Anti-diuretic action of a CAPA neuropeptide against a subset of diuretic hormones in the disease vector,Aedes aegypti.

Abstract

The mosquito is a vector responsible for transmitting various pathogens to humans, and their prominence as chief vectors of human disease is largely due to their anthropophilic blood feeding behaviour. Larval stage mosquitoes must deal with the potential dilution of their haemolymph in freshwater, whereas the haematophagus female faces the challenge of excess ion and water intake after a blood meal. The excretory system, composed of the Malpighian tubules (MTs) and hindgut, is strictly controlled by neuroendocrine factors, responsible for the regulation of diuresis across all developmental stages. The highly studied insect MTs are influenced by a variety of diuretic hormones and, in some insects, anti-diuretic factors. In the present study, we investigated the effects of CAPA-1 neuropeptide on larval and adult female MTs stimulated with various diuretic factors including serotonin (5-HT), a corticotropin-related factor (CRF) diuretic peptide, a calcitonin-related diuretic hormone (DH) and a kinin-related diuretic peptide. Overall, our findings establish that CAPA-1 specifically inhibits secretion of larval and adult MTs stimulated by 5-HT and DH, whilst having no activity on MTs stimulated by other diuretic factors. Furthermore, although CAPA-1 acts as an anti-diuretic, it does not influence the relative proportions of cations transported by adult MTs, thus maintaining the kaliuretic activity of 5-HT and natriuretic activity of DH In addition, we tested the effects of the second messenger cGMP in adult MTs. We established that cGMP has similar effects to CAPA-1, strongly inhibiting 5-HT- and DH-stimulated fluid secretion, but with only minor effects on CRF-stimulated diuresis. Interestingly, although CAPA-1 has no inhibitory activity on kinin-stimulated fluid secretion, cGMP strongly inhibited fluid secretion by this diuretic hormone, which targets stellate cells specifically. Collectively, these results support thatCAPA-1 inhibits select diuretic factors acting on the principal cells and this probably involves cGMP as a second messenger. Kinin-stimulated diuresis, which targets stellate cells, is also inhibited by cGMP, suggesting that another anti-diuretic factor in addition to CAPA-1 exists and may utilize cGMP as a second messenger.

An anti-diuretic hormone receptor in the human disease vector,Aedes aegypti: identification, expression analysis and functional deorphanization

“…In order to determine fluid secretion rates, a modified Ramsay secretion assay 65 was performed on isolated MTs of 3-6 day old adult female A. aegypti, as reported recently 37 . Tissue dissections were performed under physiological saline prepared as described previously 66 diluted 1:1 with Schneider’s Insect Medium (Sigma-Aldrich, Oakville, ON).…”

“…The proximal end of the MT was removed from the bathing saline and wrapped around a Minuten pin to allow for secretion measurements. Dosages of 25 nmol l −1 Drome DH 31 22 or 100 nmol l −1 5-HT 67,68 alone or in combination with 1 fmol l −1 Aedae CAPA-1 37 were applied to the isolated MTs as previously described 37 . To investigate the effects of the pharmacological blockers, a nitric oxide synthase (NOS) inhibitor, N ω -Nitro-L-arginine methyl ester hydrochloride (L-NAME), and protein kinase G (PKG) inhibitor, KT5823, were used against 5-HT- and DH 31 -stimulated MTs.…”

“…Dosages of 2 µmol l −1 L-NAME (manufacturer’s recommended dose) and 5 µmol l −1 KT5823 36 were applied to the MTs. The inhibitors were treated in conjunction with 1 fmol l −1 Aedae CAPA-1 and/or 100 nmol l −1 cyclic guanosine monophosphate, 8 bromo-cGMP (cGMP) 37 (Sigma-Aldrich, Oakville, ON, Canada). Unstimulated controls consisted of tubules bathed in physiological saline with no diuretic application.…”

“…In addition, a few anti-diuretic factors that inhibit primary urine secretion by the insect MTs have also been reported 31–36 , acting to counter the activity of the diuretic hormones that increase ion and water secretion rates. We recently identified an endogenous anti-diuretic hormone in the disease-vector mosquito, Aedes aegypti , that strongly inhibits select diuretic factors including the mosquito natriuretic peptide (a calcitonin-related diuretic hormone) 37 , which is critical for the post-prandial sodium-rich diuresis that follows blood gorging by adult females 22 . Similarly, anti-diuretic activity of CAPA neuropeptides has been reported earlier in larval A. aegypti 36 as well as in other insects 31,38–42 , with signalling involving cGMP as a second messenger 31,37,40,42,43 .…”

“…We recently identified an endogenous anti-diuretic hormone in the disease-vector mosquito, Aedes aegypti , that strongly inhibits select diuretic factors including the mosquito natriuretic peptide (a calcitonin-related diuretic hormone) 37 , which is critical for the post-prandial sodium-rich diuresis that follows blood gorging by adult females 22 . Similarly, anti-diuretic activity of CAPA neuropeptides has been reported earlier in larval A. aegypti 36 as well as in other insects 31,38–42 , with signalling involving cGMP as a second messenger 31,37,40,42,43 . In addition to their clear anti-diuretic roles, CAPA peptides have also been linked to desiccation, where desiccation stress in Drosophila melanogaster leads to upregulation of capa mRNA, which is suggested to elevate CAPA levels in the CNS 44 .…”