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

The female Aedes aegypti mosquito is a vector for several arboviral diseases, due to their blood feeding behavior and their association with urban communities. While ion transport in Ae. aegypti has been studied, much less is known about mechanisms of water transport. Rapid water and ion excretion occurs in the adult female mosquito post blood meal and involves a set of organs including the midgut, Malpighian tubules (MTs), and hindgut. The MTs are responsible for the formation of primary urine and are considered the most important site for active transport of ions. Within the cells of the MTs, along with various ion transporters, there are aquaporin water channels that aid in the transport of water across the tubule cell membrane. Six aquaporin genes have been molecularly identified in Ae. aegypti (AQP1-6) and found to be responsible for the transport of water and in some cases, small solutes such as glycerol. In this study, we used immunohistochemistry to localize AaAQP1, 2, 4, 5, and 6 in the adult female Ae. aegypti, in non-blood fed and post blood feeding (0.5 and 24hr) conditions. We further examined the main water transporting aquaporin, AaAQP1, using western blotting to determine protein abundance changes in isolated MTs pre- and post-blood feeding. Using fluorescence in situ hybridization, aqp1 mRNA was found exclusively in the principal cells of female MTs. Finally, we used immunogold staining with transmission electron microscopy to determine subcellular localization of AaAQP1 in the Malpighian tubules under non-blood fed conditions. Interestingly, AaAQP1 was found to be predominantly in the principal cells of the MTs, dispersed throughout the brush border; however, there was also evidence of some AaAQP1 localization in the stellate cells of the MTs.

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.

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.

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.

Membrane Protein Fractionation and Analysis through Western Blot in Aedes aegypti Malpighian Tubules.

Western blot analysis is a well-known and dependable technique used to quantify protein abundance in a wide variety of samples. A major consideration for running a successful western blot is ensuring that the protein to be analyzed is purified appropriately. For work with membrane-bound proteins, traditional methods of protein processing such as the use of high-frequency sonication and ultracentrifugation to separate proteins from the membrane are being replaced with less time-consuming approaches. The use of a membrane fractionation kit, which involves the separation of membrane proteins from soluble (cytosolic) proteins, is effective in allowing for the quantification and analysis of membrane-bound proteins. In this protocol, we describe use of the membrane fractionation kit to isolate membrane-bound proteins, followed by western blot analysis, to observe protein abundance. The protocol involves methods that require organ (or tissue) collection, followed by protein processing, and a 2-d western blot procedure.

Octopamine and tyramine signalling in Aedes aegypti: Molecular characterization and insight into potential physiological roles.

In insects, the biogenic amines octopamine (OA) and tyramine (TA) are involved in controlling several physiological and behavioural processes. OA and TA act as neurotransmitters, neuromodulators or neurohormones, performing their functions by binding to specific receptors belonging to the G protein-coupled receptor (GPCR) superfamily. OA and TA along with their receptors are involved in reproduction, smell perception, metabolism, and homeostasis. Moreover, OA and TA receptors are targets for insecticides and antiparasitic agents, such as the formamidine Amitraz. In the dengue and yellow fever vector, Aedes aegypti, limited research has been reported on their OA or TA receptors. Here, we identify and molecularly characterize the OA and TA receptors in A. aegypti. Bioinformatic tools were used to identify four OA and three TA receptors in the genome of A. aegypti. The seven receptors are expressed in all developmental stages of A. aegypti; however, their highest transcript abundance is observed in the adult. Among several adult A. aegypti tissues examined, including the central nervous system, antennae and rostrum, midgut, Malpighian tubules, ovaries, and testes, the type 2 TA receptor (TAR2) transcript is most abundant in the ovaries and the type 3 TA receptor (TAR3) is enriched in the Malpighian tubules, leading us to propose putative roles for these receptors in reproduction and diuresis, respectively. Furthermore, a blood meal influenced OA and TA receptor transcript expression patterns in adult female tissues at several time points post blood meal, suggesting these receptors may play key physiological roles associated with feeding. To better understand OA and TA signalling in A. aegypti, the transcript expression profiles of key enzymes in their biosynthetic pathway, namely tyrosine decarboxylase (Tdc) and tyramine ß-hydroxylase (Tßh), were examined in developmental stages, adult tissues, and brains from blood-fed females. These findings provide information for better understanding the physiological roles of OA, TA, and their receptors in A. aegypti, and additionally, may help in the development of novel strategies for the control of these human disease vectors.

