We thank Mark Vermeij, Kristen Marhaver, and the CARMABI Research Foundation in Cura&#231;ao for providing resources, support, and insight for this project. We thank the NAVFAC Atlantic Vieques Restoration Program and the Jacobs Engineering team for their substantial logistical and technical support in installing, maintaining, and monitoring the Coral Arks in Vieques. We are also grateful to Mike Anghera, Toni Luque, Cynthia Silveira, Natascha Varona, Andres Sanchez-Quinto, Lars ter Horst, and Ben Darby for their help and constructive input in the field. This research was funded by a Gordon and Betty Moore Foundation Aquatic Symbiosis Investigator Award to FLR &#60;https://doi.org/10.37807/GBMF9207&#62; and by the Department of Defense Environmental Security Technology Certification Program (RC20-5175).

The authors have no competing financial interests or other conflicts of interest.

The representative results presented above demonstrate that Coral Arks provide a habitat and improved water quality conditions for assembling reef communities on stable, in situ research platforms. Arks and seafloor control sites at the same depth displayed consistently different water quality profiles. Higher average current speeds and further distance from the coast reduced sedimentation and turbidity in the midwater environment at the Arks sites (Figure 6B), likely contributing to the lower measured dissolved organic carbon concentrations on the Arks (Figure 6F). Further, these improvements in water clarity resulted in elevated daytime light intensities on the Arks relative to the control sites (Figure 6A). Lower diel fluctuations in dissolved oxygen indicate improved oxygen availability for corals on the Arks compared to the benthos, especially at night (Figure 6G). These metrics have all been associated with improvements in coral survival42, growth43,44,45, and recovery from stress46,47 in past work and may be linked to enhanced survival outcomes of corals translocated to Arks as compared to benthic control sites (Figure 8). The fact that these conditions persist even after the accumulation of substantial biomass through biofouling indicates that natural recruitment processes do not diminish the improved water quality characteristics of the midwater environment. Arks were deployed 3 km offshore of the benthic control sites and likely benefitted from decreased inputs of terrestrially derived sediment, nutrients, and possibly fishing pressures that challenge nearshore sites. Siting Arks in areas with clean water and low human impact (such as offshore) may provide a better setting than heavily impacted coastal zones to propagate reef biodiversity for mesocosm-level experiments.
The preliminary findings also suggested that the midwater Arks experienced less microbialization, a central reef process associated with the degradation of benthic reef habitats4,48. High nutrient inputs and overfishing have been identified as drivers of reef-wide trophic feedback loops in which energetically destabilized microbial communities proliferate, resulting in the respiratory drawdown of metabolically available oxygen and the increased incidence of coral pathogens at the benthos6,49,50,51. The reduced abundance of free viruses on microbialized reefs, which serve as a primary lytic control on microbial community growth, indicate a breakdown in the trophic structure that favors further microbial expansion52. Water column-associated microbes on the Arks were both less abundant (Figure 7B) and physically smaller (Figure 7D) than at the seafloor sites. The Arks also displayed higher virus-to-microbe ratios (Figure 7A), abundance of free viruses (Figure 7C), and dissolved oxygen availability, particularly at night (Figure 6G). Taken together, these findings indicate that the midwater environment displayed less potential for microbialization relative to the seafloor sites. Arks, as mesocosms on which environmental conditions can be altered simply by vertical adjustment in the water column, offer an opportunity to mitigate and further explore the microbial and molecular mechanisms of reef degradation.
Geodesic spheres of two different frequencies were selected for the design of the Coral Arks presented here (Figure 1). Geodesic frequency (1V, 2V, 3V) indicates the number of repeating sub-elements in a geodesic sphere, with higher frequencies corresponding with a higher number of triangular sub-elements. From a structural perspective, geodesic polyhedra distribute mechanical stress throughout the structure, resulting in a high innate strength for their size53,54. These characteristics provide high durability and longevity but come at the cost of higher hydrodynamic drag, which can result in higher loadings on the mooring system. From a habitat perspective, the drag generated by an Ark system represents an indicator of the diffusion of momentum within the structure and, thus, the degree to which the internal ambient flow is reduced. The modeled and experimentally validated results indicate a 40%-70% reduction in the flow speed inside of the &#34;Shell&#34; Arks relative to the surrounding flow field due to the generation of turbulent flow inside the structures (see Section 6 of Supplemental File 1). While the optimal level of internal flow reduction is not clear (and differs with geodesic frequency), areas of reduced flow within the structure are important for creating niche habitats55,56, remineralizing nutrients57,58, and promoting the retention and settlement of larvae59,60. In general, larger and higher frequency geodesic structures, particularly at more exposed installation sites, require anchoring systems with higher holding power and more redundancy incorporated into the structural design.
