Effects of dietary calcium (Ca2+) and blood feeding on the immunochemical expression of the plasma membrane Ca2+-ATPase (PMCA) in Malpighian tubules of adult female mosquitoes (Aedes aegypti).

Insect Malpighian tubules contribute to Ca2+ homeostasis via Ca2+ storage in intracellular compartments, Ca2+ secretion into the tubule lumen, and Ca2+ reabsorption into the hemolymph. A plasma membrane Ca2+-ATPase (PMCA) is hypothesized to be a Ca2+-transporter involved in renal Ca2+ transport of insects, however few studies have investigated its immunochemical expression in Malpighian tubules. Here we characterized the abundance and localization of PMCA-like immunoreactivity in Malpighian tubules of adult female mosquitoes Aedes aegypti using an antibody against Drosophila melanogaster PMCA. Western blotting revealed expression of a relatively abundant 109 kDa isoform and a relatively sparse 115 kDa isoform. Feeding mosquitoes 10% sucrose with 50 mM CaCl2 for 7 days did not affect PMCA immunoreactivity. However, at 24, 48, and 96 h post-blood feeding (PBF), the relative abundance of the 109 kDa isoform decreased while that of the 115 kDa isoform increased. Immunolabeling of Malpighian tubules revealed PMCA-like immunoreactivity in both principal and stellate cells; principal cell labeling was intracellular, whereas stellate cell labeling was along the basal membrane. Blood feeding enhanced immunolabeling of PMCA in stellate cells but weakened that in principal cells. Moreover, a unique apicolateral pattern of PMCA-like immunolabeling occurred in principal cells of the proximal segment at 24 h PBF, suggesting potential trafficking to septate junctions. Our results suggest PMCA isoforms are differentially expressed and localized in mosquito Malpighian tubules where they contribute to redistributing tubule Ca2+ during blood meal processing.

Function and regulation of the insect NaCCC2 sodium transport proteins.

NaCCC2 transport proteins, including those from Drosophila melanogaster (Ncc83) and Aedes aegypti (aeCCC2), are an insect-specific clade with sequence similarity to Na+-K+-2Cl- cotransporters. Whereas the Na+-K+-2Cl- cotransporters and other cation-chloride cotransporters are electroneutral, recent work indicates that Ncc83 and aeCCC2 carry charge across membranes. Here, we further characterize the regulation and transport properties of Ncc83 and aeCCC2 expressed in Xenopus oocytes. In cation uptake experiments, Li+ was used as a tracer for Na+ and Rb+ was used as a tracer for K+. Li+ uptake of oocytes expressing either aeCCC2 or Ncc83 was greater than uptake in water-injected controls, activated by hypotonic swelling, and not inhibited by ouabain or ethyl cinnamate. Rb+ uptake of oocytes expressing either aeCCC2 or Ncc83 was not different than water injected controls. In oocytes expressing either aeCCC2 or Ncc83, Li+ uptake plateaued with increasing Li+ concentrations, with apparent Km values in the range of 10 to 20 mM. Following exposure to ouabain, intracellular [Na+] was greater in oocytes expressing aeCCC2 than in controls. Elevating intracellular cAMP (via 8-bromo-cAMP) in Ncc83 oocytes significantly stimulated both Li+ uptake and membrane conductances. Elevating intracellular cAMP in aeCCC2 oocytes did not affect Li+ uptake, but stimulated membrane conductances. Overall, these results confirm that the NaCCC2s resemble other cation-chloride cotransporters in their regulation and some transport properties. However, unlike other cation-chloride cotransporters, they carry charge across membranes.

The Molecular Physiology and Toxicology of Inward Rectifier Potassium Channels in Insects.