Functional insight and cell-specific expression of the adipokinetic hormone/corazonin-related peptide in the human disease vector mosquito, Aedes aegypti.

The adipokinetic hormone/corazonin-related peptide (ACP) is an insect neuropeptide structurally intermediate between corazonin (CRZ) and adipokinetic hormone (AKH). Unlike the AKH and CRZ signaling systems that are widely known for their roles in the mobilization of energy substrates and stress responses, respectively, the main role of ACP and its receptor (ACPR) remains unclear in most arthropods. The current study aimed to localize the distribution of ACP in the nervous system and provide insight into its physiological roles in the disease vector mosquito, Aedes aegypti. Immunohistochemical analysis and fluorescence in situ hybridization localized the ACP peptide and transcript within a number of cells in the central nervous system, including two pairs of laterally positioned neurons in the protocerebrum of the brain and a few ventrally localized neurons within the pro- and mesothoracic regions of the fused thoracic ganglia. Further, extensive ACP-immunoreactive axonal projections with prominent blebs and varicosities were observed traversing the abdominal ganglia. Given the prominent enrichment of ACPR expression within the abdominal ganglia of adult A. aegypti mosquitoes as determined previously, the current results indicate that ACP may function as a neurotransmitter and/or neuromodulator facilitating communication between the brain and posterior regions of the nervous system. In an effort to elucidate a functional role for ACP signaling, biochemical measurement of energy substrates in female mosquitoes revealed a reduction in abdominal fat body in response to ACP that matched the actions of AKH, but interestingly, a corresponding hypertrehalosaemic effect was only found in response to AKH since ACP did not influence circulating carbohydrate levels. Comparatively, both ACP and AKH led to a significant increase in haemolymph carbohydrate levels in male mosquitoes while both peptides had no influence on their glycogen stores. Neither ACP nor AKH influenced circulating or stored lipid levels in both male and female mosquitoes. Collectively, these results reveal ACP signaling in mosquitoes may have complex sex-specific actions, and future research should aim to expand knowledge on the role of this understudied neuropeptide.

The Peptide Hormone CNMa Influences Egg Production in the Mosquito Aedes aegypti.

Mosquito reproduction is regulated by a suite of hormones, many acting through membrane-bound receptor proteins. The Aedes aegypti G protein-coupled receptors AAEL024199 (AeCNMaR-1a) and AAEL018316 (AeCNMaR-1b) were identified as orthologs of the Drosophila melanogaster CNMa receptor (DmCNMaR). The receptor was duplicated early in the evolution of insects, and subsequently in Culicidae, into what we refer to as CNMaR-1a and CNMaR-1b. AeCNMaR-1a is only detected in male mosquito antennae while AeCNMaR-1b is expressed at high levels in mosquito ovaries. Using a heterologous cell assay, we determined that AeCNMa activates AeCNMaR-1a with a ~10-fold lower concentration than it does AeCNMaR-1b, though both receptors displayed half maximal effective concentrations of AeCNMa in the low nanomolar range. Finally, we show that injections of AeCNMa into blood-fed mated female Ae. aegypti resulted in fewer eggs laid.

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.

Production, composition, and mode of action of the painful defensive venom produced by a limacodid caterpillar, Doratifera vulnerans.

Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by D. vulnerans Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides.

A neuroendocrine pathway modulating osmotic stress in Drosophila.

