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 ...

Drosophila melanogaster is a powerful model to evaluate in vivo the potential toxic and endocrine effects of chemicals, such as endocrine disruptors, that represent a serious problem for human health and natural environments. Addressing the effect of EDCs on the hormonal life traits of the fly, such as fecundity, fertility, developmental time, and the lifespan. It's an easy, cheap, and fast way to investigate endocrine disruption in vivo.The fertility, development, and lifespan in Drosophila can be affected by several factor that have to be carefully controlled to ensure the successful outcome of this protocol. Therefore, maximum attention in rearing and handling flies is required. To assay the taste of the selected EDC, after anesthetization, transfer 15 young flies from the fly bed to four parallel vials containing cornmeal medium mixed with a food coloring.The control vial contains the solvent alone, and the three other vials are supplemented with different doses of the selected EDCs. Keep the vials in a humidified incubator for one day with a natural 12-hour light. After anesthetizing the flies again, transfer the immobilized flies to a white paper, and place it under a stereo microscope to observe.Compare the abdominal coloring of each treatment group with respect to the control group. To assay the reproductive performance of the flies, prepare three vials of flies for each EDC as parental vials, with eight females and four males in 10 milliliters of cornmeal medium supplemented with EE2. For the control, prepare six vials of flies, each with eight females and four males in 10 milliliters of cornmeal medium food supplemented with EE2 solvent.Rear flies in an incubator at 25 degrees Celsius. During their development, avoid overcrowding the larvae. After four days, invert each vial over an etherizer to remove the parents and return the vials to the incubator for five more days.In the late afternoon of day nine, invert each vial over an etherizer to remove all newly-emerged flies from the vials and put the vials in another incubator at 18 degrees Celsius overnight. On the morning of day 10, on a Drosophila carbon dioxide work station, carefully invert the culture vial to prevent anesthetized flies from falling onto medium and insert a rubber tube with a needle into the vial between the cotton stopper and the vial wall. Open the valve to deliver carbon dioxide.Within a matter of seconds, transfer the flies to a fly bed under a microscope. Collect virgin females and young males separately into two groups on the fly bed. Randomly subdivide each group of flies in small subgroups, with 10 females or 20 males per vial filled with fresh corresponding cornmeal medium.Continue incubation and collect 30 virgin females and 30 males for each EDC, and 90 virgin females and 90 males for the control. House these groups of flies at 25 degrees Celsius until they are aged four days post-eclosion. Then transfer them into new vials containing fresh corresponding medium every two days.Check there are no larvae in the vials of females. Then label vials with a different series of sequential numbers for the following treatment. After anesthetization, transfer one EE2 solvent-treated female into a small vial containing fresh cornmeal tomato medium without EE2 and add one EE2 solvent-treated male for the control across.Pair an EE2-treated male with EE2 solvent-treated female or EE2-treated female with EE2 solvent-treated male for each treatment. House 20 single crosses at 25 degrees Celsius. Transfer each mating pair to fresh cornmeal tomato medium vials without EDC every day for the subsequent 10 days.Visually inspect each vial every day for the eggs and report their number on the fertility spreadsheet. In each vial, check closely for newly-emerged flies and record the daily number of adult progenies over the 10-day period. After 10 days from the initial mating, remove the parents from the last series of vials.In eclosion assay protocols, first, for each treatment group, set up 10 vials of young, healthy flies less than two days old, each with six females and three males in 10 milliliters of cornmeal food without EDC. Rear the flies on food for 24 hours and allow them to mate. Then prepare another 10 parallel vials per treatment group with 10 milliliters each of fresh cornmeal food supplemented with different EDC concentrations or the EE2 solvent alone for the control.Transfer the mated flies to these new vials. Make a developmental spreadsheet to record the different series. Allow the flies to lay eggs for 16 hours.Then, remove the parents from the vials. Incubate the vials at 25 degrees Celsius for three to four days, or until wandering larvae are visible. Every day, count the number of new pupae in each vial by writing a number in sequence by each pupa on the outside of the vial to avoid counting the same pupa twice.Report the number of newly-emerged pupae for three to four days, or until no more pupae form. Starting from day nine, count the number of emerging adults daily, until no more adults emerge. Report it on the developmental spreadsheet and perform the rest of the eclosion assay according to the manuscript.In the lifespan protocol, first set up 20 vials with eight females and four males, each filled with 10 milliliters of cornmeal food and house them at 25 degrees Celsius. After four days, discard the flies and place the vials back into the incubator. In the late afternoon of day nine, remove all newly-emerged flies from the vials and return the vials to the incubator.After 16 to 24 hours, divide 250 one-day-old adult flies of both sexes into four groups under light carbon dioxide anesthetization and transfer them to 250-milliliter bottles containing adult medium food, three supplemented with different EDC concentrations and one with the EE2 solvent alone. Maintain the flies at 25 degrees Celsius for two to three days to allow them to mate. Then, sort each cohort of flies by sex into two groups.Within each treatment condition, for both sexes, randomly subdivide each group into five parallel vials at a density of 20 individuals per vial. Prepare a lifespan spreadsheet in which the number of dead flies is subtracted from the number of surviving flies from the previous transfer, so that the number of survivors at each transfer is automatically obtained. After three days, transfer the flies without anesthesia to new vials containing the corresponding food at the same time and check for death.Record the age of the flies and the number of dead flies. Repeat the transfer every three days until all of the flies die. In this experiment, lifespan curves were plotted as the cumulative survivorship versus the elapsed days for each EDC concentration and the control.For the control, after a long initial period in which the survivorship curve remained relatively high, it declined exponentially after about 60 days. Following EDC exposure, the survivorship curve of the treated flies was affected significantly and showed dramatic decline after 36 days. It's advised about that the parallel vials under analysis should be very similar, otherwise it will be difficult to understand which to discard.Therefore, we suggest taking great care, adopting a good experimental practice, using a large sample size. Following this protocols, it is also possible to evaluate the transgenerational impact of a selected endocrine disruptor as well as to test the potential additive effects of a combination of different endocrine disruptors.

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

Research

JoVE Journal

Developmental Biology

9.4K vistas.  National Research Council of Italy (CNR). Drosophila melanogaster es un potente modelo para evaluar in vivo los posibles efectos tóxicos y endocrinos de los productos químicos, como los disruptores endocrinos, que representan un grave problema para la salud humana y los entornos naturales. Abordar el efecto de los EDC en los rasgos hormonales de la vida de la mosca, como la fecundidad, la fertilidad, el tiempo de desarrollo y la vida útil. Es una manera fácil, barata y rápida de investigar la interrupción endocrina in vivo....