The authors thank Orsolina Petillo for technical support. The authors thank Dr. Mariarosaria Aletta (CNR) for bibliographic support. The authors thank Dr. Gustavo Damiano Mita for introducing them to the EDC world. The authors thank Leica Microsystems and Pasquale Romano for their assistance. This research was supported by Project PON03PE_00110_1. &#8220;Sviluppo di nanotecnologie Orientate alla Rigenerazione e Ricostruzione Tissutale, Implantologia e Sensoristica in Odontoiatria/oculistica&#8221; acronimo &#8220;SORRISO&#8221;; Committente: PO FESR 2014-2020 CAMPANIA; Project PO FESR Campania 2007-2013 &#8220;NANOTECNOLOGIE PER IL RILASCIO CONTROLLATO DI MOLECOLE BIO-ATTIVE NANOTECNOLOGIE&#8221;.

The authors have nothing to disclose.

The fruit fly D. melanogaster has been extensively employed as an in vivo model system to investigate the potential effects of environmental EDCs such as DBP28, BPA, 4-NP, 4-tert-OP29, MP30, EP31,32, DEHP33, and EE234. Several reasons have led its use as a model in this field of research. Apart from its undisputed advantages as a model ...

Human activities have been releasing into the environment a massive amount of chemicals, which represent a serious problem for organisms and for natural ecosystems1. Of these pollutants, it is estimated that about 1,000 different chemicals may alter the normal balance of endocrine systems; according to this property, they are classified as endocrine disrupting chemicals (EDCs). Specifically, based on a recent definition by the Endocrine Society, the EDCs are &#8220;an exogenous chemical, or mixture of chemicals, that can interfere with any aspect of hormone action&#8221;2. Over the last three decades, there has been growing scientific evidence that EDCs can affect the reproduction and development of animals and plants3,4,5,6,7,8. Further, EDC exposure has been related to the increasing prevalence of some human diseases, including cancer, obesity, diabetes, thyroid diseases, and behavioral disorders9,10,11.
General mechanisms of EDC
Due to their molecular properties, EDCs behave like hormones or hormone precursors3,4,5,6,7,8,9,10,11,12. In this sense, they can bind to a hormone&#8217;s receptor and disrupt endocrine systems either by mimicking hormone activity or by blocking endogenous hormones binding. In the first case, after binding to the receptor, they can activate it as its natural hormone would do. In the other case, binding of the EDC to the receptor prevents the binding of its natural hormone, so the receptor is blocked and can no longer be activated, even in the presence of its natural hormone3. As a consequence, EDCs can affect several processes, such as the synthesis, secretion, transport, metabolism, or peripheral action of endogenous hormones that are responsible for the maintenance of homeostasis, reproduction, development, and/or behavior of the organism. Receptor binding is not the only way of action described so far for the EDCs. It is now clear that they can also act by recruiting coactivators or corepressors in enzymatic pathways or by modifying epigenetic markers deregulating gene expression10,11,12,13,14, with consequences not only for the current generation but also for the health of generations to come8.
Drosophila hormones
The potential effects of selected EDCs have been studied widely, both in wildlife species and in several model systems in which endocrine mechanisms are reasonably well known. For invertebrates, endocrine systems that influence growth, development, and reproduction have been extensively characterized in insects for several reasons, involving their extensive use in the field of biological research, their economic importance, and finally the development of insecticides able to interfere specifically with the hormone system of pest insects.
In particular, among insects, the fruit fly D. melanogaster has proven to be a very powerful model system to evaluate the potential endocrine effects of EDCs. In D. melanogaster, as well as in vertebrates, hormones play an important role throughout the entire life cycle. In this organism, there are three main hormonal systems, which involve the steroid hormone 20-hydroxyecdysone (20E)15,16, the sesquiterpenoid juvenile hormone (JH)17, and the neuropeptides and peptide/protein hormones18. This third group consists of several peptides discovered more recently but clearly involved in a huge variety of physiological and behavioral processes, such as longevity, homeostasis, metabolism, reproduction, memory, and locomotor control. 