Name: ecotoxicological effects of microplastics on bird embryo development by hatching without eggshell
Uploaded: 2021-08-14T13:00:00
Description: Watch this Scientific Journal Video about Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell at JoVE.com

This work was supported by Key Research and Development projects in Xinjiang Uygur Autonomous Region (2017B03014, 2017B03014-1, 2017B03014-2, 2017B03014-3).

1. Preparation before exposure
<ol>
	<li>Select fertilized quail eggs born on the same day for the exposure test.</li>
	<li>Select quail eggs with similar weights. Each fertilized quail egg is about 10-12 g.</li>
	<li>Fully clean all fertilized quail eggs from external feces and other debris.</li>
	<li>Sterilize each pre-hatched fertilized quail egg and the eggs to be used (Choose eggs with similar shell shape, especially the tip of the egg) with an antibiotic solution (penicillin and streptomycin, 1:1000, room tempera...

<ol>
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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplastics+in+offshore+sediment+in+the+Yellow+Sea+and+East+China+Sea,+China.">Microplastics in offshore sediment in the Yellow Sea and East China Sea, China.</a> Environmental Pollution. 244, 827-833 (2019).</li><li>Obbard, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+warming+releases+microplastic+legacy+frozen+in+Arctic+Sea+ice.">Global warming releases microplastic legacy frozen in Arctic Sea ice.</a> Earths Future. 2 (6), 315-320 (2014).</li><li>Van Cauwenberghe, L., Vanreusel, A., Mees, J., Janssen, C. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physical+impacts+of+microplastics+on+marine+organisms:+A+review.">The physical impacts of microplastics on marine organisms: A review.</a> Environmental Pollution. 178, 483-492 (2013).</li><li>Ferreira, G. V. B., Barletta, M., Lima, A. R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+estuarine+resources+by+top+predator+fishes.+How+do+ecological+patterns+affect+rates+of+contamination+by+microplastics.">Use of estuarine resources by top predator fishes. How do ecological patterns affect rates of contamination by microplastics.</a> Science of the Total Environment. 655, 292-304 (2019).</li><li>Provencher, J. F., Vermaire, J. C., Avery-Gomm, S., Braune, B. M., Mallory, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Garbage+in+guano?+Microplastic+debris+found+in+faecal+precursors+of+seabirds+known+to+ingest+plastics.">Garbage in guano? Microplastic debris found in faecal precursors of seabirds known to ingest plastics.</a> Science of the Total Environment. 644, 1477-1484 (2018).</li><li>Baulch, S., Perry, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+impacts+of+marine+debris+on+cetaceans.">Evaluating the impacts of marine debris on cetaceans.</a> Marine Pollution Bulletin. 80 (1-2), 210-221 (2014).</li><li>Rochman, C. M., Kurobe, T., Flores, I., Teh, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+warning+signs+of+endocrine+disruption+in+adult+fish+from+the+ingestion+of+polyethylene+with+and+without+sorbed+chemical+pollutants+from+the+marine+environment.">Early warning signs of endocrine disruption in adult fish from the ingestion of polyethylene with and without sorbed chemical pollutants from the marine environment.</a> Science of the Total Environment. 493, 656-661 (2014).</li><li>Mattsson, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+damage+and+behavioural+disorders+in+fish+induced+by+plastic+nanoparticles+delivered+through+the+food+chain.">Brain damage and behavioural disorders in fish induced by plastic nanoparticles delivered through the food chain.</a> Scientific Reports. 7, 7 (2017).</li><li>Brown, D. M., Wilson, M. R., MacNee, W., Stone, V., Donaldson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-dependent+proinflammatory+effects+of+ultrafine+polystyrene+particles:+A+role+for+surface+area+and+oxidative+stress+in+the+enhanced+activity+of+ultrafines.">Size-dependent proinflammatory effects of ultrafine polystyrene particles: A role for surface area and oxidative stress in the enhanced activity of ultrafines.