Funding for this research was provided by the National Science Foundation (DEB-1457675). We thank the many participants in the Ice Storm Experiment (ISE) who helped with the ice application and associated field and laboratory work, especially Geoff Schwaner, Gabe Winant, and Brendan Leonardi. This manuscript is a contribution of the Hubbard Brook Ecosystem Study. Hubbard Brook is part of the Long-Term Ecological Research (LTER) network, which is supported by the National Science Foundation (DEB-1633026). The Hubbard Brook Experimental Forest is operated and maintained by the USDA Forest Service, Northern Research Station, Madison, WI.&#160;Video and images are by Jim Surette and Joe Klementovich, courtesy of the Hubbard Brook Research Foundation.

1. Develop the experimental design
<ol>
	<li>Determine the intensity and frequency of icing based on realistic values.</li>
	<li>Determine the size and shape of the plots.
	<ol>
		<li>If the goal is to evaluate tree responses, select a plot size that is large enough to include multiple trees and most of their root systems, which varies depending on factors such as tree species and age.</li>
		<li>For safety purposes, design the plots so that the entire plot area can be sprayed from outside the boundary.</li>
		<li>Space plots far enough apart (e.g., 10 m) so that a treatment in one plot does not affect another.</li>
		<li>Establish a buffer zone (e.g., 5 m) around plots to reduce edge effects and ensure a more even distribution of the ice coverage.</li>
		<li>Establish subplots within the larger plots for specific sampling needs.</li>
	</ol>
	</li>
	<li>Decide on the number of replicate plots.</li>
</ol>
2. Select and establish a study location
<ol>
	<li>Select a homogeneous forest stand with similar features, such as tree species composition, soils, lithology, and hydrology.</li>
	<li>Select a location for the application in an area where there is access to a water source during winter.</li>
	<li>Ensure that the supply of water is adequate for the ice application based on the pump rate and other factors such as the diameter of the hose, length of hose, nozzle used, and water pressure.</li>
	<li>Mark the boundary of the plots, buffer zone, and subplots.</li>
	<li>Conduct a complete forest inventory with descriptions of tree health conditions including assessments of dead, dying and damaged trees. Additionally, record any potential stressors (e.g., evidence of insect damage or disease) to help interpret the response to the ice treatment.</li>
	<li>If using UTVs to spray water, create passable trails along the sides of the plots while being careful to minimize disturbance.</li>
	<li>Once the plots are established, randomly assign a treatment to each plot and type of sampling that will be conducted in each subplot (e.g., coarse woody debris, fine litter, soil samples).</li>
</ol>
3. Timing of the application
<ol>
	<li>Select an appropriate window of time to perform the spraying.</li>
	<li>Perform the experiment when the weather conditions are conducive (e.g., when air temperature is less than -4 &#176;C and wind speed is less than 5 m/s).</li>
	<li>If spraying at night, deploy high powered lights around the edge of plots and run them on generators if electricity is not available.</li>
</ol>
4. Set up the water supply
<ol>
	<li>Set up a supply pump at the water source and connect a suction hose.</li>
	<li>Connect a strainer to the end of the suction hose to keep debris out of the lines.</li>
	<li>Break through any surface ice and fully submerge the strainer. The minimum depth of the water supply should be about 20 cm.</li>
	<li>Place a booster pump in the bed of a UTV to improve water pressure. In some cases, a booster pump may not be necessary, especially for low-stature vegetation.</li>
	<li>Run a firefighting hose from the supply pump to the booster pump.</li>
	<li>Use a fire-fighting monitor to enable safe, manual control over the high-pressure hose. The monitor can be free standing or mounted on the back of a UTV.</li>
