We thank our staff for help with the testing and optimization of this protocol: Ameya Belamkar, Marilyn Clark, Alexander Fitt, Emma Rose Gallant, Ethan Golditch, Matthew Lowe, Morgan Marsh, Kyle McClung, Andy Puga, Darcy Rose, Cameron Stockbridge, and Noelle Zolman. We also thank our colleagues from the Precision Toxicology Group, particularly our Exposure Group counterparts, for helping to identify the goals of the protocol.
This project received funding from the European Union&#39;s Horizon 2020 Research and Innovation program under Grant Agreement No. 965406. The work presented in this publication was performed as part of the ASPIS Cluster. This output reflects only the authors&#39; views, and the European Union cannot be held responsible for any use that may be made of the information contained therein. This publication was also made possible with support from the Indiana Clinical and Translational Sciences Institute, which is funded in part by Award Number UL1TR002529 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Portions of this project were supported by funds from Indiana University awarded to JRS and the PhyloTox consortium. JMH and EMP were supported by NIH award P40OD018537 to Bloomington Drosophila Stock Center.

<ol>
	<li>United States Occupational Safety and Health Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+industry:+OSHA+safety+and+health+standards+(29+CFR+1926/1910).">Construction industry: OSHA safety and health standards (29 CFR 1926/1910).</a> United States Occupational Safety and Health Administration. , (2022).</li><li>Chemical, Metals, Natural Toxins &amp; Pesticides Guidance Documents &amp; Regulations. United States Food &amp; Drug Administration Available from: <a target="_blank" href="https://www.fda.gov/food/guidance-documents-regulatory-information-topic-food-and-dietary-supplements/chemical-metals-natural-toxins-pesticides-guidance-documents-regulations">https://www.fda.gov/food/guidance-documents-regulatory-information-topic-food-and-dietary-supplements/chemical-metals-natural-toxins-pesticides-guidance-documents-regulations</a> (2022)</li><li>Drinking Water Contaminant Candidate List (CCL) and Regulatory Determination. United States Food &amp; Drug Administration Available from: <a target="_blank" href="https://www.epa.gov/cci">https://www.epa.gov/cci</a> (2022)</li><li>Administration Bottled Water/Carbonated Soft Drinks Guidance Documents &amp; Regulatory Information. United States Food &amp; Drug Administration Available from: <a target="_blank" href="https://www.fda.gov/food/guidance-documents-regulatory-information-topic-food-and-dietary-supplements/bottled-watercarbonated-soft-drinks-guidance-documents-regulatory-information">https://www.fda.gov/food/guidance-documents-regulatory-information-topic-food-and-dietary-supplements/bottled-watercarbonated-soft-drinks-guidance-documents-regulatory-information</a> (2022)</li><li>United States Congress. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Federal+Food,+Drug,+and+Cosmetic+Act.">Federal Food, Drug, and Cosmetic Act.</a> United States Congress. 21, 301-392 (1934).</li><li>Determining if a Cleaning Product is a Pesticide Under FIFRA. Environmental Protection Agency Available from: <a target="_blank" href="https://www.epa.gov/pesticide-registration/determining-if-cleaning-produt-pesticide-under-fifra">https://www.epa.gov/pesticide-registration/determining-if-cleaning-produt-pesticide-under-fifra</a> (2022)</li><li>United States Congress. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+Substances+Control+Act+of+1976.+H.R.+12440.">Toxic Substances Control Act of 1976. H.R. 12440.</a> Library of Congress, United States. , 1640-1707 (1976).</li><li>Wang, Z., Walker, G. W., Muir, D. C. G., Nagatani-Yoshida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+global+understanding+of+chemical+pollution:+a+first+comprehensive+analysis+of+national+and+regional+chemical+inventories.">Toward a global understanding of chemical pollution: a first comprehensive analysis of national and regional chemical inventories.</a> Environmental Science Technology. 54 (5), 2575-2584 (2020).</li><li>Pandey, U. B., Nichols, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+disease+models+in+Drosophila+melanogaster+and+the+role+of+the+fly+in+therapeutic+drug+discovery.">Human disease models in Drosophila melanogaster and the role of the fly in therapeutic drug discovery.</a> Pharmacological Reviews. 63 (2), 411-436 (2011).</li><li>New Approach Methods Work Plan. United States Environmental Protection Agency Available from: <a target="_blank" href="https://www.epa.gov/system/files/documents/2021-11/nams-work-plan_11_15_21_508-tagged.pdf">https://www.epa.gov/system/files/documents/2021-11/nams-work-plan_11_15_21_508-tagged.pdf</a> (2021)</li><li>Hartung, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+alternative+methods+to+a+new+toxicology.">From alternative methods to a new toxicology.</a> European Journal of Pharmaceutics and Biopharmaceutics. 77 (3), 338-349 (2011).