Ammonia transport in the excretory system of mosquito larvae (Aedes aegypti): Rh protein expression and the transcriptome of the rectum.

The role of the mosquito excretory organs (Malpighian tubules, MT and hindgut, HG) in ammonia transport as well as expression and function of the Rhesus (Rh protein) ammonia transporters within these organs was examined in Aedes aegypti larvae and adult females. Immunohistological examination revealed that the Rh proteins are co-localized with V-type H+-ATPase (VA) to the apical membranes of MT and HG epithelia of both larvae and adult females. Of the two Rh transporter genes present in A. aegypti, AeRh50-1 and AeRh50-2, we show using quantitative real-time PCR (qPCR) and an RNA in-situ hybridization (ISH) assay that AeRh50-1 is the predominant Rh protein expressed in the excretory organs of larvae and adult females. Further assessment of AeRh50-1 function in larvae and adults using RNAi (i.e. dsRNA-mediated knockdown) revealed significantly decreased [NH4+] (mmol l-1) levels in the secreted fluid of larval MT which does not affect overall NH4+ transport rates, as well as significantly decreased NH4+ flux rates across the HG (haemolymph to lumen) of adult females. We also used RNA sequencing to identify the expression of ion transporters and enzymes within the rectum of larvae, of which limited information currently exists for this important osmoregulatory organ. Of the ammonia transporters in A. aegypti, AeRh50-1 transcript is most abundant in the rectum thus validating our immunohistochemical and RNA ISH findings. In addition to enriched VA transcript (subunits A and d1) in the rectum, we also identified high Na+-K+-ATPase transcript (α subunit) expression which becomes significantly elevated in response to HEA, and we also found enriched carbonic anhydrase 9, inwardly rectifying K+ channel Kir2a, and Na+-coupled cation-chloride (Cl-) co-transporter CCC2 transcripts. Finally, the modulation in excretory organ function and/or Rh protein expression was examined in relation to high ammonia challenge, specifically high environmental ammonia (HEA) rearing of larvae. NH4+ flux measurements using the scanning-ion selective electrode (SIET) technique revealed no significant differences in NH4+ transport across organs comprising the alimentary canal of larvae reared in HEA vs freshwater. Further, significantly increased VA activity, but not NKA, was observed in the MT of HEA-reared larvae. Relatively high Rh protein immunostaining persists within the hindgut epithelium, as well as the ovary, of females at 24-48 h post blood meal corresponding with previously demonstrated peak levels of ammonia formation. These data provide new insight into the role of the excretory organs in ammonia transport physiology and the contribution of Rh proteins in mediating ammonia movement across the epithelia of the MT and HG, and the first comprehensive examination of ion transporter and channel expression in the mosquito rectum.

Duox activation in Drosophila Malpighian tubules stimulates intestinal epithelial renewal through a countercurrent flow.

The gut must perform a dual role of protecting the host against toxins and pathogens while harboring mutualistic microbiota. Previous studies suggested that the NADPH oxidase Duox contributes to intestinal homeostasis in Drosophila by producing reactive oxygen species (ROS) in the gut that stimulate epithelial renewal. We find instead that the ROS generated by Duox in the Malpighian tubules leads to the production of Upd3, which enters the gut and stimulates stem cell proliferation. We describe in Drosophila the existence of a countercurrent flow system, which pushes tubule-derived Upd3 to the anterior part of the gut and stimulates epithelial renewal at a distance. Thus, our paper clarifies the role of Duox in gut homeostasis and describes the existence of retrograde fluid flow in the gut, collectively revealing a fascinating example of inter-organ communication.

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.

Proteotranscriptomic analyses of the midgut and Malpighian tubules after a sublethal concentration of Cry1Ab exposure on Spodoptera litura.

