This work was supported by the Young Scientists Fund of the National Natural Science Foundation of China (51709082) and the Fundamental Research Funds for the Central Universities (2018B13014).

1. Laboratory safety check
<ol>
	<li>Review the safety rules relating to the use of the laser and flume system.</li>
	<li>Ensure that the safety training requirements of the laboratory have been met. 
	NOTE: In this experiment, a set of 5W air-cooling continuous wave laser with a wavelength of 532 nm and a glass-sided straight flume (Figure 1) with dimensions of 11 m length, 0.6 m width, and 0.6 m depth are used. The basic safety recommendations for these two apparatuses are as follows:
	<ol>
		<li>Check the potential reflection surfaces in the laser line-of-sight prior to testing; wear safety goggles when operating the laser device.</li>
		<li>Avoid having eyes at the level of the laser beam during the experiments and be careful of reflected laser lights when handling the optical elements or reflective tools.</li>
		<li>Ensure that the water hose does not fall off and that there is always no water overflowing from the flume.</li>
	</ol>
	</li>
</ol>
2. Flume and seabed model setup
<ol>
	<li>Prepare the erodible seabed model located in the middle of the flume. 
	NOTE: The sediment material used in this study was a uniformly distributed medium sand with a median grain size d50 = 0.45 mm, relative submerged particle density &#916; = 1.65 and geometric standard deviation &#963;g = 1.30.</li>
	<li>Compact and level the seabed using a sand leveler.</li>
	<li>Slowly fill the flume with a water hose and make sure that a flat seabed surface is intact during the filling process; stop filling when the water level has reached a depth of 0.4 m above the seabed.</li>
	<li>Clear the flume top platform and glass for setting up the pipeline model and PIV system.</li>
</ol>

3. Pipeline model and vibration system setup

<ol>
	<li>Use a prefabricated pipeline model in the form of an acrylic cylinder with a diameter of 35 mm and a length of 0.56 m.</li>
	<li>Mount the pipeline model on an aluminum supporting frame, which, in turn, is connected by two springs to a moveable pole on another fixed frame that is locked on the top rails of the flume, as illustrated in Figure 2. Fix the supporting frame inside the fixed frame by using four bearings to ensure that the supporting frame could freely vibrate only along the vertical direction (Figure 2).</li>
	<li>Use a connecting rod to tie the moveable pole to a servo motor mounted on the top of the fixed frame. In this study, the weight of the assembled vibrating system, including the pipeline model and the aluminum frames, is 1.445 kg, which has an equivalent mass ratio (m*) of 2.682; a natural frequency (fN) of 0.82 Hz; and damping ratio (&#950;) of 0.124.</li>
	<li>Adjust the moveable pole and the supporting frame to obtain a certain gap ratio between the pipeline and seabed. &#160;In this study, G/D =1, where G is the vertical distance between the bottom of the pipeline and initial seabed surface; and D is the pipeline diameter.</li>
	<li>Turn on the servo motor to induce a forced vibration on the pipeline; adjust the supporting frames and four bearings to ensure that the pipeline vibration is along the vertical direction. Turn off the servo motor when adjustments of the supporting frames have completed.</li>
	<li>Compact and level the seabed again before running the experiment if the seabed model is disturbed in 3.5.</li>
</ol>