Inward rectifier K+ (Kir) channels have been studied extensively in mammals, where they play critical roles in health and disease. In insects, Kir channels have recently been found to be key regulators of diverse physiological processes in several tissues. The importance of Kir channels in insects has positioned them to serve as emerging targets for the development of insecticides with novel modes of action. In this article, we provide the first comprehensive review of insect Kir channels, highlighting the rapid progress made in understanding their molecular biology, physiological roles, pharmacology, and toxicology. In addition, we highlight key gaps in our knowledge and suggest directions for future research to advance our understanding of Kir channels and their roles in insect physiology. Further knowledge of their functional roles will also facilitate their exploitation as targets for controlling arthropod pests and vectors of economic, medical, and/or veterinary relevance.

Stop the crop: Insights into the insecticidal mode of action of cinnamodial against mosquitoes.

Cinnamodial (CDIAL) is a drimane sesquiterpene dialdehyde found in the bark of Malagasy medicinal plants (Cinnamosma species; family Canellaceae). We previously demonstrated that CDIAL was insecticidal, antifeedant, and repellent against Aedes aegypti mosquitoes. The goal of the present study was to generate insights into the insecticidal mode of action for CDIAL, which is presently unknown. We evaluated the effects of CDIAL on the contractility of the ventral diverticulum (crop) isolated from adult female Ae. aegypti. The crop is a food storage organ surrounded by visceral muscle that spontaneously contracts in vitro. We found that CDIAL completely inhibited spontaneous contractions of the crop as well as those stimulated by the agonist 5-hydroxytryptamine. Several derivatives of CDIAL with known insecticidal activity also inhibited crop contractions. Morphometric analyses of crops suggested that CDIAL induced a tetanic paralysis that was dependent on extracellular Ca2+ and inhibited by Gd3+, a non-specific blocker of plasma membrane Ca2+ channels. Screening of numerous pharmacological agents revealed that a Ca2+ ionophore (A23187) was the only compound other than CDIAL to completely inhibit crop contractions via a tetanic paralysis. Taken together, our results suggest that CDIAL induces a tetanic paralysis of the crop by elevating intracellular Ca2+ through the activation of plasma membrane Ca2+ channels, which may explain the insecticidal effects of CDIAL against mosquitoes. Our pharmacological screening experiments also revealed the presence of two regulatory pathways in mosquito crop contractility not previously described: an inhibitory glutamatergic pathway and a stimulatory octopaminergic pathway. The latter pathway was also completely inhibited by CDIAL.

Malpighian tubules of Trichoplusia ni: recycling ions via gap junctions and switching between secretion and reabsorption of Na+ and K+ in the distal ileac plexus.

The functional kidney in insects consists of the Malpighian tubules and hindgut. Malpighian tubules secrete ions and fluid aiding in hydromineral homeostasis, acid-base balance and metabolic waste excretion. In many insects, including lepidopterans, the Malpighian tubule epithelium consists of principal cells (PCs) and secondary cells (SCs). The SCs in the Malpighian tubules of larvae of the lepidopteran Trichoplusia ni have been shown to reabsorb K+, transporting it in a direction opposite to that in the neighbouring PCs that secrete K+ One of the mechanisms that could enable such an arrangement is a gap junction (GJ)-based coupling of the two cell types. In the current study, we have immunolocalized GJ protein Innexin-2 to the PC-PC and SC-PC cell-cell borders. We have demonstrated that GJs in the SC-containing region of the Malpighian tubules enable Na+ and K+ reabsorption by the SCs. We also demonstrated that in ion-loaded animals, PCs switch from Na+/K+ secretion to reabsorption, resulting in an ion-transporting phenotype similar to that of tubules with pharmacologically blocked GJs. Concomitantly, mRNA abundance encoding GJ proteins was downregulated. Finally, we observed that such PC-based reabsorption was only present in the distal ileac plexus connected to the rectal complex. We propose that this plasticity in the PC function in the distal ileac plexus is likely to be aimed at providing an ion supply for the SC function in this segment of the tubule.

Differential expression of putative sodium-dependent cation-chloride cotransporters in Aedes aegypti.

