This work was supported by the National Research, Development and Innovation Office (NKFIH) from the National Research, Development and Innovation Fund (NKFIA); Grant Agreement: NVKP_16-1-2016-0003, EFOP-3.6.3-VEKOP-16-2017-00008 project co-financed by the European Union, and the Thematic Excellence Program NKFIH-831-10/2019 of Szent Istv&#225;n University, awarded by Ministry for Innovation and Technology.

The Animal Protocol was approved under the Hungarian Animal Welfare Law and all studies were completed before the treated individuals would have reached the free feeding stage.
1. Embryo harvest and treatment
<ol>
	<li>Maintain Tg(vtg1:mCherry) zebrafish at 25.5 &#177; 0.5 &#176;C, pH = 7 &#177; 0.2, conductivity between 525 &#177; 50 &#956;S/m, oxygen level &#8805;80% of saturation, and 14 h light and 10 h dark cycle.</li>
	<li>Fill the mating tanks with system water and set up the fish for mating the afternoon before harvesting eggs.</li>
	<li>Place the male and female fish into the tank and separate them with the help of a divider.</li>
	<li>Remove the divider from the tanks as the light switches on next morning. Check the mating tanks for eggs every 15&#8211;20 min.</li>
	<li>Harvest all embryos using a tea strainer or densely woven fine mesh and combine them into one large Petri dish with E3 buffer (5 mM sodium chloride, 0.17 mM potassium chloride, 0.33 mM calcium chloride, 0.33 mM magnesium sulfate) or clear system water.</li>
	<li>Place the embryos in the incubator set to 25.5 &#177; 0.5 &#176;C.</li>
	<li>After 1&#8211;1.5 h, remove and discard unfertilized or inadequately dividing eggs with a plastic transfer pipet under a dissecting microscope. 
	NOTE: Unfertilized eggs are opaque; fertilized eggs are transparent. To start the treatment in a later stage of development, pay attention to the normal development of the individuals being treated.</li>
	<li>Place the selected embryos in the treatment vessels (e.g., in Petri dishes or tissue culture plates) that have already been labeled and filled with different concentrations of the test substance.</li>
	<li>Incubate the embryos at 25.5 &#177; 0.5 &#176;C until the end of the experiment. 
	NOTE: Embryos can react with significant individual sensitivity to various estrogenic compounds, so it is recommended to use at least 15 embryos in at least three repeat treatments in order to evaluate the experiment properly. When selecting the container, keep in mind that the development of an embryo requires at least 200 &#181;L of water26. In this experiment the embryos were incubated until 120 hpf at 25.5 &#177; 0.5 &#176;C.</li>
	<li>Refresh the test solution if it is neccesary to maintain the treatment concentration. Be careful when changing the test solution in order to avoid damaging the embryos. 
	NOTE: It is also possible to investigate mortality or sublethal symptoms during the experiment. The concentrations used should remain as stable as possible to obtain reliable results. This can be easily achieved by refreshing the test solutions. The frequency of refreshing may vary depending on the test substance. Therefore, it is advisable to check the concentration of the test substance by analytical measurements to determine the frequency of the solution updates.</li>
</ol>
2. Larvae preparation for photography
<ol>
	<li>Prepare 4% methylcellulose with MS-222 (tricaine-methane-sulfonate) beforehand.
	<ol>
		<li>To do so, add 4 mg/mL MS-222 to 100 mL double distilled water to a final concentration of 0.168 mg/mL, and bring the solution to 4 &#730;C. Then add 4 g of methylcellulose. Stir it with a magnetic stirrer, then leave it in 4 &#730;C overnight.</li>
		<li>Next day, stir it again and the solution should be ready to use. If the methylcellulose is not completely dissolved put it back at 4 &#730;C and wait a few more hours.</li>
	</ol>
	</li>
	<li>Place 5 day old larvae into a 5 cm Petri dish per treatment group with a Pasteur pipette.</li>
	<li>Remove the treatment solution from the larvae with a plastic pipette, then fill the Petri dish with 2 mL of 0.02% MS-222 anesthetic solution. 
	NOTE: Anesthesia is usually effective in less than a minute. The larvae are anesthetized if they do not swim away in response to touch. An overdose of MS-222 can kill the larvae.</li>
	<li>Fill each square of a specially designed 10 cm Petri dish with 4% methylcellulose with MS-222 (Figure 3). 
	NOTE: Glue two 1.5 x 1.5 cm square areas at the bottom of the Petri dish using plastic sheets (1 mm height, 5 mm wide). Twenty larvae can be placed next to each other in a square. Instead of a specially designed Petri dish, another low edge container can be used to fix the position of the embryos.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60462/60462fig03.jpg" /> 
