Optimization of DNA extraction for advancing coral microbiota investigations

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Microbiome 5 (2017): 18, doi:10.1186/s40168-017-0229-y.We designed a two-phase study in order to propose a comprehensive and efficient method for DNA extraction from microbial cells present in corals and investigate if extraction method influences microbial community composition. During phase I, total DNA was extracted from seven coral species in a replicated experimental design using four different MO BIO Laboratories, Inc., DNA Isolation kits: PowerSoil®, PowerPlant® Pro, PowerBiofilm®, and UltraClean® Tissue & Cells (with three homogenization permutations). Technical performance of the treatments was evaluated using DNA yield and amplification efficiency of small subunit ribosomal RNA (SSU ribosomal RNA (rRNA)) genes. During phase II, potential extraction biases were examined via microbial community analysis of SSU rRNA gene sequences amplified from the most successful DNA extraction treatments. In phase I of the study, the PowerSoil® and PowerPlant® Pro extracts contained low DNA concentrations, amplified poorly, and were not investigated further. Extracts from PowerBiofilm® and UltraClean® Tissue and Cells permutations were further investigated in phase II, and analysis of sequences demonstrated that overall microbial community composition was dictated by coral species and not extraction treatment. Finer pairwise comparisons of sequences obtained from Orbicella faveolata, Orbicella annularis, and Acropora humilis corals revealed subtle differences in community composition between the treatments; PowerBiofilm®-associated sequences generally had higher microbial richness and the highest coverage of dominant microbial groups in comparison to the UltraClean® Tissue and Cells treatments, a result likely arising from using a combination of different beads during homogenization. Both the PowerBiofilm® and UltraClean® Tissue and Cells treatments are appropriate for large-scale analyses of coral microbiota. However, studies interested in detecting cryptic microbial members may benefit from using the PowerBiofilm® DNA treatment because of the likely enhanced lysis efficiency of microbial cells attributed to using a variety of beads during homogenization. Consideration of the methodology involved with microbial DNA extraction is particularly important for studies investigating complex host-associated microbiota.This project was supported by NSF award OCE-1233612 to AA and NSF GRFP award to LW

Diversity and function of prevalent symbiotic marine bacteria in the genus Endozoicomonas

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Applied Microbiology and Biotechnology 100 (2016): 8315–8324, doi:10.1007/s00253-016-7777-0.Endozoicomonas bacteria are emerging as extremely diverse and flexible symbionts of numerous marine hosts inhabiting oceans worldwide. Their hosts range from simple invertebrate species, such as sponges and corals, to complex vertebrates, such as fish. Although widely distributed, the functional role of Endozoicomonas within their host microenvironment is not well understood. In this review, we provide a summary of the currently recognized hosts of Endozoicomonas and their global distribution. Next, the potential functional roles of Endozoicomonas, particularly in light of recent microscopic, genomic, and genetic analyses, are discussed. These analyses suggest that Endozoicomonas typically reside in aggregates within host tissues, have a free-living stage due to their large genome sizes, show signs of host and local adaptation, participate in host-associated protein and carbohydrate transport and cycling, and harbour a high degree of genomic plasticity due to the large proportion of transposable elements residing in their genomes. This review will finish with a discussion on the methodological tools currently employed to study Endozoicomonas and host interactions and review future avenues for studying complex host-microbial symbioses.This work was supported by a KAUST-WHOI Post-doctoral Partnership Award to MJN and a KAUST-WHOI Special Academic Partnership Funding Reserve Award to CRV and AA. Research in this study was further supported by baseline research funds to CRV by KAUST and NSF award OCE-1233612 to AA

Multifaceted impacts of the stony coral Porites astreoides on picoplankton abundance and community composition

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Limnology and Oceanography 62 (2017): 217–234, doi:10.1002/lno.10389.Picoplankton foster essential recycling of nutrients in the oligotrophic waters sustaining coral reef ecosystems. Despite this fact, there is a paucity of data on how the specific interactions between corals and planktonic bacteria and archaea (picoplankton) contribute to nutrient dynamics and reef productivity. Here, we utilized mesocosm experiments to investigate how corals and coral mucus influence picoplankton and nutrients in reef waters. Over 12 days, we tracked nutrient concentrations, picoplankton abundances and taxonomic composition of picoplankton using direct cell-counts, sequencing of SSU rRNA genes and fluorescent in situ hybridization-based abundances of dominant lineages in the presence or absence of Porites astreoides corals and with mucus additions. Our results demonstrate that when corals are present, Synechococcus, SAR11 and Rhodobacteraceae cells are preferentially removed. When corals were removed, their exudates enhanced the growth of diverse picoplankton, including SAR11 and Rhodobacteraceae. A seven-fold increase in nitrate concentration, possibly caused by nitrogen remineralization (ammonification coupled to nitrification) within the coral holobiont, may have further facilitated the growth of these taxa. In contrast, the addition of mucus resulted in rapid initial growth of total picoplankton and Rhodobacteraceae, but no measurable change in overall community structure. This study presents evidence of the multifaceted influences of corals on picoplankton, in which the coral holobiont selectively removes and promotes the growth of diverse picoplankton and remineralizes nitrogen.NSF Grant Number: OCE-1233612; NSF Oceanic Microbial Observatory Grant Number: OCE-080199