The yellow fever mosquito, Aedes aegypti, has three genes that code for proteins with sequence similarity to vertebrate Na+-K+-Cl- cotransporters (NKCCs) of the solute-linked carrier 12 superfamily of cation-chloride cotransporters (CCCs). We hypothesized that these mosquito NKCC orthologues have diverged to perform distinct roles in salt secretion and absorption. In phylogenetic analyses, one protein (aeNKCC1) groups with a Drosophila melanogaster NKCC that mediates salt secretion whereas two others (aeCCC2 and aeCCC3) group with a Drosophila transporter that is not functionally characterized. The aeCCC2 and aeCCC3 genes probably result from a tandem gene duplication in the mosquito lineage; they have similar exon structures and are consecutive in genomic DNA. Predicted aeCCC2 and aeCCC3 proteins differ from aeNKCC1 and vertebrate NKCCs in residues from the third transmembrane domain known to influence ion and inhibitor binding. Quantitative PCR revealed that aeNKCC1 and aeCCC2 were approximately equally expressed in larvae and adults, whereas aeCCC3 was approximately 100-fold more abundant in larvae than in adults. In larval tissues, aeCCC2 was approximately 2-fold more abundant in Malpighian tubules compared to anal papillae. In contrast, aeCCC3 was nearly 100-fold more abundant in larval anal papillae compared to Malpighian tubules, suggesting a role in absorption. Western blots with polyclonal antibodies against isoform-specific peptides revealed stronger aeCCC2 immunoreactivity in adults versus larvae, whereas aeCCC3 immunoreactivity was stronger in larvae versus adults. The differential expression pattern of aeCCC2 and aeCCC3, and their sequence divergence in transmembrane domains, suggests that they may have different roles in transepithelial salt transport.

The excretion of NaCl and KCl loads in mosquitoes. 1. Control data.

The handling of Na(+) and K(+) loads was investigated in isolated Malpighian tubules and in whole mosquitoes of Aedes aegypti. Isolated Malpighian tubules bathed in Na(+)-rich Ringer solution secreted Na(+)-rich fluid, and tubules bathed in K(+)-rich Ringer solution secreted K(+)-rich fluid. Upon Na(+) loading the hemolymph, the mosquito removed 77% the injected Na(+) within the next 30 min. The rapid onset and magnitude of this diuresis and the excretion of more Na(+) than can be accounted for by tubular secretion in vitro is consistent with the release of the calcitonin-like diuretic hormone in the mosquito to remove the Na(+) load from the hemolymph. Downstream, K(+) was reabsorbed with water in the hindgut, which concentrated Na(+) in excreted urine hyperosmotic to the hemolymph. Upon K(+) loading the hemolymph, the mosquito took 2 h to remove 100% of the injected K(+) from the hemolymph. The excretion of K(+)-rich isosmotic urine was limited to clearing the injected K(+) from the hemolymph with a minimum of Cl(-) and water. As a result, 43.3% of the injected Cl(-) and 48.1% of the injected water were conserved. The cation retained in the hemolymph with Cl(-) was probably N-methyl-d-glucamine, which replaced Na(+) in the hemolymph injection of the K(+) load. Since the tubular secretion of K(+) accounts for the removal of the K(+) load from the hemolymph, the reabsorption of K(+), Na(+), Cl(-), and water must be inhibited in the hindgut. The agents mediating this inhibition are unknown.

Excretion of NaCl and KCl loads in mosquitoes. 2. Effects of the small molecule Kir channel modulator VU573 and its inactive analog VU342.

