The authors wish to thank Drs. Sung-wan An and Mike O&#8217;Donnell for practical advice on establishing this assay, Dr. Chih-Jen Cheng for helpful discussions on the use of ion-specific electrodes, and Dr. Chou-Long Huang for his mentorship and support. This work was supported by the National Institutes of Health (K08DK091316 to ARR) and the American Society of Nephrology Gottschalk Award to ARR.

<ol>
	<li>Ramsay, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+Transport+of+Water+by+the+Malpighian+Tubules+of+the+Stick+Insect,+Dixippus-Morosus+(Orthoptera,+Phasmidae).">Active Transport of Water by the Malpighian Tubules of the Stick Insect, Dixippus-Morosus (Orthoptera, Phasmidae).</a> J Exp Biol. 31, 104-113 (1954).</li><li>Dow, J. A., 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=The+malpighian+tubules+of+Drosophila+melanogaster:+a+novel+phenotype+for+studies+of+fluid+secretion+and+its+control.">The malpighian tubules of Drosophila melanogaster: a novel phenotype for studies of fluid secretion and its control.</a> J Exp Biol. 197, 421-428 (1994).</li><li>Brand, A. H., 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, 401-415 (1993).</li><li>Sozen, M. A., Armstrong, J. D., Yang, M., Kaiser, K., Dow, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+domains+are+specified+to+single-cell+resolution+in+a+Drosophila+epithelium.">Functional domains are specified to single-cell resolution in a Drosophila epithelium.</a> Proc Natl Acad Sci U S A. 94, 5207-5212 (1997).</li><li>Rosay, P., 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=Cell-type+specific+calcium+signalling+in+a+Drosophila+epithelium.">Cell-type specific calcium signalling in a Drosophila epithelium.</a> J Cell Sci. 110 (15), 1683-1692 (1997).</li><li>Dow, J. T., Davies, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+physiology+and+functional+genomics+of+epithelial+function+in+a+genetic+model+organism.">Integrative physiology and functional genomics of epithelial function in a genetic model organism.</a> Physiol Rev. 83, 687-729 (2003).</li><li>Beyenbach, K. W., Skaer, H., Dow, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental,+molecular,+and+transport+biology+of+Malpighian+tubules.">The developmental, molecular, and transport biology of Malpighian tubules.</a> Annu Rev Entomol. 55, 351-374 (2010).</li><li>Donnell, M. J., 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=Hormonally+controlled+chloride+movement+across+Drosophila+tubules+is+via+ion+channels+in+stellate+cells.">Hormonally controlled chloride movement across Drosophila tubules is via ion channels in stellate cells.</a> Am J Physiol. 274, 1039-1049 (1998).</li><li>Linton, S. M., O'Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+K+:Cl-+cotransport+and+Na+/K+-ATPase+to+basolateral+ion+transport+in+malpighian+tubules+of+Drosophila+melanogaster.">Contributions of K+:Cl- cotransport and Na+/K+-ATPase to basolateral ion transport in malpighian tubules of Drosophila melanogaster.</a> J Exp Biol. 202, 1561-1570 (1999).</li><li>Rheault, M. R., O'Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+epithelial+K(+)+transport+in+Malpighian+tubules+of+Drosophila+melanogaster:+evidence+for+spatial+and+temporal+heterogeneity.">Analysis of epithelial K(+) transport in Malpighian tubules of Drosophila melanogaster: evidence for spatial and temporal heterogeneity.</a> J Exp Biol. 204, 2289-2299 (2001).</li><li>Donnell, M. J., Dow, J. A., Huesmann, G. R., Tublitz, N. J., Maddrell, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separate+control+of+anion+and+cation+transport+in+malpighian+tubules+of+Drosophila+Melanogaster.">Separate control of anion and cation transport in malpighian tubules of Drosophila Melanogaster.</a> J Exp Biol. 199, 1163-1175 (1996).</li><li>Cabrero, P., 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=Chloride+channels+in+stellate+cells+are+essential+for+uniquely+high+secretion+rates+in+neuropeptide-stimulated+Drosophila+diuresis.">Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis.</a> Proc Natl Acad Sci U S A. 111, 14301-14306 (2014).</li><li>Torrie, L. 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=Resolution+of+the+insect+ouabain+paradox.">Resolution of the insect ouabain paradox.</a> Proc Natl Acad Sci U S A. 101, 13689-13693 (2004).</li><li>Rodan, A. R., Baum, M., Huang, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+NKCC+Ncc69+is+required+for+normal+renal+tubule+function.">The Drosophila NKCC Ncc69 is required for normal renal tubule function.</a> Am J Physiol Cell Physiol. 303, 883-894 (2012).</li><li>Ianowski, J. P., Christensen, R. J., O'Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na++competes+with+K++in+bumetanide-sensitive+transport+by+Malpighian+tubules+of+Rhodnius+prolixus.">Na+ competes with K+ in bumetanide-sensitive transport by Malpighian tubules of Rhodnius prolixus.</a> J Exp Biol. 207, 3707-3716 (2004).</li><li>Naikkhwah, W., O'Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+stress+alters+fluid+and+ion+transport+by+Malpighian+tubules+of+Drosophila+melanogaster:+evidence+for+phenotypic+plasticity.">Salt stress alters fluid and ion transport by Malpighian tubules of Drosophila melanogaster: evidence for phenotypic plasticity.</a> J Exp Biol. 214, 3443-3454 (2011).</li><li>Donini, A., 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=Secretion+of+water+and+ions+by+malpighian+tubules+of+larval+mosquitoes:+effects+of+diuretic+factors,+second+messengers,+and+salinity.">Secretion of water and ions by malpighian tubules of larval mosquitoes: effects of diuretic factors, second messengers, and salinity.</a> Physiol Biochem Zool. 79, 645-655 (2006).</li><li>Maddrell, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretion+by+Malpighian+Tubules+of+Rhodnius+movements+of+Ions+and+Water.">Secretion by Malpighian Tubules of Rhodnius movements of Ions and Water.