Coupled x-ray fluorescence and x-ray absorption spectroscopy for microscale imaging and identification of sulfur species within tissues and skeletons of scleractinian corals

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Analytical Chemistry 90 (2018): 12559–12566, doi:10.1021/acs.analchem.8b02638.Identifying and mapping the wide range of sulfur species within complex matrices presents a challenge for under-standing the distribution of these important biomolecules within environmental and biological systems. Here, we present a coupled micro X-ray fluorescence (μXRF) and X-ray absorption near edge structure (XANES) spectroscopy method for determining the presence of specific sulfur species in coral tissues and skeletons at high spatial resolution. By using multiple energy stacks and principal component analysis of a large spectral database, we were able to more accurately identify sulfur species components and distinguish different species and distributions of sulfur formerly unresolved by previous studies. Specifically, coral tissues were domi-nated by more reduced sulfur species, such as glutathione disulfide, cysteine and sulfoxide, as well as organic sulfate as represented by chondroitin sulfate. Sulfoxide distributions were visually correlated with the presence of zooxanthellae endosymbionts. Coral skeletons were composed primarily of carbonate-associated sulfate (CAS), along with minor contributions from organic sulfate and a separate inorganic sulfate likely in the form of adsorbed sulfate. This coupled XRF-XANES approach allows for a more accurate and informative view of sulfur within biological systems in situ, and holds great promise for pairing with other techniques to allow for a more encompassing understanding of elemental distributions within the environment.We thank Ray Dalio for funding the Micronesian expedition and K. Hughen,This material is based upon work supported by the Na-tional Science Foundation Graduate Research Fellowship under Grant No. 1122374 and a Ford Foundation Dissertation Fellowship for Gabriela Farfan

Incidence of lesions on Fungiidae corals in the eastern Red Sea is related to water temperature and coastal pollution

Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Elsevier for personal use, not for redistribution. The definitive version was published in Marine Environmental Research 98 (2014): 29-38, doi:10.1016/j.marenvres.2014.04.002.As sea surface temperatures rise and the global human population increases, large-scale field observations of marine organism health and water quality are increasingly necessary. We investigated the health of corals from the family Fungiidae using visual observations in relation to water quality and microbial biogeochemistry parameters along 1300 km of the Red Sea coast of Saudi Arabia. At large scales, incidence of lesions caused by unidentified etiology showed consistent signs, increasing significantly from the northern to southern coast and positively correlated to annual mean seawater temperatures. Lesion abundance also increased to a maximum of 96% near the populous city of Jeddah. The presence of lesioned corals in the region surrounding Jeddah was strongly correlated with elevated concentrations of ammonium and changes in microbial communities that are linked to decreased water quality. This study suggests that both high seawater temperatures and nutrient pollution may play an indirect role in the formation of lesions on corals.This research was supported by Award No. USA 00002 to K. Hughen by King Abdullah University of Science and Technology (KAUST) and a WHOI Ocean Life Institute postdoctoral scholar fellowship to A. Apprill

Impact of prawn farming effluent on coral reef water nutrients and microorganisms

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Aquaculture Environment Interactions 9 (2017): 331-346, doi:10.3354/aei00238.Tropical coral reefs are characterized by low-nutrient waters that support oligotrophic picoplankton over a productive benthic ecosystem. Nutrient-rich effluent released from aquaculture facilities into coral reef environments may potentially upset the balance of these ecosystems by altering picoplankton dynamics. In this study, we examined how effluent from a prawn (Litopenaeus vannamei) farming facility in Al Lith, Saudi Arabia, impacted the inorganic nutrients and prokaryotic picoplankton community in the waters overlying coral reefs in the Red Sea. Across 24 sites, ranging 0-21 km from the effluent point source, we measured nutrient concentrations, quantified microbial cell abundances, and sequenced bacterial and archaeal small subunit ribosomal RNA (SSU rRNA) genes to examine picoplankton phylogenetic diversity and community composition. Our results demonstrated that sites nearest to the outfall had increased concentrations of phosphate and ammonium and elevated abundances of non-pigmented picoplankton (generally heterotrophic bacteria). Shifts in the composition of the picoplankton community were observed with increasing distance from the effluent canal outfall. Waters within 500 m of the outfall harbored the most distinct picoplanktonic community and contained putative pathogens within the genus Francisella and order Rickettsiales. While our study suggests that at the time of sampling, the Al Lith aquaculture facility exhibited relatively minor influences on inorganic nutrients and microbial communities, studying the longer-term impacts of the aquaculture effluent on the organisms within the reef will be necessary in order to understand the full extent of the facility’s impact on the reef ecosystem.This research was supported by a Woods Hole Oceanographic Institution (WHOI) Ocean Life Institute postdoctoral scholar fellowship to A.A., the Semester at WHOI Program supporting C.B., and Award No. USA 00002 to K.H. made by King Abdullah University of Science and Technology (KAUST)