The effect of two small molecules VU342 and VU573 on renal functions in the yellow fever mosquito Aedes aegypti was investigated in vitro and in vivo. In isolated Malpighian tubules, VU342 (10 µM) had no effect on the transepithelial secretion of Na(+), K(+), Cl(-), and water. In contrast, 10 µM VU573 first stimulated and then inhibited the transepithelial secretion of fluid when the tubules were bathed in Na(+)-rich or K(+)-rich Ringer solution. The early stimulation was blocked by bumetanide, suggesting the transient stimulation of Na-K-2Cl cotransport, and the late inhibition of fluid secretion was consistent with the known block of AeKir1, an Aedes inward rectifier K(+) channel, by VU573. VU342 and VU573 at a hemolymph concentration of about 11 µM had no effect on the diuresis triggered by hemolymph Na(+) or K(+) loads. VU342 at a hemolymph concentration of 420 µM had no effect on the diuresis elicited by hemolymph Na(+) or K(+) loads. In contrast, the same concentration of VU573 significantly diminished the Na(+) diuresis by inhibiting the urinary excretion of Na(+), Cl(-), and water. In K(+)-loaded mosquitoes, 420 µM VU573 significantly diminished the K(+) diuresis by inhibiting the urinary excretion of K(+), Na(+), Cl(-), and water. We conclude that 1) the effects of VU573 observed in isolated Malpighian tubules are overwhelmed in vivo by the diuresis triggered with the coinjection of Na(+) and K(+) loads, and 2) at a hemolymph concentration of 420 µM VU573 affects Kir channels systemically, including those that might be involved in the release of diuretic hormones.

Evidence for intercellular communication in mosquito renal tubules: a putative role of gap junctions in coordinating and regulating the rapid diuretic effects of neuropeptides.

Adult female mosquitoes require a blood meal from a vertebrate host to successfully reproduce. During a single blood feeding, a female may ingest more than the equivalent of her own body mass, resulting in an acute stress to osmotic and ionic homeostasis. In response to this stress, the renal (Malpighian) tubules mediate a rapid diuresis that commences as soon as blood is ingested. The diuresis is regulated by neuropeptides (e.g., kinins, calcitonin-like peptide) that act on receptors in the Malpighian tubule epithelium. Interestingly, the expression of these receptors is discontinuous throughout the epithelium, which raises the question as to how Malpighian tubules mount such a rapid and synchronized response to neuropeptide stimulation. Here we propose a hypothesis that gap junctions functionally couple the epithelial cells of Malpighian tubules, resulting in a coordinated physiological response to the binding of neuropeptides. We review recent, relevant literature on the electrophysiology, physiology, and molecular biology of mosquito Malpighian tubules that indicate the presence of gap junctions in the epithelium. We also provide new physiological and immunochemical data that are consistent with the proposed hypothesis.

Larvicidal Activity of Hemp Extracts and Cannabidiol against the Yellow Fever Mosquito Aedes aegypti.

To mitigate pyrethroid resistance in mosquito vectors of emerging and re-emerging human pathogens, there is an urgent need to discover insecticides with novel modes of action. Natural alternatives, such as extracts derived from plants, may serve as substitutes for traditional synthetic insecticides if they prove to be sustainable, cost-effective, and safe for non-target organisms. Hemp (Cannabis sativa) is a sustainable plant known to produce various secondary metabolites with insecticidal properties, including terpenoids and flavonoids. The goal of this study was to assess the larvicidal activity of hemp leaf extract on mosquito larvae from both pyrethroid-susceptible (PS) and pyrethroid-resistant (PR) strains of Aedes aegypti. Another goal was to identify which components of the extract were responsible for any observed larvicidal activity. We found that a methanol extract of hemp leaves induced similar concentration-dependent larvicidal activity against PS (LC50: 4.4 ppm) and PR (LC50: 4.3 ppm) strains within 48 h. Partitioning of the leaf extract between methanol and hexane fractions revealed that full larvicidal activity was restricted to the methanol fraction. Analysis of this fraction by gas chromatography-mass spectrometry and nuclear magnetic resonance showed it to be dominated by cannabidiol (CBD). Larvicidal assays using authentic CBD confirmed this compound was primarily responsible for the toxicity of the hemp leaf extract against both strains. We conclude that hemp leaf extracts and CBD have the potential to serve as viable sources for the development of novel mosquito larvicides.

