Karen M. Qiang was the 2016 recipient of the George J. Schroepfer Research Award at Rice University. Fanli Zhou is the recipient of a Teaching Fellowship from Rice University. The services of the Bloomington Drosophila Stock Center, the Harvard TRiP facility, the Vienna Drosophila Resource Center are gratefully acknowledged.

1. Preparation of larvae
<ol>
	<li>Two or more days before beginning the assay, set up crosses of the desired experimental and control combinations (minimum 20 females and 10 males each) in egg-lay dishes as described by Wieschaus and Nusslein-Volhard 13 but using 50 mL polypropylene beakers and 6.0 cm disposable Petri dishes containing grape agar and smeared with yeast paste just before use. Keep flies in egg-lay dishes in darkness at room temperature until needed.</li>
	<li>Grape agar recipe &#8211; combine 100 mL frozen 100% grape juice concentrate, 350 mL deioinized water, 5 mL glacial acetic and 13 g D. melanogaster agar in a 1L glass beaker. Microwave in 1-2 min bursts, stirring between bursts, until agar is all dissolved. Cool the solution for a few minutes then add 10 mL of 10% (w/v) of Nipagen (methyl-p-hydroxybenzoate) in 95% ethanol. Mix, then pipette 7 mL per plate into disposable 6.0 cm Petri dishes, allow to gel and dry at room temperature for 3-4 h. Store at 4 0C in plastic bags.</li>
	<li>Yeast paste recipe &#8211; 7 g dried yeast, 10 mL deionized water, mix with a spatula until uniform consistency.</li>
	<li>Timed egg collections to generate experimental and control larvae. Using new, yeast-smeared, grape plates, collect eggs at room temperature for 4 h in darkness during the morning hours. Remove these 4 h collection plates and replace with fresh plates. Incubate the 4 h collection plates overnight at 25 0C until the afternoon of the next day, when most of the larvae will have hatched. Collect these first instar larvae and use them to set up the experiment.</li>
</ol>
2. Setting up assay plates
<ol>
	<li>Preparation of assay plates. For a single run of the assay, prepare five assay plates each of 10 experimental larvae and five plates of 10 control larvae. Use a 1.5 cm cork borer to remove a central core of agar from each plate creating a hole for the food. Put yeast paste (0.8 - 0.9 g) into the hole and pat gently with a spatula to make a mound filling the hole.</li>
	<li>Agar plate preparation &#8211; combine 700 mL deionized water with 16 g D. melanogaster agar in a 2L glass beaker and microwave for 5 min. Remove from microwave and stir with a glass rod to bring undissolved agar into the solution. Add 17.5 mL of 10% (w/v) Nipagen in 95% ethanol, mix, then continue microwave heating in 30 s bursts followed by stirring, until all agar is completely dissolved. Cool for a minute or two, then pipette 15 mL aliquots into 10 cm plastic Petri dishes. Allow agar to gel, cover plates with lids and let them dry at room temperature for a few hours or overnight. Store plates in sealed plastic bags at 18 &#176;C. 
	Note 1: To avoid formation of Nipagen crystals on the surface of the plates due to excess dessiccation, use plates within a few days of preparation. If necessary, crystals can be re-dissolved by dropping a few microliters of 95% alcohol onto them. 
	Note 2: batches of agar can have different gelling properties. The dry gel plates should be firm to the touch and sufficiently resilient that a clean, intact circle of agar can be removed when using the cork borer to create the food hole (see above).&#160;</li>
	<li>Setting up larvae in assay plates. Use mechanical pressure to curve the tip of a plastic microspatula and dip the tip in yeast paste to provide &#8220;adhesive&#8221; for picking up larvae. Pick up first instar larvae individually and place them on the agar plate close to the food mound. Prepare at least five replicate plates of 10 larvae for both experimental and control genotypes. Once completed, cap and label each plate and set the plates (lid side up) in a dark space at room temperature (in our lab this is 22 &#176;C).</li>
</ol>
3. Monitoring assay plates
<ol>