The results from the field-based measurements of the drag component of tension on the &#34;Shell&#34; Ark mooring system closely matched those results generated from the modeled and experimental towing estimates (Figure 4) and were well within the expected design ranges. These results indicate that the assumptions of the hydrodynamic model are valid and that the model can predict drag forces over the background current ranges. However, while the deviations in the modeled and experimental data were small, the range of flows during the testing period, which were typical of ambient, non-storm flow speeds at the site, did not enable a rigorous validation over the full modeling spectrum. In predicting the design requirements of Coral Arks systems, modeling efforts should be combined with information on storm frequency and exposure at the planned deployment sites to design structures and mooring systems that can survive the anticipated hydrodynamic forces. The modeling work presented here can be used to design Ark systems at other sites with minimal inputs (desired Ark size, frequency, and average current speeds at the deployment site) by providing drag coefficients and maximum expected forces on the mooring and anchoring system.
Arks and ARMS systems are modular and may be built at different scales and with alternative materials than those described here. Although their ultimate longevity has not yet been determined, Coral Arks were designed to have an approximately 10 year life cycle. The material composition of the Arks and ARMS affects the longevity of the structures, the weight of the systems, and, therefore, the required buoyancy to offset the weight and may affect the response of early fouling communities (Supplemental File 1-Figure S7). For example, limestone provides a more natural substrate for biological colonization on the ARMS and is readily and inexpensively sourced on most carbonate reef islands, but it is more fragile and heavier than other materials such as PVC and fiberglass. These factors should be considered against site-specific characteristics to design ARMS, Arks, and mooring systems that best address the desired project outcomes.
The deployment sites for Coral Arks should also be selected based on the intended project goals (i.e., research, mitigation, or restoration). Factors to consider for site selection include the access to materials, reef state or condition, community investment/involvement, resource limitation, institutional support, and permit requirements. Coral Arks may provide opportunities to meet specific needs at sites that (1) contain living coral reefs that are in relatively poor condition and would benefit from restoration activities to enhance the coral recruitment, coral cover, coastal protection, or human food resources; (2) have a need for the translocation of corals to another location, which may occur, for example, when there are legal requirements to move living corals off of debris items slated for removal (at these sites, Coral Arks can be used in collaboration with, or in support of, existing restoration and outplanting efforts to improve translocation outcomes); (3) require research into novel conservation and restoration technologies using Coral Arks to improve the success of local efforts; or (4) have sufficiently distinct local conditions (i.e., different magnitude of anthropogenic impact), meaning standardized mesocosms could yield meaningful comparisons about reef processes and interventions. The specific approaches for monitoring aspects of the Coral Arks ecosystem such as biological growth, diversity, and water chemistry will vary between projects based on the project goals and site-specific variables.A representative outline for the scientific monitoring of Coral Arks conducted to date is provided in Section 5 of Supplemental File 1.
The design of Coral Arks structures can accommodate corals of nearly any species, size, and age and should provide improved conditions relative to those on a disturbed reef benthos. Depending on the growth and calcification rates observed on a given system, the addition of positive buoyancy to the Arks structures may be required to compensate for biological growth and to reduce the risk of sinking. Positively buoyant midwater structures can be weighed using a tension/compression load cell, or strain gauge, to determine if the in-water weight of the community is increasing (Figure 5). Periodic or long-term measurements using the load cell can complement other finer-resolution coral growth metrics to generate a metric of community-level growth/calcification and have been included as a regular maintenance task to determine if the system has sufficient positive buoyancy to compensate for this biological growth over time. In the case that an installed Ark can no longer be monitored or maintained, it could be relocated and/or the buoyancy could be removed to allow the Ark to be firmly attached to the benthos.
The methods described here provide researchers with a versatile toolkit for assembling midwater reef communities that can be sited at locations with improved water quality. By altering the depth or location of the Arks structures, changes in water quality parameters can be experimentally linked to changes in reef community structure and successional trajectories. This design feature allows researchers to exploit the abundant and underutilized space in the midwater environment to assemble and study coral reef mesocosms. The use of seeded ARMS to translocate cryptic biodiversity and deliver a &#34;boost&#34; to the natural recruitment of mobile grazing invertebrates provides a functional solution for reducing algal biofouling and, thus, benthic competition for corals. Using established and standardized sampling structures as components of this system provides added value by enabling the long-term monitoring of cryptic communities on Arks and comparison to datasets generated using ARMS as a global biodiversity census tool.
Coral Arks can serve as a more holistic, integrated, and self-regulating platform for propagating coral and invertebrate biomass that can then be outplanted to nearby degraded reefs and can provide a safe haven for corals to grow and reproduce in improved water quality conditions. As is currently being demonstrated in Puerto Rico, Arks can yield improved survival outcomes for mitigation projects involving the relocation of corals and reef biodiversity from debris items or degraded areas. Arks have relevance in long-term projects as a method to replace habitats for fish populations, test novel conservation strategies, and preserve native reef biodiversity. In the process, Arks provide versatile tools for conducting in situ studies of reef assemblies and ecological succession and may generate novel insights into reef connectivity.