</li><li>Mahony, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+confidence+in+non-animal+methods:+Practical+examples+of+collaboration+between+regulators,+researchers+and+industry.">Building confidence in non-animal methods: Practical examples of collaboration between regulators, researchers and industry.</a> Computational Toxicology. 10, 78-80 (2019).</li><li>Kavlock, R. J., 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=Accelerating+the+pace+of+chemical+risk+assessment.">Accelerating the pace of chemical risk assessment.</a> Chemical Research in Toxicology. 31 (5), 287-290 (2018).</li><li>European Chemicals Agency. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Approach+Methodologies+in+Regulatory+Science,+Proceedings+of+the+Scientific+Workshop.">New Approach Methodologies in Regulatory Science, Proceedings of the Scientific Workshop.</a> European Chemicals Agency. , (2016).</li><li>Wambaugh, J. 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=New+approach+methodologies+for+exposure+science.">New approach methodologies for exposure science.</a> Current Opinion in Toxicology. 15, 76-92 (2019).</li><li>Rand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophotoxicology:+The+growing+potential+for+Drosophila+in+neurotoxicology.">Drosophotoxicology: The growing potential for Drosophila in neurotoxicology.</a> Neurotoxicology and Teratology. 32 (1), 74-83 (2010).</li><li>Ong, C., Yung, L. Y. L., Cai, Y., Bay, B. H., Baeg, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+as+a+model+organism+to+study+nanotoxicity.">Drosophila melanogaster as a model organism to study nanotoxicity.</a> Nanotoxicology. 9 (3), 396-403 (2015).</li><li>Peterson, E. K., Long, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+protocol+for+using+Drosophila+as+an+invertebrate+model+system+for+toxicity+testing+in+the+laboratory.">Experimental protocol for using Drosophila as an invertebrate model system for toxicity testing in the laboratory.</a> Journal of Visualized Experiments. (137), e57450 (2018).</li><li>Rand, M. D., Montgomery, S. L., Prince, L., Vorojeikina, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+toxicity+assays+using+the+Drosophila+model.">Developmental toxicity assays using the Drosophila model.</a> Current Protocols in Toxicology. 59, 11-20 (2014).</li><li>Misra, J. R., Horner, M. A., Lam, G., Thummel, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+xenobiotic+detoxification+in+Drosophila.">Transcriptional regulation of xenobiotic detoxification in Drosophila.</a> Genes &amp; Development. 25 (17), 1796-1806 (2011).</li><li>Rocha, J. B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+as+a+promising+model+organism+in+toxicological+studies.">Drosophila melanogaster as a promising model organism in toxicological studies.</a> Archives of Basic and Applied Medicine. 1 (1), 33-38 (2013).</li><li>Affleck, J. G., Walker, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+developmental+toxicology:+using+and+extending+the+drosophotoxicology+model.">Drosophila as a model for developmental toxicology: using and extending the drosophotoxicology model.</a> Methods in Molecular Biology. 1965, 139-153 (2019).</li><li>Ugur, B., Chen, K., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+tools+and+assays+for+the+study+of+human+diseases.">Drosophila tools and assays for the study of human diseases.</a> Disease Models &amp; Mechanisms. 9 (3), 235-244 (2016).</li><li>Chien, S., Reiter, L. T., Bier, E., Gribskov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homophila:+human+disease+gene+cognates+in+Drosophila.">Homophila: human disease gene cognates in Drosophila.</a> Nucleic Acids Research. 30 (1), 149-151 (2002).</li><li>Wangler, M. 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=Model+organisms+facilitate+rare+disease+diagnosis+and+therapeutic+research.">Model organisms facilitate rare disease diagnosis and therapeutic research.</a> Genetics. 207 (1), 9-27 (2017).</li><li>Colbourne, J. 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=Toxicity+by+descent:+A+comparative+approach+for+chemical+hazard+assessment.">Toxicity by descent: A comparative approach for chemical hazard assessment.</a> Environmental Advances. 9, 100287 (2022).</li><li>Hales, K. G., Korey, C. A., Larracuente, A. M., Roberts, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+on+the+fly:+a+primer+on+the+Drosophila+model+system.">Genetics on the fly: a primer on the Drosophila model system.</a> Genetics. 201 (3), 815-842 (2015).</li><li>Vang, L. L., Medvedev, A. V., Adler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+ways+to+measure+behavioral+responses+of+Drosophila+to+stimuli+and+use+of+these+methods+to+characterize+a+novel+mutant.">Simple ways to measure behavioral responses of Drosophila to stimuli and use of these methods to characterize a novel mutant.</a> PLoS One. 7 (5), 37495 (2012).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B., Byfield, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> Journal of Visualized Experiments. (61), e3795 (2012).</li><li>Bloomington Drosophilia Stock Center. BDSC Cornmeal Food Available from: <a target="_blank" href="https://bdsc.indiana.edu/information/recipes/bloomfood.html">https://bdsc.indiana.edu/information/recipes/bloomfood.html</a> (2022)</li><li>Martins, R. R., McCracken, A. W., Simons, M. J. P., Henriques, C. M., Rera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+catch+a+Smurf?