20E is homologous to cholesterol-derived steroid hormones such as estradiol, while JH shares some similarities with retinoic acid; both of them are the better-known hormones in Drosophila19,20. Their balance is vital in coordinating molting and metamorphosis, as well as in controlling several postdevelopmental processes, such as reproduction, lifespan, and behavior21, thus offering different possibilities for testing endocrine disruption in Drosophila. Further, ecdysteroid hormones and JHs are the main targets of the so-called third-generation insecticides, developed to interfere with developmental and reproductive endocrine-mediated processes in insects. The agonist or antagonist mode of action of these chemicals is well known, and thus they can serve as reference standards for evaluating the effects of potential EDCs on the growth, reproduction, and development of insects22. For example, methoprene, which has been widely used in controlling mosquitoes and other aquatic insects23,24, works as a JH agonist and represses 20E-induced gene transcription and metamorphosis.
In addition to hormones, the nuclear receptor (NR) superfamily in Drosophila is also well known; it consists of 18 evolutionarily conserved transcription factors involved in controlling hormone-dependent developmental pathways, as well as reproduction and physiology25. These hormone NRs belong to all six NR superfamily subtypes, including those involved in neurotransmission26, two for retinoic acid NRs, and those for steroid NRs that, in vertebrates, represent one of the primary targets of EDCs27.
Drosophila as a model system for studying EDCs
Currently, on the basis of molecular properties, several environmental agencies around the world are attributing the potential to interfere with the endocrine systems to different man-made chemicals. Given that the EDCs are a global and ubiquitous problem for the environment and for organisms, the overall goal of the research in this field is to reduce their disease burden, as well as to protect living organisms from their adverse effects. In order to deepen the understanding about the potential endocrine effects of a chemical, it is necessary to test it in vivo. To this end, D. melanogaster represents a valid model system. To date, the fruit fly has been extensively used as in vivo model to evaluate the effects of several environmental EDCs; it has been reported that exposure to several EDCs, such as dibutyl phthalate (DBP)28, bisphenol A (BPA), 4-nonylphenol (4-NP), 4-tert-octylphenol (4-tert-OP)29, methylparaben (MP)30, ethylparaben (EP)31,32, bis-(2-ethylhexyl) phthalate (DEHP)33, and 17-&#945;-ethinylestradiol (EE2)34, influences metabolism and endocrine functions as in vertebrate models. Several reasons have led to its use as a model in this field of research. Beyond an excellent knowledge of its endocrine systems, further advantages include its short lifecycle, low cost, easily manipulable genome, a long history of research, and several technical possibilities (see the FlyBase website, http://flybase.org/). D. melanogaster also provides a powerful model for easily studying transgenerational effects and population responses to environmental factors8 and avoids ethical issues relevant for in vivo studies in higher animals. In addition, the fruit fly shares a high degree of gene conservation with humans which might make it possible for Drosophila EDC assays to help in predicting or suggesting potential effects of these chemicals for human health. Besides expanding the understanding about human health effects, Drosophila can help to assess risks of EDC exposure to the environment, such as biodiversity loss and environmental degradation. Finally, the fruit fly offers the additional advantage of being used in laboratories, where the factors potentially affecting its development, reproduction, and lifespan can be kept under control in order to attribute any variation to the substance to be tested.
With this in mind, we have optimized simple and robust fitness assays for determining EDC effects on some Drosophila hormonal traits, such as fecundity/fertility, developmental timing, and adult lifespan. These assays have been widely used for some EDCs23,24,25,26,27. In particular, we have used the following protocols to evaluate the effects of the exposure to the synthetic estrogen EE234 and to BPA and to bisphenol AF (BPA F) (unpublished data). These protocols may be easily modified to investigate the effects of a given EDC at a time, as well as the combined effects of multiple EDCs in D. melanogaster.

