Mechanisms and Control of Ion Transport in the Gut of Larval Yellow Fever Mosquito (Aedes aegypti)

Washington State University, Pullman WA

Investigators

Abstract

In contrast to the acid stomach of vertebrates, the anterior stomach of mosquito larvae is very alkaline (pH10). More acidic conditions are restored in the posterior stomach. These observations suggest that, while the animal is feeding, the stomach epithelium continuously recycles alkali between the gut of the animal and its physiological interior. Disturbance that causes the animal to stop feeding and initiate escape behavior interrupts gut alkalinization, suggesting a tight neural and/or endocrine control of the process. It is already clear that some transport proteins characteristic of other well-studied acid or alkali secreting cells are present in the cells of the mosquito larval stomach. Although this group of researchers has identified a vacuolar-type H+ ATPase and at least one anion exchanger, other transporters involved in this system are not known. The gut is innervated by axons that express immunoreactivity to the neurotransmitter serotonin and to nitric oxide synthase, the enzyme responsible for synthesis of the transmitter nitric oxide. The gut epithelium contains endocrine cells that show immunoreactivity to peptide hormones belonging to the FMRFamide family. The effects of serotonin on the anterior stomach have been partly described, and it appears that it, plus an additional messenger or messengers, constitute a sufficient stimulatory signal for gut alkalinization. It is probable that there are also inhibitory signals that come from the CNS and/or the gut itself. The major questions of the project are aimed at the cellular mechanisms that drive alkalinization of the anterior gut, reacidification in the posterior stomach, and the identity of the neural and endocrine control pathways. Development of isolated, perfused preparations of the stomach by this team of investigators was a critical basis for the projected studies, because they allow experiments that would be impossible in the whole animal: electrophysiological characterization of transport mechanisms by measurement of the transepithelial voltage of the tissue, the intracellular voltages and ionic concentrations using ion-specific intracellular microelectrodes, and the transepithelial ionic fluxes using isotopes. The perfused preparations also will be used to assay the activities of candidate control neuropeptides. In a parallel experimental approach, the presence and cellular locations of known transport proteins will be determined by fluorescence immunohistochemistry, a technique in which antibodies generated against defined molecular targets are localized by fluorescently tagged secondary antibodies. Mosquitoes are by far the world's most medically significant insects. They are potential vectors for approximately 100 arboviruses that cause human disease, including yellow fever, dengue and a number of forms of encephalitis. They also transmit nematodes that cause elephantiasis, and plasmodia that cause malaria. The larval mosquito is an aquatic form that feeds on detritus. Although the larval form does not transmit disease, it may be more vulnerable to 3rd generation control measures than adults, because larvae are generally concentrated in aquatic breeding sites whereas the winged adults disperse widely. Gut alkalinization is believed to protect the animal from infection by killing ingested microbes and viruses. Weakening of this mechanism by a specific attack on the cellular processes, or their control signals, could make the larvae more susceptible to endemic or applied microbial pathogens. This project could provide the knowledge base for such an approach.