Dietary calcium (Ca2+) impacts Ca2+ content and molecular expression of Ca2+-transporters in Malpighian tubules of the yellow fever mosquito, Aedes aegypti.

The renal (Malpighian) tubules of insects play important roles in hemolymph Ca2+ regulation. Here we investigated how dietary Ca2+ loads from sucrose or blood meals affect the Ca2+ content and mRNA expression of Ca2+ transporters in Malpighian tubules of adult female mosquitoes. Using the yellow fever mosquito Aedes aegypti we found that feeding females 10% sucrose with elevated Ca2+ concentration ad libitum for 6 days led to increased Ca2+ content in Malpighian tubules. The increases of Ca2+ content correlated with up-regulations of mRNAs encoding intracellular Ca2+-ATPases (SERCA and SPCA), a plasma membrane Ca2+-ATPase (PMCA), and a K+-dependent Na+/Ca2+ exchanger (NCKX1). We also found that when adult females were fed blood, tubule Ca2+ content changed dynamically over the next 72 h in a manner consistent with redistribution of tubule Ca2+ stores to other tissues (e.g., ovaries). The changes in tubule Ca2+ were correlated with dynamic changes in mRNA abundances of SERCA, SPCA, PMCA, and NCKX1. Our results are the first to demonstrate that Malpighian tubules of adult female mosquitoes have a remarkable capacity to handle high dietary Ca2+ loads, most likely through the combination of storing excess Ca2+ within intracellular compartments and secreting it into the tubule lumen for excretion. Our results also suggest that the Malpighian tubules play key roles in supplying Ca2+ to other tissues during the processing of blood meals.

Larvicidal Activity of Carbon Black against the Yellow Fever Mosquito Aedes aegypti.

The yellow fever mosquito Aedes aegypti is one of the deadliest animals on the planet because it transmits several medically important arboviruses, including Zika, chikungunya, dengue, and yellow fever. Carbon-based nanoparticles (CNPs) derived from natural sources have previously been shown to have toxic effects on mosquito larvae and offer a potential alternative to chemical insecticides such as pyrethroids, for which mosquitoes have evolved resistance. However, CNPs derived from industrial sources, such as carbon black, have not previously been evaluated as larvicides. Here, we evaluate the effects of a commercially-available carbon black, EMPEROR® 1800 (E1800), on mortality and development of pyrethroid-susceptible (PS) and pyrethroid-resistant (PR) strains of Ae. aegypti. We found that E1800 exhibited concentration-dependent mortality against 1st instar larvae of both strains within the first 120 h after exposure, but after this period, surviving larvae did not show delays in their development to adults. Physical characterization of E1800 suspensions suggests that they form primary particles of ~30 nm in diameter that fuse into fundamental aggregates of ~170 nm in diameter. Notably, larvae treated with E1800 showed internal accumulation of E1800 in the gut and external accumulation on the respiratory siphon, anal papillae, and setae, suggesting a physical mode of toxic action. Taken together, our results suggest that E1800 has potential use as a larvicide with a novel mode of action for controlling PS and PR mosquitoes.

Sequence analysis and function of mosquito aeCCC2 and Drosophila Ncc83 orthologs.

Dipteran insects have genes that code for two different Na+-dependent cation-chloride cotransporter (CCC) paralogs. Aedes aegypti aeNKCC1 is an ortholog of Drosophila melanogaster Ncc69, a bumetanide-sensitive Na+-K+-2Cl- cotransporter (NKCC). Aedes aegypti aeCCC2 and aeCCC3 are orthologs of Drosophila Ncc83. Prior work suggests that the transport properties of aeCCC2 differ from canonical NKCCs. In particular, Xenopus oocytes expressing aeCCC2 have increased Na+-dependent membrane currents compared to controls, whereas NKCCs are electroneutral. Here, we further evaluated the function and localization of aeCCC2 and Ncc83. In oocytes expressing aeCCC2 or Ncc83, membrane potential (Vm) hyperpolarized upon Na+ removal; following hypotonic exposure the change in Vm was greater than it was in controls. In voltage-clamp experiments, membrane currents were concentration dependent on Na+ with an apparent affinity (Km) of approximately 4.6 mM. In Malpighian tubules of larval and adult mosquitoes, aeCCC2 was localized along the basolateral aspect of principal cells. Sequence comparisons among transporters from Drosophila, Aedes, Anopheles, and Culex revealed 33 residues within the transmembrane domains (TMDs) that are fully conserved within paralogs but that differ between orthologs of NKCC1 and orthologs of aeCCC2/Ncc83. These residues are distributed across all 12 TMDs. Our results provide a foundation for further exploration of the structural basis for functional differences between insect Na+-dependent CCCs.