+-+ageing+and+beyond&#8230;+in+vivo+assessment+of+intestinal+permeability+in+multiple+model+organisms.">How to catch a Smurf? - ageing and beyond&#8230; in vivo assessment of intestinal permeability in multiple model organisms.</a> Bio-Protocol. 8 (3), 2722 (2018).</li><li>Rera, M., 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=Modulation+of+longevity+and+tissue+homeostasis+by+the+Drosophila+PGC-1+homolog.">Modulation of longevity and tissue homeostasis by the Drosophila PGC-1 homolog.</a> Cell Metabolism. 14 (5), 623-634 (2011).</li><li>Benchmark Dose Software (BMDS) (Build 3.3; Model Library Version 2022.10) [Computer Software]. United States Environmental Protection Agency Available from: <a target="_blank" href="https://www.epa.gov/bmds/download-bmds">https://www.epa.gov/bmds/download-bmds</a> (2022)</li><li>Ortiz, J. G., Opoka, R., Kane, D., Cartwright, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+arsenic+susceptibility+from+a+genetic+perspective+in+Drosophila+reveals+a+key+role+for+glutathione+synthetase.">Investigating arsenic susceptibility from a genetic perspective in Drosophila reveals a key role for glutathione synthetase.</a> Toxicological Sciences. 107 (2), 416-426 (2009).</li><li>Pickett, A. D., Patterson, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenates:+effect+on+fecundity+in+some+Diptera.">Arsenates: effect on fecundity in some Diptera.</a> Science. 140 (3566), 493-494 (1963).</li><li>Goldstein, S. H., Babich, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+arsenite+and+arsenate+to+Drosophila+melanogaster+in+a+combined+adult/developmental+toxicity+assay.">Differential effects of arsenite and arsenate to Drosophila melanogaster in a combined adult/developmental toxicity assay.</a> Bulletin of Environmental Contamination and Toxicology. 42 (2), 276-282 (1989).</li><li>Polak, M., Opoka, R., Cartwright, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+fluctuating+asymmetry+to+arsenic+toxicity:+support+for+the+developmental+selection+hypothesis.">Response of fluctuating asymmetry to arsenic toxicity: support for the developmental selection hypothesis.</a> Environmental Pollution. 118 (1), 1928 (1928).</li><li>Zhou, S., 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=A+Drosophila+model+for+toxicogenomics:+Genetic+variation+in+susceptibility+to+heavy+metal+exposure.">A Drosophila model for toxicogenomics: Genetic variation in susceptibility to heavy metal exposure.</a> PLoS Genetics. 13 (7), 1006907 (2017).</li><li>Anholt, R. R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensation+and+evolution+of+Drosophila+host+plant+selection.">Chemosensation and evolution of Drosophila host plant selection.</a> iScience. 23 (1), 100799 (2020).</li><li>Depetris-Chauvin, A., Galagovsky, D., Grosjean, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemicals+and+chemoreceptors:+ecologically+relevant+signals+driving+behavior+in+Drosophila.">Chemicals and chemoreceptors: ecologically relevant signals driving behavior in Drosophila.</a> Frontiers in Ecology and Evolution. 3, 41 (2015).</li><li>Aryal, B., 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=Protocol+for+binary+food+choice+assays+using+Drosophila+melanogaster.">Protocol for binary food choice assays using Drosophila melanogaster.</a> STAR Protocols. 3 (2), 101410 (2022).</li><li>Shimada, I., Nakao, M., Kawazoe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+differential+sensitivity+and+role+of+the+central+nervous+system+in+the+feeding+behavior+of+Drosophila+melanogaster.">Acute differential sensitivity and role of the central nervous system in the feeding behavior of Drosophila melanogaster.</a> Chemical Senses. 12 (3), 481-490 (1987).</li><li>Tanimura, T., Isono, K., Takamura, T., Shimada, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dimorphism+in+the+taste+sensitivity+to+trehalose+in+Drosophila+melanogaster.">Genetic dimorphism in the taste sensitivity to trehalose in Drosophila melanogaster.</a> Journal of Comparative Physiology. 147 (4), 433-437 (1982).</li><li>Ro, J., Harvanek, Z. M., Pletcher, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLIC:+high-throughput,+continuous+analysis+of+feeding+behaviors+in+Drosophila.">FLIC: high-throughput, continuous analysis of feeding behaviors in Drosophila.</a> PLoS One. 9 (6), 101107 (2014).</li><li>National Research Council. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+Precision+Medicine:+Building+a+Knowledge+Network+for+Biomedical+Research+and+a+New+Taxonomy+of+Disease.">Toward Precision Medicine: Building a Knowledge Network for Biomedical Research and a New Taxonomy of Disease.</a> National Academy of Sciences. , (2011).</li><li>Li, H., Tennessen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Drosophila+larval+samples+for+gas+chromatography-mass+spectrometry+(GC-MS)-based+metabolomics.">Preparation of Drosophila larval samples for gas chromatography-mass spectrometry (GC-MS)-based metabolomics.</a> Journal of Visualized Experiments. (136), e57847 (2018).</li></ol>

