Bioenergetics on the Coral Reef
Global climate change is ultimately a matter of thermodynamics; where, when, and how much thermal energy is moved and stored. The total metabolism of all life on Earth must ultimately dissipate energy as heat. This process starts as solar radiation is captured by photosynthetic microbes and plants, and photonic energies are stored as chemical bonds to generate biomass, which is consumed by heterotrophs and decomposers. At each trophic level the majority of energy is lost, radiated from the Earth as heat. Microbial food webs are the most energetically active components of the biosphere. Although feedbacks between these microscopic life forms and physical forcing (e.g., CO2 loading) will ultimately determine how the Earth system evolves, there are currently zero direct measurements of the thermal flux, storage, and efficiency of microbial food webs.
The Rohwer Lab Bioenergetics Group has recently shown evidence that global changes have resulted in microbial communities utilizing less thermodynamically efficient metabolic pathways (McDole et al., 2012; McDole Somera et al., 2016; Haas et al., 2016; Roach et al., 2017). This reduction in the efficiency of microbial metabolism results in more energy being dissipated as heat, and leads to feedback mechanisms which enhance global heat production and coral reef decline. Current work in our group has begun utilizing isothermal calorimetry, the essential methodology for measuring the flux and storage of energy and heat. Calorimetry can be used to measure the thermal flux of actively metabolizing microbial communities and determine how much energy is dissipated as heat. However, we have currently reached the point where we need even more sensitive calorimeters than anything previously developed. Thus, our lab is engineering the most sensitive isothermal calorimeter built to date. By utilizing this new calorimeter in combination with other methods such as optical oxygen sensing, and isotope tracking we will be able to produce energetic models of coral reefs with an unprecedented level of resolution.
Coral Reef Metabolomics
Animals, plants, and all of life make molecules to defend themselves, such as when an immune system mounts a response to a pathogen. As we continue to better understand how coral reefs work and how the rapid spread of algae on reefs effect corals, our lab has started to use another ‘omics’ of the multi-omics approach: metabolomics of small molecules. Analytical instruments such as mass spectrometers can detect an incredible number of molecules produced by organisms, but science only understands what a handful of them are and what they do. This lack of information is particularly noteworthy on coral reefs, where the diversity of organisms living there are known for the wide range of molecules they create, some of which have been turned into life-saving drugs.
In collaboration with Pieter Dorrestein’s laboratory at UC San Diego, we are using metabolomes to better understand the biology and ecology of coral reefs. The lab started with analyzing the competitive interactions of corals of the Southern Line Islands. There we identified that corals have relatively high abundances of lyso-platelet activating factor and platelet activating factor, two molecules well-known for their important roles in human inflammatory pathways. We hypothesized that corals produce these molecules when physically interacting with each other during competition for space on the reef (Quinn et al., 2016).
Although only a few molecules can be identified within a dataset, with the use of the metabolomic networking techniques from the Dorrestein lab we can gain insights into the relatedness between unknown molecules based on their molecular fingerprints. Using these relationships we have developed and applied a new metabolomics approach to glean substantially more information from existing molecular datasets. The approach illuminates the relationships between molecules based on their molecular fingerprints. This resulted in meta-mass shift chemical profiling (MeMSChem, Hartmann et al., In Press) which examines mass shifts between unknown molecules throughout metabolomes to gain insight into potential modifications (adding or removing groups) between molecules, processes that make molecules biologically active or inactive.
By understanding how molecules are related and change, we can better understand how they are created and what they do for the organisms that make them. Our approach is now being used to address an unanswered question in coral reef ecology: why are coral reefs such incredible sources of molecular diversity?
- Hartmann, Aaron C., Daniel Petras, Robert A. Quinn, Ivan Protsyuk, Frederick I. Archer, Emma Ransome, Gareth J. Williams, Barbara A. Bailey, Mark J. A. Vermeij, Theodore Alexandrov, Pieter C. Dorrestein, & Forest L. Rohwer (2017) Meta-mass shift chemical (MeMSChem) profiling of metabolomes from coral reefs. Proceedings of the National Academy of the Sciences. doi: 10.1073/pnas.1710248114
Hunting for Coral Stem Cells
The aim of our current research is to not only identify and isolate stem cells residing in the scleractinian coral tissue, which has yet to be achieved (Gold and Jacobs, 2012; Lecointe et al., 2016; Rosental et al., 2017), but to elucidate the general rules governing stem cell identification, fate, and renewal across multiple phylum. Scleractinian corals are the major reef-building organism on coral reefs, one of the most biodiverse ecosystems on the planet. Due to global stressors, corals reefs around the world are transitioning from net accretion to net erosion, accompanied with the subsequent loss of biodiversity, these shifts in the environment have compounding effects on local and global economic revenue (Pandolfi et al., 2003; Yates et al., 2017). Current coral reef preservation projects rely on the ability of scleractinian coral to be fragmented and produce new colonies or the collection of gametes during coral spawning events (Forsman et al., 2015; Hagedorn et al., 2012). While successful, these methods take time and rely on specific timing of natural events. Our aim to utilize the tools and ideas developed over decades for studying mammalian cells and apply these techniques to corals, with hopes of developing a stable coral cell line that would expedite our understanding of the molecular consequences of human impact and prevent further degradation of the reef to ensure the future success of coral reefs around the world.