Role of an apical K,Cl cotransporter in urine formation by renal tubules of the yellow fever mosquito (Aedes aegypti).

The K,Cl cotransporters (KCCs) of the SLC12 superfamily play critical roles in the regulation of cell volume, concentrations of intracellular Cl(-), and epithelial transport in vertebrate tissues. To date, the role(s) of KCCs in the renal functions of mosquitoes and other insects is less clear. In the present study, we sought molecular and functional evidence for the presence of a KCC in renal (Malpighian) tubules of the mosquito Aedes aegypti. Using RT-PCR on Aedes Malpighian tubules, we identified five alternatively spliced partial cDNAs that encode putative SLC12-like KCCs. The majority transcript is AeKCC1-A(1); its full-length cDNA was cloned. After expression of the AeKCC1-A protein in Xenopus oocytes, the Cl(-)-dependent uptake of (86)Rb(+) is 1) activated by 1 mM N-ethylmaleimide and cell swelling, 2) blocked by 100 µM dihydroindenyloxyalkanoic acid (DIOA), and 3) dependent upon N-glycosylation of AeKCC1-A. In Aedes Malpighian tubules, AeKCC1 immunoreactivity localizes to the apical brush border of principal cells, which are the predominant cell type in the epithelium. In vitro physiological assays of Malpighian tubules show that peritubular DIOA (10 µM): 1) significantly reduces both the control and diuretic rates of transepithelial fluid secretion and 2) has negligible effects on the membrane voltage and input resistance of principal cells. Taken together, the above observations indicate the presence of a KCC in the apical membrane of principal cells where it participates in a major electroneutral transport pathway for the transepithelial secretion of fluid in this highly electrogenic epithelium.

Further SAR on the (Phenylsulfonyl)piperazine Scaffold as Inhibitors of the Aedes aegypti Kir1 (AeKir) Channel and Larvicides.

Zika virus (ZIKV), dengue fever (DENV) and chikungunya (CHIKV) are arboviruses that are spread to humans from the bite of an infected adult female Aedes aegypti mosquito. As there are no effective vaccines or therapeutics for these diseases, the primary strategy for controlling the spread of these viruses is to prevent the mosquito from biting humans through the use of insecticides. Unfortunately, the commonly used classes of insecticides have seen a significant increase in resistance, thus complicating control efforts. Inhibiting the renal inward rectifier potassium (Kir) channel of the mosquito vector Aedes aegypti has been shown to be a promising target for the development of novel mosquitocides. We have shown that Kir1 channels play key roles in mosquito diuresis, hemolymph potassium homeostasis, flight, and reproduction. Previous work from our laboratories identified a novel (phenylsulfonyl)piperazine scaffold as potent AeKir channel inhibitors with activity against both adult and larval mosquitoes. Herein, we report further SAR work around this scaffold and have identified additional compounds with improved in vitro potency and mosquito larvae toxicity.

A SLC4-like anion exchanger from renal tubules of the mosquito (Aedes aegypti): evidence for a novel role of stellate cells in diuretic fluid secretion.