	<li>Avoid situations that may interrupt the flow of water such as kinks in the hose, water drawdown at the supply source, and running out of gasoline for the pumps.</li>
</ol>
5. Creating the ice
<ol>
	<li>Create ice by spraying water vertically through gaps in the canopy. Make sure the water extends above the height of the canopy so that it is deposited vertically and freezes on contact with sub-freezing surfaces. Avoid stripping branches and bark from trees as water is sprayed upwards.</li>
	<li>Evenly distribute spray over the forest canopy by slowly driving the UTV back-and-forth along the edge of the application area. If free-standing monitors are used, move these manually to ensure that the coverage is even.</li>
	<li>Keep track of the timing of the application to help determine factors such as the weather conditions during application and the volume of water sprayed.</li>
</ol>
6. Measure ice accretion
<ol>
	<li>Make ground-based caliper measurements of radial ice thickness on lower-level branches or twigs near the edge of the application area to monitor ice accretion during application and determine when the target thickness has been attained.</li>
	<li>Obtain more accurate estimates of ice accretion with passive ice collectors after the application (Figure 1).
	<ol>
		<li>Before the application, construct passive ice collectors with two dowels oriented on three cardinal axes30 to create collectors with six component arms.</li>
		<li>Cut 2.54 cm dowels at a length of 30 cm.</li>
		<li>Join the dowels with a 6-way steel connector.</li>
		<li>Use an arborist throw weight to string parachute cord over sturdy branches that can withstand the ice load.</li>
		<li>Attach the passive ice collectors to the cord and raise them up into the canopy.</li>
		<li>Once the application is completed, lower the collectors to the ground, being careful not to lose any ice from the collector.</li>
		<li>Make vertical and horizontal measurements of ice thickness with calipers at multiple locations on the collector (e.g., three vertical and three horizontal measurements at three locations along each arm) before and immediately after ice application.</li>
		<li>Calculate ice thickness on each collector as the difference between the measurements before and after the application.</li>
		<li>To determine ice thickness with the water volume method, use a reciprocating saw to cut each dowel.</li>
		<li>Bring the dowels to a heated building, place them in buckets, and let the ice melt off at room temperature.</li>
		<li>Measure the volume of meltwater with a graduated cylinder.</li>
		<li>Calculate ice thickness based on the water volume and density of ice31.</li>
	</ol>
	</li>
</ol>
7. Safety considerations
<ol>
	<li>Stay well outside of the ice treatment area during spraying because ice loads can cause branches and limbs to break and fall.</li>
	<li>Wear hard hats or helmets to provide protection while the ice is being applied and during any sampling that occurs in the treated area after the application.</li>
	<li>Use a monitor to stabilize the hose during spraying.</li>
	<li>Dress appropriately for hazardous conditions and sub-freezing weather. Wear bright, visible clothing. Be prepared to spend long periods in wet, cold conditions by wearing rain gear and layers of warm clothes. Bring multiple changes of clothes, especially for personnel who are designated to spray.</li>
	<li>If working in a remote location, set up a temporary warming tent equipped with a portable heater.</li>
	<li>Allow personnel to have adequate time for breaks, changing out of wet clothes, and addressing problems that arise with equipment, etc.</li>
	<li>Use radios to communicate among personnel during the experiment. Maintain contact with personnel at a base station.</li>
	<li>Develop a safety plan in case of medical emergencies. Have medical personnel (e.g., Emergency Medical Technicians) and emergency equipment and supplies on site during the experiment.</li>
</ol>

