carried out via a laboratory technique called polymerase chain reaction (PCR). In order for PCR to amplify a gene, short sequences of DNA called primers need to be created. Primers act as a start and finish line by marking the beginning and end of the DNA sequence that needs to be amplified. The second step of my research project was to create primers that would be used to amplify the bindin gene of E. calamaris. I created primers using the software Primer3 Input. Important considerations in designing primers are the length and quality of the primer. As a result of the long length of the gene, I created three sets of primers, depicted by the yellow, blue, and green highlighted regions in Figure 6. The three sets of primers would ensure that I maximized the number of nucleotides included in the copied sequences. Finally, to ensure the quality of my primers, I used the software Primer Premier. By using algorithms Primer Premier rates PCR primers by reviewing ideal melting temperature and screening for additional physical characteristics. A sample result of my analysis for the blue primers is displayed in Figure 7. As shown in the first row of the table, both blue primers received a rating of 100 which indicates that they are highly likely to successfully amplify the sequence of DNA (a rating of 90 or above is excellent). The yellow and green primers received scores ranging from 91 to 100. The three PCR primers have been ordered from a local supplier and are on their way to the lab.
Figure 7: Results of the Primer Premier analysis for the blue primers
In the third step of my research, I extracted tissue from mature E. calamaris collected from the Red Sea and young E. calamaris from the Indo-Pacific (Figure 8). I collected tube feet from the Red Sea E. calamaris and muscle tissue under the spines of the Indo-Pacific E. calamaris. To extract the DNA from the collected tissue, I used the DNeasy Blood & Tissue Kit from QIAGEN. The collected DNA is currently being stored in the freezer where it is awaiting amplification.
Figure 8: Four individuals of E. calamaris collected from the Indo-Pacific used for DNA extraction and analysis
The next step of my research will be to amplify the bindin gene in the E. calamaris DNA samples through PCR and then begin my analysis. The lab skills that I have acquired thus far, including collaborating with a team of researchers, designing primers, and performing DNA extractions, as well as the mistakes I have made along the way have provided me with invaluable hands-on research experience. I look forward to gaining more confidence in the lab and increasing my knowledge about DNA amplification and genetic analysis in the coming weeks.
Jamie Gabbard, 1st Year Science, Queen’s University (Canada).
[1] R. Dirscherl, ‘Sea Urchins are Mysteriously Dying off across the Caribbean, Scientists say’, NBCNews.com, https://www.nbcnews.com/science/environment/sea-urchins-are-mysteriously-dying-caribbean-scientists-say-rcna25424 [last accessed 24/05/2022].
[2] H. A. Lessios, ‘The Great Diadema antillarum Die-Off: 30 Years Later’, Annual Review of Marine Science, 8:1 (2016) 267–283. https://doi.org/10.1146/annurev-marine-122414-033857
[3] Ibid.
[4] Ibid.
[5] Dirscherl.
[6] R. C. Carpenter, ‘Mass Mortality of Diadema Antillarum’, Marine Biology, 104 : 1 (1990), 67–77. https://doi.org/10.1007/bf01313159
[7] T. P. Hughes, D. C. Reed and M. J. Boyle, ‘Herbivory on coral reefs: Community structure following mass mortalities of Sea Urchins’, Journal of Experimental Marine Biology and Ecology, 113: 1 (1987), 39–59. https://doi.org/10.1016/0022-0981(87)90081-5
[8] National Oceanic and Atmospheric Administration, ‘The importance of coral reefs – corals: NOAA’s National Ocean Service Education. National Ocean Service’, https://oceanservice.noaa.gov/education/tutorial_corals/coral07_importance.html [last accessed 24/05/2022].
[9] S. E. Coppard and A. C. Campbell, ‘Grazing Preferences of Diadematid Echinoids in Fiji’, Aquatic Botany, 86 : 3 (2006), 204–212, https://doi.org/10.1016/j.aquabot.2006.10.005
[10] S. E. Coppard, H. Jessop and H. A. Lessios, ‘Phylogeography, Colouration, and Cryptic Speciation across the Indo-Pacific in the Sea Urchin Genus Echinothrix’, Scientific Reports, 11: 1 (2021), https://doi.org/10.1038/s41598-021-95872-0
[11] S. E. Coppard and A. C. Campbell, ‘Lunar Periodicities of Diadematid Echinoids Breeding in Fiji’, Coral Reefs, 24 : 2 (2005), 324–332, https://doi.org/10.1007/s00338-005-0491-5; S. E. Coppard and A. C Campbell, ‘Distribution and Abundance of Regular Sea Urchins on Two Coral Reefs in Fiji’, Micronesica, 37 : 2 (2005), 249–269.
[12] R. G. Harrison and E. L. Larson, ‘Hybridization, Introgression, and the Nature of Species Boundaries’, Journal of Heredity, 105: S1 (2014), 795–809, https://doi.org/10.1093/jhered/esu033
[13] Encyclopedia Britannica, https://www.britannica.com/science/backcross [last accessed 24/05/2022].
[14] S. Jagadeeshan, S. E. Coppard and H. A. Lessios, ‘Evolution of Gamete Attraction Molecules: Evidence for Purifying Selection in Speract and its Receptor, in the Pantropical Sea Urchin Diadema’, Evolution & Development, 17: 1 ,(2015), 92–108, https://doi.org/10.1111/ede.12108