Name: a direct and simple method to assess drosophila melanogaster's viability from embryo to adult
Uploaded: 2019-08-27T15:00:00.000+00:00
Duration: 4 s
Description: Watch this Scientific Journal Video about A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult at JoVE.com

Our work is funded by NIH 1R15GM1131-01 to CFH and NIH PA -12-149 TO CFH.

1. Media Preparation
<ol>
	<li>Prepare grape agar according to manufacturer instructions (see Table of Materials) and pour into a 35 mm Petri dish to half-full (Figure 5).&#160; Allow to solidify for approximately 1 h. After the grape agar solidifies, immediately use or store at 4 &#176;C.</li>
	<li>Gently make scratches across the agar plate using a small plastic knife (flies like to lay on uneven surfaces) leaving the middle of the plate without scratches (Figure 5). Place a small amount of yeast paste (made fresh; see Table of Materials) at the center of plate (Figure 5).</li>
</ol>
2. Embryo Collection Mini Cage Set Up
<ol>
	<li>Set up a cross using two virgin females and one young male inside the embryo collection cage containing the grape agar plate supplemented with yeast paste (Figure 5).</li>
	<li>After 24 h, inspect the cages for laid embryos without opening the cage by looking at the bottom of the agar plate.&#160; If embryos have been laid, remove the agar plate from the chamber and place it inside a humid chamber for microscopic observation (Figure 6).&#160; If several days of laying are scored (e.g., fertility/longevity assays), keep the breeding parents and replace the plate with a fresh one prepared as described. Early collections of less than 24 h can be done but, when using virgin females, be aware that they will not lay until 48 h after emerging from their pupa case.</li>
	<li>Cover the agar plate containing embryos with the Petri dish lid to avoid dehydration and place it immediately inside a humid chamber (Figure 7). Observe under a dissecting microscope and record hatched embryos and L1.&#160; Replace lid and store at room temperature in humid chamber until all embryos had hatched and developed into L1 larvae (Figure 6).</li>
</ol>
3. Counting Embryos and Larvae
<ol>
	<li>After 48 h, observe the plates under the dissecting microscope and record the numbers one last time before transfer of agar disc to food vial.&#160; Fertilized/viable embryos should become L1 by then.&#160; Longer incubation periods prior to transfer are possible but be aware that plates may start losing moisture and the agar may crack compromising a clean transfer to food vials (Figure 7).&#160; To ensure the plates remain hydrated and embryo viability is not compromised while counting, avoid using a direct gooseneck light over the agar surface (Figure 7E shows an appropriate distance).</li>
	<li>After counting, cover the plate and store it in the humid chamber until ready to transfer. Record findings.</li>
</ol>
4. Transfer the Grape Agar Disc to a Food Vial to monitor Viability During Development
<ol>
	<li>Once the numbers are recorded (hatched embryos/L1/L2), use a spatula to carefully transfer the grape agar disc to a vial large enough to accommodate a 35 mm disc containing Drosophila food media prepared according to manufacturer&#8217;s instructions (refer to list of reagents). Place the grape agar disc L1-side down on the food (Figure 7). &#160;After transferring the agar disc with larvae to the food vial, carefully inspect the empty Petri dish for any larvae left behind (Figure 7E).</li>
	<li>Set up a schedule to inspect food vials daily, at roughly the same time each day, to ensure L2/L3 larvae are observed making their way to the food in the vial (Figure 8).</li>
	<li>Record the number of pupae and adult Drosophila (Figure 9). Keep counting until no more adults are observed and avoid counting the following generation (do not count past 9 days after observing the first adults).</li>
	<li>Observe and record the findings. Adjust the time frame of embryo collection, counting, and recording accordingly to the mutant stock being used.</li>
	<li>Perform a chi square analysis. The null hypothesis assumes 100% viability such that the number of adults will be equal to the number of hatched embryos and L1 originally recorded and transferred to the food vials</li>
</ol>

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There are no conflicts of interest.