Across the globe, coral reef ecosystems are undergoing transitions from high-biodiversity, coral-dominated benthic communities to lower-diversity communities dominated by turf- and fleshy macroalgae1,2,3. Decades of progress in characterizing the mechanisms of coral reef degradation have revealed how links between microbial and macro-organismal communities enhance the pace and severity of these transitions. For example, the overfishing of reefs by human populations initiates a trophic cascade in which excess photosynthetically derived sugars from ungrazed algae shunt energy into the reef microbial communities, thus driving pathogenesis and causing coral decline4,5,6. This trophic downgrading is reinforced by the loss of biodiversity on reefs that results from water quality decline7,8. Mesocosm-level experiments can be used to better understand and mitigate the trophic downgrading of coral reef communities by enhancing biodiversity and improving water quality, but logistical challenges make these studies difficult to implement in situ.
A consequence of trophic downgrading on reefs is the widespread loss of cryptic biodiversity, much of which remains uncharacterized7,9. Corals rely on a diverse suite of cryptic reef organisms (&#34;cryptobiota&#34;) that support their health by playing integral roles in predator defense10, cleaning11, grazing competing algae12,13, and the regulation of reef water chemistry14,15. Until recently and due to the methodological limitations of visual surveys, reef cryptobiota have been underrepresented and poorly understood in the context of reef ecology, and they are, thus, rarely considered in efforts to restore or rebuild reefs. In the past decade, the use of standardized settlement units called Autonomous Reef Monitoring Structures (ARMS) combined with high-throughput sequencing approaches has enabled the better collection and characterization of reef cryptobiota16,17. ARMS passively recruit representatives of almost all known coral reef biodiversity and have helped reveal numerous functional roles of cryptic organisms in reef-scale processes9,18,19,20,21,22,23. These settlement units, therefore, provide a mechanism to translocate cryptic reef biota alongside corals in order to assemble more intact reef communities with biologically mediated mechanisms, such as grazing, defense, and enhancement of local water quality, that are essential to maintaining the trophic structure.
Coral-dominated reefs thrive in high-light, low-nutrient, and well-oxygenated environments. Human activities such as urbanization, agriculture, and overfishing have reduced the water quality on many coral reefs by increasing the sediment, nutrients, metals, and other compounds in runoff24,25 and by altering biogeochemical cycling26. In turn, these activities degrade reef communities through smothering, energy depletion, the delivery of pollutants associated with sedimentation27,28, enhancing the growth of macroalgae that compete with corals29, increasing the abundance of microbial pathogens6,30,31, and creating hypoxic zones that kill cryptic invertebrates32,33. These and other &#34;local impacts&#34; are compounded by regional and global changes in ocean conditions, including increasing temperatures and decreasing pH, further worsening the conditions for corals and other reef organisms34,35. At the benthic-water interface, specifically, the respiratory and photosynthetic dynamics of benthic communities cause diel fluctuations in the pH and dissolved oxygen, which become more pronounced on highly degraded reefs, thus creating conditions that benthic invertebrates cannot tolerate32,36,37,38. Providing appropriate water quality conditions is, therefore, essential for assembling functioning reef communities, but this remains challenging because an increasing number of reefs are trapped in various states of degradation.
Many of the challenges faced by corals and foundational cryptic taxa on the benthos may be overcome via relocation to the midwater, defined here as the water column setting between the ocean surface and the seafloor. In the midwater environment, water quality is improved39,40, sedimentation is reduced, and the distance from the seafloor dampens fluctuations in the parameters associated with benthic metabolism. These characteristics are improved further by moving offshore, where land-based anthropogenic impacts, such as terrestrially derived runoff, become increasingly diluted with distance from the coast. Here, we introduce and provide protocols to build, deploy, and monitor Coral Reef Arks, an approach that leverages improved water quality conditions in the midwater and incorporates cryptic biodiversity on anchored, positively buoyant structures for the assembly of coral reef communities.
Coral Reef Arks systems, or &#34;Arks,&#34; are comprised of two primary components: (1) a suspended rigid geodesic platform elevated above the benthos and (2) organism-covered or &#34;seeded&#34; ARMS that translocate reef cryptobiota from nearby benthic areas, thereby supplementing the natural recruitment processes to provide the translocated corals with a more diverse and functional reef community. A geodesic structure was selected to maximize the strength and minimize the building material (and, thus, the weight), as well as to create an internal, turbulent flow environment analogous to the reef matrix.
Two designs of Arks were successfully installed at two Caribbean field sites and are currently being used for research into reef community establishment and ecological succession (Figure 1). Coral Arks structures are intended to be long-term research platforms, and as such, a primary focus of this manuscript is to describe protocols to site, install, monitor, and maintain these structures to maximize their stability and longevity in the midwater environment. A combination of modeling and in-water testing was used to evaluate the drag characteristics of the structures and adjust the design to withstand the anticipated hydrodynamic forces. After installation, reef communities were established on the Arks and on nearby benthic control sites at the same depth through a combination of active translocation (corals and seeded ARMS units) and natural recruitment. Water quality conditions, microbial community dynamics, and coral survival on the Arks were documented at several time points throughout the early successional period and compared against the benthic control sites. To date, the conditions associated with the midwater Coral Arks environment have been consistently more favorable for corals and their associated cryptic consortia relative to the neighboring benthic control sites at the same depths. The methods below describe the steps required to replicate the Coral Arks approach, including how to select sites and design and deploy Coral Arks structures. Suggested approaches for monitoring Coral Arks are included in Supplemental File 1.