Transepithelial fluid secretion across the renal (Malpighian) tubule epithelium of the mosquito (Aedes aegypti) is energized by the vacuolar-type (V-type) H(+)-ATPase and not the Na(+)-K(+)-ATPase. Located at the apical membrane of principal cells, the V-type H(+)-ATPase translocates protons from the cytoplasm to the tubule lumen. Secreted protons are likely to derive from metabolic H(2)CO(3), which raises questions about the handling of HCO(3)(-) by principal cells. Accordingly, we tested the hypothesis that a Cl/HCO(3) anion exchanger (AE) related to the solute-linked carrier 4 (SLC4) superfamily mediates the extrusion of HCO(3)(-) across the basal membrane of principal cells. We began by cloning from Aedes Malpighian tubules a full-length cDNA encoding an SLC4-like AE, termed AeAE. When expressed heterologously in Xenopus oocytes, AeAE is both N- and O-glycosylated and mediates Na(+)-independent intracellular pH changes that are sensitive to extracellular Cl(-) concentration and to DIDS. In Aedes Malpighian tubules, AeAE is expressed as two distinct forms: one is O-glycosylated, and the other is N-glycosylated. Significantly, AeAE immunoreactivity localizes to the basal regions of stellate cells but not principal cells. Concentrations of DIDS that inhibit AeAE activity in Xenopus oocytes have no effects on the unstimulated rates of fluid secretion mediated by Malpighian tubules as measured by the Ramsay assay. However, in Malpighian tubules stimulated with kinin or calcitonin-like diuretic peptides, DIDS reduces the diuretic rates of fluid secretion to basal levels. In conclusion, Aedes Malpighian tubules express AeAE in the basal region of stellate cells, where this transporter may participate in producing diuretic rates of transepithelial fluid secretion.

The single kinin receptor signals to separate and independent physiological pathways in Malpighian tubules of the yellow fever mosquito.

In the past, we have used the kinins of the cockroach Leucophaea (the leucokinins) to evaluate the mechanism of diuretic action of kinin peptides in Malpighian tubules of the yellow fever mosquito Aedes aegypti. Now using the kinins of Aedes (the aedeskinins), we have found that in isolated Aedes Malpighian tubules all three aedeskinins (1 microM) significantly 1) increased the rate of fluid secretion (V(S)), 2) hyperpolarized the basolateral membrane voltage (V(bl)), and 3) decreased the input resistance (R(in)) of principal cells, consistent with the known increase in the Cl(-) conductance of the paracellular pathway in Aedes Malpighian tubules. Aedeskinin-III, studied in further detail, significantly increased V(S) with an EC(50) of 1.5 x 10(-8) M. In parallel, the Na(+) concentration in secreted fluid significantly decreased, and the K(+) concentration significantly increased. The concentration of Cl(-) remained unchanged. While the three aedeskinins triggered effects on V(bl), R(in), and V(S), synthetic kinin analogs, which contain modifications of the COOH-terminal amide pentapeptide core sequence critical for biological activity, displayed variable effects. For example, kinin analog 1578 significantly stimulated V(S) but had no effect on V(bl) and R(in), whereas kinin analog 1708 had no effect on V(S) but significantly affected V(bl) and R(in). These observations suggest separate signaling pathways activated by kinins. One triggers the electrophysiological response, and the other triggers fluid secretion. It remains to be determined whether the two signaling pathways emanate from a single kinin receptor via agonist-directed signaling or from a differentially glycosylated receptor. Occasionally, Malpighian tubules did not exhibit a detectable response to natural and synthetic kinins. Hypothetically, the expression of the kinin receptor may depend on developmental, nutritional, and/or reproductive signals.

Semi-synthetic cinnamodial analogues: Structural insights into the insecticidal and antifeedant activities of drimane sesquiterpenes against the mosquito Aedes aegypti.

