Experiment 1: Molecular Biodiversity and Interactions
Questions
We will collect and analyze water samples from different areas of the Blue Hole. The volume of the Hole will be divided into approximately 1000 smaller volumes each of which will constitute a collection location. Using a ROV that is being designed for us by the robotics team, we will collect water samples over the course of the six-month research period. Collection will occur in spurts, with the ROV(s) out for four days, in for minor maintenance for 1, out for 4, in for 1, out for 4, and so on and so forth. Collection rounds will also be made in the face of inclement weather, storm fronts, or unusual current changes. The water samples will be analyzed at the time of collection for temperature, turbidity, pH, conductivity, dissolved oxygen, and salinity. Recommended instrument: Horiba U-10. Also light intensity: method developed by robotics team. The ROV(s) will also note the coordinates and depth of collection using GPS. The water samples will then be brought back to the lab for further analysis.
Lab
Filter water samples to isolate biomass. Use phenol extraction of DNA sample to isolate DNA. Concentrate DNA with ethanol precipitation. Use restriction enzymes to digest the DNA into smaller pieces. Run the DNA, along with undigested DNA and a size marker, in a 0.7% or, if greater accuracy is needed, a 1% to 2% agarose gel. Elute different sized DNA fragments from low-melting temperature gel. Isolate and concentrate each segment. Run a PCR to amplify segments. Send resulting DNA samples to a sequencing facility like University of California, Berkeley's Department of Molecular and Cell Biology to be sequenced (~$7000). Use different restriction enzymes to obtain DNA segments of different sizes and different sequences. Search for overlap in sequences to determine the complete genetic code of the microorganism. Once genomes are determined, compare with existing genome libraries, with genomes of other Blue Hole microorganisms and with those of microorganisms taken from right outside the Blue Hole. This data can be used to determine evolutionary relationships between microorganisms and provide evidence towards the theory that the relative isolation of the Blue Hole makes it likely that there will have been an evolutionary divergence in the organisms found there.
This research will be ongoing over the course of the six months. Also, by noting which organisms were found in which environments, it may be possible to determine trends and relationships between the types of life found somewhere and some environmental characteristics of that location.
Reasons
Gauge state of overall ecosystem: A healthy reef can be expected to have
a certain level of biological diversity. By studying the various forms of
life in the Blue Hole, we may be able to gain a general understanding of
the health of the system.
Experiment 2: Macroscropic Biodiversity and Interactions
Questions
The macroscopic organisms in the Blue Hole tend to be restricted to the shallower, less stagnant waters, near the living reef. The organisms living there are similar to the macroscopic life in other reefs in the region. However, there is the possibility that, because of the fact that it is difficult to leave the Blue Hole, they may have adapted to the reef environment in a different way that their counterparts from outside the hole. Also we will be interested in documenting the species variety in the hole to gauge reef health, as well as noting organisms that may have potential in Experiment 3.
Lab
We will be collecting and studying members of the Grouper and Bluefish populations for research, which will consist of documenting physiological characteristics and comparing them to those of Grouper and Bluefish populations outside the Hole. If differences are noted, we will then attempt to determine if this is due to certain environmental stresses present only in the Blue Hole, and to identify what those stresses might be. The samples will be collected using a "fish-vac", a manned submersible equipped with a vacuum pump that will suck the water surrounding the organism of interest, and bring the organism with it. Environmental data, like salinity, turbidity, and temperature will be collected on location as well. Exact location, using the grid system for Experiment 1, will be noted as well. The species diversity will be noted by the scientists in planned dive trips to areas of the reef. The scientists will also take data on, and perhaps collect samples of, invertebrates living on the reef that may have potential in medical research.
Reasons
Studies of species diversity in the Blue Hole can be compared to expected levels for a healthy reef and that information, when forwarded to the Belizean government, can help them plan environmentally conscious coastal development standards to preserve their vital tourist industry.
Experiment 3: Pharmaceutical Research
Questions
We will use data collected through Experiments 1 and 3 on the biological diversity present in the reef system to identify potentially beneficial organisms for further study and analysis. Our focus will be on marine invertebrates and microorganisms. Many marine invertebrates are soft-bodied and sessile, with no visible, physical means of self-protection. Yet they manage to avoid being made extinct by larger potential predators. Their mechanism of protection is generally chemical. Marine invertebrates have been found to produce some of the most toxic organic chemicals on earth, and are therefore excellent candidates for biomedical research. Marine microorganisms are as diverse as terrestrial microorganisms and have been far less studied. However, their environments are vastly different from those of their terrestrial or even fresh-water counterparts, and as a result, it can be expected that their chemical extracts will be just as different and unique. After three months of collecting and studying the diversity of the Blue Hole organisms, the scientists will identify those organisms that seem to be chemically unique enough for biomedical study. At which point, samples of those organisms will be collected and chemical extracts taken for research.
Lab: Microorganisms
Culture the strain of interest in a liquid agar medium. Filter the broth. Percolate broth over Chromatography resin to extract very crude chemical sample. Rinse with distilled water and elute with methylated solvent. Remove solvent to obtain a coarse extract. To purify the extract, fractionate using Si gel flash chromatography using an EtOAc and MeOH gradient. Purify by HPLC using either EtOAc or MeOH. Send samples to Bioanalytical Systems, Inc. for mass spectroscopy and liquid chromatography to identify chemical characteristics of samples. Send data and samples to interested pharmaceutical companies for medical testing.
Lab: Macroorganisms
Collect sample of interest. Freeze-dry specimens. Extract with methanol solution. For samples cultivated in a liquid medium, filter broth. Extract broth with Ethyl acetate. Remove solvents from extracts. Fractionate using Si gel flash chromatography using an EtOAc and MeOH gradient. Purify by HPLC using either EtOAc or MeOH. Send samples to Bioanalytical Systems, Inc. for mass spectroscopy and liquid chromatography to identify chemical characteristics of samples. Send data and samples to interested pharmaceutical companies for medical testing.
Reasons
Researcher Requirements
We will be sending three researchers to Atlantis 1 for conducting the biological research. They must:
Equipment