coral reef arks: an in situ mesocosm and toolkit for assembling reef communities

Coral reefs thrive and provide maximal ecosystem services when they support a multi-level trophic structure and grow in favorable water quality conditions that include high light levels, rapid water flow, and low nutrient levels. Poor water quality and other anthropogenic stressors have caused coral mortality in recent decades, leading to trophic downgrading and the loss of biological complexity on many reefs. Solutions to reverse the causes of trophic downgrading remain elusive, in part because efforts to restore reefs are often attempted in the same diminished conditions that caused coral mortality in the first place. 
Coral Arks, positively buoyant, midwater structures, are designed to provide improved water quality conditions and supportive cryptic biodiversity for translocated and naturally recruited corals to assemble healthy reef mesocosms for use as long-term research platforms. Autonomous Reef Monitoring Structures (ARMS), passive settlement devices, are used to translocate the cryptic reef biodiversity to the Coral Arks, thereby providing a "boost" to natural recruitment and contributing ecological support to the coral health. We modeled and experimentally tested two designs of Arks to evaluate the drag characteristics of the structures and assess their long-term stability in the midwater based on their response to hydrodynamic forces. 
We then installed two designs of Arks structures at two Caribbean reef sites and measured several water quality metrics associated with the Arks environment over time. At deployment and 6 months after, the Coral Arks displayed enhanced metrics of reef function, including higher flow, light, and dissolved oxygen, higher survival of translocated corals, and reduced sedimentation and microbialization relative to nearby seafloor sites at the same depth. This method provides researchers with an adaptable, long-term platform for building reef communities where local water quality conditions can be adjusted by altering deployment parameters such as the depth and site.