Environmental factors challenge the physiological homeostasis in animals, thereby evoking stress responses. Various mechanisms have evolved to counter stress at the organism level, including regulation by neuropeptides. In recent years, much progress has been made on the mechanisms and neuropeptides that regulate responses to metabolic/nutritional stress, as well as those involved in countering osmotic and ionic stresses. Here, we identified a peptidergic pathway that links these types of regulatory functions. We uncover the neuropeptide Corazonin (Crz), previously implicated in responses to metabolic stress, as a neuroendocrine factor that inhibits the release of a diuretic hormone, CAPA, and thereby modulates the tolerance to osmotic and ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Mapping of the Crz receptor (CrzR) expression identified three pairs of Capa-expressing neurons (Va neurons) in the ventral nerve cord that mediate these effects of Crz. We show that Crz acts to restore water/ion homeostasis by inhibiting release of CAPA neuropeptides via inhibition of cAMP production in Va neurons. Knockdown of CrzR in Va neurons affects CAPA signaling, and consequently increases tolerance for desiccation, ionic stress and starvation, but delays chill-coma recovery. Optogenetic activation of Va neurons stimulates excretion and simultaneous activation of Crz and CAPA-expressing neurons reduces this response, supporting the inhibitory action of Crz. Thus, Crz inhibits Va neurons to maintain osmotic and ionic homeostasis, which in turn affects stress tolerance. Earlier work demonstrated that systemic Crz signaling restores nutrient levels by promoting food search and feeding. Here we additionally propose that Crz signaling also ensures osmotic homeostasis by inhibiting release of CAPA neuropeptides and suppressing diuresis. Thus, Crz ameliorates stress-associated physiology through systemic modulation of both peptidergic neurosecretory cells and the fat body in Drosophila.

Receptor Characterization and Functional Activity of Pyrokinins on the Hindgut in the Adult Mosquito, Aedes aegypti.

Pyrokinins are structurally related insect neuropeptides, characterized by their myotropic, pheromonotropic and melanotropic roles in some insects, but their function is unclear in blood-feeding arthropods. In the present study, we functionally characterized the pyrokinin-1 and pyrokinin-2 receptors (PK1-R and PK2-R, respectively), in the yellow fever mosquito, Aedes aegypti, using a heterologous cell system to characterize their selective and dose-responsive activation by members of two distinct pyrokinin subfamilies. We also assessed transcript-level expression of these receptors in adult organs and found the highest level of PK1-R transcript in the posterior hindgut (rectum) while PK2-R expression was enriched in the anterior hindgut (ileum) as well as in reproductive organs, suggesting these to be prominent target sites for their peptidergic ligands. In support of this, PRXa-like immunoreactivity (where X = V or L) was localized to innervation along the hindgut. Indeed, we identified a myoinhibitory role for a PK2 on the ileum where PK2-R transcript was enriched. However, although we found that PK1 did not influence myoactivity or Na+ transport in isolated recta, the PRXa-like immunolocalization terminating in close association to the rectal pads and the significant enrichment of PK1-R transcript in the rectum suggests this organ could be a target of PK1 signaling and may regulate the excretory system in this important disease vector species.

Expression Profiling, Downstream Signaling, and Inter-subunit Interactions of GPA2/GPB5 in the Adult Mosquito Aedes aegypti.

GPA2/GPB5 and its receptor constitute a glycoprotein hormone-signaling system native to the genomes of most vertebrate and invertebrate organisms. Unlike the well-studied gonadotropins and thyrotropin, the exact function of GPA2/GPB5 remains elusive, and whether it elicits its functions as heterodimers, homodimers or as independent monomers remains unclear. Here, the glycoprotein hormone signaling system was investigated in adult mosquitoes, where GPA2 and GPB5 subunit expression was mapped and modes of its signaling were characterized. In adult Aedes aegypti mosquitoes, GPA2 and GPB5 transcripts co-localized to bilateral pairs of neuroendocrine cells, positioned within the first five abdominal ganglia of the central nervous system. Unlike GPA2/GPB5 homologs in human and fly, GPA2/GPB5 subunits in A. aegypti lacked evidence of heterodimerization. Rather, cross-linking analysis to determine subunit interactions revealed A. aegypti GPA2 and GPB5 subunits may form homodimers, although treatments with independent subunits did not demonstrate receptor activity. Since mosquito GPA2/GPB5 heterodimers were not evident by heterologous expression, a tethered fusion construct was generated for expression of the subunits as a single polypeptide chain to mimic heterodimer formation. Our findings revealed A. aegypti LGR1 elicited constitutive activity with elevated levels of cAMP. However, upon treatment with recombinant tethered GPA2/GPB5, an inhibitory G protein (Gi/o) signaling cascade is initiated and forskolin-induced cAMP production is inhibited. These results further support the notion that heterodimerization is a requirement for glycoprotein hormone receptor activation and provide novel insight to how signaling is achieved for GPA2/GPB5, an evolutionary ancient neurohormone.