	<li>Examine plates daily under a dissecting microscope and record the number of larvae on top of the food or out on the agar surface. Note any visible dead or dying larvae. Note when, and if, tunneling into the agar substratum is seen as larvae move into third instar. Note dates on which pupae begin to form and note any pupal abnormalities. Continue daily observations until all larvae have died or pupated.</li>
	<li>Remove pupae carefully from the plate with a bent teasing needle and transfer to a grape plate. If desired, continue monitoring pupae to determine how many eclose as adults.</li>
</ol>
4. Preparing assay plates for imaging
<ol>
	<li>Carefully remove and discard all yeast food from the central well of each plate.</li>
	<li>Gently flood each plate with tap water and use a soft artist&#8217;s paint brush to carefully rub off debris that has been tracked onto the agar surface. Replace the water several times and continue gentle brushing until each plate is completely clean. Give plates a final rinse with deionized water, cap them and leave at room temperature overnight to dry. 
	Note: Tunneling into the agar is most intense around the food mound (see Figure 2). As a result, the rim of the food hole is usually expanded beyond the initial 1.5 cm diameter. In addition, the fragility of the worked agar in this region may cause small pieces of tunneled agar to be lost from the circumference of the hole during cleaning. Increasing the gel agar concentration could help with these problems, but corrections are possible during the Image J quantitation steps (see below).</li>
</ol>
5. Quantitation of tunneling
<ol>
	<li>Image the plates. Prepare an image of each plate taken against a black background so as to show the tunnels in the agar as bright white lines. 
	NOTE: We use a system commonly used to image DNA gels for this step (see Table of Materials and Reagents). Alternative imaging systems capable of generating tiff files, such as digital cameras or cell phone cameras could be adjusted to produce suitable images.</li>
	<li>Use the public domain program NIH Image J to quantitate larval tunneling. Download the program from http://rsb.info.nih.gov/ij/download.html. For more information about the program go to http://imagej.nih.gov/ij/docs/intro.html. 
	NOTE: Other imaging systems could be used to quantitate tunneling. The instructions below apply to NIH Image J.</li>
	<li>After opening a tiff file image of a tunneling plate, use the Oval tool to define an area for quantitation that represents the entire agar surface but crops out the plastic edge of the plate.</li>
	<li>Apply the automatic threshold to produce a reverse image of the plate, with the tunnels showing as black lines. Use the Color picker and Paintbrush tools to white out any black areas that represent damage to the agar rather than tunnels.</li>
	<li>Under the Analyze pull-down menu, select Set scale | Click to remove scale and then on the Set measurements window (also under Analyze), check Area and Limit to threshold.</li>
	<li>Press Cntrl key + M key to show the black area (tunnels) in pixels.</li>
	<li>Copy and paste the pixel value for each plate is into a spreadsheet program (such as Excel) that will permit quantitative analysis.</li>
	<li>The central hole is usually expanded due to intense tunneling in this region. To quantitate the missing tunneling, uncheck &#8220;Limit to threshold&#8221; and use the Wand tool to define the central hole. Press Cntrl key + M key to obtain the pixel value of this area. Subtract from this value the pixel value of a 1.5 cm hole and add the difference to the tunneling value obtained in step 5.6.</li>
	<li>For some plates, the central hole may be missing a piece of tunneled agar that includes a region of untunneled agar adjacent to the hole. When quantitating the central hole for these plates use the Color picker and Paint brush tools to add a black bar across the missing region to define the tunneled region and exclude the non-tunneled area from quantitation.&#160; 
	Note 1: If the experimental larvae perform any tunneling, this quantitation permits calculation of average tunneling values for sets of plates and statistical comparison of control and experimental values.</li>
</ol>