BACKGROUND: Cry1Ab has emerged as a bio-insecticide to control Spodoptera litura (Lepidoptera: Noctuidae). However, the sublethal effects of Cry1Ab on the physiological changes and molecular level of S. litura have not been well documented. Our aims in this study were to assess the sublethal effect of Cry1Ab on S. litura, including midgut and Malpighian tubules as targets. RESULTS: After sublethal Cry1Ab exposure, distinct histological alterations were mainly observed in the midgut. Furthermore, the results of comparative RNA sequencing and tandem mass tag-based proteomics showed that, in the midgut, most differential expression genes (DEGs) were up-regulated and significantly enriched in the serine protease activity pathway, and up-regulated differential expression proteins (DEPs) were mainly associated with the oxidative phosphorylation pathway, whereas the down-regulated involved in the ribosome pathways. In the Malpighian tubules, DEGs and DEPs were significantly enriched in the ribosome pathway. We proposed that ribosome may act as a universal target in energy metabolism with other pathways via the results of protein-protein interaction analysis. Further, by verification of the mRNA expression of some Cry protein receptor and detoxification genes after Cry1Ab treatment, it was suggested that the ribosomal proteins (RPs) possibly participate in influencing the Bt-resistance of S. litura larvae under sublethal Cry1Ab exposure. CONCLUSION: Under sublethal Cry1Ab exposure, the midgut of S. litura was damaged, and the proteotranscriptomic analysis elucidated that Cry1Ab disrupted the energy homeostasis of larvae. Furthermore, we emphasized the potential role of ribosomes in sublethal Cry1Ab exposure. © 2024 Society of Chemical Industry.

Voltage-gated ion channels are expressed in the Malpighian tubules and anal papillae of the yellow fever mosquito (Aedes aegypti), and may regulate ion transport during salt and water imbalance.

Vectors of infectious disease include several species of Aedes mosquitoes. The life cycle of Aedes aegypti, the yellow fever mosquito, consists of a terrestrial adult and an aquatic larval life stage. Developing in coastal waters can expose larvae to fluctuating salinity, causing salt and water imbalance, which is addressed by two prime osmoregulatory organs - the Malpighian tubules (MTs) and anal papillae (AP). Voltage-gated ion channels (VGICs) have recently been implicated in the regulation of ion transport in the osmoregulatory epithelia of insects. In the current study, we: (i) generated MT transcriptomes of freshwater-acclimated and brackish water-exposed larvae of Ae. aegypti, (ii) detected expression of several voltage-gated Ca2+, K+, Na+ and non-ion-selective ion channels in the MTs and AP using transcriptomics, PCR and gel electrophoresis, (iii) demonstrated that mRNA abundance of many altered significantly following brackish water exposure, and (iv) immunolocalized CaV1, NALCN, TRP/Painless and KCNH8 in the MTs and AP of larvae using custom-made antibodies. We found CaV1 to be expressed in the apical membrane of MTs of both larvae and adults, and its inhibition to alter membrane potentials of this osmoregulatory epithelium. Our data demonstrate that multiple VGICs are expressed in osmoregulatory epithelia of Ae. aegypti and may play an important role in the autonomous regulation of ion transport.

Identification of inducible CYP3 and CYP4 genes associated with abamectin tolerance in the fat body and Malpighian tubules of Spodoptera litura.

Abamectin, as a broad-spectrum bioinsecticide, has been widely used for the control of Lepidoptera insects, resulting in different levels of resistance to abamectin in Spodoptera litura. Cytochrome P450 monooxygenases (P450s) are known for their important roles in insecticide detoxification. In this study, the expression of SlCYP6B40, SlCYP4L12 and SlCYP9A32 in the fat body, and SlCYP4S9, SlCYP6AB12, SlCYP6AB58, SlCYP9A75a and SlCYP9A75b in Malpighian tubules was found to be significantly upregulated after abamectin exposure. SlCYP6AE44 and SlCYP6AN4 were simultaneously upregulated in these two tissues after abamectin exposure. Ectopically overexpressed SlCYP6AE44, SlCYP9A32 and SlCYP4S9 in transgenic Drosophila conferred tolerance to abamectin. In addition, homology modeling and molecular docking results suggested that SlCYP6AE44, SlCYP9A32 and SlCYP4S9 may be capable of binding with abamectin. These results demonstrate that upregulation of CYP3 and CYP4 genes may contribute to abamectin detoxification in S. litura and provide information for evidence-based insecticide resistance management strategies.