In summary, this method provides an accurate and simple assessment of viability in Drosophila. The entire protocol takes approximately 14 days to complete. The procedure does not require expert technical skills; however, proper timing, a schedule of daily observations, and careful agar transferring is important for accuracy and reproducibility.
In addition to the placement of the grape-agar disc embryo-side down in the food vial, another crucial step in the procedure is transferring the agar disc to a food vial no later than 48 h after removing the grape agar plate from the embryo collection mini cages. Transferring the agar after 48 h resulted in embryo and larvae loss, likely due to dehydration of the agar disc. To count L2/L3 transitions, the plate needs to incubate for 72 h. If this stage is crucial, grape agar plates should be poured thicker and a humid chamber must be used to prevent desiccation. The crosses were set up in embryo collection cages that accommodate 35 mm plates; however, this procedure can be performed using larger embryo collection cages as well, such as one that accommodates 60 mm or 100 mm Petri plates. &#160;Food bottles must then be big enough to accommodate those sizes.&#160;
There are steps that can be added to this protocol. As mentioned above, L2/L3 transitions are challenging to quantify on plates due to the time required and the potential dehydration of the agar. Additionally, larvae become highly mobile on the surface of the agar, posing a challenge to accurately count them. Placing the plates in the refrigerator for 30 min or adding a few drops of a mild anesthetic (lidocaine solution) on the surface of the agar prior to counting L2/L3 larvae can help slow down their movements to count them more accurately. &#160;A caveat to these modifications is that they can introduce variables (cold sensitivity, anesthetic sensitivity) and confound the viability results.&#160; Even without these steps, by using colored food, researchers can quantify wandering L3s/prepupae as they settle on the side of the food vials to initiate pupation. A limitation to this method is that it requires the adults used in the crosses to survive being anesthetized with CO2 for phenotyping to set up the crosses in the embryo collection cages. Dm ime4 homozygous mutant males do not recover well and die a few hours after waking up from CO2 treatment. &#160;Other methods to immobilize adults for sorting can be explored, such as placing vials in the refrigerator for a few minutes and then transfer vials to crushed ice to slow movement and sort quickly and efficiently.&#160;
Apart from comparing allelic strengths and their effects on viability, this method can be used to screen sensitivity or resistance to defined pharmaceutical compounds. Unlike other methods that screen compound toxicity in cell culture15,16,17,18, this method uses whole organisms, making assessment of developmental effects easier to analyze. In sum, using this protocol and modifications therein, will allow to measure allelic strengths as well as the effects of environmental factors and chemical compounds on Drosophila viability, fecundity, fertility, lifespan, and duration of developmental cycle.

Lifespan is affected by genetic and non-genetic factors. In standard lab growth conditions at room temperature, our lab has observed significant variation of fitness and viability among different Dm ime4 alleles grown under identical conditions (Figure 1 and Supplemental Figures). Viability studies are frequently done to investigate the effects of a certain allele combination or growth condition in population genetic studies1,2,3,4. However, detailed analyses of viability within a non-complementary group of mutations are hard to find in the scientific literature. An allele is usually labeled &#34;non-essential&#34;&#160;if the researcher finds a few individuals homozygous for that allele within the food vial that houses the balanced stock5,6. However, accurate Chi-square analyses to assess whether these homozygotes arise at the expected Mendelian ratios are not reported5,6. The most permissive temperature for any Drosophila stock is room temperature (22-23 &#176;C) and, with appropriate nutrients, the life cycle of wild-type flies takes approximately twelve days to complete7,8. As the duration of each developmental stage of wild-type Drosophila is known7,8, the method described in this report can be used to examine whether the Drosophila strain under study is fit at each stage in comparison to a control appropriate for the genetic background tested. In contrast to studies that focus on one specific aspect of development9, this protocol provides a practical way to assess viability at different developmental stages.
In our lab, this protocol is used to assess the viability of stocks that are deficient for Drosophila Inducer of Meiosis 4 (Dm ime4). Dm ime4 is an essential gene10 that encodes an RNA methyltransferase with critical roles in RNA metabolism in Drosophila and other multicellular organisms5,6,10,11,12,13,14. To quickly evaluate novel alleles of Dm ime4 generated via CRISPR/Cas9 (Supplemental Figures), an end-point viability assay was performed that only counted adult progeny produced within vials of balanced stocks (Figure 1). Some of the stocks used were described in previous Dm ime4 reports5,6. Homozygous mutants emerged at sub-Mendelian levels, as determined by chi-square analyses (Figure 1 and Supplemental Materials). To assess whether these lower-than-expected numbers were due to fewer embryos being laid, or fewer hatched embryos, or loss of viability in L1/L2 or pupae, we expanded the tracking to include counts at each of these developmental stages (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, and Figure 9).
Here, we describe the method using wild-type (OreR) flies. To empirically test this method for use with other genetic backgrounds or Drosophila species, we recommend using OreR as a reference and adjust timepoints according to the experimental organism.&#160; The protocol was further evaluated to assess viability of progeny generated by crossing virgin wild-type females with males from a heterozygous Dm ime4 mutant stock5,6 (Figure 4).