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The authors have nothing to disclose. Reference herein to any specific commercial products, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government, and shall not be used for advertising or product endorsement purposes.

It is critical to perform experimental simulations of ice storms under appropriate weather conditions to ensure their success. In a previous study30, we found that the optimal conditions for spraying are when air temperatures are below -4 &#176;C and wind speeds are less than 5 m/s. Natural ice storms most commonly occur when air temperatures are slightly less than freezing (-1 to 0 &#176;C), and although the ideal temperatures for ice storm simulations are colder, they are still within the temper...

Ice storms are an important natural disturbance that can have both short- and long-term impacts on the environment and society. Intense ice storms are problematic because they damage trees and crops, disrupt utilities, and impair roads and other infrastructure1,2. The hazardous conditions that ice storms create can cause accidents resulting in injuries and fatalities2. Ice storms are costly; financial losses average $313 million per year in the United States (US)3, with some individual storms exceeding $1 billion4. In forest ecosystems, ice storms can have negative consequences including reduced growth and tree mortality5,6,7, increased risk of fire, and proliferation of pests and pathogens8,9,10. They can also have positive effects on forests, such as enhanced growth of surviving trees5 and increased biodiversity11. Improving our ability to predict impacts from ice storms will enable us to better prepare for and respond to these events.
Ice storms occur when a layer of moist air, that is above freezing, overrides a layer of subfreezing air closer to the ground. Rain falling from the warmer layer of air supercools as it passes through the cold layer, forming glaze ice when deposited on sub-freezing surfaces. In the US, this thermal stratification can result from synoptic weather patterns that are characteristic of specific regions12,13. Freezing rain is most commonly caused by Arctic fronts that move southeastward across the US ahead of strong anticyclones13. In some regions, topography contributes to the atmospheric conditions necessary for ice storms through cold air damming, a meteorological phenomenon that occurs when warm air from an incoming storm overrides cold air that becomes entrenched alongside a mountain range14,15.
In the US, ice storms are most common in the &#8220;ice belt&#8221; that extends from Maine to western Texas16,17. Ice storms also occur in a relatively small region of the Pacific Northwest, especially around the Columbia River Basin of Washington and Oregon. Much of the US experiences at least some freezing rain, with the greatest amounts in the Northeast where the most ice prone areas have a median of seven or more freezing rain days (days during which at least one hourly observation of freezing rain occurred) annually16. Many of these storms are relatively minor, although more intense ice storms do occur, albeit with much longer recurrence intervals. For example, in New England, the range in radial ice thickness is 19 to 32 mm for storms with a 50-year recurrence interval18. Empirical evidence indicates that ice storms are becoming more frequent at northern latitudes and less frequent to the south19,20,21. This trend is expected to continue based on computer simulations using future climate change projections22,23. However, the lack of data and physical understanding make it more difficult to detect and project trends in ice storms than other types of extreme events24.
Since major ice storms are relatively rare, they are challenging to study. It is difficult to predict when and where they will occur, and it is generally impractical to &#8220;chase&#8221; storms for research purposes. Consequently, most ice storm studies have been unplanned post hoc assessments occurring in the wake of major storms. This research approach is not ideal because of the inability to collect baseline data before a storm. Additionally, it can be difficult to find unaffected areas for comparison with damaged areas when ice storms cover a large geographic extent. Rather than waiting for natural storms to occur, experimental approaches may offer advantages because they enable close control over the timing and intensity of icing events and allow for appropriate reference conditions to clearly evaluate effects.
Experimental approaches also pose challenges, especially in forested ecosystems. The height and width of trees and the canopy makes them difficult to experimentally manipulate, as compared to lower-stature grasslands or shrublands. Additionally, disturbance from ice storms is diffuse, both vertically through the forest canopy and across the landscape, which is difficult to simulate. We know of only one other study that attempted to simulate ice storm impacts in a forest ecosystem25. In this case, a rifle was used to remove up to 52% of the crown in a loblolly pine stand in Oklahoma. Although this method produced results that are characteristic of ice storms, it is not effective at removing larger branches and does not cause the trees to bend over, which is common with natural ice storms. While no other experimental methods have been used to study ice storms specifically, there are some parallels between our approach and other types of forest disturbance manipulations. For example, gap dynamics have been studied by felling individual trees26, forest pest invasions by girdling trees27, and hurricanes by pruning28 or pulling down whole trees with a winch and cable29. Of these approaches, pruning most closely imitates ice storm impacts but is labor intensive and costly. The other approaches cause mortality of whole trees, rather than the partial breakage of limbs and branches that is typical of natural ice storms.
The protocol described in this paper is useful for closely mimicking natural ice storms and involves spraying water over the forest canopy during sub-freezing conditions to simulate glaze ice events. The method offers advantages over other means because the damage can be distributed relatively evenly throughout forests over a large area with less effort than pruning or downing whole trees. Additionally, the amount of ice accretion can be regulated through the volume of water applied and by selecting a time to spray when weather conditions are conducive for optimal ice formation. This novel and relatively inexpensive experimental approach enables control over the intensity and frequency of icing, which is essential for identifying critical ecological thresholds in forest ecosystems.

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simulating impacts of ice storms on forest ecosystems

Ice storms can have profound and lasting effects on the structure and function of forest ecosystems in regions that experience freezing conditions. Current models suggest that the frequency and intensity of ice storms could increase over the coming decades in response to changes in climate, heightening interest in understanding their impacts. Because of the stochastic nature of ice storms and difficulties in predicting when and where they will occur, most past investigations of the ecological effects of ice storms have been based on case studies following major storms. Since intense ice storms are exceedingly rare events it is impractical to study them by waiting for their natural occurrence. Here we present a novel alternative experimental approach, involving the simulation of glaze ice events on forest plots under field conditions. With this method, water is pumped from a stream or lake and sprayed above the forest canopy when air temperatures are below freezing. The water rains down and freezes upon contact with cold surfaces. As the ice accumulates on trees, the boles and branches bend and break; damage that can be quantified through comparisons with untreated reference stands. The experimental approach described is advantageous because it enables control over the timing and amount of ice applied. Creating ice storms of different frequency and intensity makes it possible to identify critical ecological thresholds necessary for predicting and preparing for ice storm impacts.