No conflicts of interest to disclose.

The fruit fly Drosophila melanogaster is emerging as a powerful system for NAMs16,18,19,21. By leveraging the unparalleled genetic resources available to the fly community, combined with recent advances in genomics and metabolomics, chemical safety studies using Drosophila are capable of quickly identifying the molecular mechanisms by which individual compounds interfere with metabolism, physiology, and cell signaling (for example, see39). This inexpensive protocol is designed to rapidly define dose-response curves and subsequently generate samples for RNA-seq and metabolomics analysis. Moreover, this flexible protocol can be adapted for use with any genotype and can accommodate many classes of chemicals.
A notable aspect of this protocol is the choice of liquid food used in the chemical exposure, which is based on a previous study, but differs from the solid media used by most toxicological studies of Drosophila18,22. This specific liquid media was selected to reflect the nutritional content of the standard, solid BDSC media that the flies are also fed in this protocol, to ensure the flies receive consistent nutrition. The simplicity of liquid feeding media has many advantages. Liquid media is easier to handle than solid food, which needs to be either melted and resolidified or reconstituted from powder. Liquid media also increases the system&#39;s throughput, ensures even chemical distribution throughout the feeding media, and decreases the time spent working with hazardous compounds. Additionally, the media does not require solutions to be heated, which facilitates the testing of volatile test compounds. Finally, because of the relatively few components included in the food solution, undesirable side reactions are minimized between the test chemical and other dietary components. The yeast used in the food is also inactive, further limiting the reactivity of the feeding medium. However, please note that the method is not suitable for testing developmental or larval toxicity.
Some of the materials used in the protocol can be substituted, such as using glass fly vials rather than polypropylene. However, the materials used were selected to be both inert and disposable to avoid unwanted chemical reactions between reagents and chemical exposures that could result from cleaning glassware.
The use of liquid food necessitates a vehicle for food delivery. Cellulose acetate filter paper was selected for this purpose due to its flexibility and inert nature28. Other researchers used similar protocols but with other vehicles, such as delicate task wipes or glass fiber filter29,30. The cellulose acetate filter paper suited these needs because it is an inert vehicle which can be cut to the ideal shape to fit it into the bottom of the fly vials without large gaps between the paper and vial wall, preventing death due to flies becoming stuck in media or the vehicle itself.
An important limitation of this system is that the maximum testable concentration of a chemical is tied to the solubility of the chemical. Non-water-soluble compounds require an additional solvent, which can lead to additional or synergistic effects with the chemical of interest. This can also create situations in which it is not possible to prepare stock solutions that are concentrated enough to achieve the desired endpoint in all organisms, therefore limiting analysis of the resulting data31. To address this, chemicals with low water solubility can be tested by adding up to 0.5% dimethyl sulfoxide to the food solution. Other solvents could be used as well, but additional research is needed for each solvent of interest to determine the maximum acceptable solvent concentration within the solution to maximize solubility while minimizing solvent effects on the organism.
Extensive characterization of the olfaction response in Drosophila has described how flies avoid consuming toxic compounds40,41, leading to reduced feeding on treated media. The blue dye assay addresses this phenomenon by allowing researchers to efficiently screen the feeding behaviors of the flies fed each concentration of experimental chemical42,43,44. The presence or absence of blue in the fly&#39;s gastrointestinal tract indicates if the fly has been eating the toxicant-containing medium. Although more sophisticated methods of assessing fly feeding behaviors exist, such as the Fly Liquid-Food Interaction Counter45, this qualitative method is better suited for higher-throughput screening.
A notable aspect of this protocol is that it has been optimized for a 48 h exposure period without the need to transfer flies or add additional liquid to the exposure vial. Using a humidity chamber and placing the chambers in an incubator kept at high humidity prevented the filter paper containing the feeding media from drying out during this timeframe. The protocol can be adapted for longer exposure durations, but the method must be adjusted to ensure that the filter paper does not become dry and cause significant changes in solution concentration or lethality due to desiccation.
Finally, an important characteristic of this protocol is that it can readily accommodate genetic variants, which allows researchers to utilize the vast array of genetic tools for Drosophila to expand these preliminary studies on wild-type organisms to better understand mechanisms of chemical action in vivo. In this regard, the protocol outlined above could be easily modified to complement a previously described JoVE&#160;protocol by Peterson and Long that allows for toxicological analysis of wild-caught flies18.
Because of the wide variety of previous studies on the toxicity of sodium arsenite in Drosophila32,33,34,35,36, Oregon-R flies were treated with this compound to demonstrate the efficacy of our system. Male flies exhibited an LD50 of 0.65 mM, and females exhibited an LD50 of 0.90 mM. This aligns with previous studies of sodium arsenite-treated adult Drosophila. For example, Goldstein and Babich37 found that 50% of flies (mixed sexes) died after 7 days of exposure to 0.5 mM NaAsO2. Although this is a slightly lower dose than was presently observed, the differences between their methods and this method (including the use of solid exposure media, a longer time scale, and mixed sexes) likely account for this difference. Importantly, both methods resulted in overall similar LD50 values.
Observations from experiments using this protocol can be used to find genetic and molecular targets for subsequent behavioral or mechanistic studies. The exposure method can also be used to treat Drosophila for sampling for metabolomics and proteomics, making this protocol well suited to the growing field of precision toxicology (modeled from the precision medicine field46). In this regard, exposed flies can be collected after step 8 for subsequent genomic and metabolomics analysis. Samples collected in step 8 can then be processed, as described by Li and Tennessen47, starting with step 3.
Ultimately, the data acquired from the experiments described above, as well as any subsequent metabolomics and proteomics data, would ideally be used in cross-species comparisons. As previously noted26, such cross-species studies are powerful and capable of determining how individual chemicals interfere with conserved biological pathways. Thus, the protocol described above can be used to find evolutionary commonalities in response to individual toxicants across phyla and help inform chemical safety regulation.