Figure 3: A 10 cm Petri dish with glued 1.5 x 1.5 cm wide, 1 mm thick plastic sheet squares for larvae preparation for photography. <a href="https://www.jove.com/files/ftp_upload/60462/60462fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Transfer the anesthetized larvae to methylcellulose with a little water in one of the two squares. From the first square transfer the larvae into the second square. 
	NOTE: With the help of this transfer, the 4% methylcellulose used for photography will not be diluted and the larvae will not rotate during imaging.</li>
	<li>In the second square, rotate and orient larvae to their left side and gently press them down to the bottom of the cellulose with a microloader pipette tip cut up to 2 cm. 
	NOTE: Do not use other pipette tips because they cause injury to the larvae.</li>
</ol>
3. Microscopy
NOTE: Photography does not kill the animals. Animals can be awakened by removing them from the methylcellulose and placing them in fresh system water or treatment solution, so the same individual can be examined several times during the treatment.
<ol>
	<li>In order to evaluate the signal of the expressed reporter, image the embryos with the same view and settings. 
	NOTE: See step 2.6 for optimal embryo positioning. The photos in the manuscript were taken under the conditions described. Bright field: 60x magnification, 6 ms exposition, Iris 100%, Gain:1.1, fluorescence photos: 60x magnification, 300 ms exposition, Iris: 100%, Gain:1, mCherry filter, fluorescent light source: mercury metal halide bulb. For photo taking a fluorescent stereomicroscope, dedicated camera, and software for microscope were used.</li>
	<li>Place the Petri dish on the stage of the microscope.</li>
	<li>Focus on the liver of the embryo and capture a bright field image using the associated software.</li>
	<li>Switch the microscope to the mCherry filter and take a fluorescent image of the liver under fluorescent light using the associated software. 
	NOTE: Perform all the fluorescence steps in the dark. Do not change the magnification, the focus, or the position of the embryo between capturing the bright field and the fluorescent images, as this will assist in the analysis of the images.</li>
	<li>Repeat steps 3.3 and 3.4 until all the embryos in the experiment have been imaged.</li>
</ol>
4. Determining integrated density
NOTE: One of the best indicators for comparing fluorescent signal strength is the integrated density value (i.e., the product of the area and mean gray value). One of the easiest ways to determine integrated density is to use the ImageJ program27. The program is available on the internet and can be installed on the computer.
<ol>
	<li>Open ImageJ then upload the fluorescent image to be analyzed by either dragging and droping the image or clicking on File|Open.</li>
	<li>Click on Image|Color|Split Channels to split the image made by the fluorescent filter according to the RGB color chart.</li>
	<li>Work with the red channel image spectrum, close the other channels.</li>
	<li>Designate a similarly sized elliptical area in the image so false signals do not interfere with the evaluation. Using the Oval tools, draw an ellipse over the highlighted liver area as accurately as possible.</li>
	<li>If the signal is weak use light microscopy images (i.e., the bright field pair of the fluorescent image) to determine the location of the liver.</li>
	<li>Click on Analyze|Measure to determine the signal strength and the size of the affected area. The integrated density value is automatically calculated by the software (IntDen column in the chart).</li>
	<li>Continue the analysis by repeating steps 4.1&#8211;4.6 until all the fluorescent images of all embryos in the treatment group are analyzed.</li>
	<li>Save the data and then analyze the integrated density values. 
	NOTE: Always select the same area size in the images during analysis. The size of the area to be analyzed depends on the magnification, image resolution, other settings for shooting, etc. The analysis can be accelerated or made more accurate by using personal macros for analysis. For example, macros can be used to ensure that the selected area is always the same in the examined images. Detailed descriptions for creating macros that allow the best image analysis according to particular photo settings are found on the ImageJ website.</li>
</ol>

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The authors have nothing to disclose.

