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>

<ol>
	<li>O'Farrell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+responses+to+oxygen+deprivation.">Conserved responses to oxygen deprivation.</a> J. Clin. Invest. 107 (6), 671-673 (2001).</li><li>Lavista-Llanos, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+the+hypoxic+response+in+D.+melanogaster+by+the+basic+helix-loop-helix+pas+protein+Similar.">Control of the hypoxic response in D. melanogaster by the basic helix-loop-helix pas protein Similar.</a> Mol. Cell. Biol. 22 (19), 6842-6853 (2002).</li><li>Reiling, J. 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. M., Dekanty, A., 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=Cellular+and+developmental+adaptations+to+hypoxia:+A+D.+melanogaster+perspective.">Cellular and developmental adaptations to hypoxia: A D. melanogaster perspective.</a> Meth. Enzymol. 435, 123-144 (2007).</li><li>Dijkers, P. F., O'Farrell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+a+hypoxia-induced,+nitric+oxide-mediated+signaling+cascade.">Dissection of a hypoxia-induced, nitric oxide-mediated signaling cascade.</a> Mol. Biol.Cell. 20 (18), 4083-4090 (2009).</li><li>Zhou, F., Qiang, K. M., Beckingham, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+to+burrow+and+tunnel+reveals+roles+for+jim+lovell+in+the+growth+and+endoreplication+of+the+D.+melanogaster+larval+tracheae.">Failure to burrow and tunnel reveals roles for jim lovell in the growth and endoreplication of the D. melanogaster larval tracheae.</a> PLOS ONE. 11, e0160233 (2016).</li><li>Wingrove, J. A., O'Farrell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide+contributes+to+behavioral,+cellular,+and+developmental+responses+to+low+oxygen+in+D.+melanogaster.">Nitric oxide contributes to behavioral, cellular, and developmental responses to low oxygen in D. melanogaster.</a> Cell. 98 (1), 105-114 (1999).</li><li>Zhang, L., Ward, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uninflatable+encodes+a+novel+ectodermal+apical+surface+protein+required+for+tracheal+inflation+in+D.+melanogaster.">Uninflatable encodes a novel ectodermal apical surface protein required for tracheal inflation in D. melanogaster.</a> Dev. Biol. 336 (2), 201-212 (2009).</li><li>Langlais, K. K., Stewart, J. A., Morton, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+characterization+of+two+atypical+soluble+guanylyl+cyclases+in+the+central+and+peripheral+nervous+system+of+D.+melanogaster+melanogaster.">Preliminary characterization of two atypical soluble guanylyl cyclases in the central and peripheral nervous system of D. melanogaster melanogaster.</a> J. Expt. Biol. 207 (13), 2323-2338 (2004).</li><li>Morton, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+responses+to+hypoxia+and+hyperoxia+in+D.+melanogaster+larvae.">Behavioral responses to hypoxia and hyperoxia in D. melanogaster larvae.</a> Fly. 5 (2), 119-125 (2011).</li><li>Brand, A., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118 (2), 401-415 (1993).</li><li>Wieschaus, E., Nusslein-Volhard, C., Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+at+embryos.">Looking at embryos.</a> Drosophila: a practical approach. , 199-227 (1986).</li><li>Callier, V., Shingleton, A. W., Brent, C. S., Ghosh, S. M., Kim, J., Harrison, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+reduced+oxygen+in+the+developmental+physiology+of+growth+and+metamorphosis+initiation+in+D.+melanogaster.">The role of reduced oxygen in the developmental physiology of growth and metamorphosis initiation in D. melanogaster.</a> J. Expt. Biol. 216 (23), 4334-4340 (2013).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hif-+and+non-Hif-regulated+hypoxic+responses+require+the+estrogen-related+receptor+in+D.+melanogaster.">Hif- and non-Hif-regulated hypoxic responses require the estrogen-related receptor in D. melanogaster.</a> PLoS genetics. 9 (1), e1003230 (2013).</li><li>Grifoni, D., Sallazzo, M., Fontana, E., Froldi, F., Pession, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+strategies+of+oxygen+supply+in+Drosophila+malignancies+identify+tracheogenesis+as+a+novel+cancer+hallmark.">Multiple strategies of oxygen supply in Drosophila malignancies identify tracheogenesis as a novel cancer hallmark.</a> Scient. Rep. 5 (9061), (2015).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="890">
	
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">REAGENTS</td>
			<td style="width:223px;"></td>
			<td style="width:117px;"></td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dehydrated yeast&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Frozen grape juice concentrate</td>
			<td>Welch&#39;s</td>
			<td></td>
			<td>&#160;Available at most large supermarkets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glacial acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>320099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drosophila agar</td>
			<td>Apex Bioresearch Products</td>
			<td>66-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methyl-para-hydroxybenzoate</td>
			<td>Apex Bioresearch Products</td>
			<td>20-658</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml polypropylene beakers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6.0 cm disposable Petri dishes</td>
			<td>Falcon</td>
			<td>08757100B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 cm disposable plastic Petri dishes</td>
			<td>E+K Scientific</td>
			<td>EK-24104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic microspatulas</td>
			<td>Corning Incorporated</td>
			<td>3012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bent teasing needle</td>
			<td>Nasco</td>
			<td>S08848MH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ...

