First published online December 15, 2004
Journal of Experimental Biology 208, 93-104 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01374
Ion-selective microelectrode analysis of salicylate transport by the Malpighian tubules and gut of Drosophila melanogaster
Michael J. O'Donnell* and
Mark R. Rheault
Department of Biology, McMaster University, 1280 Main Street West,
Hamilton, Ontario, Canada L8S 4K1
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Fig. 1. The arrangements for recording salicylate flux using salicylate-selective
microelectrodes based on the ion exchanger tridodecylmethylammonium chloride.
(A) The tip of a salicylate self-referencing microelectrode (Sal-SeR)
microelectrode is moved from a position 10 µm away from the surface of the
Malpighian tubule epithelium to a position 100 µm farther away by
computer-controlled stepper motors. The differential signal between the two
positions is amplified 10x in the head stage and a further 100x in
the connected amplifier for a total voltage amplification of 1000x. An
associated PC-based data acquisition system running ASET software records the
voltages and controls the stepper motors for microelectrode positioning and
movement. The relative locations of the three segments of an anterior pair of
Malpighian tubules and the connecting common ureter are indicated. Further
details in text.
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Fig. 2. Use of salicylate-selective microelectrodes for measurement of the
concentration of salicylate in droplets of fluid secreted by isolated
Malpighian tubules in the Ramsay assay. An isolated pair of Malpighian tubules
is placed in a droplet of bathing saline under paraffin oil. One Malpighian
tubule remains in the saline, and the other is pulled out and wrapped around a
stainless steel pin embedded in the Sylgard-line base of a Petri dish.
Secreted fluid droplets collect at the ureter which is positioned just outside
the bathing saline droplet. Secreted fluid droplets are collected on glass
rods and placed on the bottom of the dish adjacent to calibration droplets
containing known concentrations of salicylate in Drosophila saline.
For each droplet, the potential difference between the salicylate-selective
microelectrode (Sal ME) and the reference microelectrode is measured by a
unity gain high impedance (>1015  ) operational amplifier.
Voltages are digitized and recorded on a PC-based data acquisition system.
Salicylate concentration in the secreted droplets is calculated from the
voltage difference between the secreted droplet and the calibration droplets,
as described in the methods.
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Fig. 3. Sal-SeR microelectrode voltage recorded over an excursion of 100 µm in
Drosophila saline containing 0.5 mmol l-1 salicylate. Each
point indicates the differential signal recorded between the excursion limits
of 100 µm, using the `move, wait, sample' protocol described in Materials
and methods. In the absence of any gradients in salicylate concentration the
voltage is indicative of the noise of the electrode and measurement system.
Variation in the signal is reduced when individual measurements (0-20 min) are
compared with averages of three (30-45 min) or five (52-60 min)
measurements.
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Fig. 4. (A) Representative scan showing spatial and temporal heterogeneity of
salicylate flux at seven locations along the main segment of the
Drosophila Malpighian tubule. At each location, the position of the
Sal-SeR microelectrode at the epithelial surface is indicated by the arrowhead
of the left-most arrow. The arrows corresponding to 11 additional scans made
at intervals of 10 min at the same seven sites are indicated by the adjacent
arrows, which have been offset to the right for clarity. ASET software
calculated the salicylate-specific signal differences between the two limits
of microelectrode excursion by subtracting the voltage at the outer limit of
the excursion from that measured at the inner limit. The length of each arrow
corresponds to the mean differential signal based on three measurements at
each time interval. (B,C) Plots of differential signal of the Sal-SeR
microelectrode as a function of time at four sites in the main segments of two
Malpighian tubules. The positions of sites 2-4 relative to site 1 are
indicated in the legend. Each point is the mean of three measurements.
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Fig. 5. Salicylate flux (right axis) and differential signal (left axis) in three
segments of the Malpighian tubule, and in the midgut, the ileum and the
rectum. The height of each bar is the mean
±S.E.M. for the number of sites
indicated at the bottom of each bar. The number of different preparations is
indicated in brackets. At each site, the salicylate flux was calculated as the
mean of five replicate measurements.
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Fig. 6. Sample recording showing the change in electrical potential of a
salicylate-selective microelectrode positioned in droplets of secreted fluid
or calibration solutions. Microelectrode voltage was sampled at 3 Hz by the
data acquisition system. The labels 5, 0.5 and 0.05 refer to calibration
solutions containing 5, 0.5 or 0.05 mmol l-1 salicylate,
respectively, in Drosophila saline. d1 indicates the voltage recorded
in a droplet that was secreted over 30 min prior to the addition of salicylate
to the bathing droplet and indicates the background voltage recorded due to
endogenously secreted compounds. d2 indicates the voltage recorded in a
droplet secreted 30 min after the addition of 0.05 mmol l-1
salicylate to the bathing saline.
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Fig. 7. The effects of bathing saline salicylate concentration on (A) fluid
secretion rate, (B) secreted fluid salicylate concentration and (C) salicylate
flux. Each point is the mean ±S.E.M.
for 6-12 tubules. The inset in B shows the relationship between bathing saline
salicylate concentration (mmol l-1) and S/M, the ratio of
salicylate concentration in the secreted fluid to that in the bathing medium.
Curves in B and C were fitted to the Michaelis-Menten equation using nonlinear
regression analysis. Jmax refers top the maximum rate of
transport and Kt to the concentration of salicylate
required to produce 50% of the maximum flux.
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Fig. 8. Salicylate concentration in haemolymph samples collected at the times
indicated after injection of 0.1 µl of 100 mmol l-1 salicylate
into the haemocoel of 3rd instar larvae. Each line indicates a different
larva. The dashed line was fit by linear regression
(r2=0.998) and describes the decline in concentration of
salicylate in four samples collected from a single larva. The corresponding
equation is: [Salicylate]h=-0.0105t+3.78, where
[Salicylate]h is haemolymph salicylate concentration in mmol
l-1 and t is in minutes.
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© The Company of Biologists Ltd 2005