4. PIV setup

<ol>
	<li>Place the laser device on the top of the flume and install the laser sheet forming optics.</li>
	<li>Turn on the laser device and adjust the laser sheet forming optics so that an illuminated flat sheet inside the field-of-interest is formed. 
	NOTE: In this study, the illuminated green laser sheet is 1.5 mm thick, parallel to the flume glass walls and is cast downward into the water along the centerline of the flume. The field-of-interest of this study refers to the interaction region of the pipeline-fluid-seabed and is confined to the right half side of the pipeline. The shadow of the pipeline will be seen on the left half side of the pipeline.</li>
	<li>Set up the high-speed camera. 
	NOTE: For this study, a high-speed camera with 12-gigabyte-memory storage and a maximum resolution of 2.3 Mpx (1920&#215;1200) is used (e.g., Phantom Miro LAB 320). The detailed operation procedures are as follows:
	<ol>
		<li>Mount the lens with appropriate focal length on the high-speed camera. Screw the high-speed camera onto a height-adjustable tripod; adjust the camera to the level of the observation region with its axis perpendicular to the illuminated laser sheet. 
		NOTE: This study uses a 60 mm prime lens at its maximum aperture of f/2.8.</li>
		<li>Connect the camera to the computer by using an Ethernet cable and turn on the camera control software (e.g., Phantom PCC 2.6); turn on the camera and connect it to the computer in the camera control software interface.</li>
		<li>Adjust the tripod to ensure that the field-of-view of the camera covers the pipeline-fluid-seabed interaction region; level the camera using the built-in bubble level on the tripod; tune the focus ring on the lens to ensure that the laser sheet is clear on the focal plane.</li>
	</ol>
	</li>
</ol>

5. Experimental setup optimization and calibration

<ol>
	<li>Add PIV seeding particles to the test section of the flume. 
	NOTE: The seeding particles used in this study were aluminum powders with a diameter of 10 &#956;m and a specific density of 2.7.</li>
	<li>Enhance the light intensity of the laser sheet if necessary.</li>
	<li>Verify the focus of the camera by observing the illuminated seeding particles on the laser sheet through a live camera view on the computer; fine-tune the focus ring, if necessary, to ensure that the seeding particles are sharp and in focus.</li>
	<li>Place a calibration ruler inside the field-of-view on the plane of the laser sheet and capture one calibration image. 
	NOTE: The adopted resolution of the image in this study was 1600 &#215; 1200 pixels.</li>
	<li>Select a proper sampling rate for data collection. 
	NOTE: The chosen sampling rate should ensure that the seeding particle displacement within a pair of images is less than 50% of the maximum interrogation window length. In this study, the maximum interrogation window size is 32 &#215; 32 pixels and the adopted sampling rate is 200 frames per second.</li>
	<li>Turn off the laser and camera when steps 5.1-5.5 are completed.</li>
</ol>

6. Running the experiment and data collection

<ol>
	<li>Place a transparent acrylic plate (20 mm thick) below the laser source and on the water surface, to suppress water surface fluctuations, and ensure tranquil optical access for the laser light.</li>
	<li>Turn on the servo motor to induce forced vibrations on the pipeline model. 
	NOTE: In this study, the induced frequency of the servo motor is f0 = 0.3 Hz.</li>
	<li>Keep the vibration system running for (t =)1440 min to obtain a quasi-equilibrium scour hole beneath the vibrating pipeline.</li>
	<li>Turn on the laser and adjust the output power to the optimized intensity. Turn on the camera and camera control software and apply the calibrated settings to the camera. Turn off the background lights in the laboratory.</li>
	<li>Start recording the seeding particle-laden flow field image with the sampling rate selected in 5.6 by clicking the Capture Bottom in the camera software control software. 
	NOTE: For every single recording in this study, the camera storage allows 1,000 images to be captured.</li>
	<li>Once the data collection is completed, review the recorded image quality and check if the seeding particle density per interrogation window (32&#215;32 pixels) is greater than 8. Save the recorded file if satisfied, otherwise, the seeding density is increased by slowly injecting seeding solutions in the observation region, and repeat steps 6.3-6.5.</li>
	<li>Repeat steps 6.3-6.5 to collect more data sets. 
	NOTE: For this study, more than 20,000 images were taken to ensure that enough raw data are obtained for calculating the flow velocities, vorticities, turbulence, and near-boundary stresses.</li>
	<li>Turn off the laser device, camera, and server motor when all the data collections are completed; turn on the background lights in the laboratory.</li>
</ol>