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7.7K Views.  Rice University. 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.

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

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate drug-seeking behavior and prompt relapse in abstinent drug users. We have developed a procedure to study the behavioral and neural processes that mediate the impact of context on alcohol-seeking behavior in rats. Following acclimation to the taste and pharmacological effects of 15% ethanol in the home cage, male Long-Evans rats receive Pavlovian discrimination training (PDT) in conditioning chambers. In each daily (Mon-Fri) PDT session, 16 trials each of two different 10 sec auditory conditioned stimuli occur. During one stimulus, the CS+, 0.2 ml of 15% ethanol is delivered into a fluid port for oral consumption. The second stimulus, the CS-, is not paired with ethanol. Across sessions, entries into the fluid port during the CS+ increase, whereas entries during the CS- stabilize at a lower level, indicating that a predictive association between the CS+ and ethanol is acquired. During PDT each chamber is equipped with a specific configuration of visual, olfactory and tactile contextual stimuli. Following PDT, extinction training is conducted in the same chamber that is now equipped with a different configuration of contextual stimuli. The CS+ and CS- are presented as before, but ethanol is withheld, which causes a gradual decline in port entries during the CS+. At test, rats are placed back into the PDT context and presented with the CS+ and CS- as before, but without ethanol. This manipulation triggers a robust and selective increase in the number of port entries made during the alcohol predictive CS+, with no change in responding during the CS-. This effect, referred to as context-induced renewal, illustrates the powerful capacity of contexts associated with alcohol consumption to stimulate alcohol-seeking behavior in response to Pavlovian alcohol cues.

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

Environmental contexts in which drugs of abuse are consumed can trigger craving, a subjective Pavlovian-conditioned response that can facilitate ...

The Social Dimension of Stress: Experimental Manipulations of Social Support and Social Identity in the Trier Social Stress Test

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations supportive behavior of other people as well as positive social relationships can act as powerful resources to cope with stress. In order to study the interplay between these variables, this protocol describes two effective experimental manipulations of social relationships and supportive behavior in the laboratory. In the present article, these two manipulations are implemented in the Trier Social Stress Test (TSST)&#8212;a standard stress induction paradigm in which participants are subjected to a simulated job interview. More precisely, we propose (a) a manipulation of the relationship between different protagonists in the TSST by making a shared social identity salient and (b) a manipulation of the behavior of the TSST-selection committee, which acts either supportively or unsupportively. These two experimental manipulations are designed in a modular fashion and can be applied independently of each other but can also be combined. Moreover, these two manipulations can also be integrated into other stress protocols and into other standardized social interactions such as trust games, negotiation tasks, or other group tasks.

Previous research on the social dimension of stress has focused on two important variables: social identity and social support. This protocol introduces an effective experimental manipulation of these two social variables and describes their implementation in a standard stress induction paradigm (Trier Social Stress Test).

In many situations humans are influenced by the behavior of other people and their relationships with them. For example, in stressful situations ...

Automatic Detection of Highly Organized Theta Oscillations in the Murine EEG

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable electroencephalography (EEG) radiotelemetry or tether system approaches. Pharmacologically, hippocampal theta is heterogeneous (see dualistic theory) and can be differentiated into type I and type II theta. These individual EEG subtypes are related to specific cognitive and behavioral states, such as arousal, exploration, learning and memory, higher integrative functions, etc. In neurodegenerative diseases such as Alzheimer&#39;s, structural and functional alterations of the septohippocampal system can result in impaired theta activity/oscillations. A standard quantitative analysis of the hippocampal EEG includes a Fast-Fourier-Transformation (FFT)-based frequency analysis. However, this procedure does not provide details about theta activity in general and highly-organized theta oscillations in particular. In order to obtain detailed information on highly-organized theta oscillations in the hippocampus, we have developed a new analytical approach. This approach allows for time- and cost-effective quantification of the duration of highly-organized theta oscillations and their frequency characteristics.

Theta activity in the hippocampus is related to specific cognitive and behavioral stages. Here, we describe an analytical method to detect highly-organized theta oscillations within the hippocampus using a time-frequency (i.e., wavelet analysis)-based approach.