The use of biomonitors/bioindicators for estrogenic effects has been spreading in toxicological studies. In vivo models play an outstanding role, because unlike in vitro tests, they not only provide information about the response of a cell or a receptor, but also allow the investigation of complex processes in the organism. Several transgenic lines for studying estrogenic effects have been produced from zebrafish, one of which Tg(vtg1:mCherry) was used for these studies. The method described here illustrates a p...

There is a significant number of endocrine disrupting compounds (EDC) that are among the most hazardous substances in our environment. These are mainly estrogenic compounds that contaminate water from natural resources. The chemical diversity of the substances belonging to the group makes testing for their presence difficult, as different analytical methods are required for their detection. Based on their chemical structure it is very difficult to determine whether a substance is actually able to act as an estrogen. In addition, these substances are never present in a pure form in the environment, so their effects may be affected by other compounds, too1. This problem can be solved by effect-detecting methods, such as the use of biomonitor/bioindicator organisms that show estrogenic effects2,3,4,5.
Recently, a variety of cell line6 and yeast-based test systems2,3 have been developed to detect estrogenic effects. However, these are generally only able to detect the binding of the substance to the estrogen receptor2,3. In addition, they are unable to model complex physiological processes in the organism, or to detect hormone-sensitive phases of life stages; thus, they often lead to false results.
It is known that certain genes react sensitively to estrogen in living organisms7. The detection of gene products by molecular biology methods is also possible at the protein or mRNA level8,9, but usually involves animal sacrifice. Animal protection laws have become stricter, and there is a growing demand for alternative test systems that minimize the number and suffering of animals used in experiments or the replacement of the animal model with another model system10. With the discovery of fluorescent proteins and the creation of biomarker lines, transgenic technologies provide a good alternative11. With these lines, the activation of an estrogen-sensitive gene can be tested in vivo.
Among vertebrates, the potential of fish in environmental risk assessment is outstanding. They offer many advantages over mammalian models: being aquatic organisms, they are able to absorb pollutants through their entire body, produce a large number of offspring, and some of their species are characterized by short generation time. Their endocrine system and physiological processes show great similarities with other vertebrates and even with mammals, including humans12.
Several genes for the detection of estrogenic effects in fish are also known. The most important are the estrogen receptors aromatase-b, choriogenin-H, and vitellogenin (vtg)7,13. Recently, several estrogen-producing biosensor lines have also been created from fish models used in the laboratory, such as from zebrafish (Danio rerio)4,5,14,15,16,17. The main advantage of zebrafish in creating biosensor lines is the transparent body of the embryos and larvae, because the fluorescent reporter signal can then be easily studied in vivo without sacrificing the animal10. In addition to animal protection, it is also a valuable feature as it allows for studying the reaction of the same individual at different times of the treatment18.
These experiments use a vitellogenin reporter transgenic zebrafish line15. The transgene construct used for the development of Tg(vtg1:mCherry) has a long (3.4 kbp) natural vitellogenin-1 promoter. The estrogen receptor (ER) is an enhancer protein activated by ligands that is a representative of the steroid/nuclear receptor superfamily. ER binds to specific DNA sequences called estrogen response elements (EREs) with high affinity and transactivates gene expression in response to estradiol and other estrogenic substances, so the more ERE in the promoter causes a stronger response19. There are 17 ERE sites in the promoter region of the Tg(vtg1:mCherry) transgene construct and they are expected to mimic the expression of the native vtg gene15. There is a continuous expression of the fluorescent signal in sexually matured females. However, in males and embryo the expression in the liver is only visible upon treatment with estrogenic substances (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60462/60462fig01.jpg" /> 
Figure 1: Red fluorescent signal in the liver of vtg1:mCherry transgenic adult zebrafish and 5 dpf embryos, following 17-&#223;-estradiol (E2) induction.&#160;In female and in male treated with E2 (25 &#181;g/L exposure time:48hrs) strong fluorescence of the liver is visible even through the pigmented skin. No fluorescent signal is visible in untreated male (A). Following E2 induction (50 &#181;g/L exposure time: 0-120 hpf), a red fluorescent signal in the liver of 5 dpf embryos can also be observed, which is not visible in control embryos (B). While the fluorescent signal is continuously present in adult females, primarily males and embryos of the line are suitable for detecting estrogenic effects. (BF: bright field, mCherry: red fluorescent filter view, single plain images, Scale bar A: 5mm, scale bar B: 250 &#181;m) <a href="https://www.jove.com/files/ftp_upload/60462/60462fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Similar to the endogenous vitellogenin, the mCherry reporter is only expressed in the liver. Because vitellogenin is only produced in the presence of estrogen, there is no fluorescent signal in the controls. Because the expression is only in the liver, the evaluation of the results is much easier15.
The sensitivity and usability of this line&#39;s embryos have been investigated on various estrogenic compound mixtures and also on environmental samples15,20, and in most cases dose-response relationships were documented (Figure 2). However, in the case of highly toxic, mainly hepatotoxic, substances (e.g., zearalenone), only a very weak fluorescent signal may be visible in the liver of treated embryos and the maximum intensity fluorescent signal caused can be reached within a very small concentration range, which makes it difficult to establish dose-effect relationships20.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60462/60462fig02.jpg" /> 
Figure 2: Dose-response diagram (A) and fluorescent images (mCherry) of the liver (B) exposed to 17-&#945;-ethynilestradiol (EE2), in 5 dpf vtg1:mCherry larvae.&#160;Results are expressed as integrated density generated from the signal strength and the size of the affected area (&#177;SEM, n = 60). 100% refers to the observed maximum. Fluorescent signal intensity increased gradually with concentration. Scale bar = 250 &#181;m. <a href="https://www.jove.com/files/ftp_upload/60462/60462fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
There are several estrogenic substances present in the environment, such as 17-&#946;-estradiol (environmental concentration: 0.1&#8211;5.1 ng/L)21, 17-&#945;-ethynylestradiol (environmental concentration: 0.16&#8211;0.2 &#181;g/L)22, zearalenone (environmental concentration: 0.095&#8211;0.22 &#181;g/L)23, bisphenol-A (environmental concentration: 0.45&#8211;17.2 mg/L)24. When testing these substances in a pure active form with the help of mCherry transgenic embryos, the lowest observed effect concentrations (LOEC) for fluorescent sign detection were 100 ng/L for 17-&#223;-estradiol, 1 ng/L for 17-&#945;-ethynilestradiol, 100 ng/L for zearalenone, and 1 mg/L for bisphenol-A (96&#8211;120 hpf treatment), which is very close to or within the range of environmental concentrations of the substances15. The Tg(vtg1:mCherry) transgenic line can help detect estrogenicity in wastewater samples after direct exposure. The line is as sensitive as the commonly used yeast estrogen test, the bioluminiscent yeast estrogen (BLYES) assay15. With the help of this line, the protective effects of beta-cyclodextrins against zearalenone-induced toxicity has been confirmed using chemical mixtures20.
In a recent report, the in vivo use of the transgenic line was demonstrated with the help of two estrogenic zearalenone (ZEA) metabolites, &#945;- and &#946;-zearalenol (&#945;-ZOL and &#946;-ZOL)25. The protocol baseline is appropriate to study the estrogenic effects of several compounds or environmental samples on Tg(vtg1:mCherry) embryos.