7. Data processing

<ol>
	<li>Open the software; click the File folder button on the toolbar and load the calibration image taken in step 5.4. 
	NOTE: Use the data processing program for pipeline displacement tracking and flow field calculations software (e.g., PISIOU).</li>
	<li>Click the Scale setup button on the toolbar; measure a known distance on the calibration image to calculate the scale of the image. 
	NOTE: The calculated image scale was 0.1694 mm/pixel.</li>
	<li>Click the Origin button on the toolbar; set the origin of the coordinates on each image.</li>
	<li>Extract the displacement time-series of the vibrating pipeline from the recorded images.
	<ol>
		<li>Load the raw images taken in step 6. Then click on the Parameter panel, enter the number of data files and the sample rate.</li>
		<li>Apply the Low pass filter in the Image filter menu. 
		NOTE: This operation will allow the edge of the pipeline (target to be tracked) to be readily recognized on the processed images (see Figure 3a).</li>
		<li>In the toolbar, click the PTV module. Then click Tracing point button, select the center point of the pipeline. Go to PTV tools, adjust Gamma, Light Gate and Median Filter to single out the pipeline outline in the image. Click the Object tracking button on the toolbar; select the target region (i.e. the pipeline) on the processed image and track the displacement of the vibrating pipeline from consecutive processed images; record the displacement time-series, &#951;(t), of the vibrating pipeline for subsequent flow field data processes (see Figure 4).</li>
		<li>Export and save the pipeline displacement time-series data for further calculations.</li>
	</ol>
	</li>
	<li>Determine instantaneous velocity fields from the recorded images.
	<ol>
		<li>Go to PTV tools, click Default button to resume the raw image for subsequent PIV analysis. Deactivate the PTV module by clicking the PTV module. Open the Parameter panel on the toolbar; specify the velocity vector calculation parameter. 
		NOTE: In this study, a multi-pass iteration process is adopted as the interrogation windows, which started from 32 &#215; 32 pixels, then passed with 16 &#215; 16 pixels, and ended with 8 &#215; 8 pixels; all passes use a 50% overlap between adjacent sub-windows.</li>
		<li>Apply the Laplacian filter function in the Image filter menu to the raw images to highlight the seeding particles and filter out undesirable scattering light (see Figure 3c).</li>
		<li>Click the Boundary button on the toolbar, set the geometric mask on the images to exclude the seabed region for further calculation.&#160;Click Boundary Save button to save the boundary data.</li>
		<li>Click the Run button on the toolbar to calculate the instantaneous velocity fields for different vibrating phases using the cross-correlation method. 
		NOTE: In this study, a multi-time interval algorithm is adopted to reduce the bias error due to high velocity gradient in the flow field (see Figure 5). The adopted multiple-time intervals for cross-correlation calculations are &#916;t, 3&#916;t, 9&#916;t and 21&#916;t (&#916;t = 5 ms). The satisfactory correlation criterion is greater than 70%.</li>
		<li>Export and save the instantaneous velocity fields data for further analysis.</li>
	</ol>
	</li>
	<li>Determine the phase-averaged velocity fields from the calculated instantaneous velocity fields with the algorithm as described in Newland 199429,30&#160; and Hsieh 200828. 
