This publication was supported by a grant from the Department of Education (PR Award #P031C160025-17, Project title: 84.031C) to the Colorado State University-Pueblo (CSU-Pueblo) Communities to Build Active STEM Engagement (C-BASE). We thank Current Zoology and Elsevier for providing the rights to use the representative results published in previous papers, as well as the editors of JoVE for providing us with the opportunity to publish this protocol. We would also like to thank the C-BASE Program, Dr. Brian Vanden Heuvel (C-BASE and Department of Biology, CSU-Pueblo), CSU-Pueblo Biology department, Thomas Graziano, Dr. Bernard Possidente (Department of Biology, Skidmore College), and Dr. Claire Varian Ramos (Department of Biology, Colorado State University-Pueblo) for their support and assistance.

The following protocol is an experimental protocol used to rear species in the Drosophila genus on contaminated medium when oral ingestion of a toxin is appropriate; other forms of exposure are possible using the Drosophila model11,15,16,26. The methods described in this protocol have been previously described by Hirsch et al.19 and Peterson <e...

Drosophila melanogaster has been established as a powerful model for a range of biological processes due to the extensive conservation of genes and pathways between D. melanogaster and humans13,14. For the same reasons that it&#39;s a powerful model for medical science, Drosophila has emerged as a suitable model system to study the impact of anthropogenic pollution on a range of toxicological endpoints. Several laboratories are successf...