<table>
	<tbody>
		<tr>
			<td>24 well tissue culture plate</td>
			<td>Jet Biofil</td>
			<td>TCP011024</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium-chloride (CaCl2)</td>
			<td>Reanal Laborvegyszer Ltd.</td>
			<td>16383-0-27-39</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 6.01 software</td>
			<td>GraphPad Software Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health, USA</td>
			<td></td>
			<td>Public access software, downloadable from: http://imagej.nih.gov/</td>
		</tr>
		<tr>
			<td>Leica Application Suite X calibrated software</td>
			<td>Leica Microsystems GmbH.</td>
			<td></td>
			<td>We used the softver described in the experiments, but any photographic software complies with the tests</td>
		</tr>
		<tr>
			<td>Leica M205 FA stereomicroscope, Leica DFC 7000T camera</td>
			<td>Leica Microsystems GmbH.</td>
			<td></td>
			<td>We used the equipments described in the experiments, but any fluorescent stereomicroscope is suitable for the tests</td>
		</tr>
		<tr>
			<td>Magnesium-sulphate (MgSO4)</td>
			<td>Reanal Laborvegyszer Ltd.</td>
			<td>20342-0-27-38</td>
			<td></td>
		</tr>
		<tr>
			<td>mCherry filter</td>
			<td>Leica Microsystems GmbH.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mehyl-cellulose</td>
			<td>Sigma Aldrich Ltd.</td>
			<td>274429</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader pipette tip</td>
			<td>Eppendorf GmbH.</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>VWR International LLC.</td>
			<td>612-1684</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri-dish</td>
			<td>Jet Biofil</td>
			<td>TCD000060</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium-chloride (KCl)</td>
			<td>Reanal Laborvegyszer Ltd.</td>
			<td>18050-0-01-33</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium-chloride (NaCl)</td>
			<td>Reanal Laborvegyszer Ltd.</td>
			<td>24640-0-01-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricane-methanesulfonate (MS-222)</td>
			<td>Sigma Aldrich Ltd.</td>
			<td>E10521</td>
			<td></td>
		</tr>
	</tbody>
</table>