<table><tbody><tr><td>Antimold additive</td><td>Carolina</td><td></td><td></td></tr><tr><td>Beaker</td><td>Fisher Scentific</td><td>S76100J</td><td>Beaker</td></tr><tr><td>Benchmark scientific digital hotplate</td><td>The Lab Depot</td><td>H3760H</td><td>Hot plate</td></tr><tr><td>Britta-filtered tap water to prepare grape agar and instant fly food</td><td>Any tap water filtration system</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; pistol or FlyNap</td><td>Carolina</td><td></td><td>Anesthetic to sort/count adult flies</td></tr><tr><td>Dissecting microscope/stereoscope</td><td>Amscope</td><td>SM-1TSZ-V203</td><td>Dissecting microscope</td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; culture vials and stoppers</td><td>Carolina</td><td>173120</td><td>Food vials for growing &lt;em&gt;Drosophila&lt;/em&gt;</td></tr><tr><td>Embryo collection mini cage</td><td>Genesee Scientific</td><td>59-105</td><td>chamber used to gather embryos</td></tr><tr><td>Erlenmeyer flask</td><td>Sigma-Aldrich</td><td>70980</td><td>Erlenmeyer flask</td></tr><tr><td>Formula 4-24 Instant &lt;em&gt;Drosophila&lt;/em&gt; Medium, Blue</td><td>Carolina</td><td></td><td>https://www.carolina.com/drosophila-fruit-fly-genetics/formula-4-24-drosophila-medium-blue/FAM_173210.pr</td></tr><tr><td>Grape agar</td><td>Genesee Scientific</td><td>47-102</td><td>Media used for agar plates</td></tr><tr><td>Instant fly flood</td><td>Carolina</td><td>173210</td><td>Blue media for food vials</td></tr><tr><td colspan="4">Large Petri dish or any clear container to be used as a humid chamber by placing wet paper towels to keep the grape-agar plates moist during incubation/pre and post microscopic observation</td></tr><tr><td>Metal spatula (small)</td><td>Carolina</td><td></td><td></td></tr><tr><td>Microwave</td><td>Any</td><td></td><td>To cook grape agar</td></tr><tr><td>Petri dish</td><td>Kord-Valmark</td><td>2901</td><td>Petri dish for grape agar</td></tr><tr><td>Stir bar</td><td>The Lab Depot</td><td>58948-981-EA</td><td>Magnetic stir bar</td></tr></tbody></table>

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;