</a> J Exp Biol. 51, 71-97 (1969).</li><li>Maddrell, S. H., Overton, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+sodium+transport+and+fluid+secretion+by+ouabain+in+an+insect+malpighian+tubule.">Stimulation of sodium transport and fluid secretion by ouabain in an insect malpighian tubule.</a> J Exp Biol. 137, 265-276 (1988).</li><li>Williams, J. C., Beyenbach, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+secretagogues+on+Na+and+K+secretion+in+the+Malpighian+tubules+of+Aedes+Aegypti+(L).">Differential effects of secretagogues on Na and K secretion in the Malpighian tubules of Aedes Aegypti (L).</a> J Comp Physiol. 149, 511-517 (1983).</li><li>Maddrell, S. H., O'Donnell, M. J., Caffrey, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+haemolymph+potassium+activity+during+initiation+and+maintenance+of+diuresis+in+fed+Rhodnius+prolixus.">The regulation of haemolymph potassium activity during initiation and maintenance of diuresis in fed Rhodnius prolixus.</a> J Exp Biol. 177, 273-285 (1993).</li><li>Messerli, M. A., Kurtz, I., Smith, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+optimized+Na++and+Cl-+liquid+membranes+for+use+with+extracellular,+self-referencing+microelectrodes.">Characterization of optimized Na+ and Cl- liquid membranes for use with extracellular, self-referencing microelectrodes.</a> Anal Bioanal Chem. 390, 1355-1359 (2008).</li><li>Ianowski, J. P., O'Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basolateral+ion+transport+mechanisms+during+fluid+secretion+by+Drosophila+Malpighian+tubules:+Na++recycling,+Na+:K+:2Cl-+cotransport+and+Cl-+conductance.">Basolateral ion transport mechanisms during fluid secretion by Drosophila Malpighian tubules: Na+ recycling, Na+:K+:2Cl- cotransport and Cl- conductance.</a> J Exp Biol. 207, 2599-2609 (2004).</li><li>Neufeld, D. S., Leader, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrochemical+characteristics+of+ion+secretion+in+malpighian+tubules+of+the+New+Zealand+alpine+weta+(Hemideina+maori).">Electrochemical characteristics of ion secretion in malpighian tubules of the New Zealand alpine weta (Hemideina maori).</a> J Insect Physiol. 44, 39-48 (1997).</li><li>Greenspan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fly Pushing: The Theory and Practice of Drosophila Genetics. , (1997).</li><li>Jayakannan, M., Babourina, O., Rengel, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+measurements+of+Na++fluxes+in+plants+using+calixarene-based+microelectrodes.">Improved measurements of Na+ fluxes in plants using calixarene-based microelectrodes.</a> J Plant Physiol. 168, 1045-1051 (2011).</li><li>Wu, Y., Schellinger, J. N., Huang, C. L., Rodan, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypotonicity+Stimulates+Potassium+Flux+through+the+WNK-SPAK/OSR1+Kinase+Cascade+and+the+Ncc69+Sodium-Potassium-2-Chloride+Cotransporter+in+the+Drosophila+Renal+Tubule.">Hypotonicity Stimulates Potassium Flux through the WNK-SPAK/OSR1 Kinase Cascade and the Ncc69 Sodium-Potassium-2-Chloride Cotransporter in the Drosophila Renal Tubule.</a> J Biol Chem. 289, 26131-26142 (2014).</li><li>Blumenthal, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+tyramine+signaling+by+osmolality+in+an+insect+secretory+epithelium.">Modulation of tyramine signaling by osmolality in an insect secretory epithelium.</a> Am J Physiol Cell Physiol. 289, 1261-1267 (2005).</li><li>Dow, J. A., Maddrell, S. H., Davies, S. A., Skaer, N. J., Kaiser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+the+nitric+oxide-cGMP+signaling+pathway:+the+control+of+epithelial+function+in+Drosophila.">A novel role for the nitric oxide-cGMP signaling pathway: the control of epithelial function in Drosophila.</a> Am J Physiol. 266, 1716-1719 (1994).</li><li>Dube, K., McDonald, D. G., O'Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+transport+by+isolated+anterior+and+posterior+Malpighian+tubules+of+Drosophila+melanogaster:+roles+of+sequestration+and+secretion.">Calcium transport by isolated anterior and posterior Malpighian tubules of Drosophila melanogaster: roles of sequestration and secretion.</a> J Insect Physiol. 46, 1449-1460 (2000).</li><li>Efetova, M., 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=Separate+roles+of+PKA+and+EPAC+in+renal+function+unraveled+by+the+optogenetic+control+of+cAMP+levels+in+vivo.">Separate roles of PKA and EPAC in renal function unraveled by the optogenetic control of cAMP levels in vivo.</a> J Cell Sci. 126, 778-788 (2013).</li><li>Rheault, M. R., O'Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+cation+transport+by+Malpighian+tubules+of+Drosophila+melanogaster:+application+of+two+novel+electrophysiological+methods.">Organic cation transport by Malpighian tubules of Drosophila melanogaster: application of two novel electrophysiological methods.</a> J Exp Biol. 207, 2173-2184 (2004).</li><li>Donnell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Too+much+of+a+good+thing:+how+insects+cope+with+excess+ions+or+toxins+in+the+diet.">Too much of a good thing: how insects cope with excess ions or toxins in the diet.</a> J Exp Biol. 212, 363-372 (2009).</li><li>Cheng, C. J., Truong, T., Baum, M., Huang, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney-specific+WNK1+inhibits+sodium+reabsorption+in+the+cortical+thick+ascending+limb.">Kidney-specific WNK1 inhibits sodium reabsorption in the cortical thick ascending limb.</a> Am J Physiol Renal Physiol. 303, 667-673 (2012).</li><li>Cheng, C. J., Yoon, J., Baum, M., Huang, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STE20/SPS1-related+Proline/alanine-rich+Kinase+(SPAK)+is+Critical+for+Sodium+Reabsorption+in+Isolated+Perfused+Thick+Ascending+Limb.">STE20/SPS1-related Proline/alanine-rich Kinase (SPAK) is Critical for Sodium Reabsorption in Isolated Perfused Thick Ascending Limb.</a> Am J Physiol Renal Physiol. , (2014).</li></ol>

