Molecular characterization, localization, and physiological roles of ITP and ITP-L in the mosquito, Aedes aegypti.

The insect ion transport peptide (ITP) and its alternatively spliced variant, ITP-like peptide (ITP-L), belong to the crustacean hyperglycemic hormone family of peptides and are widely conserved among insect species. While limited, studies have characterized the ITP/ITP-L signaling system within insects, and putative functions including regulation of ion and fluid transport, ovarian maturation, and thirst/excretion have been proposed. Herein, we aimed to molecularly investigate Itp and Itp-l expression profiles in the mosquito, Aedes aegypti, examine peptide immunolocalization and distribution within the adult central nervous system, and elucidate physiological roles for these neuropeptides. Transcript expression profiles of both AedaeItp and AedaeItp-l revealed distinct enrichment patterns in adults, with AedaeItp expressed in the brain and AedaeItp-l expression predominantly within the abdominal ganglia. Immunohistochemical analysis within the central nervous system revealed expression of AedaeITP peptide in a number of cells in the brain and in the terminal ganglion. Comparatively, AedaeITP-L peptide was localized solely within the pre-terminal abdominal ganglia of the central nervous system. Interestingly, prolonged desiccation stress caused upregulation of AedaeItp and AedaeItp-l levels in adult mosquitoes, suggesting possible functional roles in water conservation and feeding-related activities. RNAi-mediated knockdown of AedaeItp caused an increase in urine excretion, while knockdown of both AedaeItp and AedaeItp-l reduced blood feeding and egg-laying in females as well as hindered egg viability, suggesting roles in reproductive physiology and behavior. Altogether, this study identifies AedaeITP and AedaeITP-L as key pleiotropic hormones, regulating various critical physiological processes in the disease vector, A. aegypti.

Mapping Transcript Cell-Specific Localization and Protein Subcellular Localization in the Adult Mosquito Aedes aegypti.

This introduction reviews techniques used to examine the distribution and expression of gene transcripts and proteins in a variety of tissues/organs in the medically important global disease vector mosquito, Aedes aegypti Specifically, these methods allow the detection of cell-specific transcript expression by fluorescent in situ hybridization; facilitate immunohistochemical mapping of a protein of interest in whole-mount small tissue/organ samples; examine the subcellular localization of proteins, such as membrane transporters, through sectioning of paraffin-embedded tissue/organ samples; and finally, enable the efficient separation of cytosolic and membrane proteins for western blot analysis without the need for specialized equipment (e.g., ultracentrifuge) in the mosquito Ae. aegypti Such techniques are useful to help answer fundamental questions in mosquito scientific research including (but not limited to) the identification of specific cells in an organ responsible for expressing a receptor of particular interest and necessary for eliciting a response to exogenous signals, including hormones. Moreover, changes in the subcellular localization of specific targets of interest can be assessed both qualitatively and quantitatively, providing insight into transient or long-term physiologically relevant regulation necessary for activity under experimental treatments or varied internal (e.g., development) or external (e.g., environmental stress) factors that might be normally experienced by the organism.

Examining Cell-Specific Localization in Aedes aegypti Tissues with Fluorescence In Situ Hybridization.

Fluorescence in situ hybridization (FISH) is a macromolecular recognition tool that uses RNA or DNA fragments combined with fluorophore- or digoxigenin-coupled nucleotides as probes to examine transcript localization through the presence or absence of complementary sequences in fixed tissues or samples under a fluorescent microscope. FISH technology has been highly effective for mapping genes and constructing a visual map of animal genomes. Here, we describe the application of FISH technology in the Aedes aegypti mosquito, where it is specifically used to localize receptor transcripts in gut tissues/organs. The methods presented highlight the synthesis of RNA probes and describe the 2-d process of incubating the tissues/organs with the RNA probes. We also describe tyramide signal amplification for improved signal detection.

Examining Protein Localization in Aedes aegypti Cells, Tissues, and Organs: Whole-Mount Immunohistochemistry.

Immunohistochemistry (IHC) is a powerful technique used for visualizing cellular components and determining the presence and/or location of proteins or other macromolecules in tissue samples. The classical IHC process involves the detection of epitopes using a highly specific primary antibody. This is followed by a secondary antibody that is coupled to a reporter molecule or fluorophore and capable of binding to the primary antibody and allowing for protein immunodetection. Although IHC does not routinely provide quantitative results compared to an enzyme-linked immunoassay or western blotting, it permits the localization of the proteins in intact tissues. This protocol describes an IHC assay for whole-body Aedes aegypti mosquito tissues that is used to detect small proteins, specifically neuropeptide hormones. This method is useful for protein detection in whole-mount preparations; however, cross-section IHC is recommended to determine if a protein is localized in the apical versus basolateral membrane of tissues/organs or to visualize immunological distribution in larger, more complex preparations.

Examining Apical or Basolateral Protein Localization in Aedes aegypti Tissues: Cross-Section Immunohistochemistry.

