Microfluidics Expanding the Frontiers of Microbial Ecology

Microfluidics has significantly contributed to the expansion of the frontiers of microbial ecology over the past decade by allowing researchers to observe the behaviors of microbes in highly controlled microenvironments, across scales from a single cell to mixed communities. Spatially and temporally varying distributions of organisms and chemical cues that mimic natural microbial habitats can now be established by exploiting physics at the micrometer scale and by incorporating structures with specific geometries and materials. In this article, we review applications of microfluidics that have resulted in insightful discoveries on fundamental aspects of microbial life, ranging from growth and sensing to cell-cell interactions and population dynamics. We anticipate that this flexible multidisciplinary technology will continue to facilitate discoveries regarding the ecology of microorganisms and help uncover strategies to control microbial processes such as biofilm formation and antibiotic resistance.National Science Foundation (U.S.) (Grant OCE-0744641-CAREER)National Science Foundation (U.S.) (Grant IOS-1120200)National Science Foundation (U.S.) (Grant CBET-1066566)National Science Foundation (U.S.) (Grant CBET-0966000)National Institutes of Health (U.S.) (NIH grant 1R01GM100473-0)Human Frontier Science Program (Strasbourg, France)Human Frontier Science Program (Strasbourg, France) (award RGY0089)Gordon and Betty Moore Foundation (Microbial Initiative Investigator Award

Resilience of Coral-Associated Bacterial Communities Exposed to Fish Farm Effluent

10.1371/journal.pone.0007319PLoS ONE410

Baseline shifts in coral skeletal oxygen isotopic composition: a signature of symbiont shuffling?

Decades-long records of the stable isotopic composition of coral skeletal cores were analyzed from four sites on the Mesoamerican Reef. Two of the sites exhibited baseline shifts in oxygen isotopic composition after known coral bleaching events. Changes in pH at the calcification site caused by a change in the associated symbiont community are invoked to explain the observed shift in the isotopic composition. To test the hypothesis that changes in symbiont clade could affect skeletal chemistry, additional coral samples were collected from Belize for paired Symbiodinium identification and skeletal stable isotopic analysis. We found some evidence that skeletal stable isotopic composition may be affected by symbiont clade and suggest this is an important topic for future investigation. If different Symbiodinium clades leave consistent signatures in skeletal geochemical composition, the signature will provide a method to quantify past symbiont shuffling events, important for understanding how corals are likely to respond to climate change

Vortical ciliary flows actively enhance mass transport in reef corals

The exchange of nutrients and dissolved gasses between corals and their environment is a critical determinant of the growth of coral colonies and the productivity of coral reefs. To date, this exchange has been assumed to be limited by molecular diffusion through an unstirred boundary layer extending 1–2 mm from the coral surface, with corals relying solely on external flow to overcome this limitation. Here, we present direct microscopic evidence that, instead, corals can actively enhance mass transport through strong vortical flows driven by motile epidermal cilia covering their entire surface. Ciliary beating produces quasi-steady arrays of counterrotating vortices that vigorously stir a layer of water extending up to 2 mm from the coral surface. We show that, under low ambient flow velocities, these vortices, rather than molecular diffusion, control the exchange of nutrients and oxygen between the coral and its environment, enhancing mass transfer rates by up to 400%. This ability of corals to stir their boundary layer changes the way that we perceive the microenvironment of coral surfaces, revealing an active mechanism complementing the passive enhancement of transport by ambient flow. These findings extend our understanding of mass transport processes in reef corals and may shed new light on the evolutionary success of corals and coral reefs.Human Frontier Science Program (Strasbourg, France) (Award RGY0089)National Science Foundation (U.S.) (Grant OCE-0744641-CAREER)National Institutes of Health (U.S.) (Grant 1R01GM100473-01)Gordon and Betty Moore Foundation (Investigator Grant GBMF3783

Visualization of coral host--pathogen interactions using a stable GFP-labeled Vibrio coralliilyticus strain

The bacterium Vibrio coralliilyticus has been implicated as the causative agent of coral tissue loss diseases (collectively known as white syndromes) at sites across the Indo-Pacific and represents an emerging model pathogen for understanding the mechanisms linking bacterial infection and coral disease. In this study, we used a mini-Tn7 transposon delivery system to chromosomally label a strain of V. coralliilyticus isolated from a white syndrome disease lesion with a green fluorescent protein gene (GFP). We then tested the utility of this modified strain as a research tool for studies of coral host–pathogen interactions. A suite of biochemical assays and experimental infection trials in a range of model organisms confirmed that insertion of the GFP gene did not interfere with the labeled strain’s virulence. Using epifluorescence video microscopy, the GFP-labeled strain could be reliably distinguished from non-labeled bacteria present in the coral holobiont, and the pathogen’s interactions with the coral host could be visualized in real time. This study demonstrates that chromosomal GFP labeling is a useful technique for visualization and tracking of coral pathogens and provides a novel tool to investigate the role of V. coralliilyticus in coral disease pathogenesis.Human Frontier Science Program (Strasbourg, France) (No. RGY0089RS

Deciphering coral disease dynamics: integrating host, microbiome, and the changing environment