The authors have nothing to disclose.

The use of the Ramsay assay, together with ion-specific electrodes, allows the measurement of fluid secretion rates and ion fluxes in isolated insect Malpighian (renal) tubules. Twenty or more tubules can be assayed at a time, allowing higher throughput compared to the assay of individual in vitro microperfused tubules. In addition, ion-specific electrodes allow the determination of ion concentrations within the secreted fluid in situ, limiting errors that may be introduced in the transfer of the small volumes of fluid to a second apparatus. Critical steps within the protocol include ensuring that tubules are dissected without rents or tears (which will cause fluid to be secreted from the tear, rather than the cut end of the ureter), and ensuring that the ISE is working well by determining its slope prior to starting the experiment. Even with careful dissections, it is not unusual for occasional tubules to fail to secrete (up to ~10% of tubules dissected), but if a high proportion of tubules do not secrete this may be for biological reasons (i.e., very low secretion rates under that particular condition). Some pitfalls that may lead to an ISE not working well include: ionophore leaking out (which may be due to the electrode tip breaking or inadequate silanization), or introduction of an air bubble into the tip. These problems can be ascertained by visualization of the electrode under a compound microscope and usually require filling a new ISE with ionophore before proceeding. Additionally, an ISE may work well for several days, or even weeks, but will eventually fail, again mandating construction of a new ISE. It is also critical that the assay dish is thoroughly washed at the end of the experiment, because residual salts will alter the ionic and osmotic composition of the bathing saline in subsequent experiments.
In our experience, the Na+ ISE described in this protocol is less stable than the K+ ISE, often exhibiting deterioration of its slope over the course of the experiment. Indeed, Jayakannan et al. described an improved Na+ ISE based on the same ionophore with better selectivity and stability. Their cocktail included 3.5% 4-tert-butylcalix[4]arene-tetraacetic acid tetraethylester, 95.9% 2-nitrophenyl octyl ether, and 0.6% potassium tetrakis (4-chlorophenyl) borate26.
Once the experimenter is comfortable performing this assay, many permutations can be introduced to probe the underlying biology of epithelial ion transport. For example, tubules of differing genotypes may be examined to determine the role of a specific transporter or ion channel on transepithelial fluid and ion transport14, or of extracellular or intracellular signaling pathways regulating these transporters or channels14,27 . The bathing droplet composition can be modified to look at the role of its specific components, such as ions, glucose or amino acids9, or osmolarity27,28. Or, hemolymph itself obtained from animals reared under different conditions may be used in place of the bathing saline16. Drugs, hormones (or their second messengers) or peptides can be added to ascertain their effects2,9,14,23,29. In the current protocol, fluid and ion fluxes in the main segment are examined, since the initial segment does not secrete fluid2, and the lower segment is outside the bathing drop. However, the protocol can be modified to examine activity of the initial or lower segments30. Transgenic reporters, such as the calcium-sensing aequorin transgene5, or the pH- and chloride-sensing ChlopHensor transgene12, can allow correlation of intracellular ion concentrations with transepithelial flux. Similarly, Efetova et. al. demonstrated cell-specific manipulation of cAMP levels using optogenetic control of a photoactive adenylyl cyclase, demonstrating differential effects of cAMP on stellate and principal cells31.
While the current protocol is focused on the Malpighian tubules of adult Drosophila&#160;melanogaster, the techniques used have been applied in other insects, including Rhodnius prolixus21, Hemideina maori24, and multiple mosquito species17, as well as on larval Drosophila tubules16. Similarly, other ions besides Na+ and K+, such as H+, Ca2+ or tetraethylammonium may be assayed30,32, as well as toxins such as salicylate33. Finally, the ion-specific electrode technique can also be used to measure ion concentrations in microperfused mammalian tubules, obviating the need for radiolabeled ions34,35.
In summary, use of the Ramsay assay with ion-specific electrodes allows measurement of transepithelial fluid and ion fluxes from isolated Drosophila melanogaster renal tubules. Together with the powerful Drosophila genetic tools available, molecular mechanisms of epithelial ion transport can be elucidated.