This method accurately and reproducibly allows one to gauge viability from embryos to emerged adults when coupled to chi square analyses.
In initial studies, after counting embryos and larvae, the grape agar was placed inside the vial upright on the side of the vial. Unfortunately, when the agar was placed on the side of the vial many of the embryos and larvae did not mature to adults. This was likely due to the grape agar disc drying out (Figure 3). This placement introduced an environmental variable (hydration of agar disc) that could confound the results. Approximately 39% of the progeny was lost between embryos to adult as only 61% of adults emerged. The biggest losses occurred between the L1 larvae (counted on surface of grape agar plates) and pupae (counted on walls of food vials) counts. However, when the grape agar was placed faced down inside the vial in direct contact with the food surface, less than 6% of the progeny was lost (Figure 2). The agar remained hydrated as the entire grape agar disc remained in contact with the moisture of the instant food inside the vial (Figure 7, Figure 8). These results indicate that the position of the agar inside the vial is important for obtaining reliable data and minimize contribution of environmental variables. Further data to support the effectiveness of the method was collected by comparing wild-type progeny used in initial method validation and progeny produced by crossing virgin wild-type females to males from a balanced Dm ime4 mutant stock (Supplemental Figure 2, ime4&#916;null/TM3sb). Approximately 91% of the progeny matured to adulthood compared to 94% of the progeny from wildtype (Figure 4).&#160; This difference was not statistically significant.
Homozygous Dm ime4 mutant males5 were also used to provide further validation of the method. However, the homozygous mutants were too sick to reproduce and died before embryos were deposited. Therefore, one limitation of the experiment is that males need to be healthy enough to reproduce to generate a starting embryo population to track.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59996/59996fig1.jpg" /> 
Figure 1: Dm ime4 mutant stocks emerge at sub-Mendelian levels. Stocks in which portions of the catalytic domain or Ado-Met binding domain were mutated are shown (refer to Supplemental Figure 1 for details). Some stocks had portions of either domain replaced with Ala (alanine-scanning mutagenesis), while other stocks had either or both domains deleted. The ratio of observed number of homozygotes to the expected numbers is represented in percentages (compared to heterozygous sibling controls, refer to Supplemental Figure 3 for sorting scheme and chi square analysis). Chi square analyses were performed to determine if the difference between observed and expected was statistically significant and not due to chance alone (p-values &#60; 0.01). Error bars represent standard error of the mean (minimum of three trials per cross indicated).&#160; A spreadsheet with all the data can be found in Supplemental Materials. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig1large.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/59996/59996fig2.jpg" /> 
Figure 2: Agar position inside the vial can introduce unintended environmental variables. Histogram of the number of individuals at different stages of development counted from grape agar disc embryo side down in food vial or agar on the side of the food vial. Placing the agar disc embryo-side down in the vial resulted in 94% (SD &#177; 0.02) of initial embryos surviving until adulthood. Placing the agar on the side of the food vial resulted in only 61% (SD &#177; 0.07) of the originally counted embryos reaching adulthood. A spreadsheet with all the data can be found in the Supplemental Materials. The expected numbers at each stage is set as the initial number of embryos/L1 counted on the grape agar disc prior to transfer to food vial (Null hypothesis: 100% of hatched embryos develop). The observed numbers are the actual number of individuals counted for the developmental stage indicated. Thus, each stage is compared to the initial number of hatched embryos counted on the grape agar plate. Chi square analyses were performed to determine if the difference between observed and expected was statistically significant and not due to chance alone using a p-value threshold of 0.05. The difference was significant for embryos grown with agar on the side of the food vial, but not for those grown in a disc embryo side down. Errors bars represent standard error (minimum of three trials per cross indicated). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59996/59996fig3.jpg" /> 
Figure 3: Cartoon showing two ways grape agar discs carrying embryos/L1s can be placed inside the food vial. &#160;There was a significant difference between the total number of embryos and adult progeny based on the position of the agar in the food vial. The difference stems from an environmental variable (hydration) created by the difference in disc placement inside the food vial:&#160; there is a statistically significant difference between expected and observed numbers in the larvae and pupae counts when the agar disc was placed on the side of the vial versus no significant difference in expected versus observed numbers when the agar disc was in contact with the food. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59996/59996fig4.jpg" /> 
Figure 4: Histogram showing the number of individuals at different stages of development from wild-type males versus males from a Dm ime4 mutant balanced stock. Balanced males crossed to virgin wild-type females as described in 2.1.&#160; Agar was transferred embryo-side down into food vials.&#160; The average ratio of adults emerging from the original embryos counted in grape agar discs from wild-type X wild-type was 94%, SD &#177; 0.02, whilst that ratio for Dm ime4 mutant/+ X wild-type was 91%, SD &#177; 0.01. Statistical analysis was performed as described for Figure 2; differences were not significant for either group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59996/59996fig5.jpg" /> 
Figure 5: Experimental setup. (A) Embryo collection mini-cages and grape-agar mini-Petri dishes. Plates are placed inside a humid chamber (standard Petri dish with absorbent paper disc saturated with water) to prevent dehydration.&#160; A small amount of yeast paste was placed at the center of each grape-agar plate.&#160; (B) Detach the embryo collection plate holder lid (red) and place a prepared plate, yeast side up.&#160; (C) Transfer the cross to the embryo cage and immediately place a Petri dish lid to hold flies inside.&#160; (D) Quickly remove cover and flip the embryo cage so opening contacts grape-agar plate.&#160; Incubate for 24-48 h, inspecting daily. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59996/59996fig6.jpg" /> 
Figure 6: Embryo development on grape-agar plates. (A) Wildtype embryos deposited on the grape-agar mini-Petri dishes are counted under a dissecting microscope. Keep plates inside humid chamber when not visualizing and avoid direct light to prevent dehydration.&#160; (B) Grape-agar plates are kept in a humid chamber and hatched wildtype embryos are recorded.&#160; This photo shows three wildtype L1 larvae that developed from embryos shown in A.&#160; (C) Example of recorded numbers of embryos (day 1) and L1 larvae (day 2) for the two plates shown in Figure 5.&#160; The &#34;expected number&#34;&#160;for the null hypothesis is determined by the number of embryos that hatched and became L1 larvae at the time of transfer to the food vial (table below).&#160; For wildtype flies, almost all embryos hatch and develop into L1 larvae.&#160; This may not be the case for other genetic backgrounds and this method is used to determine these differences in viability. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59996/59996fig7.jpg" /> 
Figure 7: Transferring larvae.&#160; (A) After recording L1 counts, the grape-agar discs are carefully removed from the Petri dish using a clean (ethanol-wiped) spatula (B,C) and transferred L1 side-down to contact the food in the vials as shown in D.&#160; (E)&#160; The empty Petri dish is carefully inspected to detect any larvae left behind.&#160; If larvaee left behind are alive and can be transferred to the food vial, do so carefully and immediately (F).&#160; If larvae left behind are dead or cannot be transferred, record this fact to correct the &#34;expected number&#34;&#160;for subsequent calculations. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/59996/59996fig8.jpg" /> 
Figure 8: L2 larvae make their way into the food. See crevices and grooves between the grape-agar disc and the blue food.&#160; Set up a schedule to observe the vials daily, preferably at the same time of day. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/59996/59996fig9.jpg" /> 
Figure 9: Adult emergence. (A)&#160; L3 larvae make their way out of the food to become pupae. &#160;(B) Adults start to emerge on day 11.&#160; Set up a schedule to observe the vials daily, preferably at the same time of day and carefully record your observations.&#160; Stop counting when all the pupae have emerged (count empty pupae cases) and avoid counting the next generation of flies by stopping the experiment on day 15 and count dead pupae if any (black/dried pupae).&#160; Mutants with lengthier life cycles may need longer periods of time, which need to be determined empirically. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figures.&#160;&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/Supplemental_Figures.zip" target="_blank">Please click here to download these files.</a>
Supplemental Materials.&#160;&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/Supplemental_Materials.zip" target="_blank">Please click here to download these files.</a>