An ice storm simulation was performed in a 70&#8210;100 year-old northern hardwood forest at the Hubbard Brook Experimental Forest in central New Hampshire (43&#176; 56&#8242; N, 71&#176; 45&#8242; W). The stand height is approximately 20 m and the dominant tree species in the area of the ice application are American beech (Fagus grandifolia), sugar maple (Acer saccharum), red maple (Acer rubrum) and yellow birch (Betula alleghaniensis). Ten 20 m x 30 m plots were established and rando...

Watch this Scientific Journal Video about Simulating Impacts of Ice Storms on Forest Ecosystems at JoVE.com

Simulating Impacts of Ice Storms on Forest Ecosystems

Ice storms are important weather events that are challenging to study because of difficulties in predicting their occurrence. Here, we describe a novel method for simulating ice storms that involves spraying water over a forest canopy during sub-freezing conditions.

Ice storms can have profound and lasting effects on the structure and function of forest ecosystems in regions that experience freezing conditions. ...

simulating-impacts-of-ice-storms-on-forest-ecosystems

Research

JoVE Journal

Environment

6.9K Views.  U.S. Forest Service, Durham, NH. Ice storms are important weather events that are challenging to study because of difficulties in predicting their occurrence. Here, we describe a novel method for simulating ice storms that involves spraying water over a forest canopy during sub-freezing conditions.

Video: Simulating Impacts of Ice Storms on Forest Ecosystems

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events often have devastating effects on agricultural production and can also play an important role in shaping community structure in natural populations of plants, especially in alpine, sub-arctic, and arctic ecosystems. Therefore, a better understanding of the freezing process in plants can play an important role in the development of methods of frost protection and understanding mechanisms of freeze avoidance. Here, we describe a protocol to visualize the freezing process in plants using high-resolution infrared thermography (HRIT). The use of this technology allows one to determine the primary sites of ice formation in plants, how ice propagates, and the presence of ice barriers. Furthermore, it allows one to examine the role of extrinsic and intrinsic nucleators in determining the temperature at which plants freeze and evaluate the ability of various compounds to either affect the freezing process or increase freezing tolerance. The use of HRIT allows one to visualize the many adaptations that have evolved in plants, which directly or indirectly impact the freezing process and ultimately enables plants to survive frost events.

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

Here we present a protocol that allows one to visualize sites of ice formation and avenues of ice propagation in plants utilizing high resolution infrared thermography (HRIT).

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. Yet the in situ study of DOM is difficult as the molecular complexity of the DOM pool cannot be easily reproduced for experimental purposes. Nutrient additions to streams however, have been shown to repeatedly alter the in situ and ambient DOM pool. Here we demonstrate an easily replicable field-based method for manipulating the ambient pool of DOM at the ecosystem scale. During nutrient pulse experiments changes in the concentration of both dissolved organic carbon and dissolved organic nitrogen can be examined across a wide-range of nutrient concentrations. This method allows researchers to examine the controls on the DOM pool and make inferences regarding the role and function that certain fractions of the DOM pool play within ecosystems. We advocate the use of this method as a technique to help develop a deeper understanding of DOM biogeochemistry and how it interacts with nutrients. With further development this method may help elucidate the dynamics of DOM in other ecosystems.

Understanding Dissolved Organic Matter Biogeochemistry Through In Situ Nutrient Manipulations in Stream Ecosystems

Dissolved organic matter provides an important source of energy and nutrients to stream ecosystems. Here we demonstrate a field-based method to manipulate the ambient pool of dissolved organic matter in situ through easily replicable nutrient pulses.

Dissolved organic matter (DOM) is a highly diverse mixture of molecules providing one of the largest sources of energy and nutrients to stream ecosystems. ...

Methods of Soil Resampling to Monitor Changes in the Chemical Concentrations of Forest Soils

Recent soils research has shown that important chemical soil characteristics can change in less than a decade, often the result of broad environmental changes. Repeated sampling to monitor these changes in forest soils is a relatively new practice that is not well documented in the literature and has only recently been broadly embraced by the scientific community. The objective of this protocol is therefore to synthesize the latest information on methods of soil resampling in a format that can be used to design and implement a soil monitoring program. Successful monitoring of forest soils requires that a study unit be defined within an area of forested land that can be characterized with replicate sampling locations. A resampling interval of 5 years is recommended, but if monitoring is done to evaluate a specific environmental driver, the rate of change expected in that driver should be taken into consideration. Here, we show that the sampling of the profile can be done by horizon where boundaries can be clearly identified and horizons are sufficiently thick to remove soil without contamination from horizons above or below. Otherwise, sampling can be done by depth interval. Archiving of sample for future reanalysis is a key step in avoiding analytical bias and providing the opportunity for additional analyses as new questions arise.

