image: A coral disease called growth anomalies (GAs) is depicted here in the coral species Porites compressa, a reef building species found off the coast of Hawaii. GAs are a tumor-like disease that can cause tumor-like protrusions that affect both the coral skeleton and its soft tissues.
Coral reefs are a favorite spot for scuba divers and are among the world's most diverse ecosystems. For example, the Hawaiian coral reefs, known as the "rainforests of the sea," host over 7,000 species of marine animals, fishes, birds and plants. But coral reefs are facing serious threats, including a number of diseases that have been linked to human activity.
To understand the connection between human activity and a type of tumorlike disease called growth anomalies (GAs), researchers at the National Institute of Standards and Technology (NIST) have collaborated with the U.S Geological Survey (USGS) and the National Oceanic and Atmospheric Administration (NOAA) to use an emerging molecular profiling method to identify 18 small molecules that promise to help them better understand the series of molecular reactions that lead to the disease.
GAs affect both the coral skeleton and its soft tissues. Scientists don't know the cause of the disease or how it spreads but have hypothesized that there is a strong correlation between GA prevalence in coral colonies and human population density nearby.
Almost all types of corals are made of hundreds to millions of individual soft-bodied animals called polyps. The polyps secrete calcium carbonate to form a hard skeleton that lays the foundation for the coral colony. GAs affect corals through irregular and accelerated growth of their skeleton, causing it to be less dense and filled with holes. This results in a tumorlike mass in the skeleton of a coral colony with fewer polyps and a diminished ability to reproduce.
Shallow water corals receive food like carbohydrates and oxygen as a byproduct of photosynthesis from the symbiotic relationship they have with zooxanthellae, photosynthetic algae that live inside coral tissues. GAs can lead to fewer symbiotic zooxanthellae and therefore less energy being absorbed from photosynthesis.
Even though GAs do not typically directly lead to coral death, they do affect the overall health of coral colonies and can pose an ecological threat to coral populations. To analyze the disease, NIST researchers chose the coral species Porites compressa as their target sample.
This coral species is known as the "finger" or "hump" coral and is part of the stony coral family, which is "one of the important reef-building species in Hawaii," said NIST chemist Tracey Schock. "They lay the foundation for the coral reef."
P. compressa is found in shallow lagoons off the Hawaiian Islands, and the researchers obtained their coral samples from Kaneohe Bay, Oahu. The bay has been studied widely as a site affected by human activity such as sewage discharge and metal pollution. GAs have previously been observed in the coral species there.
In order to analyze and study GAs in P. compressa, researchers turned to the field of metabolomics, which is the study of small molecules, such as those making up living organisms found in tissues, blood or urine. These small molecules, known as metabolites, are the intermediate and end products in a linked series of biochemical reactions known as molecular pathways in an organism.
Some examples of such small molecules include sugars like glucose, amino acids, lipids and fatty acids. Their production can be influenced by genetic and environmental factors and can help researchers better understand the biochemical activity of tissue or cells. In this case, chemical analysis of metabolites provides significant information that helps researchers understand the physiology of the disease.
For their study, researchers sampled a coral colony that had both healthy and diseased tissue. They split up their samples so they could assess the healthy coral and diseased coral separately. They also had a separate adjacent sample that was free of diseased tissue.
The samples were frozen in liquid nitrogen, and then freeze-dried for practical sample processing while maintaining metabolic integrity. The researchers then separated the diseased parts from the healthy colony using a hammer and stainless-steel chisel and collected the tissue from the skeleton with a brush. In one of the final stages of the sample preparation, they chemically extracted the metabolites from the coral tissue using a combination of methanol, water and chloroform.
"The method is novel for coral studies," said Schock. "With metabolomics, it is critical to preserve the state of all metabolites in a sample at the time of collection. This requires halting all biochemical activity using liquid nitrogen and maintaining this state until chemical extraction of the metabolome. The complexity of a coral structure necessitates stringent collection and processing protocols."
The researchers then produced a metabolomic analysis of the coral samples by using a reproducible profiling technique known as proton nuclear magnetic resonance (1H NMR).
The 1H NMR technique exposes the coral extract to electromagnetic fields and measures the radio frequency signals released by the hydrogens in the sample. The various kinds of metabolites are revealed by their unique signals which inform of their chemical environment. NMR detects all signals from the magnetic nuclei within a sample, making it an unbiased "all-in-one" technique. Two-dimensional NMR experiments that can identify both hydrogens (1H) and their directly bound carbon (13C) atoms provide more chemical information, giving confidence in the accuracy of the identities of the various metabolites within a sample.
The study identified 18 different metabolites and a new GA morphological form in P. compressa. The researchers found that GA tumors have distinct metabolite profiles compared with healthy areas of the same coral colony and detected specific metabolites and metabolic pathways that may be important for these profile differences. They also discovered that the loss of internal pH regulation is seemingly responsible for the hollow skeletons that are a characteristic of GAs.
"We have not only characterized new aspects of GA physiology, but have also discovered candidate pathways that provide a clear path forward for future research efforts aiming to further understand GA formation and coral metabolism, in general," said Schock.
As studies of this type accumulate, the researchers envision a database that could pull together coral metabolite information from multiple coral species into an accessible location for all scientists.
Collaborating with other researchers in different fields could increase understanding of the biological impacts of this disease on coral colonies. "We are going to learn which species are tolerant and which species are sensitive to stresses, and the physiological adaptations or mechanisms of both types will be important to conservation efforts," said Schock.
For now, the researchers hope these findings will be helpful for other scientists analyzing coral species and ultimately be beneficial for the coral reefs themselves, potentially aiding efforts to better preserve them.