Watch this Scientific Journal Video about A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult at JoVE.com

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.

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 ...

a-direct-simple-method-to-assess-drosophila-melanogaster-s-viability

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.3K Views.  Clarkson University. Het belangrijkste voordeel van deze test is de eenvoud en de nauwkeurigheid, omdat het niet vereist dat een embryo spoelt om over te brengen naar het voedsel flesje en dat voorkomt verliezen door technische fouten. Dit protocol vereist geen deskundige technische vaardigheden. Echter, de juiste timing en zorgvuldige wateroverdracht zijn belangrijk voor de nauwkeurigheid en reproduceerbaarheid.Het aantonen van deze procedure zal Antonio Rockwell, een onlangs afgestudeerde promovendus van...

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

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as self-fertilizing hermaphrodites, which produce large broods of progeny-when males are present, they can produce even larger broods of cross progeny. Errors in meiosis, fertilization, and embryogenesis can be rapidly assessed as phenotypes of sterility, reduced fertility, or embryonic lethality. This article describes a method to determine embryonic viability and brood size in C. elegans. We demonstrate how to set up this assay by picking a worm onto an individual Modified Youngren's, Only Bacto-peptone (MYOB) plate, establish the appropriate timeframe to count viable progeny and non-viable embryos, and explain how to accurately count live worm specimens. This technique can be used to determine viability in self-fertilizing hermaphrodites as well as cross-fertilization by mating pairs. These relatively simple experiments are easily adoptable for new researchers, such as undergraduate students and first-year graduate students.

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Here, we present a general method to determine the embryonic viability and total number of embryos produced (brood) using the model organism C. elegans.

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as ...

Genetics

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 ...

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 ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Biology

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Assessing Differences in Sperm Competitive Ability in Drosophila

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1. Sperm competition represents the competition between males after copulating with the same female 2, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males 4. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5.
Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9, which is assumed to be reflective of different competence attributes.
Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.

Assessing Differences in Sperm Competitive Ability in Drosophila

Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male.

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the ...

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 ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

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 ...

Drosophila melanogaster Embryo and Larva Harvesting and Preparation

Drosophila melanogaster embryos and larvae are easy to manipulate and develop rapidly by mechanisms that are analogous to other organisms, including mammals. For these reasons, many researchers utilize fly embryos and larvae to answer questions in diverse fields ranging from behavioral to developmental biology. Prior to experimentation, however, the embryos and larvae must first be collected.
This video will first demonstrate how "egg-laying cups" are used to collect Drosophila embryos on agar plates. The harvest and dechorionation of embryos will then be described. Next, the video will demonstrate how to identify and manipulate Drosophila in one of the three larval stages that follow the embryo stage. Finally, examples of some of the ways in which fly embryos and larvae are used in biological research are provided.

Drosophila melanogaster Embryo and Larva Harvesting and Preparation

Drosophila melanogaster embryos and larvae are easy to manipulate and develop rapidly by mechanisms that are analogous to other organisms, including ...

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

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