Immunohistochemistry (IHC) is an important technique that permits visualization of cellular components and for determining the presence and/or distribution of proteins or other macromolecules in tissue samples. Normally, IHC involves the detection of epitopes using an antigen-specific primary antibody and a secondary antibody coupled with a reporter molecule or fluorophore that can bind to the primary antibody, allowing for the spatial distribution of a protein of interest to be detected. Although normally IHC does not provide quantitative results compared to techniques such as enzyme-linked immunoassay or western blotting, it permits the localization, expression mapping, and distribution of target proteins in intact tissues. Here, we describe an IHC protocol for examining apical versus basolateral protein staining through sectioning tissue samples from fixed, embedded tissues (e.g., IHC-paraffin) and adding primary antibodies against a target protein. This IHC protocol provides a guide for tissue fixation, sectioning, and staining of tissue samples.

The V-type H+-ATPase is targeted in antidiuretic hormone control of the Malpighian "renal" tubules.

Like other insects, secretion by mosquito Malpighian tubules (MTs) is driven by the V-type H+-ATPase (VA) localized in the apical membrane of principal cells. In Aedes aegypti, the antidiuretic neurohormone CAPA inhibits secretion by MTs stimulated by select diuretic hormones; however, the cellular effectors of this inhibitory signaling cascade remain unclear. Herein, we demonstrate 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 through a NOS/cGMP/PKG signaling pathway. A critical feature of VA activation involves the reversible association of the cytosolic (V1) and membrane (Vo) complexes. Indeed, higher V1 protein abundance was found in membrane fractions of DH31-treated MTs, whereas CAPA significantly decreased V1 abundance in membrane fractions while increasing it in cytosolic fractions. V1 immunolocalization was observed strictly in the apical membrane of DH31-treated MTs, whereas immunoreactivity was dispersed following CAPA treatment. VA complexes colocalized apically in female MTs shortly after a blood meal consistent with the peak and postpeak phases of diuresis. Comparatively, V1 immunoreactivity in MTs was more dispersed and did not colocalize with the Vo complex in the apical membrane at 3 h post blood meal, representing a time point after the late phase of diuresis has concluded. Therefore, CAPA inhibition of MTs involves reducing VA activity and promotes complex dissociation hindering secretion. Collectively, these findings reveal a key target in hormone-mediated inhibition of MTs countering diuresis that provides a deeper understanding of this critical physiological process necessary for hydromineral balance.

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito.

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

Hormonal regulation and functional role of the "renal" tubules in the disease vector, Aedes aegypti.

The Aedes aegypti mosquito is a vector responsible for transmitting various arboviruses including dengue and yellow fever. Their ability to regulate the ionic and water composition of their hemolymph is a major physiological phenomenon, allowing the mosquito to adapt to a range of ecological niches. Hematophagus insects, including the female A. aegypti, face the challenge of excess salt and water intake after a blood meal. Post-prandial diuresis is under rigorous control by neuroendocrine factors, acting on the Malpighian "renal" tubules (MTs), to regulate primary urine production. The MTs are made up of two cell types; mitochondria-rich principal cells, which facilitate active transport of Na+ and K+ cations across the membrane, and thin stellate cells, which allows for transepithelial Cl- secretion. The active driving force responsible for ion transport is the apical V-type H+ ATPase, which creates a proton gradient allowing for Na+ and/or K+ cation exchange through cation/H+ antiporters. Additionally, the basolaterally localized Na+-K+-2Cl- cotransporter (NKCC) is responsible for the transport of these ions from the hemolymph into the principal cells. Numerous studies have examined hormonal regulation of the mosquito MTs and identified several diuretics including serotonin (5HT), a calcitonin-related diuretic hormone 31 (DH31), a corticotropin-related factor like diuretic peptide (DH44), a kinin-related diuretic peptide, as well as anti-diuretic factors including CAPA peptides, all of which are known to regulate fluid and ion transport by the MTs. This review therefore focuses on the control of ionic homeostasis in A. aegypti mosquitoes, emphasizing the importance of the MTs, the channels and transporters involved in maintaining hydromineral balance, and the neuroendocrine regulation of both diuresis and anti-diuresis.

CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti.

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.

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

The mosquito Aedes aegypti 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 A. aegypti 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 AedaeCAPA-1 neuropeptide on larval and adult female A. aegypti MTs stimulated with various diuretic factors including serotonin (5-HT), a corticotropin-related factor (CRF) diuretic peptide, a calcitonin-related diuretic hormone (DH31) and a kinin-related diuretic peptide. Overall, our findings establish that AedaeCAPA-1 specifically inhibits secretion of larval and adult MTs stimulated by 5-HT and DH31, whilst having no activity on MTs stimulated by other diuretic factors. Furthermore, although AedaeCAPA-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 DH31 In addition, we tested the effects of the second messenger cGMP in adult MTs. We established that cGMP has similar effects to AedaeCAPA-1, strongly inhibiting 5-HT- and DH31-stimulated fluid secretion, but with only minor effects on CRF-stimulated diuresis. Interestingly, although AedaeCAPA-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 that AedaeCAPA-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 AedaeCAPA-1 exists and may utilize cGMP as a second messenger.