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H., Hafen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hypoxia-induced+paralogs+Scylla+and+Charybdis+inhibit+growth+by+down-regulating+S6K+activity+upstream+of+Tsc+in+D.+melanogaster.">The hypoxia-induced paralogs Scylla and Charybdis inhibit growth by down-regulating S6K activity upstream of Tsc in D. melanogaster.</a> Genes Dev. 18 (23), 2879-2892 (2004).</li><li>Gorr, T. A., Gassmann, M., Wappner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+and+responding+to+hypoxia+via+Hif+in+model+invertebrates.">Sensing and responding to hypoxia via Hif in model invertebrates.</a> J. Insect Physiol. 52 (4), 349-364 (2006).</li><li>Romero, N. 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The authors declare they have no competing financial interests.

We have presented here a simple assay designed to detect tissue hypoxia in D. melanogaster larvae. Diagnosis is based on decreased burrowing into food mounds in early larval life and the absence of substratum tunneling late in larval life. Larval crowding can cause premature migration away from a food source and thus one critical aspect of the assay is that a small number of larvae are assayed in the presence of a large excess food. Inclusion of the fungicide methyl-p hydroxyl benzoate (Nipagen) into the agar plates is also essential to prevent mold growth during the assay.
We find that the agar plates can be a source of variability in the assay. Typically, larvae of the same genotype, from different batches of parents, or from different collections of larvae, show relatively limited variation in their behavior in the assay. In contrast, agar plates made on different days or with different batches of agar can yield differences in tunneling. One stipulation therefore is that control and experimental larvae should all be tested using agar plates from the same batch preparation. Agar from different manufacturers or even shipments from the same manufacture can vary in their gelling strength, and so it may be necessary to the adjust the agar concentration upwards from the 2.2% used here to achieve an optimal gel. We have found that wild type larvae can readily burrow through 3% agar gels.
In order to demonstrate the value of this assay, we have used it to investigate potential tissue hypoxia in larvae with suppressed function of uninflatable in the tracheae. Our findings provide strong support for the hypothesis that loss of tracheal expression of this gene can produce hypoxia: btl-Gal4 &#62;UAS-uif RNAi larvae showed failure to burrow into food and complete absence of substratum tunneling during the third instar. In our prior studies of other genotypes, we observed that hypoxia-induced loss of food burrowing is not as complete as loss of substratum tunneling and the btl-Gal4&#62;UAS-uif RNAi larvae studied here behaved similarly. The failed tunneling component of this assay therefore provides the strongest indication of hypoxia.
Although the btl-Gal4&#62;uif RNAi larvae showed behavioral traits diagnostic of hypoxia, the cut(ue)-Gal4&#62;UAS-uif RNAi did not exhibit these abnormalities. The btl and cut(ue) Gal4 drivers are expressed at different stages, and in different patterns, within the larval tracheae. The btl-Gal4 driver is expressed throughout the tracheal system starting from its development in embryogenesis and continuing through larval life. In contrast, Gal4 expression from the cut(ue)-Gal4 driver only begins at the end of embryonic life, after morphogenesis of the tracheae, and is limited to the extreme posterior sections of the dorsal trunks, the major longitudinal vessels of the tracheal system. uif knockdown with this Gal4 line may not therefore reduce uif expression early enough or broadly enough to produce some threshold level of hypoxia required to trigger the behaviors measured in this assay.
A prior study found that third instar larvae exposed to low (10%) oxygen levels show decreased growth and delayed onset of pupariation 14. The btl-Gal4&#62;UAS-uif RNAi larvae studied here progressed to the third instar but the effects on their growth and pupation rates were more pronounced: they were considerably smaller than controls with very little fatty tissue under the epidermis (Figure 3) and only a minor fraction (~10 %) attempted pupariation. These differences suggest the btl-Gal4&#62;UAS-uif RNAi larvae experienced a greater degree of hypoxia, either because uif knockdown in the tracheae was present throughout their entire larval lives, or because it produced a more severe oxygen deprivation in third instar. How loss of uif function in the tracheae might prevent oxygen transport is unclear at this point. The tracheae of the btl-Gal4&#62; UAS-uif RNAi larvae were readily visible through the cuticle (Figure 3), an indication that they contained air and were not damaged to the point that fluid entry compromised function. It is formally possible therefore that the tracheal damage created by loss of uif function does not elicit hypoxia but rather some other defect that inhibits tunneling. For the genotypes studied previously, we determined that failure to tunnel is associated with elevated levels of LDH mRNA7, the canonical indicator of glycolysis and hypoxia in late third instar larvae 15. Thus, final confirmation of hypoxia for the btl-Gal4&#62;UAS-uif RNAi larvae (and in larvae examined in future use of this assay) would involve RT-PCR to assess LDH mRNA levels or use of a commercially available indicator to measure intracellular oxygen levels (for example see 16).