Every year, approximately 1,000 new chemicals are introduced by the chemical industry1,2; however, the environmental impacts of only a small percentage of these chemicals are tested before distribution2,3. Although large-scale catastrophes are uncommon, sublethal and chronic exposure to a large variety of pollutants are widespread in both humans and wildlife4,5. The historical focus of ecotoxicology and environmental toxicology was to test lethality, single chemical exposure, acute exposure, and the physiological effects of exposure, as a means of measuring the impact of pollutants on survival6,7,8,9,10. Although there is a shift towards ethical and non-invasive approaches to animal testing, current approaches are limiting because of the role that development, emergent properties, and external factors (such as population-level and ecosystem-level interactions) play in mediating ecologically-important endpoints8. Therefore, there is a need for methods that incorporate a more holistic approach without sacrificing wildlife and/or vertebrates in the laboratory.
Invertebrate model systems, such as Drosophila melanogaster, are an attractive alternative to address the need for a more holistic approach to toxicity testing. D. melanogaster, was originally developed as an invertebrate model system for human-related genetic research about a century ago11.D. melanogaster is now prominently used as a vertebrate model alternative for several reasons: 1) the conservation of genes and pathways between D. melanogaster and humans; 2) short generation time compared to vertebrate models; 3) inexpensive cost of maintenance; 4) ease in generating large sample sizes; and 5) plethora of phenotypic- and ecologically-relevant endpoints available for testing11,12,13,14,15,16,17.
Several laboratories11,15,16,17,18,19,20,21,22,23,24,25 are now using D. melanogaster as a vertebrate model alternative for toxicity testing to understand the impacts of pollution on humans. Local wild species of Drosophila can be utilized, as well, as toxicity models for wildlife (and humans) to answer ecologically-, behaviorally-, and evolutionarily-relevant questions at multiple biological levels of organization. Using species within the Drosophila genus as a model, several measurable endpoints are possible11,15,16,18,19,20,21,22,23,24,25. In addition, using the Drosophila model, toxicologists can: 1) ethically link effects at multiple biological levels of the organization; 2) incorporate the role of emergent factors and development; 3) study ecologically-important endpoints (in addition to medically-important endpoints); 4) test multiple stressors simultaneously; 5) and test long-term multigenerational (e.g. evolutionary and transgenerational) implications of stressors. Therefore, using Drosophila as a model system enables a multitude of approaches, not limited to studying mechanistic approaches with inbred strains of D. melanogaster in the laboratory.
In this paper, we present the methods for rearing and collecting Drosophila to answer various toxicological questions. More specifically, we describe the methodology for 1) rearing Drosophila in medium laced with one or more pollutants; 2) collecting Drosophila throughout development (e.g. wandering third-instar larvae, pupal cases, newly-eclosed adults, and mature adults); and 3) rearing Drosophila in the contaminated medium to test intergenerational and transgenerational transmission, as well as evolutionary implications of long-term toxicant exposure. Using this protocol, previous authors18,19,20,21,22,23,24,25 have reported different physiological, genetic, and behavioral effects of developmental lead (Pb2+) exposure. This protocol enables toxicologists to use a more holistic toxicological approach, which is essential to understanding how pollutants are risk factors for both humans and wildlife in an ever increasingly polluted environment.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Carolina Biological Instant Drosophila Medium Formula 4-24</td>
			<td>Carolina Biological</td>
			<td>173204</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila vials, Narrow (PS), Polystyrene, Superbulk, 1000 vials/unit</td>
			<td>Genessee Scientific</td>
			<td>32-116SB</td>
			<td>Used to store flies</td>
		</tr>
		<tr>
			<td>Flugs Closures for vials and bottles, Narrow plastic vials</td>
			<td>Genessee Scientific</td>
			<td>49-102</td>
			<td>Used to store flies</td>
		</tr>
		<tr>
			<td>Cardboard trays, trays only, narrow</td>
			<td>Genessee Scientific</td>
			<td>32-124</td>
			<td>Used to organize populations of flies</td>
		</tr>
		<tr>
			<td>Cardboard trays, dividers only, narrow</td>
			<td>Genessee Scientific</td>
			<td>32-126</td>
			<td>Used to organize populations of flies</td>
		</tr>
		<tr>
			<td>Thermo Scientific&#160;Nalgene Square Wide-Mouth HDPE Bottles with Closure</td>
			<td>Fischer Scientific</td>
			<td>03-312D</td>
			<td>Useful for storage of contaminants</td>
		</tr>
		<tr>
			<td>Thermo Scientific&#160;Nalgene Color-Coded LDPE Wash Bottles</td>
			<td>Fischer Scientific</td>
			<td>03-409-17C</td>
			<td>Useful for storage of contaminants</td>
		</tr>
		<tr>
			<td>Eppendorf&#160;Repeater M4 Manual Handheld Pipette Dispenser</td>
			<td>Fischer Scientific</td>
			<td>14-287-150</td>
			<td>Used to prepare medium</td>
		</tr>
		<tr>
			<td>Combitips Advanced Pipetter Tips - Standard, Eppendorf Quality Tips</td>
			<td>Fischer Scientific</td>
			<td>13-683-708</td>
			<td>Used to prepare medium</td>
		</tr>
		<tr>
			<td>Flypad, Standard Size (8.1 X 11.6cm)</td>
			<td>Genessee Scientific</td>
			<td>59-114</td>
			<td>Used to anesthetize flies</td>
		</tr>
		<tr>
			<td>Flystuff foot valve</td>
			<td>Genessee Scientific</td>
			<td>59-121</td>
			<td>Used to anesthetize flies</td>
		</tr>
		<tr>
			<td>Tubing, green (1 continguous foot/unit)</td>
			<td>Genessee Scientific</td>
			<td>59-124G</td>
			<td>Used to anesthetize flies</td>
		</tr>
		<tr>
			<td>Mineral Oil, Light, White, High Purity Grade, 500 mL HDPE Bottle</td>
			<td>VWR</td>
			<td>97064-130</td>
			<td>Used to make a morgue</td>
		</tr>
		<tr>
			<td>Glass Erlenmeyer Flask Set - 3 Sizes - 50, 150 and 250ml, Karter Scientific 214U2</td>
			<td>Walmart</td>
			<td>Not applicable</td>
			<td>Used to make a morgue</td>
		</tr>
		<tr>
			<td>BGSET5 Glass Beaker Set Of 5</td>
			<td>Walmart</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inbred or wildtype line of Drosophila</td>
			<td>Bloomington Drosophila Stock Center at Indiana University</td>
			<td></td>
			<td>https://bdsc.indiana.edu</td>
		</tr>
		<tr>
			<td>Wild popultions of Drosophila</td>
			<td>UC San Diego Drosophila Stock Center</td>
			<td></td>
			<td>https://stockcenter.ucsd.edu/info/welcome.php</td>
		</tr>
	</tbody>
</table>

experimental protocol for using drosophila as an invertebrate model system for toxicity testing in the laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

By orally exposing Drosophila to a contaminant(s) throughout development, various toxicological questions can be tested by exposing Drosophila at different levels of biological organization. This section presents representative results obtained using this protocol in previously published papers23,24. In particular, this protocol was previously used to evaluate the accumulation, elimination and sequestration of le...

