Insects have ingenious ways of ridding themselves of unwanted toxins. Multidrug resistance genes, for example, code for transporters in some insects' Malpighian tubules that pump a wide range of noxious molecules out of insects' bodies. Two of these transporters are p-glycoprotein (p-gp) and multidrug resistance associated protein 2 (MRP2). Nestled in cell membranes,p-gp and MRP2 pump toxins out of insect tissues and are suspected to help insects resist insecticides. If we know which insects have these transporters,we might be in a better position to foil pest species' defences. Curious to find out whether fruit flies and the cricket Teleogryllus commodus, a pest species in New Zealand, possess these two toxin transporters, John Leader and Mike O'Donnell designed a clever technique to measure the transport rates of toxic fluorescent compounds(p. 4363).
To calculate how quickly fruit flies and crickets excrete the fluorescent compounds that are transported by p-gp and MRP2, the pair decided to measure fluorescence intensity in the insects' Malpighian tubules. After bathing Malpighian tubules in Texas Red, a fluorescent compound that's transported by MRP2, Leader and O'Donnell stimulated fluorescence by shining a laser through the tubules and then tried to measure the resulting fluorescence intensity. But they couldn't measure the fluorescence intensity because the laser light kept bouncing off small clumps of inorganic material sequestered in the Malpighian tubules.
Pondering how to measure the fluorescence intensity of the insects'secretions, Leader decided to extract a sample of the secreted fluid to analyze it outside the Malpighian tubules. Leader and O'Donnell suspended a drop of fluid containing fluorescent compounds in a layer of paraffin oil and bathed Malpighian tubules in the drop. Carefully pulling the open end of a tubule into the surrounding paraffin, they made a small cut in the tubule to collect a sample of secreted fluid. Realizing that measuring the sample's fluorescence in a round capillary wouldn't work, as the light would bounce off the capillary's curved edges, the pair inserted a rectangular-shaped capillary into the secreted droplet to suck up a fluid sample. Placing the flat capillary under a microscope, they shone a laser light through the secreted fluid sample to measure the sample's fluorescence intensity. They compared this to a calibration curve to calculate the concentration of the secreted fluorescent compound. To their delight, `the technique worked beautifully the first time we tried it, and every time after that', O'Donnell recalls. Now that they could reliably measure the concentration of the secreted fluorescent compounds, the pair set out to show that both fruit flies and crickets possess p-gp and MRP2 in their Malpighian tubules. Sure enough, when the team added known inhibitors of the two transporters to the bathing fluid, they noticed that the fruit fly and cricket tubules stopped secreting the fluorescent compounds; both species clearly possess these two transporters.
Using their new technique, Leader and O'Donnell calculated the transport rates of various fluorescent toxins, concluding that p-gp and MRP2 play a big part in toxin excretion for fruit flies and crickets. They hope that their simple, accurate technique to measure the activity of these two transporters will inspire researchers to apply the technique in other research areas.