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

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

Enriching the Vision of Campus Kitchen: A Recipe for Justice

Campus Kitchen provides an environment that is ripe for community-based, experiential-learning experiences, especially on the topic of Eco-Justice. Student volunteers have substantive opportunities to investigate and promote various food justice and hunger advocacy initiatives, as well as form meaningful personal relationships with those whom they serve. Volunteers are encouraged to learn everything from the practical skills of food preparation to the social forces that underlie food insecurity in the community. Still, many Campus Kitchen participants remain unaware of the seriousness of food waste and “throwaway” cultural attitudes that perpetuate hunger. This paper presents data illustrating the different levels of understanding and action-motivations for volunteer involvement at the kitchen, concluding that a richer vision of Eco-Justice is needed. Findings from the research led program leaders to re-envision their approach to, and understanding of, justice education. Empowering students to be in the driver’s seat of their education, as well as imbuing people with tools to be in charge of their own nutrition, is the best recipe for personal growth and social change

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

Temporal and regional variability in the skin microbiome of humpback whales along the Western Antarctic Peninsula

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Applied and Environmental Microbiology 84 (2018): e02574-17, doi:10.1128/AEM.02574-17.The skin is the first line of defense between an animal and its environment, and disruptions in skin-associated microorganisms can be linked to an animal's health and nutritional state. To better understand the skin microbiome of large whales, high-throughput sequencing of partial small subunit ribosomal RNA genes was used to study the skin-associated bacteria of 89 seemingly healthy humpback whales (Megaptera novaeangliae) sampled along the Western Antarctic Peninsula (WAP) during early (2010) and late (2013) austral summers. Six core genera of bacteria were present in 93% or more of all humpback skin samples. A shift was observed in the average relative abundance of these core genera over time, with the emergence of four additional core genera corresponding to a decrease in water temperature, possibly caused by seasonal or foraging related changes in skin biochemistry that influenced microbial growth, or other temporal-related factors. The skin microbiome differed between whales sampled at several regional locations along the WAP, suggesting that environmental factors or population may also influence the whale skin microbiome. Overall, the skin microbiome of humpback whales appears to provide insight into animal and environmental-related factors and may serve as a useful indicator for animal health or ecosystem alterations.This project was supported by 67 donors to the “Whale Bacterial Buddies” crowdfunded project supported by WHOI, the Edna Bailey Sussman Fund, and the Michael K. Orbach Enrichment Fund awarded to K. C. Bierlich

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)

Extensive core microbiome in drone-captured whale blow supports a framework for health monitoring

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in mSystems 2 (2017): e00119-17, doi:10.1128/mSystems.00119-17.The pulmonary system is a common site for bacterial infections in cetaceans, but very little is known about their respiratory microbiome. We used a small, unmanned hexacopter to collect exhaled breath condensate (blow) from two geographically distinct populations of apparently healthy humpback whales (Megaptera novaeangliae), sampled in the Massachusetts coastal waters off Cape Cod (n = 17) and coastal waters around Vancouver Island (n = 9). Bacterial and archaeal small-subunit rRNA genes were amplified and sequenced from blow samples, including many of sparse volume, as well as seawater and other controls, to characterize the associated microbial community. The blow microbiomes were distinct from the seawater microbiomes and included 25 phylogenetically diverse bacteria common to all sampled whales. This core assemblage comprised on average 36% of the microbiome, making it one of the more consistent animal microbiomes studied to date. The closest phylogenetic relatives of 20 of these core microbes were previously detected in marine mammals, suggesting that this core microbiome assemblage is specialized for marine mammals and may indicate a healthy, noninfected pulmonary system. Pathogen screening was conducted on the microbiomes at the genus level, which showed that all blow and few seawater microbiomes contained relatives of bacterial pathogens; no known cetacean respiratory pathogens were detected in the blow. Overall, the discovery of a shared large core microbiome in humpback whales is an important advancement for health and disease monitoring of this species and of other large whales.Funding for sample analysis was provided through a grant to A.A., M.J.M., and J.W.D. from the Ocean Life Institute of the Woods Hole Oceanographic Institution. Attachments for collection surfaces on the hexacopter were constructed with funding support from NOAA’s UAS Program

Body size data collected non-invasively from drone images indicate a morphologically distinct Chilean blue whale (Blaenoptera musculus) taxon

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Leslie, M. S., Perkins-Taylor, C. M., Durban, J. W., Moore, M. J., Miller, C. A., Chanarat, P., Bahamonde, P., Chiang, G., & Apprill, A. Body size data collected non-invasively from drone images indicate a morphologically distinct Chilean blue whale (Blaenoptera musculus) taxon. Endangered Species Research, 43, (2020): 291-304, https://doi.org/10.3354/esr01066.The blue whale Balaenoptera musculus (Linnaeus, 1758) was the target of intense commercial whaling in the 20th century, and current populations remain drastically below pre-whaling abundances. Reducing uncertainty in subspecific taxonomy would enable targeted conservation strategies for the recovery of unique intraspecific diversity. Currently, there are 2 named blue whale subspecies in the temperate to polar Southern Hemisphere: the Antarctic blue whale B. m. intermedia and the pygmy blue whale B. m. brevicauda. These subspecies have distinct morphologies, genetics, and acoustics. In 2019, the Society for Marine Mammalogy’s Committee on Taxonomy agreed that evidence supports a third (and presently unnamed) subspecies of Southern Hemisphere blue whale subspecies, the Chilean blue whale. Whaling data indicate that the Chilean blue whale is intermediate in body length between pygmy and Antarctic blue whales. We collected body size data from blue whales in the Gulfo Corcovado, Chile, during the austral summers of 2015 and 2017 using aerial photogrammetry from a remotely controlled drone to test the hypothesis that the Chilean blue whale is morphologically distinct from other Southern Hemisphere blue whale subspecies. We found the Chilean whale to be morphologically intermediate in both overall body length and relative tail length, thereby joining other diverse data in supporting the Chilean blue whale as a unique subspecific taxon. Additional photogrammetry studies of Antarctic, pygmy, and Chilean blue whales will help examine unique morphological variation within this species of conservation concern. To our knowledge, this is the first non-invasive small drone study to test a hypothesis for systematic biology.We are thankful to Foundation MERI (Melimoyu Ecosystem Research Institute) for logistical and funding support. Cruise support in 2017 was provided by the Dalio Foundation (now ‘OceanX’)

Improved bacterial 16S rRNA gene (V4 and V4-5) and fungal internal transcribed spacer marker gene primers for microbial community surveys

© The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in mSystems 1 (2015): e00009-15, doi:10.1128/mSystems.00009-15.Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies.J.A.F. and A.P. are supported by the Gordon and Betty Moore Foundation (GMBF3779) and NSF grant 1136818. A.P. is supported by an NSF Graduate Fellowship. A.A. is supported by NSF grant OCE-1233612. J.K.J. is supported by the Microbiomes in Transition Initiative LDRD Program at the Pacific Northwest National Laboratory, a multiprogram national laboratory operated by Battelle for the DOE under contract DE-AC06-76RL01830. J.A.G. is supported by the U.S. Department of Energy under contract DE-AC02-06CH11357. J.G.C., J.A.G., and R.K. are supported by the Alfred P. Sloan Foundation. R.K. is supported by the Howard Hughes Medical Institute