Renal epithelial ion transport underlies organismal iono- and osmoregulation. The Drosophila&#160;melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport. This is due to the combination of the powerful genetics of Drosophila, paired with the accessibility of its renal tubules to physiological study. The Ramsay assay, named after the investigator who pioneered the technique1, measures fluid secretion rates from isolated Malpighian tubules, and was established in Drosophila in 1994 by Dow and colleagues2. This paved the way for further studies using Drosophila genetic tools, such as the GAL4-UAS system3,4, to define cell-specific signaling pathways regulating fluid secretion. An example includes calcium signaling in response to a peptide hormone5, amongst many others6,7.
A combination of genetic techniques and classical physiological study has shown that urine generation in the fly occurs through the secretion of a potassium chloride-rich fluid from the main segment of the tubule. This occurs through the parallel transepithelial secretion of cations, primarily K+ but also Na+, through the principal cell, and Cl- secretion through the stellate cell8-12. The ability to separately measure transepithelial K+ and Na+ fluxes allows a more detailed characterization of transport mechanisms than the measurement of fluid secretion alone. For example, in unstimulated Drosophila tubules, the Na+/K+-ATPase inhibitor ouabain has no effect on fluid secretion2, even when its uptake into principal cells is inhibited by the organic anion transporter inhibitor taurocholate13. However, Linton and O&#8217;Donnell showed that ouabain depolarizes the basolateral membrane potential, and increases Na+ flux9. As shown in the Representative Results, we replicated these findings, and showed that K+ flux is concomitantly decreased14; the increased Na+ flux and decreased K+ flux have opposing effects on fluid secretion, resulting in no net change in secretion. Thus, there are two resolutions to the &#8220;ouabain paradox,&#8221; i.e., the initial observation that ouabain has no effect on fluid secretion in the Drosophila tubule: first, in stimulated tubules, the effect of ouabain on fluid secretion is not apparent due to its uptake by the organic anion transporter13; and second, in unstimulated tubules, ouabain has opposing effects on transepithelial Na+ and K+ flux, resulting in no net change in fluid secretion (see Representative Results and ref. 9). Therefore, the primary role of the Na+/K+-ATPase in unstimulated tubules is to lower intracellular Na+ concentration to generate a favorable concentration gradient for Na+-coupled transport processes across the basolateral membrane. Indeed, by separately measuring Na+ and K+ fluxes, we demonstrated that tubules lacking the fly sodium-potassium-2-chloride cotransporter (NKCC) have decreased transepithelial K+ flux, with no further decrease after ouabain addition, and no change in transepithelial Na+ flux14. These findings supported our conclusion that Na+ entering the cell through the NKCC is recycled through the Na+/K+-ATPase. In another example, Ianowski et al. observed that lowering bath K+ concentration from 10 mM to 6 mM decreased transepithelial K+ flux and increased transepithelial Na+ flux in tubules from Rhodnius prolixus, with no net change in fluid secretion15. Differential effects on Na+ flux and K+ flux across larval tubules have also been observed in Drosophila tubules in response to varying salt diets16 and in two mosquito species in response to rearing salinity17.
The greatest challenge in the measurement of transepithelial ion flux in the Ramsay assay preparation is the determination of ion concentrations within the secreted fluid. This challenge has been met with varying solutions, including flame photometery18, use of radioactive ions19, and electron probe wavelength dispersive spectroscopy20. These techniques require transfer of the secreted fluid drop to an instrument for measurement of ion concentrations. Since the volume of fluid secreted by the unstimulated Drosophila tubule is small, typically ~0.5 nl/min, this poses a technical challenge and also introduces error if some of the secreted fluid is lost upon transfer. In contrast, the use of ion-specific electrodes allows the measurement of ion activity (from which ion concentration can be calculated) in situ. The current protocol was adapted from that used by Maddrell and colleagues to measure transepithelial K+ flux across the Rhodnius tubule using valinomycin as the K+ ionophore21, and also describes the use of a 4-tert-butylcalix[4]arene-tetraacetic acid tetraethyl ester-based Na+-specific ion-specific electrode characterized by Messerli et. al.22. Ion-specific electrodes have also been used to measure ion concentrations in fluid secreted by Malpighian tubules in the Ramsay assay in adult9,23 and larval16Drosophila melanogaster, the New Zealand Alpine Weta (Hemideina maori)24 and in mosquitoes17.
Here, we describe in detail the use of the Ramsay assay to measure fluid secretion rates in Malpighian tubules from Drosophila melanogaster, as well as the use of ion-specific electrodes to determine the concentrations of K+ and Na+ within the secreted fluid and thus the calculation of transepithelial ion fluxes. An overview of the assay is provided in Figure 1.
<img alt="Figure 1" src="/files/ftp_upload/53144/53144fig1.jpg" />
Figure 1.&#160;Schematic of the Malpighian Tubules and the Ramsay Assay with Use of Ion-specific Electrodes to Measure Ion Concentrations. This figure illustrates the setup for the Ramsay assay. (A) Each fly has four tubules, a pair of anterior tubules and a pair of posterior tubules, that float in the abdominal cavity surrounded by hemolymph. In each pair, the two tubules join at the ureter, which then empties the urine at the junction of the midgut and hindgut. The tubules are blind-ended. Urine is generated by the fluid-secreting main segment (shown in red), and flows toward the ureter and out into the gut. After dissection, the tubule pair is dissociated from the gut by transecting the ureter. (B) The pair of tubules is then transferred into a droplet of bathing saline within a well of the assay dish. One of the two tubules, referred to here as the &#8220;anchor tubule,&#8221; is wrapped around a metal pin and is inert. The other tubule is the secreting tubule. The initial segment (which does not secrete fluid) and main segment of the secreting tubule remain within the droplet of bathing saline. Ions and water move from the bathing saline and into the tubule lumen of the main segment, and then move toward the ureter, as would occur in vivo. The lower segment (blue) is outside the bathing saline and therefore inert. Since the ureter is cut, the secreted fluid emerges as a droplet from the cut end of the ureter. The secreted fluid droplet enlarges over time as secretion continues, and its diameter is measured using an ocular micrometer. A layer of mineral oil prevents evaporation of the secreted fluid. The reference and ion specific electrodes measure the ion concentration of the secreted fluid. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53144/53144fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Ellsworth Adhesives</td><td>http://www.ellsworth.com/dow-corning-sylgard-184-silicone-encapsulant-0-5kg-kit-clear/</td><td>May be purchased from multiple distributors</td></tr><tr><td>Petri dish, polystyrene, 100 mm x 15 mm</td><td>Fisher</td><td>FB0875712</td><td>Specific brand is not important</td></tr><tr><td>Petri dish, polystyrene, 35 mm x 10 mm</td><td>Corning Life Sciences</td><td>Fisher 08-757-100A</td><td>Specific brand is not important</td></tr><tr><td>Scalpel Handle #3</td><td>Fine Science Tools</td><td>10003-12</td><td>Specific brand is not important</td></tr><tr><td>Scalpel Blades #1</td><td>Fine Science Tools</td><td>10011-00</td><td>Specific brand is not important; use appropriate sharps precautions</td></tr><tr><td>Needle, 30 G x 1/2</td><td>Becton Dickinson</td><td>305106</td><td>Use appropriate sharps precautions</td></tr><tr><td>Minutien pins, black anodized, 0.15 mm</td><td>Fine Science Tools</td><td>26002-15</td><td></td></tr><tr><td>Stereomicroscope with ocular micrometer</td><td>Nikon</td><td>SMZ800</td><td>Specific brand is not important; this is given as an example</td></tr><tr><td>Sheet of black stained glass, 3 mm (1/8 inch) thick</td><td>Hobby shop</td><td></td><td>Example includes Spectrum Black Opal by Spectrum Glass (http://www.delphiglass.com/spectrum-glass/opalescent/spectrum-black-opal)</td></tr><tr><td>Glass cutting tools (glass cutter, glass cutting pliers)</td><td>Hobby shop</td><td></td><td>Examples include the Studio Pro Lightweight Running Pliers by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/pliers-nippers/studio-pro-lightweight-running-pliers) and the Studio Pro Brass Glass Cutter by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/glass-cutters/studio-pro-brass-glass-cutter). Use appropriate safety precautions when cutting glass</td></tr><tr><td>Borosilicate glass capillary tube, unfilamented, GC120-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td><td>Warner Instruments</td><td>30-0042</td><td></td></tr><tr><td>Borosilicate glass capillary tube, filamented, GC120F-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td><td>Warner Instruments</td><td>30-0044</td><td></td></tr><tr><td>Nitric acid, 70%</td><td>Sigma</td><td>438073</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines. Specific brand is not important</td></tr><tr><td>Cimarec 7 in x 7 in hotplate</td><td>Fisher</td><td>11675911Q</td><td>Specific brand is not important; caution when heated</td></tr><tr><td>Selectophore dichlorodimethylsilane</td><td>Sigma</td><td>40136-1ML</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Two-step vertical pipet puller</td><td>Narishige</td><td>PC-10</td><td>Other pipet pullers can be used; this is given as an example</td></tr><tr><td>Glass petri dish, 150 mm diameter x 15 mm height</td><td>Fisher</td><td>08-748E</td><td>Specific brand is not important; only one dish needed</td></tr><tr><td>World Precision Instruments E210 1 mm micropipette storage jar</td><td>Fisher</td><td>50-821-852</td><td>May be available from other distributors. Useful to have two jars. Note that although this jar is specified for 1 mm pipets, and the pipets used here are 1.2 mm, in our experience the 1 mm jar works best for the 1.2 mm pipets.</td></tr><tr><td>Silica Gel, Tel-Tale Desiccant, indicating, 10-18 mesh</td><td>Fisher</td><td>S161-500</td><td>Indicating silica useful for determining whether silica gel retains desiccating ability</td></tr><tr><td>World Precision Instruments MicroFil, 34G</td><td>Fisher</td><td>50-821-914</td><td>May be available from other distributors.</td></tr><tr><td>1 ml syringe with luer lock</td><td>Becton Dickinson</td><td>309659</td><td>May be available from other distributors.</td></tr><tr><td>3 ml syringe with luer lock</td><td>Becton Dickinson</td><td>309657</td><td>May be available from other distributors.</td></tr><tr><td>D300 3-way stopcock with female luer lock inlet port, male luer outlet port with rotating collar and guard</td><td>Cole-Parmer</td><td>UX-30600-02</td><td>Specific brand is not important</td></tr><tr><td>Female Luer Locking Connector</td><td>4 Medical Solutions</td><td>ADC 9873-10</td><td>Specific brand is not important; barbed end is ~4 mm at narrowest point and ~7 mm at widest point.</td></tr><tr><td>Silicone Tubing I.D. x O.D. x Wall: 1/16 x 1/8 x 1/32 in. (1.59 x 3.18 x 0.79 mm)</td><td>Fisher</td><td>14-179-110</td><td>Specific brand is not important</td></tr><tr><td>E-3603 tubing, I.D. x O.D.: 1/32 x 3/32 in</td><td>Fisher</td><td>14171208</td><td>Specific brand is not important</td></tr><tr><td>Modeling clay</td><td></td><td></td><td>Specific brand is not important</td></tr><tr><td>Selectophore potassium ionophore I, cocktail B</td><td>Sigma</td><td>99373</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Selectophore sodium ionophore X</td><td>Sigma</td><td>71747</td><td>Sodium ionophore X = 4-tert-butylcalix[4]arene-tetraacetic acid tetraethylester</td></tr><tr><td>Selectophore 2-nitrophenyl octyl ether</td><td>Sigma</td><td>73732</td><td></td></tr><tr><td>Selectophore sodium tetraphenylborate</td><td>Sigma</td><td>72018</td><td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td></tr><tr><td>Schneider&#039;s Drosophila medium</td><td>Life Technologies</td><td>21720024</td><td></td></tr><tr><td>High impedance electrometer</td><td>World Precision Instruments</td><td>FD223a</td><td></td></tr><tr><td>Microelectrode holder 1 mm with 45&amp;deg; body, vented, with handle</td><td>Warner Instruments</td><td>64-1051</td><td></td></tr><tr><td>Microelectrode holder 1 mm with straight body, vented</td><td>Warner Instruments</td><td>64-1007</td><td></td></tr><tr><td>Silver wire</td><td>Warner Instruments</td><td>64-1318</td><td></td></tr><tr><td>Micromanipulators, pair</td><td>Leitz</td><td></td><td>Various brands/models will work; this is an example</td></tr><tr><td>Faraday cage</td><td>Technical Manufacturing Corporation</td><td>81-334-03</td><td>This is an example; any Faraday cage will work</td></tr><tr><td>Single gooseneck fiberoptic light</td><td>Nikon</td><td></td><td>Specific brand is not important</td></tr><tr><td>mineral oil</td><td>Fisher</td><td>BP-2629</td><td>Specific brand is not important</td></tr><tr><td>forceps, Dumont #5 with Biologie tip</td><td>Fine Science Tool</td><td>11295-10</td><td>May be available from other distributors.</td></tr></tbody></table>