</a> Toxicology and Applied Pharmacology. 175 (3), 191-199 (2001).</li><li>Salvati, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+theoretical+comparison+of+intracellular+import+of+polymeric+nanoparticles+and+small+molecules:+toward+models+of+uptake+kinetics.">Experimental and theoretical comparison of intracellular import of polymeric nanoparticles and small molecules: toward models of uptake kinetics.</a> Nanomedicine-Nanotechnology Biology and Medicine. 7 (6), 818-826 (2011).</li><li>Frohlich, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+polystyrene+nanoparticles+of+different+sizes+on+lysosomal+function+and+integrity.">Action of polystyrene nanoparticles of different sizes on lysosomal function and integrity.</a> Particle and Fibre Toxicology. 9, 13 (2012).</li><li>Bexiga, M. G., Kelly, C., Dawson, K. A., Simpson, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-mediated+inhibition+of+apoptosis+fails+to+prevent+cationic+nanoparticle-induced+cell+death+in+cultured+cells.">RNAi-mediated inhibition of apoptosis fails to prevent cationic nanoparticle-induced cell death in cultured cells.</a> Nanomedicine. 9 (11), 1651-1664 (2014).</li><li>Lehner, R., Weder, C., Petri-Fink, A., Rothen-Rutishauser, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+Nanoplastic+in+the+Environment+and+Possible+Impact+on+Human+Health.">Emergence of Nanoplastic in the Environment and Possible Impact on Human Health.</a> Environmental Science, Technology. 53 (4), 1748-1765 (2019).</li><li>Pinsino, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino-modified+polystyrene+nanoparticles+affect+signalling+pathways+of+the+sea+urchin+(Paracentrotus+lividus)+embryos.">Amino-modified polystyrene nanoparticles affect signalling pathways of the sea urchin (Paracentrotus lividus) embryos.</a> Nanotoxicology. 11 (2), 201-209 (2017).</li><li>El-Ghali, N., Rabadi, M., Ezin, A. M., De Bellard, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Methods+for+Chicken+Embryo+Manipulations.">New Methods for Chicken Embryo Manipulations.</a> Microscopy Research and Technique. 73 (1), 58-66 (2010).</li><li>Rashidi, H., Sottile, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chick+embryo:+hatching+a+model+for+contemporary+biomedical+research.">The chick embryo: hatching a model for contemporary biomedical research.</a> Bioessays. 31 (4), 459-465 (2009).</li><li>Faez, T., Skachkov, I., Versluis, M., Kooiman, K., de Jong, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+characterization+of+ultrasound+contrast+agents:+microbubble+spectroscopy+in+a+chicken+embryo.">In vivo characterization of ultrasound contrast agents: microbubble spectroscopy in a chicken embryo.</a> Ultrasound in Medicine and Biology. 38 (9), 1608-1617 (2012).</li><li>Yamamoto, F. Y., Neto, F. F., Freitas, P. F., Ribeiro, C. A. O., Ortolani-Machado, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadmium+effects+on+early+development+of+chick+embryos.">Cadmium effects on early development of chick embryos.</a> Environmental Toxicology and Pharmacology. 34 (2), 548-555 (2012).</li><li>Li, X. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caffeine+interferes+embryonic+development+through+over-stimulating+serotonergic+system+in+chicken+embryo.">Caffeine interferes embryonic development through over-stimulating serotonergic system in chicken embryo.</a> Food and Chemical Toxicology. 50 (6), 1848-1853 (2012).</li><li>Lokman, N. A., Elder, A. S. F., Ricciardelli, C., Oehler, M. 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This paper provides an effective experimental scheme to evaluate quail embryo development by detecting the basic development indexes. However, there are still some limitations to this experiment. 
First, the mortality of quail embryos in the later stage of hatching is higher because of the shell-less hatching. There are artificially uncontrollable factors such as the destruction of normal protein ratio in the experimental process. We limited the exposure time of embryos to ensure the accuracy ...