Expression of the non-neuronal cholinergic system components in Malpighian tubules of Mythimna separata and evidence for non-neuronal acetylcholine synthesis.

The non-neuronal cholinergic system, widely distributed in nature, is an ancient system that has not been well studied in insects. This study aims to investigate the key components of the cholinergic system and to identify the non-neuronal acetylcholine (ACh)-producing cells and the acting sites of ACh in the Malpighian tubules (MTs) of Mythimna separata. We found that non-neuronal ACh in MTs is synthesized by carnitine acetyltransferase (CarAT), rather than choline acetyltransferase (ChAT), as confirmed by using enzyme inhibitors and high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). Fluorescence in situ hybridization revealed the presence of CarAT mRNA within MTs, specifically localized in the principal cells. Immunohistochemistry showed strong staining for A-mAChR, a muscarinic acetylcholine receptor, in the principal cells. Pharmacological analysis further demonstrated that ACh acts through A-mAChR in the principal cells to increase the intracellular Ca2+ concentration. These findings provide compelling evidence for the existence of a non-neuronal cholinergic system in the MTs of M. separata, and the principal cells play a crucial role in ACh synthesis via CarAT.

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.

A novel antidiuretic hormone governs tumour-induced renal dysfunction.

Maintenance of renal function and fluid transport are essential for vertebrates and invertebrates to adapt to physiological and pathological challenges. Human patients with malignant tumours frequently develop detrimental renal dysfunction and oliguria, and previous studies suggest the involvement of chemotherapeutic toxicity and tumour-associated inflammation1,2. However, how tumours might directly modulate renal functions remains largely unclear. Here, using conserved tumour models in Drosophila melanogaster3, we characterized isoform F of ion transport peptide (ITPF) as a fly antidiuretic hormone that is secreted by a subset of yki3SA gut tumour cells, impairs renal function and causes severe abdomen bloating and fluid accumulation. Mechanistically, tumour-derived ITPF targets the G-protein-coupled receptor TkR99D in stellate cells of Malpighian tubules-an excretory organ that is equivalent to renal tubules4-to activate nitric oxide synthase-cGMP signalling and inhibit fluid excretion. We further uncovered antidiuretic functions of mammalian neurokinin 3 receptor (NK3R), the homologue of fly TkR99D, as pharmaceutical blockade of NK3R efficiently alleviates renal tubular dysfunction in mice bearing different malignant tumours. Together, our results demonstrate a novel antidiuretic pathway mediating tumour-renal crosstalk across species and offer therapeutic opportunities for the treatment of cancer-associated renal dysfunction.

Inducible Cytochrome P450s in the Fat Body and Malpighian Tubules of the Polyphagous Pests of Spodoptera litura Confer Xenobiotic Tolerance.

Cytochrome P450 plays vital roles in detoxifying xenobiotics. In this study, SlCYP340A and SlCYP340L expression in the Spodoptera litura fat body and SlCYP332A1, SlCYP6AB12, SlCYP6AB58, SlCYP6AB59, and SlCYP6AN4 expression in the Malpighian tubules were significantly upregulated after cyantraniliprole exposure, and SlCYP6AB58 and SlCYP6AB59 expression levels were simultaneously increased in the Malpighian tubules after gossypol treatment. Drosophila ectopically expressing candidate P450 genes showed that SlCYP332A1, SlCYP6AB12, SlCYP6AB59, SlCYP6AN4, and SlCYP340A conferred cyantraniliprole tolerance. The overexpression of SlCYP6AB58 and SlCYP6AB59 in Drosophila increased the number of eggs laid under the gossypol treatment. Moreover, the knockdown of SlCYP332A1, SlCYP6AB12, SlCYP6AB59, SlCYP6AN4, and SlCYP340A increased S. litura mortality under the cyantraniliprole treatment. Homology modeling and molecular docking results suggested that candidate P450 has the potential to bind with cyantraniliprole. These results indicate that the CYP3 and CYP4 genes participate in cyantraniliprole detoxification and that SlCYP6AB59 may be simultaneously involved in the gossypol tolerance of S. litura.