Repeated soil sampling has recently been shown to be an effective way to monitor forest soil change over years and decades. To support its use, a protocol is presented that synthesizes the latest information on soil resampling methods to aid in the design and implementation of successful soil monitoring programs.

Recent soils research has shown that important chemical soil characteristics can change in less than a decade, often the result of broad environmental ...

Building Double-decker Traps for Early Detection of Emerald Ash Borer

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions of forest and landscape ash (Fraxinus spp.) trees. Several artificial trap designs to attract and capture EAB beetles have been developed to detect, delineate, and monitor infestations. Double-decker (DD) traps consist of two corrugated plastic prisms, one green and one purple, attached to a 3 m tall polyvinyl chloride (PVC) pipe supported by a t-post. The green prism at the top of the PVC pipe is baited with cis-3-hexenol, a compound produced by ash foliage. Surfaces of both prisms are coated with sticky insect glue to capture adult EAB beetles. Double-decker traps should be placed near ash trees but in open areas, exposed to sun. Double-decker trap construction and placement are presented here, along with a summary of field experiments demonstrating the efficacy of DD traps in capturing EAB beetles. In a recent study in sites with relatively low EAB densities, double-decker traps captured significantly more EAB than green or purple prism traps or green funnel traps, all of which are designed to be suspended from a branch in the canopy of ash trees. A greater percentage of double decker traps were positive, i.e., captured at least one EAB, than the prism traps or funnel traps that were hung in ash tree canopies.

Effective traps to attract and capture the emerald ash borer (EAB) are a key element of detecting and managing this invasive pest. Double-decker traps, placed in full sun near ash trees, incorporate visual and olfactory cues and were more likely to capture EAB than other trap designs in field trials.

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Wastewater Irrigation Impacts on Soil Hydraulic Conductivity: Coupled Field Sampling and Laboratory Determination of Saturated Hydraulic Conductivity

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. Rather than discharging treated wastewater to a stream, and thereby directly impacting the stream quality, the effluent is applied to forested and cropped land managed by the University. Concerns related to reductions in soil hydraulic conductivity occur when considering wastewater reuse. The methodology described in this manuscript, matching soil sample size with the size of the laboratory-based hydraulic conductivity measurement apparatus, provides the benefits of a relatively rapid collection of samples with the benefits of controlled laboratory boundary conditions. The results suggest that there may have been some impact of wastewater reuse on the soil&#39;s ability to transmit water at deeper depths in the depressional areas of the site. Most of the reductions in the soil hydraulic conductivity in the depressions appear to be related to the depth from which the sample was collected, and by inference, associated with the soil structural and textural differences.

Here we present a methodology which matches a soil sample size and a hydraulic conductivity measurement device to prevent the so-called wall flow along the inside of the soil container from being erroneously included in water flow measurements. Its use is demonstrated with samples collected from a wastewater irrigation site.

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ&#160;by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (&#60; ca. &#8722;0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2&#160;for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ&#160;by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...

Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological community or food web. Importantly, the size spectrum assumes that individual size, rather than species&#8217; behavioral or life history characteristics, is the primary determinant of abundance within an ecosystem. Thus, unlike traditional allometric relationships that focus on species-level data (e.g., mean species&#8217; body size vs. population density), size spectra analyses are &#8216;ataxic&#8217; &#8211; individual specimens are identified only by their size, without consideration of taxonomic identity. Size spectra models are efficient representations of traditional, complex food webs and can be used in descriptive as well as predictive contexts (e.g., predicting responses of large consumers to changes in basal resources). Empirical studies from diverse aquatic ecosystems have also reported moderate to high levels of similarity in size spectra slopes, suggesting that common processes may regulate the abundances of small and large organisms in very different settings. This is a protocol to model the community-level size spectrum in wadable streams. The protocol consists of three main steps. First, collect quantitative benthic fish and invertebrate samples that can be used to estimate local densities. Second, standardize the fish and invertebrate data by converting all individuals to ataxic units (i.e., individuals identified by size, irrespective of taxonomic identity), and summing individuals within log2 size bins. Third, use linear regression to model the relationship between ataxic M and D estimates. Detailed instructions are provided herein to complete each of these steps, including custom software to facilitate D estimation and size spectra modeling.

This is a protocol to model the size spectrum (scaling relationship between individual mass and population density) for combined fish and invertebrate data from wadable streams and rivers. Methods include: field techniques to collect quantitative fish and invertebrate samples; lab methods to standardize the field data; and statistical data analysis.

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological ...

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 ...