The production of plastic waste was about 6300 Mt in 2015, one-tenth of which was recycled, and the rest was burned or buried underground. It is estimated that about 12,000 Mt of plastic waste would be buried underground by 20501. With the international community&#39;s attention to plastic waste, Thompson first proposed the concept of microplastics in 20042. Microplastics (MPs) refer to small particle plastics with a particle diameter less than 5 mm. At present, researchers have detected the ubiquitous presence of MPs in the coastline of various continents, the Atlantic Islands, inland lakes, the Arctic, and deep-sea habitats3,4,5,6,7. Therefore, more researchers have begun to study the environmental hazards of MPs.
Organisms could ingest MPs in the environment. MPs were found in the digestive tract of 233 marine organisms worldwide (including 100% turtle species, 36% seal species, 59% whale species, 59% seabird species, 92 kinds of sea fish, and 6 kinds of invertebrates)8. Moreover, MPs may block the organisms&#39; digestive system, accumulate, and migrate in their bobies9. It has been found that MPs can be transferred via the food chain, and their intake differs with the changes of habitat, growth stage, feeding habits, and food sources10. Some researchers reported the existence of MPs in the droppings of seabirds11, which means that seabirds act as the carrier of MPs. In addition, ingestion of MPs can affect health of some organisms. For example, MPs can be entangled in the gastrointestinal tract, thus increasing the mortality of cetaceans12.
MPs alone have toxic effects on organisms as well as joint toxic effects on organisms with other pollutants. Ingestion of environmental-related concentrations of plastic debris may disturb the endocrine system function of adult fish13. The size of microplastics is one of the important factors that affect their uptake and accumulation by organisms14,15. The small-size plastics, especially the nanosize plastics, are prone to interaction with cells and organisms with high toxicity16,17,18,19. Although the harmful effects of nano-particle size microplastics on organisms exceed the current research level, the detection and quantification of microplastics with sizes less than several micrometers, especially the submicron/nano-plastics in the environment, is still a great challenge. In addition, nano-plastics also have some effects on embryos. Polystyrene can damage the development of sea urchin embryos by regulating protein and gene profiles20.
To explore the potential impact of MPs on organisms, we conducted this study. Due to the similarity between bird embryos and human embryos, they are usually used in developmental biology research21 including angiogenesis and antiangiogenesis, tissue engineering, biomaterial implant, and brain tumors22,23,24. Bird embryos have the advantages of low cost, a short culture cycle and easy operation25,26. Therefore, we chose quail embryos with a short growth cycle as the experimental animal in this study. Simultaneously, we can directly observe the morphological changes of quail embryos exposed to MPs during the embryonic development stage using an eggshell-free hatching technology. The experimental materials used were polypropylene (PP) and polystyrene (PS). Because PP and PS27 account for the largest proportion of polymer types obtained in sediments and water bodies worldwide, the most common polymer types extracted from captured marine organisms are ethylene and propylene28. This experimental protocol describes the whole process for visual evaluation of toxicological effects of MPs on quail embryos exposed to MPs. We can easily extend this method to examine other pollutants&#39; toxicity to embryo development of other oviparous animals.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;Multi sample tissue grinder</td>
			<td>Shanghai Jingxin Industrial Development Co., Ltd.</td>
			<td>Tissuelyser-24</td>
			<td>Grind large-sized plastics into small-sized ones at low temperature</td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>OHAUS corporation</td>
			<td>PR Series Precision</td>
			<td>Used for weighing</td>
		</tr>
		<tr>
			<td>Fertilized quail eggs</td>
			<td>Guangzhou Cangmu Agricultural Development Co., Ltd.</td>
			<td></td>
			<td>Quail eggs for hatching without shell</td>
		</tr>
		<tr>
			<td>Fluorescent polypropylene particles</td>
			<td>Foshan Juliang Optical Material Co., Ltd.</td>
			<td></td>
			<td>Types of plastics selected for the experiment</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Shandong, Bangda Incubation Equipment Co., Ltd.</td>
			<td>264 pc</td>
			<td>Provide a place for embryo growth and development</td>
		</tr>
		<tr>
			<td>Nanometer-scale polystyrene microspheres</td>
			<td>Xi&#8217;an Ruixi Biological Technology Co., Ltd.</td>
			<td>100 nm, 200 nm, 500 nm</td>
			<td>Types of plastics selected for the experiment</td>
		</tr>
		<tr>
			<td>Steel ruler</td>
			<td>Deli Group</td>
			<td>20 cm</td>
			<td>Used to measure&#160; length</td>
		</tr>
		<tr>
			<td>Vertical heating pressure steam sterilizer</td>
			<td>Shanghai Shenan Medical Instrument Factory</td>
			<td>LDZM-80KCS-II</td>
			<td>Sterilize the experimental articles</td>
		</tr>
	</tbody>
</table>