Requirement of Snakeskin for normal functions of midgut and Malpighian tubules in Henosepilachna vigintioctopunctata.

Septate junctions (SJs) are located between epithelial cells and play crucial roles in epithelial barrier formation and epithelia cell homeostasis. Nevertheless, the molecular constituents, especially those related to smooth SJs (sSJs), have not been well explored in non-Drosophilid insects. A putative integral membrane protein Snakeskin (Ssk) was identified in a Coleoptera foliar pest Henosepilachna vigintioctopunctata. RNA interference-aided knockdown of Hvssk at the third-instar larval stage arrested larval development. Most resultant larvae failed to shed larval exuviae until their death. Silence of Hvssk at the fourth-instar larvae inhibited the growth and reduced foliage consumption. Dissection and microscopic observation revealed that compromised expression of Hvssk caused obvious phenotypic defects in the midgut. A great number of morphologically abnormal columnar epithelial cells accumulated throughout the midgut lumen. Moreover, numerous vesicles were observed in the malformed cells of the Malpighian tubules (Mt). All the Hvssk depleted larvae remained as prepupae; they gradually darkened and eventually died. Furthermore, depletion of Hvssk at the pupal stage suppressed adult feeding and shortened adult lifespan. These findings demonstrated that Ssk plays a vital role in the integrity and function of both midguts and Mt, and established the conservative roles of Ssk in the formation of epithelial barrier and the homeostasis of epithelial cells in H. vigintioctopunctata.

Adaptive Changes in Detoxification Metabolism and Transmembrane Transport of Bombyx mori Malpighian Tubules to Artificial Diet.

The high adaptability of insects to food sources has contributed to their ranking among the most abundant and diverse species on Earth. However, the molecular mechanisms underlying the rapid adaptation of insects to different foods remain unclear. We explored the changes in gene expression and metabolic composition of the Malpighian tubules as an important metabolic excretion and detoxification organ in silkworms (Bombyx mori) fed mulberry leaf and artificial diets. A total of 2436 differentially expressed genes (DEGs) and 245 differential metabolites were identified between groups, with the majority of DEGs associated with metabolic detoxification, transmembrane transport, and mitochondrial function. Detoxification enzymes, such as cytochrome P450 (CYP), glutathione-S-transferase (GST), and UDP-glycosyltransferase, and ABC and SLC transporters of endogenous and exogenous solutes were more abundant in the artificial diet group. Enzyme activity assays confirmed increased CYP and GST activity in the Malpighian tubules of the artificial diet-fed group. Metabolome analysis showed increased contents of secondary metabolites, terpenoids, flavonoids, alkaloids, organic acids, lipids, and food additives in the artificial diet group. Our findings highlight the important role of the Malpighian tubules in adaptation to different foods and provide guidance for further optimization of artificial diets to improve silkworm breeding.

Aedes aegypti Malpighian tubules are immunologically activated following systemic Toll activation.

BACKGROUND: Canine heartworm is a widespread and potentially fatal mosquito-borne disease caused by infections with the parasitic nematode, Dirofilaria immitis. We have previously shown that systemic activation of the Toll immune pathway via silencing of the negative regulator Cactus in Aedes aegypti blocks parasite development in the Malpighian tubules (MT), the mosquito renal organ. However, it was not established whether the MT were directly responding to Toll activation or were alternatively responding to upregulated proteins or other changes to the hemolymph driven by other tissues. Distinguishing these possibilities is crucial for developing more precise strategies to block D. immitis while potentially avoiding the fitness cost to the mosquito associated with Cactus silencing. METHODS: This study defines the transcriptional response of the MT and changes to the hemolymph proteome of Ae. aegypti after systemic Toll activation via intra-thoracic injection of double-stranded Cactus (dsCactus) RNA. RESULTS: Malpighian tubules significantly increased expression of the Toll pathway target genes that significantly overlapped expression changes occurring in whole mosquitoes. A significant overlap between the transcriptional response of the MT and proteins upregulated in the hemolymph was also observed. CONCLUSIONS: Our data show that MT are capable of RNA interference-mediated gene silencing and directly respond to dsCactus treatment by upregulating targets of the canonical Toll pathway. Although not definitive, the strong correspondence between the MT transcriptional response and the hemolymph proteomic responses provides evidence that the MT may contribute to mosquito humoral immunity.