The Aedes aegypti mosquito serves as a major vector for viral diseases, such as dengue, chikungunya, and Zika, which are spreading across the globe and threatening public health. In addition to increased vector transmission, the prevalence of insecticide-resistant mosquitoes is also on the rise, thus solidifying the need for new, safe and effective insecticides to control mosquito populations. We recently discovered that cinnamodial, a unique drimane sesquiterpene dialdehyde of the Malagasy medicinal plant Cinnamosma fragrans, exhibited significant larval and adult toxicity to Ae. aegypti and was more efficacious than DEET-the gold standard for insect repellents-at repelling adult female Ae. aegypti from blood feeding. In this study several semi-synthetic analogues of cinnamodial were prepared to probe the structure-activity relationship (SAR) for larvicidal, adulticidal and antifeedant activity against Ae. aegypti. Initial efforts were focused on modification of the dialdehyde functionality to produce more stable active analogues and to understand the importance of the 1,4-dialdehyde and the α,ß-unsaturated carbonyl in the observed bioactivity of cinnamodial against mosquitoes. This study represents the first investigation into the SAR of cinnamodial as an insecticide and antifeedant against the medically important Ae. aegypti mosquito.

Heterologous Expression of Aedes aegypti Cation Chloride Cotransporter 2 (aeCCC2) in Xenopus laevis Oocytes Induces an Enigmatic Na⁺/Li⁺ Conductance.

The yellow fever mosquito Aedes aegypti possesses three genes encoding putative Na⁺-coupled cation chloride cotransporters (CCCs): aeNKCC1, aeCCC2, and aeCCC3. To date, none of the aeCCCs have been functionally characterized. Here we expressed aeCCC2 heterologously in Xenopus oocytes and measured the uptake of Li⁺ (a tracer for Na⁺) and Rb⁺ (a tracer for K⁺). Compared to control (H2O-injected) oocytes, the aeCCC2-expressing oocytes exhibited significantly greater uptake of Li⁺, but not Rb⁺. However, the uptake of Li⁺ was neither Cl--dependent nor inhibited by thiazide, loop diuretics, or amiloride, suggesting unconventional CCC activity. To determine if the Li⁺-uptake was mediated by a conductive pathway, we performed two-electrode voltage clamping (TEVC) on the oocytes. The aeCCC2 oocytes were characterized by an enhanced conductance for Li⁺ and Na⁺, but not K⁺, compared to control oocytes. It remains to be determined whether aeCCC2 directly mediates the Na⁺/Li⁺ conductance or whether heterologous expression of aeCCC2 stimulates an endogenous cation channel in the oocyte plasma membrane.

Insecticidal and Antifeedant Activities of Malagasy Medicinal Plant (Cinnamosma sp.) Extracts and Drimane-Type Sesquiterpenes against Aedes aegypti Mosquitoes.

The overuse of insecticides with limited modes of action has led to resistance in mosquito vectors. Thus, insecticides with novel modes of action are needed. Secondary metabolites in Madagascan plants of the genus Cinnamosma (Canellaceae) are commonly used in traditional remedies and known to elicit antifeedant and toxic effects in insect pests. Here we test the hypothesis that extracts of Cinnamosma sp. enriched in drimane sesquiterpenes are toxic and/or antifeedant to the yellow fever mosquito Aedes aegypti. We show that the bark and root extracts, which contain a higher abundance of drimane sesquiterpenes compared to leaves, were the most efficacious. Screening isolated compounds revealed cinnamodial to be the primary driver of adulticidal activity, whereas cinnamodial, polygodial, cinnafragrin A, and capsicodendrin contributed to the larvicidal activity. Moreover, an abundant lactone (cinnamosmolide) in the root extract synergized the larvicidal effects of cinnamodial. The antifeedant activity of the extracts was primarily contributed to cinnamodial, polygodial, and cinnamolide. Parallel experiments with warburganal isolated from Warburgia ugandensis (Canellaceae) revealed that aldehydes are critical for-and a hydroxyl modulates-insecticidal activity. Our results indicate that plant drimane sesquiterpenes provide valuable chemical platforms for developing insecticides and repellents to control mosquito vectors.