Humans are constantly exposed to chemicals from a variety of sources, including air1, food2, water3,4, medications5, cleaning agents6, personal care products7, industrial chemicals7, and building materials7. Moreover, thousands of new chemicals are introduced each year8, many of which are not properly vetted for health and environmental safety. This lack of adequate chemical safety testing stems in part from an over-reliance on mammalian models, such as mice and rats. While such rodent models are informative, chemical safety testing in these systems is expensive, time-consuming, and often causes unacceptable levels of suffering to the test animal9.
The financial and ethical burdens associated with mammalian chemical safety testing, as well as the time-consuming nature of mammalian studies, are major contributing factors to the paucity of data surrounding new chemicals. To address this issue, the U.S. Environmental Protection Agency (EPA), the European Chemicals Agency (ECHA), Health Canada, and other agencies are implementing measures that incorporate new approach methodologies (NAMs) into regulatory frameworks10, thus placing North American and European policy in line with international goals to replace, reduce, and refine the use of animals (the 3Rs principal)11,12,13,14. NAMs encompass a variety of assays primarily based on in vitro and in silico models that provide a mechanistic understanding of chemical toxicity instead of observing adversity inflicted on mammalian test species, thereby increasing the rate of data generation for chemical risk assessment while still producing high fidelity outputs15. However, these methods are not yet proven to safeguard against systemic toxicity, including the disruption of vital biological processes involving interorgan communication and endocrine signaling. Further, they cannot account for the bioaccumulation of chemicals within specific tissues, the ability of individual compounds to be absorbed and secreted, and the interplay between behavior and chemical exposure.
Due to the limitations of in vitro and computational models, the successful use of NAMs to reduce or replace mammalian models should also include invertebrate in vivo models, such as the fruit fly, Drosophila melanogaster. Previous studies in the fly have demonstrated that this organism is well suited for studying the conserved genetic pathways that protect animal cells against toxic molecules16,17,18,19,20,21,22. Moreover, the fly shows remarkable genetic similarity to humans, including functional homologs to over 65% of human diseases23,24,25 and an even greater conservation of important functional pathways26. These features, combined with their relatively short life cycle, low maintenance cost, and readily observable behavioral responses, make Drosophila well-suited for use as a toxicological model27,28,29,30. Moreover, flies have much higher throughput than rodent models and capture effects on metabolism, physiology, and hormone signaling that are not readily detectable by other non-organismal NAMs9.
The protocol described here represents a framework for testing the effects of chemical exposure on adult Drosophila. The method is designed to be efficient, inexpensive, and reproducible, while also minimizing the time researchers must be in contact with the test chemical and accommodating sample collection for metabolomics and other omics approaches. The protocol is optimized for testing a single chemical per experiment, but can easily accommodate other experimental parameters, such as varied solvents or combinations of chemicals.