ecotoxicological effects of microplastics on bird embryo development by hatching without eggshell

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues and organs. Ecotoxicological effects of microplastics on the development of bird embryos are not known. The bird egg is a complete development and nutrition system, and the entire embryo development occurs in the eggshell. Therefore, a direct record of bird embryo development under the stress of pollutants such as microplastics is highly limited by the opaque eggshell in traditional hatching. In this study, the effects of microplastics on quail embryo development were visually monitored by hatching without an eggshell. The main steps include the cleaning and disinfection of fertilized eggs, the incubation before exposure, the short-term incubation after exposure, and the sample extraction. The results show that compared with the control group, the wet weight and body length of the microplastics-exposed group displayed a statistical difference and the liver proportion of the whole exposed group significantly increased. Additionally, we evaluated external factors that affect the incubation: temperature, humidity, egg rotation angle, and other conditions. This experimental method provides valuable information on the ecotoxicology of microplastics and a novel way to study the adverse effects of pollutants on the development of embryos.

For the analysis of experimental data, we compared wet weight, body length, sternum length and the change of hepatosomatic index between the control group and the 6 experimental groups, measuring and reflecting the quail embryos&#39; growth and development from a macro perspective. We detected six normal quail embryos in each group. Each embryo reached the required Hamburger and Hamilton (HH) stage.
In Figure 1, we transferred the pre-hatched fertilized quail egg ...

Watch this Scientific Journal Video about Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell at JoVE.com

Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell

This paper introduces a method of hatching without using an eggshell for toxicological studies of particle pollutants such as microplastics.

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues ...

This experiment will enclose four parts.Preparation before exposure, hatching quail egg without shell, incubation process and a sample collection, And representative results.Please watch the following video for the specific experimental process.In the exposure test, the same batch of quail fertilized eggs were selected for the experiment.The eggs with similar size were selected to reduce the experimental error.At the same time all quail fertilize the eggs and eggs should be completely cleaned, because there will be feces and other substances on their shells.After that, the pre-hatched, the fertilized quail eggs, and the eggs to be used were disinfected with penicillin and a mycin When placing you need to pay attention to the top facing down and then the blunt end facing up.The egg's cleaning process isn't shown in the video.The next step is the process of grounding the eggshell.It's the most important step in preparation.The chanted egg shell is a cut in the centre egg size with a dental drill, but can't be broken.Trying to make the fracture surface of the incision smooth enough, then the incubator was disinfected with 75%Acinol solution.Quail fertilized egg placed in the incubator with a humidity of 60%and 38

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A Push-pull Protocol to Reduce Colonization of Bird Nest Boxes by Honey Bees

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. These bees select cavities that are suitable nest sites for birds, resulting in competition. The difficulty of removing bees and their defensive behavior makes a prevention protocol necessary. Here, we describe a push-pull integrated pest management protocol to deter bees from inhabiting bird boxes by applying a bird safe insecticide, permethrin, to repel bees from nest boxes, while simultaneously attracting them to pheromone-baited swarm traps. Shown here is an example experiment using Barn Owl nest boxes. This protocol successfully reduced colonization of Barn Owl nest boxes by Africanized honey bees. This protocol is flexible, allowing adjustments to accommodate a wide range of bird species and habitats. This protocol could benefit conservation efforts where AHB are located.

Preventing colonization of bird nest boxes by invasive Africanized honey bees is important for conservation efforts of nest-site limited birds. We provide an integrated pest management approach to &#34;push&#34; bees away from nest boxes with a repellant insecticide, permethrin, and &#34;pull&#34; them toward pheromone baited swarm traps.

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

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 ...

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related research fields. Thanks to its unique features, including large fecundity, embryo translucency, rapid and simultaneous development, etc., zebrafish embryos are often used for large scale toxicity assessment of chemicals and drug/compound screening. A typical screening procedure involves adult zebrafish spawning, embryos selection, and arraying the embryos into multi-well plates. From there, embryos are subjected to exposure and the toxicity of chemical, or the effectiveness of the drugs/compounds can be evaluated relatively quickly based on phenotypic observations. Among these processes, embryos arraying is one of the most time-consuming and labor-intensive steps that limits the throughput level. In this protocol, we present an innovative approach that makes use of a 3D-printed arraying template coupled with vacuum manipulation to speed up this laborious step. The protocol herein describes the overall design of the arraying template, a detailed experimental setup and step-by-step procedure, followed by representative results. When implemented, this approach should prove beneficial in a variety of research applications using zebrafish embryos as testing subjects.

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...

An Experimental Protocol for Studying Mineral Effects on Organic Hydrothermal Transformations

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the deep ocean. Roles of minerals are critical in many hydrothermal organic geochemical processes. Traditional hydrothermal methodology, which includes using reactors made of gold, titanium, platinum, or stainless-steel, is usually associated with the high cost or undesired metal catalytic effects. Recently, there is a growing tendency for using the cost-effective and inert quartz or fused silica glass tubes in hydrothermal experiments. Here, we provide a protocol for carrying out organic-mineral hydrothermal experiments in silica tubes, and we describe the essential steps in the sample preparation, experimental setup, products separation, and quantitative analysis. We also demonstrate an experiment using a model organic compound, nitrobenzene, to show the effect of an iron-containing mineral, magnetite, on its degradation under a specific hydrothermal condition. This technique can be applied to study complex organic-mineral hydrothermal interactions in a relatively simple laboratory system.

Earth-abundant minerals play important roles in the natural hydrothermal systems. Here, we describe a reliable and cost-effective method for the experimental investigation of organic-mineral interactions under hydrothermal conditions.

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the ...

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 ...

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial.&#160;

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, ...