- Forsman, Z.H., Page, C.A., Toonen, R.J., and Vaughan, D. (2015). Growing coral larger and faster: micro-colony-fusion as a strategy for accelerating coral cover. PeerJ 3.
- Gold, D.A., and Jacobs, D.K. (2012). Stem cell dynamics in Cnidaria: are there unifying principles? Dev. Genes Evol. 223, 53–66.
- Hagedorn, M., Carter, V., Martorana, K., Paresa, M.K., Acker, J., Baums, I.B., Borneman, E., Brittsan, M., Byers, M., Henley, M., et al. (2012). Preserving and Using Germplasm and Dissociated Embryonic Cells for Conserving Caribbean and Pacific Coral. PLoS ONE 7.
- Lecointe, A., Domart-Coulon, I., Paris, A., and Meibom, A. (2016). Cell proliferation and migration during early development of a symbiotic scleractinian coral. Proc. R. Soc. B-Biol. Sci. 283, 20160206.
- Pandolfi, J.M., Bradbury, R.H., Sala, E., Hughes, T.P., Bjorndal, K.A., Cooke, R.G., McArdle, D., McClenachan, L., Newman, M.J.H., Paredes, G., et al. (2003). Global Trajectories of the Long-Term Decline of Coral Reef Ecosystems. Science 301, 955–958.
- Rosental, B., Kozhekbaeva, Z., Fernhoff, N., Tsai, J.M., and Traylor-Knowles, N. (2017). Coral cell separation and isolation by fluorescence-activated cell sorting (FACS). BMC Cell Biol. 18.
- Yates, K.K., Zawada, D.G., Smiley, N.A., and Tiling-Range, G. (2017). Divergence of seafloor elevation and sea level rise in coral reef ecosystems. Biogeosciences 14, 1739–1772.
PtW Meets BAM and DDAM
Bacteriophages, viruses that infect bacteria, are the most abundant biological entities on the planet. They can control bacterial community composition and growth by infecting and lysing bacterial cells. But phages can also partner with their bacterial hosts during lysogenic infections, improving bacterial fitness and contributing to bacterial growth. Our group has shown that when microbial abundances and growth are high, the partnership between phages and bacteria is favored. Phages piggyback the growth of successful bacteria, which we call the Piggyback-the-Winner dynamic. In coral reefs under high anthropogenic pressure, where fleshy algae win the competition for space with corals, bacterial growth is stimulated by dissolved organic carbon (DOC) released by algae. These are the perfect conditions for phages to stop lysing bacteria and instead, piggyback their growth. These phages bring new genes into this partnership, including diverse virulence factors, and improve bacterial growth even further. This feedback loop of algae and bacterial dominance pushes coral reef ecosystems into a microbialized state, in which bacterial biomass and energetic demands surpass that of corals and fish.
Highly abundant bacteria in partnership with phages that carry virulence factors can attack and kill corals. Corals surfaces are covered on mucus layers formed of complex mucin molecules released by the coral epithelium. Or lab has shown that phages have the ability to recognize eukaryotic mucin molecules, a phenomenon named BAM (bacteriophage adherence to mucus) By binding to mucin molecules, phages increase their hunting efficiency, protecting the coral from bacterial infections. However, in a microbialized reef, bacterial cells with phage-derived virulence are able to invade the coral, scape predation from the coral resident phages and stablish infection. Through this mechanism, phages are important drivers of the DDAM cycle of coral reef degradation: DOC, Disease, Algae, Microbes. Currently, our lab is interested in the mechanisms by which phage modulate reef degradation in order to reverse ecosystem loss.
- Silveira C & F Rohwer (2016) Piggyback-the-Winner in host-associated microbial communities. npj Biofilms and Microbiomes : 10.1038/npjbiofilms.2016.
- Haas AF, Fairoz MF, Kelly LW, Nelson CE, Dinsdale EA, Edwards RA, Giles S, Hatay M, Hisakawa N, Knowles B, Lim YW. Global microbialization of coral reefs. Nature microbiology. 2016 1:16042.
