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.

Insect CAPA neuropeptides are homologs of mammalian neuromedin U and are known to influence ion and water balance by regulating the activity of the Malpighian ‘renal’ tubules (MTs). Several diuretic hormones are known to increase primary fluid and ion secretion by insect MTs and, in adult female mosquitoes, a calcitonin-related peptide (DH31) called mosquito natriuretic peptide, increases sodium secretion to compensate for the excess salt load acquired during blood-feeding. An endogenous mosquito anti-diuretic hormone was recently described, having potent inhibitory activity against select diuretic hormones, including DH31. Herein, we functionally deorphanized, both in vitro and in vivo, a mosquito anti-diuretic hormone receptor (AedaeADHr) with expression analysis indicating highest enrichment in the MTs where it is localized within principal cells. Characterization using a heterologous in vitro system demonstrated the receptor was highly sensitive to mosquito CAPA neuropeptides while in vivo, AedaeADHr knockdown abolished CAPA-induced anti-diuretic control of DH31-stimulated MTs. CAPA neuropeptides are produced within a pair of neurosecretory cells in each of the abdominal ganglia, whose axonal projections innervate the abdominal neurohaemal organs, where these neurohormones are released into circulation. Lastly, pharmacological inhibition of nitric oxide synthase (NOS) and protein kinase G (PKG) signaling eliminated anti-diuretic activity of CAPA, highlighting the role of the second messenger cGMP and NOS/PKG in this anti-diuretic signaling pathway.

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Like other insects, secretion by mosquito MTs is driven by the V-type H+-ATPase (VA), which is densely packed in the apical membrane of principal cells. The anti-diuretic neurohormone CAPA inhibits tubule secretion stimulated by select diuretic hormones; however, the cellular effectors of this inhibitory signaling cascade remain unclear. Herein, we show that the VA inhibitor bafilomycin selectively inhibits serotonin (5HT)- and calcitonin-related diuretic hormone (DH31)-stimulated secretion. VA activity increases in DH31-treated MTs, whereas CAPA abolishes this increase. A critical feature of VA activation involves the reversible association of the cytosolic (V1) and membrane (Vo) complexes and results revealed higher V1 protein abundance in membrane fractions of DH31-treated MTs whereas CAPA significantly decreased V1 protein abundance in membrane fractions while increasing it in cytosolic fractions. Immunolocalization of V1 was observed strictly in the apical membrane of MTs treated with DH31 alone whereas immunoreactivity was dispersed following CAPA treatment. Elevated levels of cGMP in CAPA-treated MTs suggests activation of a phosphodiesterase (PDE), resulting in decreased levels of DH31-stimulated cAMP. Therefore, CAPA inhibition of MTs involves reducing VA activity and promotes complex dissociation hindering secretion. Collectively, these findings reveal a key target necessary for hormone-mediated inhibition of MTs required in countering diuresis providing a deeper understanding of this critical physiological process.Significance StatementThe V-type H+-ATPase (VA) proton pump, provides the driving force for transepithelial ion and fluid secretion in insect Malpighian tubules (MTs). While studies have shown that diuretic stimulation activates various signaling pathways that culminate with increased VA activity, our understanding of anti-diuretic signaling and its potential regulation involving the VA remains rudimentary. Here we show that a CAPA neuropeptide acts through the NOS/cGMP/PKG pathway that inhibits VA activity, supporting the notion that anti-diuretic regulation is mediated through reversible dissociation of the VA complexes. Our results demonstrate a critical role of VA inhibition and trafficking necessary for anti-diuretic signaling, advancing our understanding of the complex neuroendocrine regulatory mechanisms of the MTs in an important human disease vector mosquito.

Insect CAPA neuropeptides, which are homologs of mammalian neuromedin U, have been described in various insect species and are known to influence ion and water balance by regulating the activity of the Malpighian ‘renal’ tubules (MTs). A number of diuretic hormones have been shown to increase primary fluid and ion secretion by the insect MTs and, in the adult female mosquito, a calcitonin-related peptide (DH31) also known as mosquito natriuretic peptide, increases sodium secretion at the expense of potassium to remove the excess salt load acquired upon blood-feeding. An endogenous mosquito anti-diuretic hormone was recently described, having inhibitory activity against select diuretic factors and being particularly potent against DH31-stimulated diuresis. In the present study, we have functionally deorphanized, both in vitro and in vivo, a mosquito anti-diuretic hormone receptor (AedaeADHr). Expression analysis by quantitative PCR indicates the receptor is highly enriched in the MTs, and fluorescent in situ hybridization confirms expression within principal cells. Characterization using a heterologous system demonstrated the receptor was highly sensitive to mosquito CAPA peptides. In adult females, AedaeADHr transcript knockdown using RNAi led to the abolishment of CAPA-peptide induced anti-diuretic control of DH31-stimulated MTs. The neuropeptidergic ligand is produced within a pair of neurosecretory cells in each of the six abdominal ganglia, whose axonal projections innervate the abdominal neurohaemal organs (known as the perivisceral organs), where these neurohormones are released into the open circulatory system of the insect. Furthermore, pharmacological inhibition of PKG/NOS signalling abolished the anti-diuretic activity of AedaeCAPA-1, which collectively confirms the role of cGMP/PKG/NOS in this anti-diuretic signalling pathway.SignificanceInsects are by far the most successful and abundant group of organisms on earth. As a result of their small size, insects have a relatively large surface area to volume ratio, raising the potential for rapid gain or loss of water, ions and other molecules including toxins – a phenomenon that applies to insects living in both aquatic and terrestrial environments. In common with many other organisms, hormones are key regulators of the excretory system in insects, and numerous factors control the clearance of excess water and ions (i.e. diuretics) or retention of these elements (i.e. anti-diuretics). Here we characterized an endogenous anti-diuretic hormone receptor in the human disease vector, Aedes aegypti, demonstrating its expression is highly enriched in the Malpighian ‘renal’ tubules and is necessary for eliciting anti-diuretic control of this key component of the mosquito excretory system.

Harding grass (Phalaris aquatica), an invasive non-native species of bunchgrass, has been introduced to grasslands in many regions of California, particularly those with a history of disturbance, such as tilling and grazing. Due do the invasive nature of Harding grass, we sought to examine whether it has an effect on small animal abundance and diversity in the grasslands, Rancho Marino Reserve (RM) and Fiscalini Ranch Preserve (FR) in California. Both grasslands have similar climate and geographic location but differ in management history. Two transects were created in each site, with eight plots per transect. Animal cameras were deployed over the course of three nights to examine the abundance and diversity of small animals. Due to the history of tilling and planting of RM, and its increase in P. aquatica coverage, there was less animal abundance and diversity compared to FR. The results indicated that the untilled/unplanted areas had more animal abundance and diversity compared to tilled/planted due to the lack of Harding Grass. This can be due to factors such as diminished soil quality, difficulty in maneuvering in the tall grass, and adaptability to native vegetative state. Invasive plants have the ability to increase rapidly in space and potentially lead to ecosystem degradation. This adds further knowledge in the relationship between small animals and their habitats and helps conservation biologists ensure mammalian populations remain stable.