Exploratory phosphoproteomics profiling of Aedes aegypti Malpighian tubules during blood meal processing reveals dramatic transition in function.

Malpighian tubules, the renal organs of mosquitoes, facilitate the rapid dehydration of blood meals through aquaporin-mediated osmosis. We performed phosphoproteomics analysis of three Malpighian tubule protein-libraries (1000 tubules/sample) from unfed female mosquitoes as well as one and 24 hours after a blood meal. We identified 4663 putative phosphorylation sites in 1955 different proteins. Our exploratory dataset reveals blood meal-induced changes in phosphorylation patterns in many subunits of V-ATPase, proteins of the target of rapamycin signaling pathway, vesicle-mediated protein transport proteins, proteins involved in monocarboxylate transport, and aquaporins. Our phosphoproteomics data suggest the involvement of a variety of new pathways including nutrient-signaling, membrane protein shuttling, and paracellular water flow in the regulation of urine excretion. Our results support a model in which aquaporin channels translocate from intracellular vesicles to the cell membrane of stellate cells and the brush border membrane of principal cells upon blood feeding.

Tryptophan regulates Drosophila zinc stores.

Zinc deficiency is commonly attributed to inadequate absorption of the metal. Instead, we show that body zinc stores in Drosophila melanogaster depend on tryptophan consumption. Hence, a dietary amino acid regulates zinc status of the whole insect­a finding consistent with the widespread requirement of zinc as a protein cofactor. Specifically, the tryptophan metabolite kynurenine is released from insect fat bodies and induces the formation of zinc storage granules in Malpighian tubules, where 3-hydroxykynurenine and xanthurenic acid act as endogenous zinc chelators. Kynurenine functions as a peripheral zinc-regulating hormone and is converted into a 3-hydroxykynurenine­zinc­chloride complex, precipitating within the storage granules. Thus, zinc and the kynurenine pathway­well-known modulators of immunity, blood pressure, aging, and neurodegeneration­are physiologically connected.

Blending physiology and RNAseq to provide new insights into regulation of epithelial transport: switching between ion secretion and reabsorption.

This Review addresses the means by which epithelia change the direction of vectorial ion transport. Recent studies have revealed that insect Malpighian (renal) tubules can switch from secreting to reabsorbing K+. When the gut of larval lepidopterans is empty (during the moult cycle) or when the larvae are reared on K+-deficient diet, the distal ileac plexus segment of the tubule secretes K+ from the haemolymph into the tubule lumen. By contrast, in larvae reared on K+-rich diet, ions and fluid are reabsorbed from the rectal lumen into the perinephric space surrounding the cryptonephridial tubules of the rectal complex. Ions and fluid are then transported from the perinephric space into the lumen of the cryptonephridial tubules, thus supplying the free segments of the tubule downstream. Under these conditions, some of the K+ and water in the tubule lumen is reabsorbed across the cells of the distal ileac plexus, allowing for expansion of haemolymph volume in the rapidly growing larvae, as well as recycling of K+ and base equivalents. RNA sequencing data reveal large-scale changes in gene transcription that are associated with the switch between ion secretion and ion reabsorption by the distal ileac plexus. An unexpected finding is the presence of voltage-gated, ligand-gated and mechanosensitive ion channels, normally seen in excitable cells, in Malpighian tubules. Transcriptomic surveys indicate that these types of channels are also present in multiple other types of vertebrate and invertebrate epithelia, suggesting that they may play novel roles in epithelial cell signalling and regulation of epithelial ion transport.

Prophenoloxidase-positive tubes derived from the hindguts may be the doorkeeper to detoxify the waste metabolites collected by Malpighian tubules in Lepidoptera insects.