<table><tbody><tr><td>1.5 inch flower lever action craft punch</td><td>Bira Craft</td><td>HCP-115-024</td><td></td></tr><tr><td>15 mL Centrifuge Tubes&amp;nbsp;</td><td>VWR</td><td>89039-666</td><td>High-Performance Centrifuge Tubes with Flat or Plug Caps, Polypropylene, 15 mL</td></tr><tr><td>2 ml Tubes&amp;nbsp;</td><td>VWR</td><td>16466-044</td><td>Micro Centrifuge Tube with Flat Screw-Cap, conical bottom</td></tr><tr><td>5 ml Tubes&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>60818-576</td><td>Culture Tubes, Plastic, with Dual-Position Caps</td></tr><tr><td>50 mL Centrifuge Tubes&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>430290</td><td>50 mL polypropylene centrifuge tubes, conical bottom with plug seal cap</td></tr><tr><td>Benchmark Dose Software version 3.2</td><td>U.S. Environmental Protection Agency</td><td></td><td></td></tr><tr><td>Cardboard trays</td><td>Genesee Scientific flystuff</td><td>32-122</td><td>trays and dividers for narrow vials&amp;nbsp;</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; gas pads</td><td>Genesee Scientific flystuff</td><td>59-114</td><td>FlyStuff flypad, CO&lt;sub&gt;2&lt;/sub&gt; anesthetizing apparatus&amp;nbsp;</td></tr><tr><td>Combitips advanced, 50 mL</td><td>Eppendorf&amp;nbsp;</td><td>0030089693</td><td>Combitips&amp;nbsp;advanced, Biopur, 50&amp;nbsp;mL, light gray, colorless tips</td></tr><tr><td>Cotton balls</td><td>Genesee Scientific flystuff</td><td>51-101</td><td>Cotton balls, large, fits narrow vials&amp;nbsp;</td></tr><tr><td>Delicate task wipes</td><td>Kimtech</td><td>34155</td><td>Kimtech Science Kimwipes Delicate Task Wipes, 1 Ply / 8.2&quot; x 4.39&quot;</td></tr><tr><td>Drosophila Vial Plugs, Cellulose Acetate (aka, Flugs)</td><td>VWR</td><td>89168-888</td><td>Wide</td></tr><tr><td>FD&amp;amp;C Blue No. 1</td><td>Spectrum Chemical</td><td>FD110</td><td>CAS number 3844-45-9</td></tr><tr><td>Flies&amp;nbsp;</td><td>BDSC</td><td>Stock #2057&amp;nbsp;</td><td>OregonR wildtype&amp;nbsp;</td></tr><tr><td>Gloves (nitrile)</td><td>Kimtech</td><td>55082/55081/55083</td><td>Kimtech purple nitrile exam gloves, 5.9 mil, ambidextrous 9.5&quot;&amp;nbsp;</td></tr><tr><td>Grade 1 CHR cellulose chromatography paper</td><td>Cytvia</td><td>3001-917</td><td>Sheet, 46 x 57 cm</td></tr><tr><td>Mesh for humidity chamber</td><td></td><td></td><td></td></tr><tr><td>Multipette / Repeater (X) stream&amp;nbsp;</td><td>Eppendorf&amp;nbsp;</td><td>022460811</td><td>Repeater Xstream&amp;nbsp;</td></tr><tr><td>Plastic grate</td><td>Plaskolite</td><td>18469 (from lowes)</td><td>Plaskolite 24 in x 48 in 7.85 sq ft louvered ceiling light panels, cut down to fit in rubbermaid tubs</td></tr><tr><td>Plastic trays for glass vials</td><td>Genesee Scientific flystuff</td><td>59-207</td><td>Narrow fly vial reload tray&amp;nbsp;</td></tr><tr><td>Polypropylene Drosophila Vial</td><td>VWR</td><td>75813-156</td><td>Wide (28.5 mm)</td></tr><tr><td>Rubbermaid tubs</td><td>Rubbermaid</td><td>3769017 (from Lowes)</td><td>Rubbermaid Roughneck Tote 10 gallon 18&quot; L x 12&quot; W x 8 1/2&quot; H</td></tr><tr><td>Sucrose ultra pure</td><td>MP Biomedicals, Inc.</td><td>821721</td><td></td></tr><tr><td>Tube racks for wide-mouthed tubes</td><td>Thermo scientific&amp;nbsp;</td><td>5970-0230</td><td>Nalgene Unwire Test tube racks, for 30 mm tubes&amp;nbsp;</td></tr><tr><td>Water Purification System</td><td>Millipore Milli-Q</td><td>ZMQ560F01</td><td>Millipore Milli-Q Biocel Water Purifier&amp;nbsp;</td></tr><tr><td>Yeast extract</td><td>Acros Organics</td><td>451120050</td><td>CAS number 84604-16-0</td></tr></tbody></table>