using tg(vtg1:mcherry) zebrafish embryos to test the estrogenic effects of endocrine disrupting compounds

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult due to their chemical diversity; therefore, increasingly more effect-detecting methods are used, such as estrogenic effect-sensitive biomonitor/bioindicator organisms. These biomonitoring organisms include several fish models. This protocol covers the use of zebrafish Tg(vtg1: mCherry) transgenic line as a biomonitoring organism, including the propagation of fish and the treatment of embryos, with an emphasis on the detection, documentation, and evaluation of fluorescent signals induced by EDC. The goal of the work is the demonstration of the use of the Tg(vtg1: mCherry) transgenic line embryos to detect estrogenic effects. This work documents the use of transgenic zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects by testing two estrogenic substances, &#945;- and &#946;-zearalenol. The described protocol is only a basis for designing assays; the test method can be varied according to the test endpoints and the samples. Moreover, it can be combined with other assay methods, thereby facilitating the future use of the transgenic line.

In the experiment presented in this manuscript, the effects of two estrogenic substances were tested at five concentrations starting at fertilization for 5 days on Tg(vtg1:mCherry) zebrafish embryos. We investigated whether fluorescent signals appeared in the liver of fish by the end of the exposure time because of the substances and whether there were differences in the estrogenicity of the two substances. Results were evaluated on the basis of the fluorescent images and integrated density values. In general, b...

Watch this Scientific Journal Video about Using TgVtg1:mcherry Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds at JoVE.com

Using TgVtg1:mcherry Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

Present here is a detailed protocol for the use of zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects. The protocol covers the propagation of the fish and treatment of embryos, and emphasizes the detection, documentation, and the evaluation of fluorescent signals induced by endocrine disrupting compounds (EDC).

Using Tg(Vtg1:mcherry) Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult ...

using-tg-vtg1mcherry-zebrafish-embryos-to-test-estrogenic-effects

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6.2K Views.  Szent István University. Present here is a detailed protocol for the use of zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects. The protocol covers the propagation of the fish and treatment of embryos, and emphasizes the detection, documentation, and the evaluation of fluorescent signals induced by endocrine disrupting compounds (EDC).