use of the ramsay assay to measure fluid secretion and ion flux rates in the drosophila melanogaster malpighian tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila&#160;melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.

Figures 7 and 8 demonstrate that use of the Ramsay assay with ion-specific electrodes to measure K+ and Na+ concentrations can distinguish genetically and pharmacologically distinct K+ and Na+ fluxes, information that is not captured by measuring fluid secretion rates alone. Figure 7 shows that decreased fluid secretion in tubules from flies carrying a homozygous null mutation in the NKCC is driven by a decrease in K+ flux in the mutants (Figure 7A), while Na+ flux is not changed (Figure 7B). Figure 8 shows that the Na+/K+-ATPase inhibitor, ouabain, has distinct effects on each of the three parameters measured: in wild-type tubules, fluid secretion rate is unchanged (Figure 8A and 8B), Na+ flux is increased (Figure 8A), and K+ flux is decreased (Figure 8B). Furthermore, ouabain has no effect on transepithelial K+ flux in the NKCCnull mutant tubules (Figure 8B). Together, these data indicate that the Na+/K+-ATPase provides the driving force for K+ uptake through the NKCC, while the Na+ is recycled through the Na+/K+-ATPase14. Between 13 and 18 tubules were analyzed in each genotype/condition; differences observed were statistically significant with a p &#60;0.01 (or less) by unpaired, two-tailed Student&#8217;s t-test. Since ~15-20 tubules can easily be analyzed in a single day&#8217;s experiment, the results shown here represent ~10 days of experimentation.
<img alt="Figure 7" src="/files/ftp_upload/53144/53144fig7.jpg" /> 
Figure 7.&#160;Fluid Secretion Rate and Transepithelial K+ Flux are Decreased in Tubules Carrying a Homozygous Null Mutation in NKCC, While Na+ Flux is Unchanged. (A) Fluid secretion rate (nl/min/tubule) and K+ flux (pmol/min/tubule) were measured in tubules from control flies (wBerlin) or from flies carrying a homozygous null mutation in the fly NKCC (w; Ncc69r2/Ncc69r2). n = 18 tubules/genotype. (B) Fluid secretion rate (nl/min/tubule) and Na+ flux (pmol/min/tubule) were measured from control flies (wBerlin, n = 13 tubules) or from flies carrying a homozygous null mutation in the fly NKCC (w; Ncc69r2/Ncc69r2, n = 14 tubules). Values shown are mean &#177; standard error of the mean (SEM). NS, not significant; **, p &#60;0.01; ***, p &#60;0.001; ****, p &#60;0.0001. This figure was adapted from Rodan et. al.14 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53144/53144fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/53144/53144fig8.jpg" /> 
Figure 8.&#160;The Na+/K+-ATPase Recycles Na+ Entering through the NKCC. (A) Fluid secretion rate (nl/min/tubule) and Na+ flux (pmol/min/tubule) were measured in tubules from control flies (wBerlin) in the absence (n = 13 tubules) or presence (n = 15 tubules) of 100 &#956;M ouabain, an inhibitor of the Na+/K+-ATPase. (B) Fluid secretion rate (nl/min/tubule) and K+ flux (pmol/min/tubule) were measured in tubules dissected from control flies (wBerlin) in the absence (n = 17 tubules) or presence (n = 17 tubules) of 100 &#956;M ouabain. K+ flux was also measured in NKCCnull&#160;tubules in the absence or presence of ouabain(w; Ncc69r2/Ncc69r2, n = 16 - 17 tubules/condition). Values shown are mean &#177; SEM. NS, not significant; **, p &#60;0.01; ***, p &#60;0.001. This figure was adapted from Rodan et. al.14 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53144/53144fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table>
	<tbody>
		<tr>
			<td colspan="2">Drosophila saline</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>For 1L:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Weigh out 3.6 g glucose and place into a beaker; final concentration = 20 mM</td>
		</tr>
		<tr>
			<td>Then add:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Volume (ml)</td>
			<td colspan="2">Final concentration (mM)</td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>~800</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 M NaCl</td>
			<td>47</td>
			<td>117.5</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M KCl</td>
			<td>20</td>
			<td>20</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M CaCl2</td>
			<td>2</td>
			<td>2</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>8.5</td>
			<td>8.5</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M NaHCO3</td>
			<td>10.2</td>
			<td>10.2</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M HEPES</td>