assessment of chemical toxicity in adult drosophila melanogaster

Human industries generate hundreds of thousands of chemicals, many of which have not been adequately studied for environmental safety or effects on human health. This deficit of chemical safety information is exacerbated by current testing methods in mammals that are expensive, labor-intensive, and time-consuming. Recently, scientists and regulators have been working to develop new approach methodologies (NAMs) for chemical safety testing that are cheaper, more rapid, and reduce animal suffering. One of the key NAMs to emerge is the use of invertebrate organisms as replacements for mammalian models to elucidate conserved chemical modes of action across distantly related species, including humans. To advance these efforts, here, we describe a method that uses the fruit fly, Drosophila melanogaster,&#160;to assess chemical safety. The protocol describes a simple, rapid, and inexpensive procedure to measure the viability and feeding behavior of exposed adult flies. In addition, the protocol can be easily adapted to generate samples for genomic and metabolomic approaches. Overall, the protocol represents an important step forward in establishing Drosophila as a standard model for use in precision toxicology.

The fly has long served as a model in studies for determining sodium arsenite (NaAsO2) toxicity35,36,37,38. To demonstrate the efficacy of the protocol, male and female flies were exposed to NaAsO2, with the goal of comparing these results with earlier studies. Using the methodology described above, adult Oregon-R (BDSC stock #2057) males and females were exposed to a range of NaAsO2 concentrations (0, 0.01, 0.02, 0.1, 0.2, 1, and 2 mM) and scored for lethality 48 h after the start of exposure (Figure 1A,B).
The purpose of this initial analysis was to identify the approximate range of concentrations that would allow a more precise characterization of NaAsO2 toxicity. In subsequent experiments, concentrations were selected (0, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 5 mM) that more precisely defined the NaAsO2 dose-response curve (Figure 1C,D). Note that the resulting analysis examined several concentrations that induced 100% lethality. Data were analyzed using the Environmental Protection Agency&#39;s publicly available Benchmark Dose Software version 3.2.0.125. The data were modeled as &#34;dichotomous&#34; and the Dichotomous Hill model was used for subsequent analyses. Based on this model, the final LD10, LD25, and LD50 of male flies fed NaAsO2 were 0.30 mM, 0.50 mM, and 0.65 mM, respectively. For female flies, these values were slightly higher, with an LD10 of 0.30 mM, an LD25 of 0.65 mM, and an LD50 of 0.90 mM. Overall, the values obtained using this method are similar to those previously reported for arsenic toxicity in the Drosophila melanogaster35,36,37,38, thus validating the methodology.
In addition to the six replicates used to calculate the dose-response curve, male and female flies were also fed NaAsO2 exposure solutions that contained 1% FD&#38;C blue, which is easily visible in the digestive tract using light microscopy. Based on the presence of blue dye within the intestine of NaAsO2-fed flies, both male and female flies continued to feed 24 h after the beginning of the chemical exposure, regardless of the NaAsO2 concentration present within the liquid media (Figure 2). However, the ingested food was observed to be occasionally regurgitated at doses above 0.2 mM for females and 0.5 mM for males (Figure 2). These findings suggest that regurgitation could serve a key role in the Drosophila response to arsenic poisoning.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65029/65029fig01.jpg" /> 
Figure 1: Dose-response curves for male and female Drosophila treated with NaAsO2 for 48 h. All graphs show the estimated proportions of dead flies at each NaAsO2 concentration tested based on the Dichotomous Hill model. (A,B) A broad range of NaAsO2 concentrations were tested to approximate the dose at which each sex of fly begins to die. (A) shows the male data, and (B) shows the female data. N = 4 vials, with 20 flies per vial. (C,D) A narrower range of NaAsO2 concentrations was tested to determine precise doses at which 10%, 25%, and 50% of each sex of flies died. These doses are indicated to the right of each graph. (C) shows the male data, and (D) shows the female data. N = 6 vials, with 20 flies per vial. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65029/65029fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65029/65029fig02.jpg" /> 
Figure 2: Representative results from the blue dye assay of male and female Drosophila treated with NaAsO2 for 24 h. Micrographs show flies fed increasing concentrations of NaAsO2. Row (A) shows male flies, and row (B) shows female flies, with the concentration of NaAsO2 increasing from left to right. The abdomen shows a small amount of blue near the thorax at low concentrations, indicating that exposure media entered the gut. At higher concentrations, blue dye begins to accumulate around the mouth, suggesting that exposure media is being regurgitated. The scale bar is 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65029/65029fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessment of Chemical Toxicity in Adult Drosophila Melanogaster at JoVE.com