The sophisticated array of molecular genetic tools available in D. melanogaster make it a valuable organism for the study of evolutionarily conserved biological processes. Key molecular responses to oxygen availability have proved to be conserved across evolution and prior studies in D. melanogaster have generated insights into the universal components of these signaling pathways 1,2,3,4,5,6.
As part of a study aimed at dissecting sensory neuron function in D. melanogaster larvae, we identified two behavioral responses that proved to be activated by tissue hypoxia at normal oxygen levels 7. One of these, failure to burrow into food, is highly related to the response to low oxygen levels reported by Wingrove and O&#39;Farrell 8. The second behavior, failure to tunnel into a soft substratum during the late third instar wandering phase, had not been previously identified as hypoxia-related. We determined that exposing wild type wandering larvae to low oxygen levels also inhibits substratum tunneling 7, thus establishing that both these behaviors originate from hypoxia - either induced by tissue damage or by low oxygen intake levels. Here we describe an assay we have developed to quantitate these two hypoxia-induced behaviors, which starts with observations immediately after larval hatching.
Hypoxic responses in the early larval stages have not been examined previously and therefore performing an analysis throughout larval life is a valuable component of our assay. Most of the obvious manifestations of hypoxia &#8211; slow development, poor growth, and locomotor sluggishness &#8211; overlap with larval phenotypes produced by many mutations. But we have found that only third instar larvae with hypoxia show a complete failure to tunnel 7. Thus, we determined that even larvae more compromised in terms of growth and locomotion than our hypoxic larvae, still performed some tunneling, whereas hypoxic larvae never tunneled 7. Another valuable element of this assay is thus that it provides a way to establish when hypoxia is the source of a particular set of pleiotropic phenotypes, as opposed to some other stress or metabolic malfunction. As a demonstration of the assay, here we describe its use in characterizing the responses of larvae with reduced tracheal expression of uninflatable, a gene that functions in the larval airways 9.
We envisage that this assay will be of value to researchers engaged in characterizing larval phenotypes that include poor growth and sluggish behavior. As a result, new genes that influence the distribution, usage, or responses to, oxygen throughout the body could be identified. Further, incorporating this assay into a mutant screening protocol would provide a direct route to identifying mutations that produce hypoxia. This assay will also be valuable in analyzing the circuitry that elicits the hypoxia-induced innate behaviors described here. Neural network analysis of this type is a focus of much current research and the simple nervous system of the D. melanogaster larva is a valuable system for dissecting out automated behaviors. Sensory neurons involved in larval oxygen perception have already been identified, providing a first step towards defining the complete circuitry for hypoxia-induced responses 10,11. Using our assay in combination with selective neural knockdown via the GAL4-UAS system12 is a clear route for delineating further components of the neural network.

<table><tbody><tr><td>&lt;strong&gt;REAGENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dehydrated yeast&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Frozen grape juice concentrate</td><td>Welch&#039;s</td><td></td><td>&amp;nbsp;Available at most large supermarkets</td></tr><tr><td>Glacial acetic acid</td><td>Sigma-Aldrich</td><td>320099</td><td></td></tr><tr><td>Drosophila agar</td><td>Apex Bioresearch Products</td><td>66-103</td><td></td></tr><tr><td>Methyl-para-hydroxybenzoate</td><td>Apex Bioresearch Products</td><td>20-658</td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50 ml polypropylene beakers</td><td></td><td></td><td></td></tr><tr><td>6.0 cm disposable Petri dishes</td><td>Falcon</td><td>08757100B</td><td></td></tr><tr><td>10 cm disposable plastic Petri dishes</td><td>E+K Scientific</td><td>EK-24104</td><td></td></tr><tr><td>Plastic microspatulas</td><td>Corning Incorporated</td><td>3012</td><td></td></tr><tr><td>Bent teasing needle</td><td>Nasco</td><td>S08848MH</td><td></td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td>Any microscope with 10-30X magnification</td></tr></tbody></table>

a burrowing/tunneling assay for detection of hypoxia in drosophila melanogaster larvae

