Exploratory phosphoproteomics profiling of Aedes aegypti Malpighian tubules during blood meal processing reveals dramatic transition in function

Abstract

Malpighian tubules, the renal organs of mosquitoes, facilitate the rapid dehydration of blood meals through aquaporin-mediated osmosis. We performed phosphoproteomics analysis of three Malpighian tubule protein-libraries (1000 tubules/sample) from unfed female mosquitoes as well as one and 24 hours after a blood meal. We identified 4663 putative phosphorylation sites in 1955 different proteins. Our exploratory dataset reveals blood meal-induced changes in phosphorylation patterns in many subunits of V-ATPase, proteins of the target of rapamycin signaling pathway, vesicle-mediated protein transport proteins, proteins involved in monocarboxylate transport, and aquaporins. Our phosphoproteomics data suggest the involvement of a variety of new pathways including nutrient-signaling, membrane protein shuttling, and paracellular water flow in the regulation of urine excretion. Our results support a model in which aquaporin channels translocate from intracellular vesicles to the cell membrane of stellate cells and the brush border membrane of principal cells upon blood feeding.

Protein localization of aquaporins in the adult female disease vector mosquito, Aedes aegypti

“…Specifically, AaAQP1 was detected on the apical membrane of the ileum and in the apical membrane of the rectal pads ( Figure 1 ) where it could play a role in absorbing water from the gut contents prior to excretion. In particular, the strong immunoreactive staining on the apical membrane of rectal pads 24hr PBM fits with the implied increased absorptive activities during this time ( 23 , 52 ). AaAQP2 was detected in the ileum at 0.5hr PBM but, we could not verify if AaAQP2 was in the rectal pads because we did not have sections of rectal pads for NBF or 0.5hr PBM.…”

“…A recent study by Kandel and colleagues examined AQP regulation in Ae. aegypti by phosphorylation and concluded that it is more likely that AQPs are regulated by membrane translocation during the post-blood feeding period in adult female mosquitoes ( 23 ).…”

“…Through the control of fluid secretion by the MTs, AaAQP regulation is also possible, however there have been limited studies on invertebrate AQP regulation. It has been proposed that AQPs can be phosphorylated during periods of stress, in addition to the possibility that they are packaged into membrane vesicles on demand, for example during diuresis ( 23 ). The goal of this study was to provide a deeper understanding of the localization of AQPs in the adult female Ae.…”

Proton-driven sodium secretion in a saline water animal

“…Instead, salt stress induction of H + ATPase expression has only been documented in salt-tolerant plants where this stress induces V-type and P-type H + ATPase expression in key tissues involved in sequestering or eliminating excess Na + 35 . Forms of post-translational regulation of VHA function have been noted in animals that involve the association or dissociation of V 0 and V 1 complexes as means to stimulate or inhibit activity respectively following an acute challenge 36 , 37 . For example, serotonin regulates VHA assembly leading to enhanced activity as described in Calliphora salivary glands 38 .…”

“…Bradley and Phillips 10 , however, found that application of serotonin, cAMP, and theophylline to in vitro preparations of A. taeniorhynchus posterior recta did not impact trans-rectal potential difference or secretion rate. More recently, using phosphoproteomics, Kandel et al 37 showed that the degree of VHA subunit phosphorylation in the Malpighian tubules of adult female Aedes aegypti did not differ between unfed and 1 h post blood meal when VHA activity is high and driving rapid urine production. Instead, VHA subunits are dephosphorylated 24 h post blood meal paralleling the reduction in ion and water transport in this organ.…”