A larval mosquito floating around in its watery home has a tough job to do,sucking up yummy nutrients from the rotting plant material it feeds on, while maintaining just the right balance of body water and salts. Adult females face a different problem: they must extract nutrients from blood meals, yet rid themselves of excess salts and water. Both adults and larvae rely on ion pumps called ATPases in the inner and outer membranes of the alimentary canal to help maintain the water and salt balance in their bodies. However, scientists aren't sure how important two types of ATPase (Na+/K+and H+-ATPase) are in this process. Marjorie Patrick and her colleagues at the University of California, Riverside, wanted to understand more about the roles of ATPases in larval and adult mosquitoes, but first they needed to know exactly where the ATPases are located. Using gene and protein expression techniques, they set out to describe where the two ATPases are found in the alimentary canal in larval and adult mosquitoes(p. 4638).
First, the team identified where Na+/K+-ATPase and H+-ATPase genes were being expressed, using a technique known as RT-PCR. `RT-PCR asks a simple question: is a gene on, yes or no?', says Patrick. They found that both ATPase genes were transcribed, producing mRNA in all the alimentary canal tissues involved in maintaining water and salt balance, in both larvae and adults. Having shown that the genes were being transcribed in all the tissues, the team needed to show where the mRNA is translated into functional protein, in this case the membrane pumps. To do this they turned to tissue-staining techniques and confocal microscopy, which show where proteins are in the cell. Having delicately extracted the alimentary canal tissue from larvae and adults, or sectioned whole larvae and adults, the team bathed the tissue samples in solution containing fluorescently labelled antibodies, designed to stick to their target ATPase. They then examined the colourfully glowing tissues under the microscope, which lit up where Na+/K+-ATPase and H+-ATPase were pumping away in the tract's inner and outer epithelial membranes.
In both larvae and adults, Patrick says the team found a surprising result,which hadn't been seen before, in the two cell types that contribute to ion secretion in the Malpighian tubules, the insect equivalent of the kidney. They found Na+/K+-ATPase in cells called stellate cells: the staining revealed the cells had extensive finger-like projections wedged between the other cell type, the principal cells. These glowed with H+-ATPase. Also, there was a difference in ATPase position in the larvae and adult midguts. Both ATPases in larval midgut were found in the inner and outer membranes, but the position of both switched between inner and outer membrane down the whole midgut's length. In the adults, this switching didn't happen, and both ATPases were distributed evenly.
Patrick speculates that these differences are due to the different lifestyles of larval and adult mosquitoes, but needs to do more experiments to be sure. The next step, she says, is to work out what the ATPases do in their different positions along the alimentary canal. She is planning to challenge mosquitoes' ability to maintain water and salt balance by giving adults a slap-up meal and changing the chemical composition of the larvae's aquatic environment to see how different parts of the alimentary canal help maintain the status quo.