Watch this Scientific Journal Video about Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory at JoVE.com

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

This protocol can be used to understand the impact of toxicants on fitness at different developmental stages and examine the effect of exposure to pollutants across generations. Demonstrating the procedure will be two undergraduate students in my research laboratory that are a part of the communities to build active stem engagement at Colorado State University Pueblo, Hugh Long and Tyler Harvey. To begin working with an environmental contaminant of interest, first determine the 50%lethal dose of the compound Drosophilia.Use established methods to create a dose response curve. Next, prepare the growth medium with the compound at the desired concentration. For instance, add the compound in the water aliquot when making instant medium.In parallel, make control medium without the added compound. To set up the culture from which to collect experimental animals, transfer the reproductive adults to the growth medium using standard methods. Standardize the number of flies per vial and avoid overcrowding the adults.Then, incubate the flies under standard conditions for one to four days. After the incubation, discard the adults, leaving behind fertilized eggs to mature for testing. Then, return the vials in the incubator.The exposure can be set up in any developmental stage. In addition, experimental subjects can be collected in any developmental stage depending on the experimental goals. Check on the developing animals daily to identify the first sign of the stage of interest, such as wandering third instar larvae.To test the effects of exposure on the wandering third instar larvae stage, collect the larvae within an hour of lights on, when they begin to emerge from the food substrate. Next, pour out the water and move the larvae to a lab tissue to gently remove any excess water. Once dried off, transfer the larvae to the appropriate tubes for collection.To test the effects of exposure on adults, collect the newly eclosed adults;carbon dioxide anesthesia is okay. Be sure to collect the flies before they are reproductively mature and begin mating. Divide the flies into sex-specific groups and transfer them to vials loaded with the control or experimental-contaminated medium to match pre-existing exposure.Later, 24 hours prior to measuring the contaminants in the adult flies, transfer the adults in both control and contaminated medium to a vial loaded with control medium to allow the adults to groom excess medium off their bodies. After 24 hours of grooming, the flies can be tested using the appropriate method. For example, behavioral changes can be tested using a mating choice assay.Using these methods, studies of transgenerational effects of contaminant exposure are very easy. Simply set up crosses between exposed adults on the control medium and study the effects of Fnot exposure on their progeny. This protocol was used to evaluate the accumulation, elimination and sequestration of lead within the same generation of offspring.Drosophila readily accumulated lead at different doses, developmental stages, and evolutionary scales. Using a multi-generational exposure, it was found that parental exposure is not transmitted to the first generation of adult offspring. Therefore, using this protocol, it is possible to test adaptive responses at different evolutionary scales, as well as transgenerational effects Fnot of exposure.Next, flies were reared from egg stage to adulthood on control or leaded medium and tested after 24 hours of depuration, mate preference was tested. Females reared on leaded medium, preferentially mated with lead-exposed males over control-exposed males. On the other hand, males did not show the same bias.After watching this video, you should have a good understanding of how to rear and collect Drosophila to answer various questions and toxicology. Once mastered, this protocol can be done in one to two months if it is performed properly. While attempting this procedure, it's important to remember to gently handle the flies, so not to induce unnecessary stress.Following this procedure, other questions like behavioral, genetic and neurological questions can be tested. Don't forget that working with contaminants can be extremely hazardous and precautions, such as wearing personal protective equipment and preparing contaminants in a hood should always be taken while performing this procedure.

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12.5K vistas.  Colorado State University-Pueblo. This protocol can be used to understand the impact of toxicants on fitness at different developmental stages and examine the effect of exposure to pollutants across generations. Demonstrating the procedure will be two undergraduate students in my research laboratory that are a part of the communities to build active stem engagement at Colorado State University Pueblo, Hugh Long and Tyler Harvey. To begin working with an environmental contaminant of interest, first determine the 50%lethal dose...