	NOTE: The calculation procedures for this step is described as follows:
	<ol>
		<li>Apply the wavelet transform function to the displacement time-series, &#951;(t), of the vibrating pipeline to obtain the instantaneous phase for each instantaneous velocity field. The wavelet transform function is defined as: 
		<img alt="Equation 1" src="/files/ftp_upload/59745/59745eq1.jpg" />&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160;(1) 
		where W&#936; is wavelet coefficient; &#945; and &#946; are the scale and translation parameters, respectively; the function &#936; is the Morlet function and is calculated as <img alt="Equation 2" src="/files/ftp_upload/59745/59745eq2.jpg" />; the superscript &#8220;*&#8221; denotes the complex conjugate. The instantaneous phases, &#934;, of the vibrating pipeline that correspond to the different pipeline displacements can be calculated from: 
		<img alt="Equation 3" src="/files/ftp_upload/59745/59745eq3.jpg" />&#160;&#160; &#160; &#160; &#160; &#160; &#160; &#160; &#160; (2)</li>
		<li>Average the instantaneous velocity fields with the same phase to obtain the phase-averaged velocity fields.</li>
		<li>Determine the flow vorticity, &#969;2, in the calculated phase-averaged velocity fields from: 
		<img alt="Equation 4" src="/files/ftp_upload/59745/59745eq4.jpg" />&#160; &#160;&#160; &#160; &#160; &#160; &#160;(3) 
		where <img alt="Equation 5" src="/files/ftp_upload/59745/59745eq5.jpg" /> and <img alt="Equation v" src="/files/ftp_upload/59745/59745eqv.jpg" /> are phase-averaged velocities along x and y directions.</li>
	</ol>
	</li>
	<li>Load the calculated phase-averaged velocity and vorticity data in Tecplot software for visualization.</li>
	<li>Determine the near-boundary shear and normal stresses from the calculated instantaneous velocity fields with the algorithm as described in Hsieh et al. 2016 9. The calculation procedures for this step is described as follows:
	<ol>
		<li>Extract the near-boundary velocity data (0-5 mm above the seabed) from the calculated phase-averaged velocity flow fields.</li>
		<li>Compute the near-boundary shear stresses, ts and normal stresses, tn, along the scour profile (approximately 2 mm above the scour hole boundary) for different phases within one vibrating cycle. Note: The calculation equations are as follows: 
		<img alt="Equation 7" src="/files/ftp_upload/59745/59745eq7.jpg" />, <img alt="Equation 8" src="/files/ftp_upload/59745/59745eq8.jpg" />&#160; &#160; &#160; &#160; &#160; &#160; &#160; (4) 
		where, &#956; = dynamic viscosity of the fluid (herein taken as 1&#215;10-3 Pa&#8729;s); up = near-boundary velocity parallel to the bed; un = near-boundary velocity perpendicular to the bed; n = normal distance from the bed.</li>
	</ol>
	</li>
	<li>Load the calculated near-boundary shear and normal stresses data in a software (e.g., Tecplot) for visualization.</li>
</ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+recirculation+zone+structure+behind+an+impulsively+started+circular+cylinder.">Characteristics of recirculation zone structure behind an impulsively started circular cylinder.</a> Journal of Engineering Mechanics. 138 (2), 184-198 (2012).</li><li>Sarpkaya, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+review+of+the+intrinsic+nature+of+vortex-induced+vibrations.">A critical review of the intrinsic nature of vortex-induced vibrations.</a> Journal of Fluids and Structures. 19 (4), 389-447 (2004).</li></ol>