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable ...

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. To understand the mechanism of these cellular events, and their causal relationships with each other, Drosophila melanogaster, with its sophisticated genetic tools and diverse behavioral features, are used as disease models for assessing neurodegenerative phenotypes. Here we describe a high-throughput method to analyze Drosophila adult negative geotaxis behavior, as an indication for possible motor defects associated with neurodegeneration. An automated machine is designed and developed to drive fly synchronization using an initial electric impulse, later allowing the recording of negative geotaxis behavior over a course of secs to mins. Images from the digitally recorded video are then processed with the self-designed RflyDetection software for statistical data manipulation. Different from the manually controlled negative geotaxis assay based on single fly, this precise, fast, and high-throughput protocol allows data acquisition from more than hundreds of flies simultaneously, providing an efficient approach to advance our understanding in the underlying mechanism of locomotor deficits associated with neurodegeneration.

An Automated Rapid Iterative Negative Geotaxis Assay for Analyzing Adult Climbing Behavior in a Drosophila Model of Neurodegeneration

This step-by-step protocol analyzes Drosophila negative geotaxis behavior using an automated multi-cylinder system that hosts hundreds of flies and synchronizes their action by an electric motor. Upon synchronization, fly negative geotaxis behavior is assayed, digitally recorded, and analyzed using the self-designed RflyDetection software.

Neurodegenerative diseases are frequently associated with a progressive loss of movement ability, reduced life span, and age-dependent neurodegeneration. ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental factors, and the toxicological and pharmacological effects of numerous substances. Few methods currently implemented are amenable to high throughput measurement. The Microplate Feeder Assay (MFA) was developed for quantifying the consumption of liquid food for individual flies using absorbance. In this assay, flies consume liquid food medium from select wells of a 1536-well microplate. By incorporating a dilute tracer dye into the liquid food medium and loading a known volume into each well, absorbance measurements of the well acquired before and after consumption reflect the resulting change in volume (i.e., volume consumed). To enable high throughput analysis with this method, a 3D-printed coupler was designed that allows flies to be sorted individually into 96-well microplates. This device precisely orients 96- and 1536-well microplates to give each fly access to up to 4 wells for consumption, thus enabling food preference quantification in addition to regular consumption. Furthermore, the device has barrier strips that toggle between open and closed positions to allow for controlled containment and release of a column of samples at a time. This method enables high throughput measurements of consumption of aqueous solutions by many flies simultaneously. It also has the potential to be adapted to other insects and to screen consumption of nutrients, toxins, or pharmaceuticals.

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

The microplate feeder assay offers an economical, high throughput method for quantifying liquid food consumption in Drosophila. A 3D-printed device connects a 96-well microplate in which flies are housed to a 1536-well microplate from which flies consume a feeding solution with a tracer dye. The solution volume decline is measured spectrophotometrically.

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental ...

A Procedure to Study Stress-Induced Relapse of Heroin Seeking after Punishment-Imposed Abstinence

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with drug-taking. This model has been implemented in experiments using different types of substances of abuse such as methamphetamine, cocaine, and alcohol. However, punishment-induced abstinence in heroin-trained animals has not been demonstrated. Furthermore, acute stress is a key trigger for relapse in humans and animal models. It was previously demonstrated that acute food deprivation robustly induced reinstatement of extinguished cocaine and heroin seeking. The procedure described here can be used to assess the effects of acute stress exposure on heroin seeking after punishment-imposed abstinence. A total of 8 rats were implanted with chronic intravenous (i.v.) catheters and trained to self-administer heroin (0.1 mg/kg/infusion) for 18 days under a seek-take chained schedule. Completing the seek link gave access to the take lever, which was paired with a heroin infusion. The seek lever was programmed with a variable interval 60 schedule of reinforcement (VI60), and the take lever was programmed with a fixed-ratio 1 reinforcement schedule (FR1). Following self-administration training, a mild foot shock was delivered on 30% of the completed seek links instead of the extension of the take lever. Footshock intensity was increased by 0.1 mA per daily session from 0.2 mA to 1.0 mA. Heroin-seeking tests were performed after 24 h of food deprivation (FD) or sated conditions. Rats under acute food deprivation condition robustly increased heroin seeking after punishment-imposed abstinence.

A procedure that demonstrates a robust acute food deprivation-induced relapse to heroin seeking after punishment-imposed abstinence is described. A punishment-imposed abstinence model was successfully implemented using the seek and take chain schedule for heroin self-administration. Heroin-seeking tests are then performed following 24 h of food-deprivation stress.

The punishment-imposed abstinence procedure models the self-imposed abstinence that humans initiate due to the adverse consequences associated with ...