Oxygen deprivation in animals can result from exposure to low atmospheric oxygen levels or from internal tissue damage that interferes with oxygen distribution. It is also possible that aberrant behavior of oxygen-sensing neurons could induce hypoxia-like behavior in the presence of normal oxygen levels. In D. melanogaster, development at low oxygen levels results in inhibition of growth and sluggish behavior during the larval phases. However, these established manifestations of oxygen deficit overlap considerably with the phenotypes of many mutations that regulate growth, stress responses or locomotion. As result, there is currently no assay available to identify i) cellular hypoxia induced by a mutation or ii) hypoxia-like behavior when induced by abnormal neuronal behavior.
We have recently identified two distinctive behaviors in D. melanogaster larvae that occur at normal oxygen levels in response to internal detection of hypoxia. First, at all stages, such larvae avoid burrowing into food, often straying far away from a food source. Second, tunneling into a soft substratum, which normally occurs during the wandering third instar stage is completely abolished if larvae are hypoxic. The assay described here is designed to detect and quantitate these behaviors and thus to provide a way to detect hypoxia induced by internal damage rather than low external oxygen. Assay plates with an agar substratum and a central plug of yeast paste are used to support animals through larval life. The positions and state of the larvae are tracked daily as they proceed from first to third instar. The extent of tunneling into the agar substratum during wandering phase is quantitated after pupation using NIH ImageJ. The assay will be of value in determining when hypoxia is a component of a mutant phenotype and thus provide insight into possible sites of action of the gene in question.

As a demonstration of the value of this assay we have used it to investigate potential hypoxia in larvae with impaired function of the gene uninflatable (uif) in the tracheae. uif encodes a large transmembrane protein that is strongly expressed on the apical surface of the larval tracheal cells. Mutants of uif were previously noted to exhibit aberrant behavior that could indicate tissue hypoxia due to tracheal malfunction 9. We specifically suppressed uif expression in the larval trachea using the Gal4-UAS system. Two Gal4 lines were used: i) breathless (btl)-Gal4, which is expressed strongly in the tracheae from the beginning of their embryonic development and ii) cut(ue)-Gal4, which begins expression in a small posterior region of the main dorsal trunk tracheae at the end of embryogenesis and continues strong expression in this region throughout larval life 7. A UAS-uif RNAi line was obtained from the Vienna Drosophila Research Center (VDRC ID #1050).
As described in the protocol above, five replicate plates of newly hatched first instar larvae were set up for each of four crosses as follows.
Experiment 1 
1) Control 1 - cut(ue)-Gal4 x Canton-S (+) 
2) Experimental 1 - cut(ue)-Gal4 x UAS-uif RNAi
Experiment 2 
3) Control 2 - btl-Gal4 x Canton-S (+) 
4) Experimental 2 - btl-Gal4 x UAS-uif RNAi
The behavior of the cut(ue)-Gal4&#62;UAS-uif RNAi larvae of Experiment 1 was comparable to that of the control cut(ue)-Gal4&#62; + animals in terms of burrowing, tunneling and survival to pupation, (Figure 1 and Figure 2). In contrast, down-regulating uif expression throughout the entire tracheal system from early in development (Experiment 2) produced markedly different responses. The btl-Gal4&#62;UAS-uif RNAi larvae displayed both behavioral manifestations of tissue hypoxia &#8211; reduced food burrowing and a total absence of substratum tunneling later in larval life (Figure 1 and Figure 2). The experimental larvae were also noticeably smaller, thinner and more sluggish than controls (Figure 3), and showed a high death rate during the course of the assay. As discussed above, reduced growth, sluggishness and organismal death are known symptoms of larval hypoxia. In addition, most of the experimental larvae failed to attempt pupation and remained as third instar larvae long after control larvae had pupated. Some larvae survived more than 15 days before eventually dying without pupating (Figure 1A). A few of the experimental larvae tried to pupate but in all cases abnormal pupae were formed that did not generate viable adults.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57131/57131fig1.jpg" /> 
Figure 1. Quantitation of food burrowing and larval development. 
(A) Percentage of live larvae outside the food mounds for the four genotypes studied here. Averages for the five assay plates used for each genotype are shown. By day 3 after hatching, ~70% of the btl-Gal4&#62;UAS uif RNAi larvae were outside the food, whereas no larvae were detected outside the food or on the agar surface for the other three genotypes. The btl-Gal4&#62;UAS uif RNAi larvae die slowly between days 4-20, with ~15% of them still alive 15 days after hatching. The black arrow along the x axis here and in (B) indicates the day by which all larvae of the three other genotypes had pupated. 
(B) Percentage of dead larvae visible outside the food for the four genotypes. Averages for the five assay plates for each genotype are shown. Dead btl-Gal4&#62;UAS uif RNAi larvae accumulate over time with most dying outside the food. The other genotypes all survive to pupation 
(C) Percentage survival to pupation for the four genotypes studied. Averages for the five assay plates for each genotype are shown. No normal pupae of the btl-Gal4&#62;UAS uif RNAi genotype were produced. Error bars throughout figure represent SEMs. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57131/57131fig1large.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/57131/57131fig2.jpg" /> 
Figure 2. Quantitation of larval tunneling. 
(A) Examples of the assay plates for all four genotypes studied after preparation for tunneling quantitation. Note complete absence of tunneling for the btl-Gal4&#62;UAS uif RNAi larvae. Assay plates are 10 cm Petri plates. 
(B) Tunneling quantitation derived from Image J for the four genotypes. Average pixel values for the five assay plates for each genotype. No tunneling was observed for the btl-Gal4&#62;UAS uif RNAi larvae. Error bars = SEMs. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57131/57131fig2large.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/57131/57131fig3.jpg" /> 
Figure 3. Comparison of btl-Gal4&#62;UAS uif RNAi and btl-Gal4&#62; + larvae 
Larvae of similar age (day 6 after hatching) for the btl-Gal4&#62;UAS uif RNAi (A) and btl-Gal4&#62; + (B) genotypes. Red arrows point to the dorsal trunk tracheae. Both larvae imaged at the same magnification. Scale bar = 0.5 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57131/57131fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae at JoVE.com

