Name: methods to test endocrine disruption in drosophila melanogaster
Uploaded: 2019-07-03T14:00:00
Description: Watch this Scientific Journal Video about Methods to Test Endocrine Disruption in Drosophila melanogaster at JoVE.com

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;.

1. Food Preparation
<ol>
	<li>For stock maintenance and for larval growth, use a cornmeal medium containing 3 % powdered yeast, 10 % sucrose, 9 % precooked cornmeal, 0.4 % agar, thereafter called Cornmeal medium (CM).
	<ol>
		<li>Put 30 g of yeast into 100 mL of tap water, bring it to a boil and let it boil for 15 min.</li>
		<li>Separately, mix well 90 g of precooked cornmeal, 100 g of sugar, and 4 g of agar into 900 mL of tap water.</li>
		<li>Bring the solution to a boil, lower the heat and cook for...

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&#945;-Ethinylestradiol</td>
			<td>Sigma</td>
			<td>E4876-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar for Drosophila 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&#39; 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>25x95 mm</td>
		</tr>
		<tr>
			<td>Drosophila Vials</td>
			<td>BIOSIGMA</td>
			<td>789009</td>
			<td>29x95 mm</td>
		</tr>
		<tr>
			<td>Drosophila Vials</td>
			<td>Kaltek</td>
			<td>187</td>
			<td>22X63</td>
		</tr>
		<tr>
			<td>Embryo collection cage</td>
			<td>Crafts</td>
			<td></td>
			<td>Plexiglass cylinder (12,5 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>250mL Bottles</td>
		</tr>
		<tr>
			<td>Glass Vials</td>
			<td>Microtech</td>
			<td>ST 10024</td>
			<td>FLAT BOTTOM TUBE 100X24</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 x 2,6 cm) in wich 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...

Watch this Scientific Journal Video about Methods to Test Endocrine Disruption in Drosophila melanogaster at JoVE.com

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 ...

Research

JoVE Journal

Developmental Biology

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

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 ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Imaging Live Brain Explants from Drosophila melanogaster Third Instar Larvae

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Investigating Asymmetric Cell Division Dynamics: A Protocol for Live-Imaging of Drosophila Larval Brain Explants (Video) | JoVE

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...