Species-specific control of external superoxide levels by the coral holobiont during a natural bleaching event

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Nature Communications 7 (2016): 13801, doi:10.1038/ncomms13801.The reactive oxygen species superoxide (O2·−) is both beneficial and detrimental to life. Within corals, superoxide may contribute to pathogen resistance but also bleaching, the loss of essential algal symbionts. Yet, the role of superoxide in coral health and physiology is not completely understood owing to a lack of direct in situ observations. By conducting field measurements of superoxide produced by corals during a bleaching event, we show substantial species-specific variation in external superoxide levels, which reflect the balance of production and degradation processes. Extracellular superoxide concentrations are independent of light, algal symbiont abundance and bleaching status, but depend on coral species and bacterial community composition. Furthermore, coral-derived superoxide concentrations ranged from levels below bulk seawater up to ∼120 nM, some of the highest superoxide concentrations observed in marine systems. Overall, these results unveil the ability of corals and/or their microbiomes to regulate superoxide in their immediate surroundings, which suggests species-specific roles of superoxide in coral health and physiology.This work was supported by a Postdoctoral Fellowship from the Ford Foundation (J.M.D.), the National Science Foundation under grants OCE 1225801 (J.M.D.) and OCE 1233612 (A.A.), the Ocean and Climate Change Institute of the Woods Hole Oceanographic Institution (C.M.H.), the Sidney Stern Memorial Trust (C.M.H. and A.A.) and an anonymous donor

Dark production of extracellular superoxide by the coral Porites astreoides and representative symbionts

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Marine Science 3 (2016): 232, doi:10.3389/fmars.2016.00232.The reactive oxygen species (ROS) superoxide has been implicated in both beneficial and detrimental processes in coral biology, ranging from pathogenic disease resistance to coral bleaching. Despite the critical role of ROS in coral health, there is a distinct lack of ROS measurements and thus an incomplete understanding of underpinning ROS sources and production mechanisms within coral systems. Here, we quantified in situ extracellular superoxide concentrations at the surfaces of aquaria-hosted Porites astreoides during a diel cycle. High concentrations of superoxide (~10's of nM) were present at coral surfaces, and these levels did not change significantly as a function of time of day. These results indicate that the coral holobiont produces extracellular superoxide in the dark, independent of photosynthesis. As a short-lived anion at physiological pH, superoxide has a limited ability to cross intact biological membranes. Further, removing surface mucus layers from the P. astreoides colonies did not impact external superoxide concentrations. We therefore attribute external superoxide derived from the coral holobiont under these conditions to the activity of the coral host epithelium, rather than mucus-derived epibionts or internal sources such as endosymbionts (e.g., Symbiodinium). However, endosymbionts likely contribute to internal ROS levels via extracellular superoxide production. Indeed, common coral symbionts, including multiple strains of Symbiodinium (clades A to D) and the bacterium Endozoicomonas montiporae LMG 24815, produced extracellular superoxide in the dark and at low light levels. Further, representative P. astreoides symbionts, Symbiodinium CCMP2456 (clade A) and E. montiporae, produced similar concentrations of superoxide alone and in combination with each other, in the dark and low light, and regardless of time of day. Overall, these results indicate that healthy, non-stressed P. astreoides and representative symbionts produce superoxide externally, which is decoupled from photosynthetic activity and circadian control. Corals may therefore produce extracellular superoxide constitutively, highlighting an unclear yet potentially beneficial role for superoxide in coral physiology and health.This work was supported by a Postdoctoral Fellowship from the Ford Foundation (JD), the National Science Foundation under grants OCE 1225801 (JD) and OCE 1233612 (AA), the Ocean and Climate Change Institute of the Woods Hole Oceanographic Institution (CH), a BIOS Grant in aid award (SM), the Sidney Stern Memorial Trust (CH and AA), as well as an anonymous donor

Soundscapes influence the settlement of the common caribbean coral porites astreoides irrespective of light conditions

Author Posting. © Royal Society, 2018. This article is posted here by permission of Royal Society for personal use, not for redistribution. The definitive version was published in Proceedings of the Royal Society Open Science 5(12) (2018): 181358. doi: 10.1098/rsos.181358.The settlement of reef-building corals is critical to the survival and recovery of reefs. Recent evidence indicates that coral larvae orient towards reef sound, yet the components of the acoustic environment that may attract coral larvae and induce settlement are unknown. Here we investigated the effects of ambient soundscapes on settlement of Porites astreoides coral larvae using in situ chambers on reefs differing in habitat quality (coral and fish abundance). Mean larval settlement was twice as high in an acoustic environment with high levels of low-frequency sounds, typical of a high-quality, healthy reef; this result was observed in both natural light and dark treatments. Overall, the enhancement of coral settlement by soundscapes typical of healthy reefs suggests a positive feedback where soundscape properties of reefs with elevated coral and fish abundance may facilitate coral recruitment.This study is funded by NSF Biological Oceanography award 15-36782 which supported all authors