Prophenoloxidase (PPO), an important immunity protein in insects, is mainly produced by hemocytes and released into the hemolymph upon cell lysis. In addition, PPO can also be produced by epidermal cells in the foregut to detoxify the toxic plant secondary metabolites and in the hindgut to kill pathogens through PPO-induced melanization. Previously, we noticed a pair of tubes extended from the larval hindgut became melanized upon staining in dopamine dissolved in 30% ethanol. However, the structure and function of these tubes are largely unknown. In this study, we performed staining of the tubes and the neighboring Malpighian tubule for further confirmation. Eventually, we detected PPO inside epidermal cells of the tubes, and called them as PPO-positive tubes. We observed that the PPO-positive tubes are physically derived from the hindgut but strongly adhere to the Malpighian tubule. Inside the PPO-positive tubes, there is an acellular peritrophic membrane to protect the epidermal cells. Furthermore, the PPO-positive tubes act like a doorkeeper to firstly detoxify the metabolite wastes collected by the Malpighian tubule from the hemolymph.

Fluid Secretion by Malpighian Tubules of Rhodnius prolixus: Neuroendocrine Control With New Insights From a Transcriptome Analysis.

Rhodnius prolixus (the kissing bug and a major vector of Chagas disease) is an obligate blood feeder that in the case of the fifth instar consumes up to 10 times its unfed body weight in a single 20-minute feed. A post-prandial diuresis is initiated, within minutes of the start of gorging, in order to lower the mass and concentrate the nutrients of the meal. Thus, R. prolixus rapidly excretes a fluid that is high in NaCl content and hypo-osmotic to the hemolymph, thereby eliminating 50% of the volume of the blood meal within 3 hours of gorging. In R. prolixus, as with other insects, the Malpighian tubules play a critical role in diuresis. Malpighian tubules are not innervated, and their fine control comes under the influence of the neuroendocrine system that releases amines and neuropeptides as diuretic or antidiuretic hormones. These hormones act upon the Malpighian tubules via a variety of G protein-coupled receptors linked to second messenger systems that influence ion transporters and aquaporins; thereby regulating fluid secretion. Much has been discovered about the control of diuresis in R. prolixus, and other model insects, using classical endocrinological studies. The post-genomic era, however, has brought new insights, identifying novel diuretic and antidiuretic hormone-signaling pathways whilst also validating many of the classical discoveries. This paper will focus on recent discoveries into the neuroendocrine control of the rapid post-prandial diuresis in R. prolixus, in order to emphasize new insights from a transcriptome analysis of Malpighian tubules taken from unfed and fed bugs.

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.

Lead (Pb2+)-induced calcium oxalate crystallization ex vivo is ameliorated via inositol 1,4,5-trisphosphate receptor (InsP3R) knockdown in a Drosophila melanogaster model of nephrolithiasis.

Nephrolithiasis causes severe pain and is a highly recurrent pathophysiological state. Calcium-containing stones, specifically calcium oxalate (CaOx), is the most common type accounting for approximately 75 % of stone composition. Genetic predisposition, gender, geographic region, diet, and low fluid intake all contribute to disease pathogenesis. However, exposure to environmental pollutants as a contribution to kidney stone formation remains insufficiently studied. Lead (Pb2+) is of particular interest as epidemiological data indicate that low-level exposure (BLL = 0.48-3.85 µM) confers a 35 % increased risk of developing CaOx nephrolithiasis. However, mechanisms underlying this association have yet to be elucidated. Drosophila melanogaster provide a useful genetic model where major molecular pathophysiological pathways can be efficiently studied. Malpighian tubules (MT) were isolated from either Wild-Type or InsP3R knockdown flies and treated with oxalate (5 mM) ± Pb2+ (2µM) for 1 h. Following exposure, MTs were imaged and crystals quantified. CaOx crystal number and total area were significantly increased (˜5-fold) in Pb2+(pre-treatment) + oxalate-exposed MTs when compared to oxalate alone controls. However, CaOx crystal number and total crystal area in Pb2+ + oxalate-exposed InsP3R knockdown MTs were significantly decreased (˜3-fold) indicating the role for principal cell-specific InsP3R-mediated Ca2+ mobilization as a mechanism for Pb2+-induced increases in CaOx crystallization inset model of nephrolithiasis.