A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae

The protocol describes a simple assay to identify Drosophila melanogaster larvae that are experiencing hypoxia under normal atmospheric oxygen levels. This protocol allows hypoxic larvae to be distinguished from other mutants that show overlapping phenotypes such as sluggishness or slow growth.

A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae

Oxygen deprivation in animals can result from exposure to low atmospheric oxygen levels or from internal tissue damage that interferes with oxygen ...

a-burrowingtunneling-assay-for-detection-hypoxia-drosophila

Nanyang Technological University

Research

JoVE Journal

Behavior

7.8K Views.  Rice University. Het protocol beschrijft een eenvoudige test om te identificeren van Drosophila melanogaster larven die hypoxie onder normale atmosferische zuurstofniveaus ondervindt. Dit protocol staat hypoxische larven te onderscheiden van andere mutanten die aantonen dat overlappende fenotypen zoals traagheid of trage groei.

Video: A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Developmental Biology

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Neuroscience

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

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 ...

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

We provide protocols for anyone with a "maker culture" mind to start building a flylab for quantitative analysis of a myriad of behavioral parameters in Drosophila melanogaster, by 3D-printing many of the necessary pieces of equipment. We also describe a high resolution respirometry protocol using larvae to combine behavioral and mitochondrial metabolism data.

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

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

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

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

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

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Biology

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model to study animal avoidance behavior. This protocol introduces a light-spot assay to investigate larval phototactic behavior. The experimental set-up includes two main parts: a visual stimulation system that generates the light spot, and an infrared light-based imaging system that records the process of larval light avoidance. This assay allows tracking of the behavior of larva before entering, during encountering, and after leaving the light spot. Details of larval movement including deceleration, pause, head casting, and turning can be captured and analyzed using this method.

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging system.

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

Drosophila Burrowing and Tunneling Assay: A Method to Assess Tissue Hypoxia in Fly Larvae

Source: Qiang, K. M., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57131/" target="_blank">A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae</a>. J. Vis. Exp. (2018).
This video shows a method to test hypoxia in Drosophila larvae by assessing their burrowing and tunneling abilities. Using this technique researchers can distinguish larvae experiencing oxygen deprivation from those with developmental deficits. The example protocol demonstrates the setup for this assay.

Source: Qiang, K. M., et al. A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae. J. Vis. Exp. (2018).
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A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae

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