<table><tbody><tr><td>17&amp;alpha;-Ethinylestradiol</td><td>Sigma</td><td>E4876-1G</td><td></td></tr><tr><td>Agar for &lt;em&gt;Drosophila&lt;/em&gt; medium</td><td>BIOSIGMA</td><td>789148</td><td></td></tr><tr><td>Bisphenol A</td><td>Sigma</td><td>239658-50G</td><td></td></tr><tr><td>Bisphenol AF</td><td>Sigma</td><td>90477-100MG</td><td></td></tr><tr><td>Cornmeal</td><td>CA&#039; BIANCA</td><td></td><td></td></tr><tr><td>Diethyl ether</td><td>Sigma</td><td></td><td></td></tr><tr><td>Drosophila Vials</td><td>BIOSIGMA</td><td>789008</td><td>25 mm x 95 mm</td></tr><tr><td>Drosophila Vials</td><td>BIOSIGMA</td><td>789009</td><td>29 mm x 95 mm</td></tr><tr><td>Drosophila Vials</td><td>Kaltek</td><td>187</td><td>22 mm x 63 mm</td></tr><tr><td>Embryo collection cage</td><td>Crafts</td><td></td><td>Plexiglass cylinder (12.5 mm x7 cm) with an open end and the other end closed by a rectangular base in which a slot allows the insertion of special trays for laying</td></tr><tr><td>Ethanol</td><td>FLUKA</td><td>2860</td><td></td></tr><tr><td>Etherizer</td><td>Crafts</td><td></td><td>cylindrical glass container with a cotton plug</td></tr><tr><td>Glass Bottle</td><td></td><td></td><td>250 mL Bottles</td></tr><tr><td>Glass Vials</td><td>Microtech</td><td>ST 10024</td><td>Flat bottom tube 100 x 24</td></tr><tr><td>Hand blender Pimmy</td><td>Ariete</td><td></td><td>food processor</td></tr><tr><td>Instant Success yeast</td><td>ESKA</td><td></td><td>Powdered yeast</td></tr><tr><td>Laying tray</td><td>Crafts</td><td></td><td>plexiglass trays (11 cm x 2.6 cm) in which to pour medium for laying</td></tr><tr><td>Methyl4-hydroxybenzoate</td><td>SIGMA</td><td>H5501</td><td></td></tr><tr><td>Petri Dish</td><td>Falcon</td><td>351016</td><td>60x5</td></tr><tr><td>Red dye no. 40</td><td>SIGMA</td><td>16035</td><td></td></tr><tr><td>Stereomicroscope with LED lights</td><td>Leica</td><td></td><td>S4E</td></tr><tr><td>Sucrose</td><td>HIMEDIA</td><td>MB025</td><td></td></tr><tr><td>Tomato sauce</td><td>Cirio</td><td></td><td></td></tr></tbody></table>

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.

In this section, key steps of the above protocols are reported in the form of simplified schemes. Given that flies tend to avoid unpalatable compounds, the first thing to do is to assay the taste of the selected EDC. This can be done by mixing a food coloring (for example, red food dye no. 40)35 with the food supplemented with the selected EDC at various doses or with the solvent alone. Flies fed on these media are examined under a stereomicroscope and the food intake is estimated by their abdomin...

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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.

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 ...

methods-to-test-endocrine-disruption-in-drosophila-melanogaster

Research

JoVE Journal

Developmental Biology

9.6K Views.  National Research Council of Italy (CNR). 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.

Video: Methods to Test Endocrine Disruption in Drosophila melanogaster

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 ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Neuroscience

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Biology

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

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 ...

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 &#176;C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

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 ...

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.

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.

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

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/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 ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

Summary

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Chapters in this video

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

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

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

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

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

Assessment of Chemical Toxicity in Adult Drosophila Melanogaster

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