Video: Using TgVtg1:mcherry Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

Exploring the Effects of Atmospheric Forcings on Evaporation: Experimental Integration of the Atmospheric Boundary Layer and Shallow Subsurface

Evaporation is directly influenced by the interactions between the atmosphere, land surface and soil subsurface. This work aims to experimentally study evaporation under various surface boundary conditions to improve our current understanding and characterization of this multiphase phenomenon as well as to validate numerical heat and mass transfer theories that couple Navier-Stokes flow in the atmosphere and Darcian flow in the porous media. Experimental data were collected using a unique soil tank apparatus interfaced with a small climate controlled wind tunnel. The experimental apparatus was instrumented with a suite of state of the art sensor technologies for the continuous and autonomous collection of soil moisture, soil thermal properties, soil and air temperature, relative humidity, and wind speed. This experimental apparatus can be used to generate data under well controlled boundary conditions, allowing for better control and gathering of accurate data at scales of interest not feasible in the field. Induced airflow at several distinct wind speeds over the soil surface resulted in unique behavior of heat and mass transfer during the different evaporative stages.

A protocol for the design and construction of a soil tank interfaced to a small climate controlled wind tunnel to study the effects of atmospheric forcings on evaporation is presented. Both the soil tank and wind tunnel are instrumented with sensor technologies for the continuous in situ measurement of environmental conditions.

Evaporation is directly influenced by the interactions between the atmosphere, land surface and soil subsurface. This work aims to experimentally study ...

A Bending Test for Determining the Atterberg Plastic Limit in Soils

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a considerable subjective judgment from the operator that carries out the test is involved during its performance, which may affect the final result significantly. Different alternative methods have been put forward, but they cannot compete with the standard rolling test in speed, simplicity and cost.
In an earlier study by the authors, a simple method with a simple device to determine the PL was presented (the &#34;thread bending test&#34; or simply &#34;bending test&#34;); this method allowed the PL to be obtained with minimal operator interference. In the present paper a version of the original bending test is shown. The experimental basis is the same as the original bending test: soil threads which are 3 mm in diameter and 52 mm long are bent until they start to crack, so that both the bending produced and its related moisture content are determined. However, this new version enables the calculation of PL from an equation, so it is not necessary to plot any curve or straight line to obtain this parameter and, in fact, the PL can be achieved with only one experimental point (but two experimental points are recommended).
The PL results obtained with this new version are very similar to those obtained through the original bending test and the standard rolling test by a highly experienced operator. Only in particular cases of high plasticity cohesive soils, there is a greater difference in the result. Despite this, the bending test works very well for all types of soil, both cohesive and very low plasticity soils, where the latter are the most difficult to test via the standard thread rolling method.

The traditional standardized test for determining the plastic limit in soils is performed by hand, and the result varies depending on the operator. An alternative method based on bending measurements is presented in this study. This allows the plastic limit to be obtained with a clear and objective criterion.

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a ...

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

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

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

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

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

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into changes in clinical standards used for treating wounds and decreasing morbidity and mortality for patients. Wound healing is a complex process that requires strategic cell and tissue interaction and function. One of the many critically important functions of wound healing is individual and collective cellular migration. Upon injury, various cells from the blood, surrounding connective, and epithelial tissues rapidly migrate to the wound site by way of chemical and/or physical stimuli. This migration response can largely dictate the outcomes and success of a healing wound. Understanding this specific cellular function is important for translational medicine that can lead to improved wound healing outcomes. Here, we describe a protocol used to better understand cellular migration as it pertains to wound healing, and how changes to the cellular environment can significantly alter this process. In this example study, dermal fibroblasts were grown in media supplemented with fetal bovine serum (FBS) as monolayer cultures in tissue culture flasks. Cells were aseptically transferred into tissue culture treated 12-well plates and grown to 100% confluence. Upon reaching confluence, the cells in the monolayer were vertically scratched using a p200 pipet tip. Arsenic diluted in culture media supplemented with FBS was added to individual wells at environmentally relevant doses ranging 0.1-10 &#956;M. Images were captured every 4 hours (h) over a 24 h period using an inverted light microscope to observe cellular migration (wound closure). Images were individually analyzed using image analysis software, and percent wound closure was calculated. Results demonstrate that arsenic slows down wound healing. This technique provides a rapid and inexpensive first screen for evaluation of the effects of contaminants on wound healing.

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

This study focuses on an in vitro model of wound healing (scratch assay) as a mechanism for determining how environmental contaminants such as arsenic influence cellular migration. The results demonstrate that this in vitro assay provides rapid and early indications of changes to cellular migration prior to in vivo experimentation.

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...