The authors have nothing to disclose.

The protocol presented in this paper describes a method for visualization of the two-dimensional flow fields and determination of the near-boundary flow stress fields around a forced vibrating pipeline in an equilibrium scour hole by using the PIV techniques. Since the designed pipeline motion is one-dimensional along the y direction, preparing and adjusting the pipeline model and vibration system to fulfill this objective are critical prerequisites for a successful outcome. Any undesirable motions of the pipeli...

Subsea pipelines are widely used in offshore environments for the purpose of fluid or hydro-carbon products conveyance. When a pipeline is placed on an erodible seabed, a scour hole around the pipeline is likely to form because of the waves, currents or dynamic motions of the pipeline itself (forced-vibration or vortex-induced-vibration)1,2. To improve the understanding of the scour mechanism around a subsea pipeline, measurements of the turbulent flow fields and estimations of the bed shear and normal stresses within the pipeline-fluid-seabed interaction region are essential in addition to measurements of the scour hole dimension1,2,3,4,5,6,7. In an environment where the bed shear and normal stresses are extremely difficult to be determined because the flow field is unsteady and the bottom boundary is rough, measured instantaneous near-boundary stresses (at approximately 2 mm above the boundary) could be used as their surrogate8,9. In the past few decades, scour around a vibrating pipeline has been studied and published without quantitatively presenting the values of the sophisticated flow fields around the pipeline within the scour hole3,4,5,10,11,12,13,14,15,16,17,18. Therefore, the goal of this method paper is to provide a novel experimental protocol for visualizing the detailed flow fields and to determine the near-boundary shear and normal stresses within an equilibrium scour hole induced by a forced vibrating pipeline. It should be noted that the pipeline-fluid-seabed interaction process in this study is in a quiescent water environment rather than those with unidirectional currents and waves.
This experimental method consists of two important components, namely, (1) simulation of pipeline (forced) vibrations; and (2) measurements of the flow fields around the pipeline. In the first component, the vibrating pipeline was simulated in an experimental flume by using a vibrating system, which has a servo motor, two connecting springs, and pipeline supporting frames. Different vibration frequencies and amplitudes can be simulated by adjusting the motor speed and location of the connecting springs. In the second component, the time-resolved particle image velocimetry (PIV) and wavelet transform techniques were adopted to obtain high temporal and spatial resolution flow field data at different pipeline vibration phases. The time-resolved PIV system consists of a continuous wave laser, a high-speed camera, seeding particles, and cross-correlation algorithms. Although PIV techniques have been widely used in obtaining steady turbulent flow fields19,20,21,22,23,24,25, applications in complex unsteady flow field conditions, such as cases of pipeline-fluids-seabed interaction, are relatively limited8,9,26,27. The reason probably is because traditional single-time-interval cross-correlation algorithm of PIV techniques is unable to accurately capture the flow features in unsteady flow fields where a relatively high velocity gradient is present9,20. The method described in this paper can solve this problem by using the multiple-time-interval cross-correlation algorithm9,28.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Camera control software</td>
			<td style="width:359px;">Vision Research</td>
			<td style="width:175px;">Phantom PCC 2.6</td>
			<td style="width:555px;">Camera control, image data acquisition and processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Camera lens</td>
			<td style="width:359px;">Nikon Chiyoda</td>
			<td style="width:175px;"></td>
			<td>Nikor&#160; 60mm, f=2.8 prime lens</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Continuous wave laser&#160;</td>
			<td style="width:359px;">Beijing Laserwave optoelectronics technology co. ltd.</td>
			<td style="width:175px;"></td>
			<td>PIV Laser source; Nd:YAG laser, 532 nm; air-cooling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">High-speed camera</td>
			<td style="width:359px;">Vision Research</td>
			<td style="width:175px;">Phantom Miro 120</td>
			<td>Image data recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Laser sheet forming optics&#160;</td>
			<td style="width:359px;">Thorlabs Inc</td>
			<td style="width:175px;"></td>
			<td>Transform the point laser to a thin laser sheet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipeline model</td>
			<td style="width:359px;">ZONCEPZ SOLUTIONS</td>
			<td style="width:175px;"></td>
			<td>Acrylic cylinder with a diameter of 35 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipeline vibration system</td>
			<td style="width:359px;">ZONCEPZ SOLUTIONS</td>
			<td style="width:175px;"></td>
			<td>Consists of a sever motor, two connecting springs and pipeline supporting frames.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">PIV calcuation software</td>
			<td>AXESEA Engineering Technology Limited Co.</td>
			<td style="width:175px;">PISIOU</td>
			<td>Image data processing for obtaining flow fields and pipeline displacements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">PIV seeding materials</td>
			<td style="width:359px;">Shimakyu</td>
			<td style="width:175px;"></td>
			<td>Aluminum powder with a diameter of 10um</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Recirculating flume&#160;</td>
			<td style="width:359px;">SZU ENGINEERING PTE LTD</td>
			<td style="width:175px;"></td>
			<td>Glass-sided, 11 m long, 0.6 m wide, and 0.6 m deep</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Tri-pod</td>
			<td style="width:359px;">MANFROTTO</td>
			<td style="width:175px;">SKU MT190GOC4US 410</td>
			<td>Camara supporting</td>
		</tr>
	</tbody>
</table>

visualization of flow field around a vibrating pipeline within an equilibrium scour hole

An experimental method is presented in this paper to facilitate visualization of the detailed flow fields and determination of the near-boundary shear and normal stresses within an equilibrium scour hole induced by a vibrating pipeline. This method involves the implementation of a pipeline vibration system in a straight flume, a time-resolved particle image velocimetry (PIV) system for pipeline displacement tracking and flow fields measurements. The displacement time-series of the vibrating pipeline are obtained by using the cross-correlation algorithms. The steps for processing raw particle laden images obtained by using the time-resolved PIV are described. The detailed instantaneous flow fields around the vibrating pipeline at different vibrating phases are calculated by using a multiple-time-interval cross-correlation algorithm to avoid displacement bias error in the flow regions with a large velocity gradient. By applying the wavelet transform technique, the captured images that have the same vibrating phase are accurately cataloged before the phase-averaged velocity fields are obtained. The key advantages of the flow measurement technique described in this paper are that it has a very high temporal and spatial resolution and can be simultaneously used to obtain the pipeline dynamics, flow fields, and near-boundary flow stresses. By using this technique, more in-depth studies of the 2-dimensional flow field in a complex environment, such as that around a vibrating pipeline, can be conducted to better understand the associated sophisticated scour mechanism.

An example of the comparison between the raw image and processed image of the pipeline displacements tracking and instantaneous velocity calculation is shown in Figure 3. As shown in Figure 3b, the seeding particles and noise in the raw image are filtered out and the shining pipeline edge is retained to obtain the displacement time series. As shown in Figures 3c, light scatters/reflections around the seeding particles, pipeline ...

Watch this Scientific Journal Video about Visualization of Flow Field Around a Vibrating Pipeline Within an Equilibrium Scour Hole at JoVE.com

Visualization of Flow Field Around a Vibrating Pipeline Within an Equilibrium Scour Hole

The goal of the protocol is to enable visualization of the detailed flow fields and determination of the near-boundary shear and normal stresses within an equilibrium scour hole induced by a vibrating pipeline.

An experimental method is presented in this paper to facilitate visualization of the detailed flow fields and determination of the near-boundary shear and ...

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5.5K Views.  Hohai University. The goal of the protocol is to enable visualization of the detailed flow fields and determination of the near-boundary shear and normal stresses within an equilibrium scour hole induced by a vibrating pipeline.

Video: Visualization of Flow Field Around a Vibrating Pipeline Within an Equilibrium Scour Hole

Surface Renewal: An Advanced Micrometeorological Method for Measuring and Processing Field-Scale Energy Flux Density Data

Advanced micrometeorological methods have become increasingly important in soil, crop, and environmental sciences. For many scientists without formal training in atmospheric science, these techniques are relatively inaccessible. Surface renewal and other flux measurement methods require an understanding of boundary layer meteorology and extensive training in instrumentation and multiple data management programs. To improve accessibility of these techniques, we describe the underlying theory of surface renewal measurements, demonstrate how to set up a field station for surface renewal with eddy covariance calibration, and utilize our open-source turnkey data logger program to perform flux data acquisition and processing. The new turnkey program returns to the user a simple data table with the corrected fluxes and quality control parameters, and eliminates the need for researchers to shuttle between multiple processing programs to obtain the final flux data. An example of data generated from these measurements demonstrates how crop water use is measured with this technique. The output information is useful to growers for making irrigation decisions in a variety of agricultural ecosystems. These stations are currently deployed in numerous field experiments by researchers in our group and the California Department of Water Resources in the following crops: rice, wine and raisin grape vineyards, alfalfa, almond, walnut, peach, lemon, avocado, and corn.

Surface renewal is a micrometeorological method that is being used increasingly to determine energy fluxes, but its technical complexity makes it inaccessible to a broad audience. We describe the steps needed to set up and calibrate a surface renewal field station, to acquire and process data, and to correctly interpret results.

Advanced micrometeorological methods have become  increasingly important in soil, crop, and environmental sciences.  For many scientists without formal ...

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 ...

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 ...

Watershed Planning within a Quantitative Scenario Analysis Framework

There is a critical need for tools and methodologies capable of managing aquatic systems within heavily impacted watersheds. Current efforts often fall short as a result of an inability to quantify and predict complex cumulative effects of current and future land use scenarios at relevant spatial scales. The goal of this manuscript is to provide methods for conducting a targeted watershed assessment that enables resource managers to produce landscape-based cumulative effects models for use within a scenario analysis management framework. Sites are first selected for inclusion within the watershed assessment by identifying sites that fall along independent gradients and combinations of known stressors. Field and laboratory techniques are then used to obtain data on the physical, chemical, and biological effects of multiple land use activities. Multiple linear regression analysis is then used to produce landscape-based cumulative effects models for predicting aquatic conditions. Lastly, methods for incorporating cumulative effects models within a scenario analysis framework for guiding management and regulatory decisions (e.g., permitting and mitigation) within actively developing watersheds are discussed and demonstrated for 2 sub-watersheds within the mountaintop mining region of central Appalachia. The watershed assessment and management approach provided herein enables resource managers to facilitate economic and development activity while protecting aquatic resources and producing opportunity for net ecological benefits through targeted remediation.

There is a critical need for tools and methodologies capable of managing aquatic systems in the face of uncertain future conditions. We provide methods for conducting a targeted watershed assessment that enables resource managers to produce landscape-based cumulative effects models for use within a scenario analysis management framework.

There is a critical need for tools and methodologies capable of managing aquatic systems within heavily impacted watersheds. Current efforts often fall ...

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. Biomass-derived biofuels have gained considerable attention in this regard, however first generation biofuels from edible crops like corn ethanol or soybean biodiesel have generally fallen out of favor. There is thus great interest in the development of methods for the production of liquid fuels from domestic and superior non-edible sources. Here we describe a detailed procedure for the production of a purified biodiesel from the marine microalgae Isochrysis. Additionally, a unique suite of lipids known as polyunsaturated long-chain alkenones are isolated in parallel as potentially valuable coproducts to offset the cost of biodiesel production. Multi-kilogram quantities of Isochrysis are purchased from two commercial sources, one as a wet paste (80% water) that is first dried prior to processing, and the other a dry milled powder (95% dry). Lipids are extracted with hexanes in a Soxhlet apparatus to produce an algal oil (&#34;hexane algal oil&#34;) containing both traditional fats (i.e., triglycerides, 46-60% w/w) and alkenones (16-25% w/w). Saponification of the triglycerides in the algal oil allows for separation of the resulting free fatty acids (FFAs) from alkenone-containing neutral lipids. FFAs are then converted to biodiesel (i.e., fatty acid methyl esters, FAMEs) by acid-catalyzed esterification while alkenones are isolated and purified from the neutral lipids by crystallization. We demonstrate that biodiesel from both commercial Isochrysis biomasses have similar but not identical FAME profiles, characterized by elevated polyunsaturated fatty acid contents (approximately 40% w/w). Yields of biodiesel were consistently higher when starting from the Isochrysis wet paste (12% w/w vs. 7% w/w), which can be traced to lower amounts of hexane algal oil obtained from the powdered Isochrysis product.

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Detailed methods are presented for the production of biodiesel along with the co-isolation of alkenones as valuable coproducts from commercial Isochrysis microalgae.

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. ...

Kinematic History of a Salient-recess Junction Explored through a Combined Approach of Field Data and Analog Sandbox Modeling

Within fold-thrust belts, the junctions between salients and recesses may hold critical clues to the overall kinematic history. The deformation history within these junctions is best preserved in areas where thrust sheets extend from a salient through an adjacent recess. We examine one such junction within the Sevier fold-thrust belt (western United States) along the Leamington transverse zone, northern Utah. Deformation within this junction took place by faulting and cataclastic flow. Here, we describe a protocol that examines these fault patterns to better understand the kinematic history of the field area. Fault data is supplemented by analog sandbox experiments. This study suggests that, in detail, deformation within the overlying thrust sheet may not directly reflect the underlying basement structure. We demonstrate that this combined field-experimental approach is easy, accessible, and may provide more details to the deformation preserved in the crust than other more expensive methods, such as computer modeling. In addition, the sandbox model may help to explain why and how these details formed. This method can be applied throughout fold-thrust belts, where upper-crustal rocks are well preserved. In addition, it can be modified to study any part of the upper crust that has been deformed via elastico-frictional mechanisms. Finally, this combined approach may provide more details as to how fold-thrust belts maintain critical-taper and serve as potential targets for natural resource exploration.

Kinematic histories of fold-thrust belts are typically based on careful examinations of high-grade metamorphic rocks within a salient. We provide a novel method of understanding fold-thrust belts by examining salient-recess junctions. We analyze the oft-ignored upper crustal rocks using a combined approach of detailed fault analysis with experimental sandbox modeling.

Within fold-thrust belts, the junctions between salients and recesses may hold critical clues to the overall kinematic history. The deformation history ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Reliable Method for Assessing Seed Germination, Dormancy, and Mortality under Field Conditions

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester fabric and glue can be folded and glued to construct a strip of compartments that house seeds and identifying information, while allowing contact with soil leachate, water, microorganisms, and ambient temperature. Strips may be constructed with a wide range of compartment numbers and sizes and allow the researcher to house a variety of genotypes within a single species, different species, or seeds that have experienced different treatments. As opposed to individual seed packets, strips are more easily retrieved as a unit. While replicate packets can be included within a strip, different strips can act as blocks or can be retrieved at different times for observation of seed behavior over time. We used a high temperature glue gun to delineate compartments and sealed the strips once the seed and tags identifying block and removal times were inserted. The seed strips were then buried in the field at the desired depth, with the location marked for later removal. Burrowing animal predators were effectively excluded by use of a covering of metal mesh hardware cloth on the soil surface. After the selected time interval for burial, strips were dug up and seeds were assessed for germination, dormancy and mortality. While clearly dead seeds can often be distinguished from ungerminated living ones by eye, dormant seeds were conclusively identified using a standard Tetrazolium chloride colorimetric test for seed viability.

Here we present a protocol for assessing seed survivorship, germination and dormancy under field conditions using buried, labeled seed strips and tetrazolium chloride (TZ) viability testing.

We describe techniques for approximating seed bank dynamics over time using Helianthus annuus as an example study species. Strips of permeable polyester ...

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify the pollination effectiveness of each pollinator for visited flowers. Pollinator observations have been one of the most common methods to identify pollinators, and bagging and cage experiments have been conducted to show the effectiveness of specific pollinators. In a previous study of Lycoris sanguinea var. sanguinea, its effective pollinators, the visitation frequencies of each floral visitor, and its reproductive strategies were not identified. This study reports the observation that small bees visited flowers that were partially opened (breaking buds). To the best of our knowledge, this phenomenon has not been reported previously. Further, this study investigates the hypothesis that small bees can pollinate at that flowering stage. This study demonstrates the basic methods of field experiments in pollination ecology using L. sanguinea var. sanguinea. Pollinator observations and digital video showed the visitation frequencies of each floral visitor. Bagging and cage experiments revealed that these flowers could be pollinated fully and that breaking-bud pollination could be important for the pollination of this plant species. The advantages and disadvantages of each method are discussed, and recent developments, including laboratory experiments, are described.

Field Experiments of Pollination Ecology: The Case of Lycoris sanguinea var. sanguinea

To reveal the pollinator effectiveness of a given plant species, multiple methods of field experiments have been developed. This study demonstrates the basic methods of field experiments for pollination ecology using the case study of Lycoris sanguinea var. sanguinea and the novel pollination mechanism, breaking-bud pollination.

Plant-pollinator interactions have been studied for approximately one hundred years. During that time, many field methods have been developed to clarify ...

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Here, we report the different stages involved in the knowledge-based development of an effective mycoinsecticide, including the isolation, identification, screening, and selection of the "best-fit" entomopathogenic fungus, Metarhizium anisopliae, for the control of insect pests in agriculture.