- McDole-Somera T, C Silveira, BE Hilton, J Grasis, J Nulton, B Bailey, B Nosrat, C Sullivan, M Hatay, K Barott, RE Brainard, F Rohwer (2016) Energetic differences between bacterioplankton trophic groups and coral reef resistance. Proceedings of the Royal Society B: 10.1098/rspb.2016.0467
- Knowles B, Silveira CB, Silva GGZ, Quistad SD, Lim YW, Sanchez SE, Coutinho FH, Green KT, Hester ER, Haggerty JM, George EE, Little M, Thompson C, Haas AF, McDole-Somera T, Young C, Hisakawa NG, Furby KA, Cantu A, McNair K, Robinett NL, Cobián-Güemes AG, Zgliczynski B, Dinsdale E, Kelly LW1, Felts B, Salamon P, Sandin S, Smith J, Sala E, Luque A, Brainard R, Gregoracci G, Bailey BA, Edwards RA, Nulton J, Thompson F, Rohwer F (2016) Lytic to Temperate Switching of Viral Communities. Nature 531:466-470 10.1038/nature17193
- Barr JJ, R Auro, N Sam-Soon, S Kassegne, G Peters, N Bonilla, M Hatay, S Mourtada, B Bailey, M Youle, B Felts, A Baljon, J Nulton, P Salamon, F Rohwer (2015) Bacteriophage subdiffusive hunting strategy in mucosal surfaces. PNAS: 10.1073/pnas.1508355112
- Kelly LW, Williams GJ, Barott KL, Carlson CA, Dinsdale EA, Edwards RA, Haas AF, Haynes M, Lim YW, McDole T, Nelson CE, Sala E, Sandin SA, Smith JE, Vermeij MJ, Youle M, Rohwer F (2014) Local genomic adaptation of coral reef-associated microbiomes to gradients of natural variability and anthropogenic stressors. Proc. Natl. Acad. Sci. U.S.A. 111(28):10227-32 (PMC4104888)
- Barr JJ, Auro R, Furlan M, Whiteson KL, Erb ML, Pogliano J, Stotland A, Wolkowicz R, Cutting AS, Doran KS, Salamon P, Youle M, Rohwer F (2013) Bacteriophage adhering to mucus provide a non-host-derived immunity. Proc. Natl. Acad. Sci. U.S.A. 110(26):10771-6 (PMC3696810)
- Barott KL, Rohwer FL (2012) Unseen players shape benthic competition on coral reefs. Trends Microbiol. 20(12):621-8
Coral reefs worldwide are in decline
The dramatic rise in incidences of coral disease over the last two decades has been instrumental in this process. We have hypothesized that most of these diseases are actually opportunistic infections instigated by anthropogenic stressors. Our research is focused around understanding the interactions between the microbial world and coral reefs, and how these systems change following perturbation.
The Coral Holobiont
Corals are host to a wide diversity of organisms, including endosymbiotic algae, protists, fungi, Bacteria, Archaea, and viruses. Together, these organisms make up the coral holobiont. In our lab, we are interested in understanding the physiological roles of these players in their interaction with the coral animal, and how this relates to coral reef health. Incidences of coral death and disease are highly correlated with human impact, and we propose that anthropogenic stresses induce microbes normally associated with the coral to become opportunistic pathogens. Alternatively, opportunistic or specific pathogens from the water column might attack the weakened coral. To differentiate between these possibilities, my lab has had to determine if healthy corals have characteristic microbiotas. To do this, we have employed a variety of techniques ranging from electron microscopy (e.g., Johnston and Rohwer 2007) to metagenomics (e.g., Wegley et al. 2007).
In order to look at the diversity and specificity of coral microbes, our lab used high-throughput sequencing of bacterial 16S rDNAs associated with three coral species. This culture-independent study of coral-associated Bacteria found 430 (mostly novel) bacterial species in 14 samples from 3 coral species. The coral-associated microbial communities were ecologically structured: different coral species had different bacterial communities, even when physically adjacent, while bacterial communities from the same coral species separated by time (~1 year) or space (3000 km) were similar. We also found that some bacterial species were present only in a subset of spatial niches within individual coral colonies (Rohwer, et al., 2002).
In order to look at the function of microbes on corals, we use metagenomic sequencing (454 Life Sciences) to identify the microbes and their functional genes. Our work found that bacteria associated with corals are primarily heterotrophic. Our metagenomic data showed an abundance of sugar and protein utilization and uptake pathways in the microbial community. These microbes are likely utilizing the complex polysaccharides and peptides from the coral mucus. Several types of cyanobacteria were also found associated with the coral, and may be providing fixed carbon and nitrogen to the coral. In addition, an abundance of fungi were associated with corals, including those involved in nitrogen cycling, indicating that fungi may be fixing nitrogen and making it available to members of the coral holobiont (Wegley et al. 2007).