Watch this Scientific Journal Video about Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities at JoVE.com

Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities

Moored midwater geodesic structures called Coral Arks provide a modular, scalable, and vertically adjustable research platform that can be used to build, monitor, and perturb coral reef communities in previously inoperative areas, including offshore.

Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities

Coral reefs thrive and provide maximal ecosystem services when they support a multi-level trophic structure and grow in favorable water quality conditions ...

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3.2K Views.  San Diego State University. Moored midwater geodesic structures called Coral Arks provide a modular, scalable, and vertically adjustable research platform that can be used to build, monitor, and perturb coral reef communities in previously inoperative areas, including offshore. 

Video: Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps&#160;and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter&#160;scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,&#160;(2) superior optical sectioning ability,&#160;and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

An integrated suite of imaging techniques has been applied to determine polyp morphology and tissue structure in the Caribbean corals Montastraeaannularis and M. faveolata. Fluorescence, serial block face,&#160;and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and distributions.

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues ...

Spotting Cheetahs: Identifying Individuals by Their Footprints

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges widely over sub-Saharan Africa and in parts of the Middle East. Cheetah conservationists face two major challenges, conflict with landowners over the killing of domestic livestock, and concern over range contraction. Understanding of the latter remains particularly poor2. Namibia is believed to support the largest number of cheetahs of any range country, around 30%, but estimates range from 2,9053 to 13,5204. The disparity is likely a result of the different techniques used in monitoring.
Current techniques, including invasive tagging with VHF or satellite/GPS collars, can be costly and unreliable. The footprint identification technique5 is a new tool accessible to both field scientists and also citizens with smartphones, who could potentially augment data collection. The footprint identification technique analyzes digital images of footprints captured according to a standardized protocol. Images are optimized and measured in data visualization software. Measurements of distances, angles, and areas of the footprint images are analyzed using a robust cross-validated pairwise discriminant analysis based on a customized model. The final output is in the form of a Ward's cluster dendrogram. A user-friendly graphic user interface (GUI) allows the user immediate access and clear interpretation of classification results.
The footprint identification technique algorithms are species specific because each species has a unique anatomy. The technique runs in a data visualization software, using its own scripting language (jsl) that can be customized for the footprint anatomy of any species. An initial classification algorithm is built from a training database of footprints from that species, collected from individuals of known identity. An algorithm derived from a cheetah of known identity is then able to classify free-ranging cheetahs of unknown identity. The footprint identification technique predicts individual cheetah identity with an accuracy of &gt;90%.

The cheetah (Acinonyx jubatus) is an iconic, endangered species, but conservation efforts are challenged by habitat shrinkage and conflict with commercial farmers. The footprint identification technique, a robust, accurate and cost-effective image classification system, is a new approach to monitoring cheetahs.

The cheetah (Acinonyx jubatus) is Africa's most endangered large felid and listed as Vulnerable with a declining population trend by the IUCN1. It ranges ...

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and organiclastic sulfate reduction (OSR) in marine sediments. However, unravelling the complex pyritization sequence is a challenge because of the coexistence of different sequentially formed pyrite phases. This manuscript describes a sample preparation procedure that enables the use of secondary ion mass spectroscopy (SIMS) to obtain in situ &#948;34S values of various pyrite generations. This allows researchers to constrain how SO4-AOM affects pyritization in methane-bearing sediments. SIMS analysis revealed an extreme range in &#948;34S values, spanning from -41.6 to +114.8&#8240;, which is much wider than the range of &#948;34S values obtained by the traditional bulk sulfur isotope analysis of the same samples. Pyrite in the shallow sediment mainly consists of 34S-depleted framboids, suggesting early diagenetic formation by OSR. Deeper in the sediment, more pyrite occurs as overgrowths and euhedral crystals, which display much higher SIMS &#948;34S values than the framboids. Such 34S-enriched pyrite is related to enhanced SO4-AOM at the sulfate-methane transition zone, postdating OSR. High-resolution in situ SIMS sulfur isotope analyses allow for the reconstruction of the pyritization processes, which cannot be resolved by bulk sulfur isotope analysis.