Diseases of tropical reef organisms is an intensive area of study, but despite significant advances in methodology and the global knowledge base, identifying the proximate causes of disease outbreaks remains difficult. The dynamics of infectious wildlife diseases are known to be influenced by shifting interactions among the host, pathogen, and other members of the microbiome, and a collective body of work clearly demonstrates that this is also the case for the main foundation species on reefs, corals. Yet, among wildlife, outbreaks of coral diseases stand out as being driven largely by a changing environment. These outbreaks contributed not only to significant losses of coral species but also to whole ecosystem regime shifts. Here we suggest that to better decipher the disease dynamics of corals, we must integrate more holistic and modern paradigms that consider multiple and variable interactions among the three major players in epizootics: the host, its associated microbiome, and the environment. In this perspective, we discuss how expanding the pathogen component of the classic host-pathogen-environment disease triad to incorporate shifts in the microbiome leading to dysbiosis provides a better model for understanding coral disease dynamics. We outline and discuss issues arising when evaluating each component of this trio and make suggestions for bridging gaps between them. We further suggest that to best tackle these challenges, researchers must adjust standard paradigms, like the classic one pathogen-one disease model, that, to date, have been ineffectual at uncovering many of the emergent properties of coral reef disease dynamics. Lastly, we make recommendations for ways forward in the fields of marine disease ecology and the future of coral reef conservation and restoration given these observations

Managing marine disease emergencies in an era of rapid change

Infectious marine diseases can decimate populations and are increasing among some taxa due to global change and our increasing reliance on marine environments. Marine diseases become emergencies when significant ecological, economic or social impacts occur. We can prepare for and manage these emergencies through improved surveillance, and the development and iterative refinement of approaches to mitigate disease and its impacts. Improving surveillance requires fast, accurate diagnoses, forecasting disease risk and real-time monitoring of disease-promoting environmental conditions. Diversifying impact mitigation involves increasing host resilience to disease, reducing pathogen abundance and managing environmental factors that facilitate disease. Disease surveillance and mitigation can be adaptive if informed by research advances and catalysed by communication among observers, researchers and decision-makers using information-sharing platforms. Recent increases in the awareness of the threats posed by marine diseases may lead to policy frameworks that facilitate the responses and management that marine disease emergencies require

Microbial Responses and Coral Reef Resilience to Organic Matter Inputs

Little attention has been given to the small-scale mechanisms relevant to microbial processes that determine the resilience of individual corals to the stress of organic matter (OM) inputs. Such mechanisms may be a critical link for predicting larger scale patterns of reef resilience. The research presented here aims to develop methods necessary to elucidate these processes and to explore the in situ responses of microbial assemblages on coral reefs experiencing persistent OM enrichment. A new method using trypsinization of coral mucus before staining for epifluorescence microscopy is described. It is then applied to a coral reef ecosystem influenced by sewage effluent to discover that corals exposed to effluent had the same number of bacteria present as reference corals; however, the size structure of the community was significantly different. Investigating the responses of microbial communities (from both the water column and corals) to OM inputs from coastal milkfish (Chanos chanos ) pens, we found that the percentage of the water bacterial community attached to particles increased by more than 50-fold near the pens. This suggested a physiological or life-strategy change may be induced by such enrichment. A clonally replicated coral transplantation experiment examined the response of naïve coral-associated bacterial communities to high and low levels of pen effluent exposure. We found that the communities on corals exposed to high levels of effluent had drastically altered community compositions after five days and the abundance of bacteria in the coral mucus-tissue slurries were ~100-fold higher controls at low effluent and reference sites. We also observed a surprising resilience of these communities in that their composition and total abundance recovered by day 22. A combination of novel imaging technology (high speed laser scanning confocal microscopy on live coral) and controlled aquaria experiments were developed and used to investigate a microscale mechanism by which such resilience may occur: that corals may release ("shed") bacteria into the surrounding water as a mechanism for controlling bacterial abundance on their surface. We observed this phenomenon in real time, and quantified an increase in the rate at which corals shed bacteria as a response to OM enrichment

Microbial responses and coral reef resilience to organic matter inputs

Little attention has been given to the small-scale mechanisms relevant to microbial processes that determine the resilience of individual corals to the stress of organic matter (OM) inputs. Such mechanisms may be a critical link for predicting larger scale patterns of reef resilience. The research presented here aims to develop methods necessary to elucidate these processes and to explore the in situ responses of microbial assemblages on coral reefs experiencing persistent OM enrichment. A new method using trypsinization of coral mucus before staining for epifluorescence microscopy is described. It is then applied to a coral reef ecosystem influenced by sewage effluent to discover that corals exposed to effluent had the same number of bacteria present as reference corals; however, the size structure of the community was significantly different. Investigating the responses of microbial communities (from both the water column and corals) to OM inputs from coastal milkfish (Chanos chanos) pens, we found that the percentage of the water bacterial community attached to particles increased by more than 50- fold near the pens. This suggested a physiological or life -strategy change may be induced by such enrichment. A clonally replicated coral transplantation experiment examined the response of naïve coral-associated bacterial communities to high and low levels of pen effluent exposure. We found that the communities on corals exposed to high levels of effluent had drastically altered community compositions after five days and the abundance of bacteria in the coral mucus-tissue slurries were ̃100- fold higher controls at low effluent and reference sites. We also observed a surprising resilience of these communities in that their composition and total abundance recovered by day 22. A combination of novel imaging technology (high speed laser scanning confocal microscopy on live coral) and controlled aquaria experiments were developed and used to investigate a microscale mechanism by which such resilience may occur: that corals may release ("shed") bacteria into the surrounding water as a mechanism for controlling bacterial abundance on their surface. We observed this phenomenon in real time, and quantified an increase in the rate at which corals shed bacteria as a response to OM enrichmen