Assessment of Chemical Toxicity in Adult Drosophila Melanogaster

This protocol describes an efficient and inexpensive method that uses liquid media to assess the effects of chemical toxicants on the viability of adult Drosophila melanogaster.

Assessment of Chemical Toxicity in Adult Drosophila Melanogaster

Human industries generate hundreds of thousands of chemicals, many of which have not been adequately studied for environmental safety or effects on human ...

assessment-of-chemical-toxicity-in-adult-drosophila-melanogaster

Nanyang Technological University

Research

JoVE Journal

Biology

3.2K Views.  Indiana University. This protocol describes an efficient and inexpensive method that uses liquid media to assess the effects of chemical toxicants on the viability of adult Drosophila melanogaster. 

Video: Assessment of Chemical Toxicity in Adult Drosophila Melanogaster

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

Behavior

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Developmental Biology

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

Neuroscience

Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster

Zinc oxide nanoparticles (ZnO NPs) have a wide range of applications, but the number of reports on ZnO NP-associated toxicity has grown rapidly in recent years. However, studies that elucidate the underlying mechanisms for ZnO NP-induced toxicity are scanty. We determined the toxicity profiles of ZnO NPs using both in vitro and in vivo experimental models. A significant decrease in cell viability was observed in ZnO NP-exposed MRC5 lung fibroblasts, showing that ZnO NPs exert cytotoxic effects. Similarly, interestingly, gut exposed to ZnO NPs exhibited a dramatic increase in reactive oxygen species levels (ROS) in the fruit fly Drosophila. More in-depth studies are required to establish a risk assessment for the increased usage of ZnO NPs by consumers.

Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster

We describe a detailed protocol for evaluating the toxicological profiles of zinc oxide nanoparticles (ZnO NPs) in particular, the type of cell death in human MRC5 lung fibroblasts and ROS formation in the fruit fly Drosophila.

Zinc oxide nanoparticles (ZnO NPs) have a wide range of applications, but the number of reports on ZnO NP-associated toxicity has grown rapidly in recent ...

Immunology and Infection

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

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.

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.

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

Lifespan Protocol for Drosophila melanogaster: Generating Survival Curves to Identify Differences in Fly Longevity

Source: Bovier, T. F., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/59535/" target="_blank">Methods to Test Endocrine Disruption in Drosophila melanogaster</a>. J. Vis. Exp. (2019).
Scientists use the Drosophila lifespan protocol to measure how different experimental conditions affect flies&#39; longevity and generate survival curves in the process to quantify the experimental outcomes. The sample protocol features the assay being applied to study the effects on longevity from exposure to varying concentrations of endocrine disruptor chemicals, or EDCs, such as 17-&#945;-ethinylestradiol (EE2).

Source: Bovier, T. F., et al. Methods to Test Endocrine Disruption in Drosophila melanogaster. J. Vis. Exp. (2019).
Scientists use the Drosophila lifespan ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Adult

Feeding Test: Assessment of Feeding Preferences in Drosophila melanogaster

Source: Bovier, T. F., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/59535/" target="_blank">Methods to Test Endocrine Disruption in Drosophila melanogaster</a>. J. Vis. Exp. (2019).
This video describes a feeding assay for fruit flies in which colored food is used to monitor feeding and the effect added compounds may have on feeding activity. The example protocol tests the effects of several endocrine disrupting chemicals (EDC).

Source: Bovier, T. F., et al. Methods to Test Endocrine Disruption in Drosophila melanogaster. J. Vis. Exp. (2019).
This video describes a feeding assay ...

Assessment of Chemical Toxicity in Adult Drosophila Melanogaster

Summary

Explore More Videos

Chapters in this video

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Methods to Test Endocrine Disruption in Drosophila melanogaster

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

Taste Preference Assay for Adult Drosophila

Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

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

Lifespan Protocol for Drosophila melanogaster: Generating Survival Curves to Identify Differences in Fly Longevity

Feeding Test: Assessment of Feeding Preferences in Drosophila melanogaster