			<td>15</td>
			<td>15</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M NaH2PO4</td>
			<td>4.3</td>
			<td>4.3</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Stir, adjust pH to 7.0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Add H2O to final volume of 1L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Filter sterilize; avoid autoclaving (glucose will caramelize)</td>
		</tr>
		<tr>
			<td>Store at 4 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Discard if evidence of bacterial or fungal growth</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Making Drosophila Saline.
<table>
	<tbody>
		<tr>
			<td>Schneider&#39;s medium</td>
			<td></td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Concentration (mM)</td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>3.33</td>
		</tr>
		<tr>
			<td>L-arginine</td>
			<td>2.3</td>
		</tr>
		<tr>
			<td>L-aspartic acid</td>
			<td>3.01</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>0.496</td>
		</tr>
		<tr>
			<td>L-cystine</td>
			<td>0.417</td>
		</tr>
		<tr>
			<td>L-glutamic acid</td>
			<td>5.44</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>12.33</td>
		</tr>
		<tr>
			<td>L-histidine</td>
			<td>2.58</td>
		</tr>
		<tr>
			<td>L-isoleucine</td>
			<td>1.15</td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>1.15</td>
		</tr>
		<tr>
			<td>L-lysine hydrochloride</td>
			<td>9.02</td>
		</tr>
		<tr>
			<td>L-methionine</td>
			<td>5.37</td>
		</tr>
		<tr>
			<td>L-phenylalanine</td>
			<td>0.909</td>
		</tr>
		<tr>
			<td>L-proline</td>
			<td>14.78</td>
		</tr>
		<tr>
			<td>L-serine</td>
			<td>2.38</td>
		</tr>
		<tr>
			<td>L-threonine</td>
			<td>2.94</td>
		</tr>
		<tr>
			<td>L-tryptophan</td>
			<td>0.49</td>
		</tr>
		<tr>
			<td>L-tyrosine</td>
			<td>2.76</td>
		</tr>
		<tr>
			<td>L-valine</td>
			<td>2.56</td>
		</tr>
		<tr>
			<td>&#946;-alanine</td>
			<td>5.62</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>5.41</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>15.06</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>21.33</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>3.31</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>36.21</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>4.76</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>4.94</td>
		</tr>
		<tr>
			<td>&#945;-ketoglutaric acid</td>
			<td>1.37</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>11.11</td>
		</tr>
		<tr>
			<td>fumaric acid</td>
			<td>0.862</td>
		</tr>
		<tr>
			<td>malic acid</td>
			<td>0.746</td>
		</tr>
		<tr>
			<td>succinic acid</td>
			<td>0.847</td>
		</tr>
		<tr>
			<td>trehalose</td>
			<td>5.85</td>
		</tr>
		<tr>
			<td>yeastolate</td>
			<td>2,000 mg/L</td>
		</tr>
	</tbody>
</table>
Table 2. Composition of Schneider&#8217;s Medium.
<table>
	<tbody>
		<tr>
			<td colspan="2">Pre</td>
			<td colspan="2">Post</td>
			<td>Interval</td>
			<td>Slope/decile</td>
		</tr>
		<tr>
			<td>mM</td>
			<td>mV</td>
			<td>mM</td>
			<td>mV</td>
			<td>15 - 150 (pre)</td>
			<td>52.8</td>
		</tr>
		<tr>
			<td>15</td>
			<td>-28.4</td>
			<td>15</td>
			<td>-29.4</td>
			<td>15 - 150 (post)</td>
			<td>54.4</td>
		</tr>
		<tr>
			<td>75</td>
			<td>9.1</td>
			<td>75</td>
			<td>9.4</td>
			<td>75 - 150 (pre)</td>
			<td>51</td>
		</tr>
		<tr>
			<td>150</td>
			<td>24.4</td>
			<td>150</td>
			<td>25</td>
			<td>75 - 150 (post)</td>
			<td>52</td>
		</tr>
		<tr>
			<td>200</td>
			<td>30.6</td>
			<td>200</td>
			<td>31.9</td>
			<td>150 - 200 (pre)</td>
			<td>49.6</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>150 - 200 (post)</td>
			<td>55.2</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>Mean</td>
			<td>52.5</td>
		</tr>
	</tbody>
</table>
Table 3.&#160;Calibration of K+ ISE.The K+ ISE is calibrated in solutions containing 15, 75, 150 and 200 mM KCl before and after the measurement of experimental secreted fluid; an example of actual measurements is shown in this table. Calculation of the slope/decile change in K+ concentration is illustrated. For the 15-150 mV interval, slope/decile = mV for the 150 mM calibration drop &#8211; mV for the 15 mM calibration drop. For the 75 - 150 interval, the difference is divided by 0.3 [log (150/75)]. For the 150 - 200 interval, the difference is divided by 0.125 [log (200/150)]. The six slopes (three pre-experiment, three post-experiment) are averaged together to get the mean slope/decile. This table was adapted from Rodan et. al.14