We have also looked at the viruses associated with healthy and bleaching corals, and find viruses with a wide variety of hosts including many of the various members of the coral holobiont. These viruses include plant and algal viruses, herpes-like viruses, and cyano- and vibriophage, to name a few (Wegley et al. 2007, Marhaver et al. 2008). Due to the abundance of viruses and the wide variety of host ranges they possess, we expect that they play an important role in coral health and structuring of the coral holobiont.
In summary, the associations of the coral animal, prokaryotes, zooxanthellae, viruses, fungi, and other undefined components will define the niche that any coral colony occupies on a reef. This system is almost certainly exemplary of many other interactions between microbes and their higher eukaryotic hosts, and our studies will make predictions that can/will be tested in other complex host-microbial flora systems.
Stressors Alter Microbial Dynamics on Corals
An important implication of the coral holobiont model is that disrupting any one of these components may cause the whole community to collapse and lead to coral death. In order to test this hypothesis, we have performed several experiments exposing corals to different stressors and then looked at the changes in microbial dynamics and diversity, as well as coral pathology. In collaboration with Dr. Nancy Knowlton and Davey Kline at the Scripps Institution of Oceanography, we applied stresses to different coral species in the presence and absence of antibiotics. Our data showed that of the many commonly cited stressors of corals, organic carbon (OC) loading is the most problematic. Coral death induced by OC can be delayed with antibiotics. Additionally, OC loading causes the coral-associated microbial communities to grow much faster then normal. This strongly suggests that changes in the bacterial community, and not the stresses themselves, are responsible for coral mortality. (Kline et al. 2006, Kuntz et al. 2005). Additionally, when corals are placed next to algae with a filter impervious to viruses and bacteria, corals mortality is high. This mortality is also inhibited by antibiotics (Smith et al. 2006).
In a separate experiment, corals were exposed to one of four types of stressors currently threatening coral reefs: elevated nutrients, temperature, and organic carbon, and lowered pH. We then isolated the microbial and viral communities and performed whole-genome sequencing (pyrosequencing, 454 Life Sciences) to look at how the diversity and function of these organisms changed following stress. Our data showed that stress led to a shift towards a more pathogenic microbial community in all cases, with pathogen-associated genes also increasing in abundance (e.g. motility, virulence, and secondary metabolite genes) (Vega Thurber et al. Env Micro 2009). The viral assemblages also changed on the coral, with viruses related to the Herpesviridae family greatly increasing in abundance (Vega Thurber et al. PNAS 2008). We found that one herpes-like virus was undetectable by quantitative PCR (qPCR) prior to stress, but then increased dramatically within 1 hr of stress exposure, indicating an increase in production of the virus under stress.
Coral Reef Microbiology in Pristine and Human-impacted Reefs
In 2005, we visited the Northern Line Islands with a group of coral reef experts to look at coral reef health across a gradient of human disturbance. The islands ranged from uninhabited to serving as a home for over 9000 residents. Surveys found that uninhabited islands had high coral cover and fish biomass (Sandin et al. 2008). The microbial community on healthy reefs was evenly split between autotrophs and heterotrophs, while on Kiritimati, the most inhabited island, the microbial community was primarily heterotrophs. Microbes were also ~10 times more abundant on these inhabited reefs, while coral cover decreased and disease was much more prevalent (Dinsdale et al. 2008).
In April 2009, we participated in a cruise to the Southern Line Islands to characterize pristine coral reefs. The group included most of the collaborators from the NLI trip, as well as a group from National Geographic to photo-document the reefs (http://ocean.nationalgeographic.com/). The islands visited on this trip lie just south of the equator, and have been uninhabited for 100 years or more. As such, they are home to some of the last remaining pristine coral reefs on the planet. We are again characterizing microbial and viral communities at these islands.
We are also trying to use metabolic theory to link performance of individual organisms to the whole community ecology of coral reefs, using data collected across the Pacific. We are interested in how fishing-related alterations in trophic structure affect community-level energy use and biomass production. Allometric power laws provide the basis for assessing the relative importance of fish vs. microbial components across coral reefs of varying degrees of health. Metabolic theory predicts that rates of energy and nutrient use should be approximately equal for all size categories within a restricted taxonomic group. However, when both fish and microbes are considered, we want to know if the flow of energy and materials through coral reef ecosystems is dominated by small or large organisms (i.e., microbes or fish).
Collaborators: Dr. Jennifer Smith, Dr. Stuart Sandin