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Analyses of the sulfur isotopic composition (&#948;34S) of pyrite from methane-bearing sediments have typically focused on bulk samples. Here, we applied secondary ion mass spectroscopy to analyze the &#948;34S values of various pyrite generations to understand the diagenetic history of pyritization.

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

Characterizing Microbiome Dynamics &#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical applications driven by microorganisms. Next generation sequencing methods are widely utilized to resolve microbiomes, but they are generally resource and time intensive and deliver mostly qualitative information. Flow cytometric microbiome analysis does not suffer from those disadvantages and can provide relative subcommunity abundances and absolute cell numbers at-line. Although it does not deliver direct phylogenetic information, it can enhance the analysis depth and resolution of sequencing approaches. In sharp contrast to medical applications in both research and routine settings, flow cytometry is still not widely used for microbiome analysis. Missing information on sample preparation and data analysis pipelines may create an entry barrier for the researchers facing microbiome analysis challenges that would often be textbook flow cytometry applications. Here, we present three comprehensive workflows for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively. We describe individual sampling and fixation procedures and optimized staining protocols for the respective sample sets. We elaborate the cytometric analysis with a complex research centered and an application focused bench top device, describe the cell sorting procedure and suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets.

Characterizing Microbiome Dynamics &amp;#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

Flow cytometric analysis has proven valuable for investigating pure cultures and monitoring microbial community dynamics. We present three comprehensive workflows, from sampling to data analysis, for pure cultures and complex communities in clear medium as well as in challenging matrices, respectively.

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical ...

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) and quantify any differences in fin cortisol levels among three main sturgeon species. Fins were harvested from 19 sacrificed sturgeons including seven beluga (Huso huso), seven Siberian (Acipenser baerii), and five sevruga (A. stellatus). The sturgeons were raised in Iranian farms for 2 years (2017-2018), and cortisol extraction analysis was conducted in South Korea (January-February 2019). Jawbones from five H. huso were also used for cortisol extraction. Data were analyzed using the general linear model (GLM) procedure in the SAS environment. The intra- and inter-assay coefficients of variation were 14.15 and 7.70, respectively. Briefly, the cortisol extraction technique involved washing the samples (300 &#177; 10 mg) with 3 mL of solvent (ultrapure water and isopropanol) twice, rotation at 80&#160;rpm for 2.5 min, air-drying the washed samples at room temperature (22-28 &#176;C) for 7 days, further drying the samples using a bead beater at 50 Hz for 32 min and grinding them into powder, applying 1.5 mL methanol to the dried powder (75 &#177; 5 mg), and slow rotation (40 rpm) for 18 h at room temperature with continuous mixing. Following extraction, samples were centrifuged (9,500 x g for 10 min), and 1 mL supernatant was transferred into a new microcentrifuge tube (1.5 mL), incubated at 38 &#176;C to evaporate the methanol, and analyzed via enzyme-linked immunosorbent assay (ELISA). No differences were observed in fin cortisol levels among species or in fin and jawbone cortisol levels between washing solvents. The results of this study demonstrate that the sturgeon jawbone matrix is a promising alternative stress indicator to solid matrices.

In this study, we present a protocol for cortisol extraction from the fin and jawbone of sturgeon species. Fin and jawbone cortisol levels were further examined by comparing two washing solvents followed by ELISA assays. This study piloted the feasibility of jawbone cortisol as a novel stress indicator.

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) ...

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous environments. It also plays a crucial role in symbiotic interactions, disease, and global processes, such as biogeochemical cycling. However, current techniques restrict chemotaxis research to the laboratory and are not easily applicable in the field. Presented here is a step-by-step protocol for the deployment of the in situ chemotaxis assay (ISCA), a device that enables robust interrogation of microbial chemotaxis directly in the natural environment. The ISCA is a microfluidic device consisting of a 20 well array, in which chemicals of interest can be loaded. Once deployed in aqueous environments, chemicals diffuse out of the wells, creating concentration gradients that microbes sense and respond to by swimming into the wells via chemotaxis. The well contents can then be sampled and used to (1) quantify strength of the chemotactic responses to specific compounds through flow cytometry, (2) isolate and culture responsive microorganisms, and (3) characterize the identity and genomic potential of the responding populations through molecular techniques. The ISCA is a flexible platform that can be deployed in any system with an aqueous phase, including marine, freshwater, and soil environments.

Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread ...