Name: development of new methods for quantifying fish density using underwater stereo-video tools
Uploaded: 2017-11-20T14:00:00
Description: Watch this Scientific Journal Video about Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools at JoVE.com

This work was funded by The Nature Conservancy and private donors, Resources Legacy Fund Foundation, Gordon and Betty Moore Foundation, Environmental Defense Fund, California Sea Grant Program, the NMFS National Cooperative Research Program, and a NOAA Saltonstall-Kennedy Grant #13-SWR-008. Marine Applied Research and Exploration (Dirk Rosen, Rick Botman, Andy Lauerman, and David Jefferies) developed, constructed and maintained the video Lander tool. We thank Jim Seager and SeaGIS&#8482; software for technical support. Captain and commercial fisherman Tim Maricich and crew onboard the F/V Donna Kathleen provided support in deploying the Lander from 2012-2015. Thank you to all who participated in video data collection or analysis (Anne Tagini, Donna Kline, Lt. Amber Payne, Bryon Downey, Marisa Ponte, Rebecca Miller, Matt Merrifield, Walter Heady, Steve Rienecke, EJ Dick, and John Field).

NOTE: Screenshots of software steps are included as Supplementary Files. Please note that the software steps described below are specific to the chosen software (see the Table of Materials). The overall approach can be extended to any stereo software platform.
1. Prepare Stereo-camera Footage for Analysis
NOTE: Calibration using a calibration cube is recommended. A calibration cube is a three-dimensional aluminum-frame with precisely ...

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The traditional MaxN metric is predicated on the idea of counting a guaranteed minimum number of individuals present during a survey. If a certain number of fish are simultaneously visible in a single video frame, there cannot be any fewer present, but because fish are mobile and heterogeneously distributed, the likelihood of seeing all individuals simultaneously during a single video frame is low. It is therefore likely that traditional MaxN underestimates true fish abundance16,<sup cl...

Along the U.S. Pacific Coast, many of the species important to commercial and recreational groundfish fisheries (e.g., the rockfish complex (Sebastes spp.) and Lingcod (Ophiodon elongatus)) are strongly associated with high-relief, hard-bottom habitats1,2,3,4,5. Stereo-video drop cameras are an attractive non-extractive tool to use in rocky habitats due to the relative ease and simplicity of operation. A variety of stereo-video camera systems have been developed and deployed in southern-hemisphere, shallow-water ecosystems6,7,8,9,10, and recently, video drop-cameras have gained traction as a management tool for deep water rocky-reef environments along the Pacific Coast11,12,13. We sought to modify these existing stereo-camera designs by using a stereo-video camera system (hereafter referred to as &#34;Lander&#34;) to more efficiently characterize fish populations in high-relief seafloors along the central Pacific Coast (see Table of Materials). The Lander used was different than existing video systems because cameras were mounted to a central rotating bar, which allowed for a full 360&#176; of coverage of the seafloor at the drop location14. The Lander completed one full rotation per minute, which allowed us to rapidly characterize the abundance and community composition of an area and achieve the same level of statistical power with fewer Lander deployments. (See Starr (2016)14 for greater detail on the specifics of the Lander configuration). Preliminary tests in the study system suggested that eight rotations of the cameras in our surveys were sufficient to characterize species abundance and richness. This determination was made by an observation of diminishing returns in species abundance and fish density over longer drops. We recommend that a pilot study including longer soak times be conducted in any new system to determine the optimal soak time for a given ecosystem/study species.
By using paired stereo cameras, both total survey area and absolute fish density can be calculated for each video survey; however, the use of rotating cameras necessitated the modification of traditional fish count metrics. Stationary video systems most often use &#34;MaxN&#34; as a conservative count of fishes on a deployment6,10. Traditional MaxN describes the maximum number of fish of a given species observed together in a single video frame, in order to avoid double counting a fish that has left and returned to frame. MaxN has therefore been an estimate of the minimum number of fish known to be present and may underestimate true fish abundance6,10. The MaxN metric was redefined to represent the greatest number of fish seen in each full rotation of the cameras.
The second modification to previous stereo video methods was to account for the fact that species of various sizes, color, and shapes have different maximum distances of reliable identification. For example, large species such as O. elongatus have a distinct elongated shape and can reliably be identified at much greater distances compared with small and cryptic species such as the Squarespot Rockfish (Sebastes hopkinsi). These different maximum ranges of detectability change the effective area sampled by the Lander for each species. Because the stereo cameras allow us to place every fish in three-dimensional space with a high degree of accuracy, one can determine the distance from the cameras that each fish was measured (i.e., the &#34;Z distance&#34;, named for the &#34;z-axis&#34; which is perpendicular to the straight line drawn between the cameras). For each species, the distance within which 95% of all individuals were observed (hereafter &#34;95% Z distance&#34;) was considered to be the radius of the survey area, and was used to calculate the total area surveyed. In addition to species-specific characteristics, identifiability will be impacted by environmental conditions such as water turbidity. Because these factors can vary in time and space, it is important to use the 95% Z statistic only in aggregate. While it will be highly accurate for large samples, any one individual survey may vary in area surveyed.
The protocol detailed below provides guidance on how to create and use these metrics. Though the focus was to characterize deep-water rocky habitat along the Pacific Coast, the methodology described for modified MaxN count is readily applicable to any rotating drop-camera system. The number of camera rotations needed to characterize fish populations will depend on local ecosystem dynamics, but the conceptualization of the modified MaxN will remain the same. Similarly, whereas we used 3D photogrammetric software to analyze stereo video, the techniques described herein are easily applied across software platforms, as long as the precise location of fish in three-dimensional space is possible. Additionally, the approach of applying a 95% Z distance value could be considered in future studies with stereo-cameras to account for species-specific ranges of detectability and to more accurately calculate fish abundance.

<table border="0" cellpadding="0" cellspacing="0" style="width:877px;" width="876">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:201px;">calibration cube</td>
			<td style="width:193px;">SeaGIS</td>
			<td style="width:279px;">http://www.seagis.com.au/hardware.html</td>
			<td style="width:204px;">1000x1000x500 mm is the preferred dimensions. Other methods of calibration are available.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:201px;">CAL calibration software</td>
			<td style="width:193px;">SeaGIS</td>
			<td style="width:279px;">http://www.seagis.com.au/bundle.html</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:201px;">EventMeasure stereo measurement software</td>
			<td style="width:193px;">SeaGIS</td>
			<td style="width:279px;">http://www.seagis.com.au/event.html</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:201px;">Statistical software</td>
			<td style="width:193px;">R Core Team 2017 (v. 3.4.0)</td>
			<td style="width:279px;"></td>
			<td style="width:204px;">Bootstrapping code can be found: https://github.com/rfields2017/JoVE-Bootstrap-Function</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:201px;">Spreadsheet Software</td>
			<td style="width:193px;">Microsoft Excel</td>
			<td style="width:279px;"></td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:201px;">2&#160; waterproof cameras</td>
			<td style="width:193px;">Deep Sea Power and Light</td>
			<td style="width:279px;"></td>
			<td style="width:204px;">HD quality preferred</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:201px;">2 depth rated, waterproof lights</td>
			<td style="width:193px;">Deep Sea Power and Light : 3000 lumen LED with 5000k color temperature</td>
			<td style="width:279px;"></td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:201px;">DVR recorder</td>
			<td style="width:193px;">Stack LTD DVR</td>
			<td style="width:279px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:201px;">standard PC</td>
			<td style="width:193px;"></td>
			<td style="width:279px;"></td>
			<td>Windows 10 preferred OS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:201px;">rotating Lander platform</td>
			<td style="width:193px;">Marine Applied Research and Engineering (MARE)</td>
			<td style="width:279px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of new methods for quantifying fish density using underwater stereo-video tools

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and estimating fish abundance. We developed and implemented a rotating stereo-video camera tool that covers a full 360 degrees of sampling, which maximizes sampling effort compared to stationary camera tools. A variety of studies have detailed the ability of static, stereo-camera systems to obtain highly accurate and precise measurements of fish; the focus here was on the development of methodological approaches to quantify fish density using rotating camera systems. The first approach was to develop a modification of the metric MaxN, which typically is a conservative count of the minimum number of fish observed on a given camera survey. We redefine MaxN to be the maximum number of fish observed in any given rotation of the camera system. When precautions are taken to avoid double counting, this method for MaxN may more accurately reflect true abundance than that obtained from a fixed camera. Secondly, because stereo-video allows fish to be mapped in three-dimensional space, precise estimates of the distance-from-camera can be obtained for each fish. By using the 95% percentile of the observed distance from camera to establish species-specific areas surveyed, we account for differences in detectability among species while avoiding diluting density estimates by using the maximum distance a species was observed. Accounting for this range of detectability is critical to accurately estimate fish abundances. This methodology will facilitate the integration of rotating stereo-video tools in both applied science and management contexts.

Between 2013 and 2014, we conducted 816 surveys with the rotating stereo-video Lander (Figure 1) along the central California coast and collected MaxN and 95% Z distance (Figure 4) data on more than 20 species. There were clear patterns in the effective detectable range of species observed, likely due to the interaction of species&#39; size, shape, and coloration (Figure 5). For instance, the Flag Ro...

Watch this Scientific Journal Video about Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools at JoVE.com

Development of New Methods for Quantifying Fish Density Using Underwater Stereo-video Tools

We describe a new method for counting fishes, and estimating relative abundance (MaxN) and fish density using rotating stereo-video camera systems. We also demonstrate how to use distance from camera (Z distance) to estimate species-specific detectability.

The use of video camera systems in ecological studies of fish continues to gain traction as a viable, non-extractive method of measuring fish lengths and ...

The overall goal of this video analysis technique is to more accurately estimate density, mean length and species composition of fish in deep rocky habitats. This method can help answer key questions in the field of fisheries management such as what is the abundance and size distribution of species inhabiting deep-water rocky reefs. The main advantage of this technique is that it provides more accurate density estimates without extracting fish from the environment.In advance of this procedure, collect field data as outlined in the text protocol. In stereophotogrammetry, knowing the exact relative position of the cameras is important for accurate measurements. Proper calibration is an important step in this process.After the field study is complete, create a new project folder containing both the video and calibration files. In the stereo measurement software, navigate to Measurement, New measurement file. Set the picture directory by navigating to Picture.Set picture directory and then choose the folder containing all of the project files. Navigate to Stereo, Cameras, Left, then Load camera file to select and load the appropriate left camera file. Repeat this process choosing Right instead to load the right camera file.Next, navigate to Picture, Define movie sequence. Select the left camera video file to define the movie sequence for the left video. Click Picture, Load picture to load the left video file into the measurement software.After this, click Stereo, Picture, Define movie sequence to define the movie sequence for the right video. Load the video file by selecting Stereo, Picture then load video. Navigate to Measurement, Attributes, Edit load species file to load the species list.Click Measurement, Information fields, Edit field values to open the information field values table. Enter the survey ID information and save the file to create an event measure observations project. If using a UTC timestamp, frame step forward in the left video until the timestamp starts a new second or until a light flash or hand clap occurs.Frame step the right video forward until the timestamp light flash or hand clap matches the left video exactly. Then, click the Lock button to ensure the videos play together and maintain synchronization. As soon as the lander starts its first rotation, right-click and select Period definitions, Add new start period to define a new sample period.Enter zero one as the first period name and click OK.While the lander rotates, mark each fish that comes into frame with a 2D point by right-clicking, select Add point and choose the correct species name. Label to the lowest possible taxonomic level and then click OK.Continue marking each new fish until the rotation completes. It's critical to identify and count each fish to get accurate estimates of MaxN.Repeat this process for an additional lander rotation ensuring that a new period is defined at the start of each. After all rotations have been enumerated, navigate to Measurement, Measurement summaries, Point measurements and save the 2D points as a TXT file. Open this file as a spreadsheet.Navigate to Insert, Pivot Table to create a pivot table. Selecting Genus and Species for the row label and Period for the column label. Select the camera rotation that has the greatest number of individuals for a given species to choose the MaxN for that species.For fish identified only to genus, select a genus level MaxN based on the rotation that had the greatest number of individuals identified to species in that particular genus. Next, use the saved 2D points to navigate to the exact same fish for the 3D measurement. Zoom in at least four times to better identify the tip of the fish snout and the edges of the caudal fins.Manually click on the tip of the snout and then the edge of the tail in the left camera. Repeat the selection in the same order in the right video. Then, right-click, select Add length and select the correct species identification.If 3D length measurement is not possible, left-click in the same position on the fish in both videos to mark a 3D point. Fill out the information fields leaving the comment Exclude from length measurement. After completing 3D measurements for all fish, navigate to Measurement, Measurement summaries and 3D point and length measurements.Save the data as a TXT file to export it for further analysis. Then, determine if adequate samples were obtained as outlined in the text protocol. In this study, underwater stereo video tools are utilized to quantify fish density.There are clear patterns in the detectable range of the species observed which is likely due to the interaction of each species'size, shape and coloration. The 95%Z distance calculations are then performed for two species in particular. For Sebastes wilsoni and Ophiodon elongatus, the 95%Z distance is seen to be 2.65 meters for Sebastes wilsoni and 3.96 meters for Ophiodon elongatus which translates into effective survey areas of 18.6 meters squared and 46 meters squared respectively.A simple bootstrap analysis confirms that sufficient sample sizes are obtained as the estimate of 95%Z distance for both samples stabilizes when over 50 surveys are sampled. MaxN counts per survey are then converted into densities. For both species, the densities are seen to be significantly greater over high and medium-relief habitats as compared to low-relief habitats.Density estimates for the pseudo-stationary lander are standardized using reduced areas of coverage. Mean densities obtained by the rotating camera are 18%greater than those obtained with stationary cameras. Additionally, the coefficient of variation is seen to be a 1.8 times greater when using stationary cameras.Once mastered, this technique can be used to count and measure fish in only a few minutes if properly performed. When performing this procedure, it's important to remember that 95%Z values are tool and survey specific. And specific values should not be used universally.Following this procedure, a variety of multivariate or ordination statistics can be performed to answer additional questions about species composition across different habitat types. The implications of this technique extend toward improved understanding of the ecology of deep-water rocky reef species as current survey mechanisms provide only a poor understanding of fish length and abundances. Thought this technique can provide insight into deep-water marine habitats, it can also be useful in other systems such as coral reefs and kelp forests.Generally, individuals new to this method will struggle because it requires an understanding of the geometry of stereo camera systems. Visual demonstration of this technique is helpful because the calculation of MaxN is derived from a variety of data and thus requires many steps in the software.

development-new-methods-for-quantifying-fish-density-using-underwater

Research

JoVE Journal

Environment

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail millet. Recently, the 510 Mb genome of foxtail millet, S. italica, has been sequenced 1,2 and a 25x coverage genome sequence of the weedy relative S. viridis is in progress. S. viridis has a number of characteristics that make it a potentially excellent model genetic system including a rapid generation time, small stature, simple growth requirements, prolific seed production 3 and developed systems for both transient and stable transformation 4. However, the genetics of S. viridis is largely unexplored, in part, due to the lack of detailed methods for performing crosses. To date, no standard protocol has been adopted that will permit rapid production of seeds from controlled crosses.
The protocol presented here is optimized for performing genetic crosses in S. viridis, accession A10.1. We have employed a simple heat treatment with warm water for emasculation after pruning the panicle to retain 20-30 florets and labeling of flowers to eliminate seeds resulting from newly developed flowers after emasculation. After testing a series of heat treatments at permissive temperatures and varying the duration of dipping, we have established an optimum temperature and time range of 48 &deg;C for 3-6 min. By using this method, a minimum of 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Therefore, an average of 45-75 outcross progeny can be produced by one person in a single day. Broad implementation of this technique will facilitate the development of recombinant inbred line populations of S. viridis X S. viridis or S. viridis X S. italica, mapping mutations through bulk segregant analysis and creating higher order mutants for genetic analysis.

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

We have developed a methodology for performing crosses in Setaria viridis (S. viridis). The method involves pruning the panicle prior to a hot water treatment to kill viable pollen. Crosses are performed following a well-controlled growth regime and typically result in the recovery of 1 to 7 cross-pollinated seed/s per panicle.

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail ...

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 ...

Protocols for Quantifying Transferable Pesticide Residues in Turfgrass Systems

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes onto humans. For this reason, transferable pesticide residue experiments are required for registration and re-registration by the United States Environmental Protection Agency (USEPA). Although such experiments are required, limited specificity is required pertaining to experimental approach. Experimental approaches used to assess pesticide transfer to humans including hand wiping with cotton gloves, modified California roller (moving a roller of known mass over cotton cloth) and soccer ball roll (ball wrapped with sorbent strip) over three treated turfgrass species (creeping bentgrass, hybrid bermudagrass and tall fescue maintained at 0.4, 5 and 9 cm, respectively) are presented. The modified California roller is the most extensively utilized approach to date, and is best suited for use at low mowing heights due to its reproducibility and large sampling area. The soccer ball roll is a less aggressive transfer approach; however, it mimics a very common occurrence in the most popular international sport, and has many implications for nondietary pesticide exposure from hand-to-mouth contact. Additionally, this approach may be adjusted for other athletic activities with limited modification. Hand wiping is the best approach to transfer pesticides at higher mowing heights, as roller-based approaches can lay blades over; however, it is more subjective due to more variable sampling pressure. Utility of these methods across turfgrass species is presented, and additional considerations to conduct transferable pesticide residue research in turfgrass systems are discussed.

Transferable pesticide residue experiments in turfgrass systems are integral components of human risk exposure assessments. Experimental approaches to measure transferable residues should be adjusted to the human interaction of interest and turfgrass system dynamics. Three transferable pesticide residue protocols are presented and the suitability across three turfgrass systems is discussed.

Plant canopies in established turfgrass systems can intercept an appreciable amount of sprayed pesticides, which can be transferred through various routes ...

High-pressure, High-temperature Deformation Experiment Using the New Generation Griggs-type Apparatus

In order to address geological processes at great depths, rock deformation should ideally be tested at high pressure (&#62; 0.5 GPa) and high temperature (&#62; 300 &#176;C). However, because of the low stress resolution of current solid-pressure-medium apparatuses, high-resolution measurements are today restricted to low-pressure deformation experiments in the gas-pressure-medium apparatus. A new generation of solid-medium piston-cylinder (&#34;Griggs-type&#34;) apparatus is here described. Able to perform high-pressure deformation experiments up to 5 GPa and designed to adapt an internal load cell, such a new apparatus offers the potential to establish a technological basis for high-pressure rheology. This paper provides video-based detailed documentation of the procedure (using the &#34;conventional&#34; solid-salt assembly) to perform high-pressure, high-temperature experiments with the newly designed Griggs-type apparatus. A representative result of a Carrara marble sample deformed at 700 &#176;C, 1.5 GPa and 10-5 s-1 with the new press is also given. The related stress-time curve illustrates all steps of a Griggs-type experiment, from increasing pressure and temperature to sample quenching when deformation is stopped. Together with future developments, the critical steps and limitations of the Griggs apparatus are then discussed.

Rock deformation needs to be quantified at high pressure. A description of the procedure to perform deformation experiments in a newly designed solid-medium Griggs-type apparatus is here given. This provides technological basis for future rheological studies at pressures up to 5 GPa.

In order to address geological processes at great depths, rock deformation should ideally be tested at high pressure (&#62; 0.5 GPa) and high temperature ...

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

Necropsy-based Wild Fish Health Assessment

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on fish populations. Adverse effects monitoring, utilizing biomarkers from the organismal to the molecular level, can be used to assess the cumulative effects on fishes and other organisms. Fish health has been used worldwide as an indicator of aquatic ecosystem health. The necropsy-based fish health assessment provides data on visible abnormalities and lesions, parasites, condition and organosomatic indices. These can be compared by site, season and sex, as well as temporally, to document change over time. Severity ratings can be assigned to various observations to calculate a fish health index for more quantitative assessment. A drawback of the necropsy-based assessment is that it is based on visual observations and condition factors, which are not as sensitive as tissue and subcellular biomarkers for sublethal effects. Additionally, it is rarely possible to identify causes or risk factors associated with observed abnormalities. So, for instance a raised lesion or "tumor" on the fins, lips or body surface may be a neoplasm. However, it could also be a response to a parasite, chronic inflammation or hyperplasia of normal cells in response to an irritant. Conversely, neoplasms, certain parasites, other infectious agents and many tissue changes are not visible and so may be underestimated. However, during the necropsy-based assessment, blood (plasma), tissues for histopathology (microscopic pathology), genomics and other molecular analyses, and otoliths for aging can be collected. These downstream analyses, together with geospatial analyses, habitat assessments, water quality and contaminant analyses can all be important in comprehensive ecosystem evaluations.

The health of wild fishes can be used as an indicator of aquatic ecosystem health. Necropsy-based fish health assessments provide documentation of visible lesions or abnormalities, data used to calculate condition indices as well as the opportunity to collect tissues for microscopic evaluation, gene expression and other more in-depth analyses.

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on ...

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...

Combined Size and Density Fractionation of Soils for Investigations of Organo-Mineral Interactions

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF relies on sequential density separation and sedimentation steps to isolate (1) the free light fraction (uncomplexed organic matter), (2) the occluded light fraction (uncomplexed organic matter trapped in soil aggregates) and (3) a variable number of heavy fractions (soil minerals and their associated organic matter) differing in composition. Provided that the parameters of the CSDF (dispersion energy, density cut-offs, sedimentation time) are properly selected, the method yields heavy fractions of relatively homogeneous mineral composition. Each of these fractions is expected to have a different complexing ability towards organic matter, rendering this a useful method to isolate and study the nature of organo-mineral interactions. Combining density and particle size separation brings an improved resolution compared to simple size or density fractionation methods, allowing the separation of heavy components according to both mineralogy and size (related to surface area) criteria. As is the case for all physical fractionation methods, it may be considered as less disruptive or aggressive than chemically-based extraction methods. However, CSDF is a time-consuming method and furthermore, the quantity of material obtained in some fractions can be limiting for subsequent analysis. Following CSDF, the fractions may be analyzed for mineralogical composition, soil organic carbon concentration and organic matter chemistry. The method provides quantitative information about organic carbon distribution within a soil sample and brings light to the sorptive capacity of the different, naturally-occurring mineral phases, thus providing mechanistic information about the preferential nature of organo-mineral interactions in soils (i.e., which minerals, what type of organic matter).

Combined size and density fractionation (CSDF) is a method to physically separate soil into fractions differing in texture (particle size) and mineralogy (density). The purpose is to isolate fractions with different reactivities towards soil organic matter (SOM), in order to better understand organo-mineral interactions and SOM dynamics.

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF ...

A Method for Quantifying Foliage-Dwelling Arthropods

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimates of density requires a method with high detection probability and a known sampling area. While most described methods provide a qualitative or semi-quantitative estimate adequate for describing species presence, richness, and diversity, few provide an adequately consistent detection probability and known or consistent sampling areas to provide an index or estimate with adequate precision to detect differences in abundance across environmental, spatial, or temporal variables. We describe how to quantify leaf-dwelling arthropods by sealing the leaves and end of branches in a bag, clipping and freezing the bagged material, and rinsing the previously frozen material in water to separate arthropods from the substrate and quantify them. As we demonstrate, this method can be used at a landscape scale to quantify leaf-dwelling arthropods with adequate precision to test for and describe how spatial, temporal, environmental, and ecological variables influence arthropod richness and abundance. This method allowed us to detect differences in density, richness, and diversity of leaf-dwelling arthropods among 5 genera of trees commonly found in southeastern deciduous forests.

We describe how to quantify leaf dwelling arthropods by sealing the leaves and end of branches in a bag, clipping and freezing the bagged material, and rinsing the previously frozen material in water to separate arthropods from the substrate for quantification.

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimates of density ...

Quantifying Corticolous Arthropods Using Sticky Traps

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimate of density requires a method with high detection probability and a consistent sampling area. We used manufactured sticky traps to compare abundance, total length (a surrogate for biomass), richness, and Shannon diversity of corticolous arthropods among the boles of 5 tree species. Efficacy of this method was adequate to detect variation in corticolous arthropods among tree species and provide a standard error of the mean that was &#60;20% of the mean for all estimates with sample sizes from 7 to 15 individual trees of each species. Our results indicate, even with these moderate sample sizes, the level of precision of arthropod community metrics produced with this approach is adequate to address most ecological questions regarding temporal and spatial variation in corticolous arthropods. Results from this method differ from other quantitative approaches such as chemical knockdown, visual inspection, and funnel traps in that they provide an indication of corticolous arthropod activity over a relatively long-term, better including temporary bole residents, flying arthropods that temporarily land on the tree bole and crawling arthropods that use the tree bole as a travel route from the ground to higher forest foliage. Furthermore, we believe that commercially manufactured sticky traps provide more precise estimates and are logistically simpler than the previously described method of directly applying a sticky material to tree bark or applying a sticky material to tape or other type of backing and applying that to the tree bark.

We describe a semi-quantitative approach of measuring characteristics of corticolous (bark-dwelling) arthropod communities. We placed commercially manufactured sticky traps on tree boles to estimate abundance, total length (a surrogate to biomass), richness, and Shannon diversity for comparison among tree species.

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimate of density ...