Watch this Scientific Journal Video about Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule at JoVE.com

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

This protocol describes the use of the Ramsay assay to measure fluid secretion rates from isolated Malpighian (renal) tubules from Drosophila melanogaster. In addition, the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid, allowing calculation of transepithelial ion flux, is described.

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

use-ramsay-assay-to-measure-fluid-secretion-ion-flux-rates-drosophila

Nanyang Technological University

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JoVE Journal

Biology

11.7K Views.  University of Texas Southwestern Medical Center. This protocol describes the use of the Ramsay assay to measure fluid secretion rates from isolated Malpighian (renal) tubules from Drosophila melanogaster. In addition, the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid, allowing calculation of transepithelial ion flux, is described.

Video: Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

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 ...

Developmental Biology

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Immunology and Infection

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Neuroscience

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 ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

This protocol outlines methodologies behind the Ramsay assay, ion-selective microelectrodes, Scanning Ion-selective Electrode Technique (SIET) and in vitro contraction assays, applied to study the adult mosquito excretory system, comprised of the Malpighian tubules and hindgut, to collectively measure ion and fluid secretion rates, contractile activity, and transepithelial ion transport.

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...

Monitoring Gut Acidification in the Adult Drosophila Intestine

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the proper functioning of the gut. One such region, the copper cell region (CCR), is localized to the middle midgut and consists, in part, of a group of cells known as copper cells. Copper cells are involved in gastric acid secretion, an evolutionarily conserved process whose precise role is poorly understood. This paper describes improvements in the current protocol used to assay for acidification of the adult Drosophila melanogaster gut and demonstrates that it can be used on other species of flies. In particular, this paper demonstrates that gut acidification is dependent on the fly's nutritional status and presents a protocol based on this new finding. Overall, this protocol demonstrates the potential usefulness of studying Drosophila copper cells to uncover general principles underlying the mechanisms of gut acidification.

Monitoring Gut Acidification in the Adult Drosophila Intestine

Here, we present a standardized protocol for monitoring gut acidification in Drosophila melanogaster with optimal output. We first use this protocol for gut acidification monitoring in Drosophila melanogaster and then demonstrate its use in non-model Drosophila species.

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the ...

Drosophila Fecal Deposit Analysis for Drug Discovery

Assessment of The Effect of Antidiarrheal Drugs and Plant Extracts on Drosophila melanogaster

To study human gastrointestinal physiology, biomedical scientists have relied on the use of model organisms. Although many researchers have used mice as a model to study intestinal function, only a few reports have focused on Drosophila melanogaster (D. melanogaster). Compared to mice, fruit flies present many advantages, such as a short life cycle, cost-effective and simple maintenance, and no ethical issues. Furthermore, the mammalian gastrointestinal physiology, anatomy, and signaling pathways are highly conserved in D. melanogaster. Plant extracts have been used traditionally to treat diarrhea and constipation. For example, Psidium guajava (P. guajava) is one of the most known antidiarrheal agents in the tropics. However, no studies have evaluated the effect of antidiarrheal and laxative drugs and plant extracts in D. melanogaster, and it remains unknown if similar effects (e.g., smaller, more concentrated, and less abundant fecal deposits in the case of antidiarrheal drugs) can occur in the fruit flies compared to mammals. In this study, an antidiarrheal effect induced by P. guajava is demonstrated in a D. melanogaster strain that presents a diarrheic phenotype. Fecal sampling produced by flies is monitored using a dye-supplemented food. This protocol outlines the method used for preparing food with drugs, evaluating the fecal deposits of flies fed on these food preparations, and interpreting the data obtained.

Author Spotlight: Advancing Antidiarrheal Research with the Drosophila Model 

Here, a method is described to feed Drosophila melanogaster with drugs and plant extracts and assess their effect on the gastrointestinal tract by analyzing the fruit flies fecal deposits. The drug-treated flies can be used as a model for further research.

To study human gastrointestinal physiology, biomedical scientists have relied on the use of model organisms. Although many researchers have used mice as a ...

Malpighian Tubule Dissection from an Adult Fly: Isolating an Osmoregulatory and Excretory Organ

Source: Rossano, A. J. and Romero, M. F. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/55698/" target="_blank">Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator</a>. J. Vis. Exp. (2017).
This article describes the dissection of the anterior Malpighian tubules (MT) from a female Drosophila fly and an example protocol from Rossano et al. (2017) in which the MTs are prepared for live imaging in a perfusion chamber.

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Summary

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Chapters in this video

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

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

Measurement of Larval Activity in the Drosophila Activity Monitor

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

Monitoring Gut Acidification in the Adult Drosophila Intestine

Assessment of The Effect of Antidiarrheal Drugs and Plant Extracts on Drosophila melanogaster

Malpighian Tubule Dissection from an Adult Fly: Isolating an Osmoregulatory and Excretory Organ