Sugar feeding protects against arboviral infection by enhancing gut immunity in the mosquito vector Aedes aegypti.

As mosquito females require a blood meal to reproduce, they can act as vectors of numerous pathogens, such as arboviruses (e.g. Zika, dengue and chikungunya viruses), which constitute a substantial worldwide public health burden. In addition to blood meals, mosquito females can also take sugar meals to get carbohydrates for their energy reserves. It is now recognised that diet is a key regulator of health and disease outcome through interactions with the immune system. However, this has been mostly studied in humans and model organisms. So far, the impact of sugar feeding on mosquito immunity and in turn, how this could affect vector competence for arboviruses has not been explored. Here, we show that sugar feeding increases and maintains antiviral immunity in the digestive tract of the main arbovirus vector Aedes aegypti. Our data demonstrate that the gut microbiota does not mediate the sugar-induced immunity but partly inhibits it. Importantly, sugar intake prior to an arbovirus-infected blood meal further protects females against infection with arboviruses from different families. Sugar feeding blocks arbovirus initial infection and dissemination from the gut and lowers infection prevalence and intensity, thereby decreasing the transmission potential of female mosquitoes. Finally, we show that the antiviral role of sugar is mediated by sugar-induced immunity. Overall, our findings uncover a crucial role of sugar feeding in mosquito antiviral immunity which in turn decreases vector competence for arboviruses. Since Ae. aegypti almost exclusively feed on blood in some natural settings, our findings suggest that this lack of sugar intake could increase the spread of mosquito-borne arboviral diseases.

A nutrient-responsive hormonal circuit mediates an inter-tissue program regulating metabolic homeostasis in adult Drosophila.

Animals maintain metabolic homeostasis by modulating the activity of specialized organs that adjust internal metabolism to external conditions. However, the hormonal signals coordinating these functions are incompletely characterized. Here we show that six neurosecretory cells in the Drosophila central nervous system respond to circulating nutrient levels by releasing Capa hormones, homologs of mammalian neuromedin U, which activate the Capa receptor (CapaR) in peripheral tissues to control energy homeostasis. Loss of Capa/CapaR signaling causes intestinal hypomotility and impaired nutrient absorption, which gradually deplete internal nutrient stores and reduce organismal lifespan. Conversely, increased Capa/CapaR activity increases fluid and waste excretion. Furthermore, Capa/CapaR inhibits the release of glucagon-like adipokinetic hormone from the corpora cardiaca, which restricts energy mobilization from adipose tissue to avoid harmful hyperglycemia. Our results suggest that the Capa/CapaR circuit occupies a central node in a homeostatic program that facilitates the digestion and absorption of nutrients and regulates systemic energy balance.

Specialized stellate cells offer a privileged route for rapid water flux in Drosophila renal tubule.

Insects are highly successful, in part through an excellent ability to osmoregulate. The renal (Malpighian) tubules can secrete fluid faster on a per-cell basis than any other epithelium, but the route for these remarkable water fluxes has not been established. In Drosophila melanogaster, we show that 4 genes of the major intrinsic protein family are expressed at a very high level in the fly renal tissue: the aquaporins (AQPs) Drip and Prip and the aquaglyceroporins Eglp2 and Eglp4 As predicted from their structure, and by their transport function by expressing these proteins in Xenopus oocytes, Drip, Prip, and Eglp2 show significant and specific water permeability, whereas Eglp2 and Eglp4 show very high permeability to glycerol and urea. Knockdowns of any of these genes result in impaired hormone-induced fluid secretion. The Drosophila tubule has 2 main secretory cell types: active cation-transporting principal cells, wherein the aquaglyceroporins localize to opposite plasma membranes, and small stellate cells, the site of the chloride shunt conductance, with these AQPs localizing to opposite plasma membranes. This suggests a model in which osmotically obliged water flows through the stellate cells. Consistent with this model, fluorescently labeled dextran, an in vivo marker of membrane water permeability, is trapped in the basal infoldings of the stellate cells after kinin diuretic peptide stimulation, confirming that these cells provide the major route for transepithelial water flux. The spatial segregation of these components of epithelial water transport may help to explain the unique success of the higher insects in regulating their internal environments.

Targeted renal knockdown of Na+/H+ exchanger regulatory factor Sip1 produces uric acid nephrolithiasis in Drosophila.

Nephrolithiasis is one of the most common kidney diseases, with poorly understood pathophysiology, but experimental study has been hindered by lack of experimentally tractable models. Drosophila melanogaster is a useful model organism for renal diseases because of genetic and functional similarities of Malpighian (renal) tubules with the human kidney. Here, we demonstrated function of the sex-determining region Y protein-interacting protein-1 (Sip1) gene, an ortholog of human Na+/H+ exchanger regulatory factor (NHERF1), in Drosophila Malpighian tubules and its impact on nephrolithiasis. Abundant birefringent calculi were observed in Sip1 mutant flies, and the phenotype was also observed in renal stellate cell-specific RNA interference Sip1 knockdown in otherwise normal flies, confirming a renal etiology. This phenotype was abolished in rosy mutant flies (which model human xanthinuria) and by the xanthine oxidase inhibitor allopurinol, suggesting that the calculi were of uric acid. This was confirmed by direct biochemical assay for urate. Stones rapidly dissolved when the tubule was bathed in alkaline media, suggesting that Sip1 knockdown was acidifying the tubule. SIP1 was shown to collocate with Na+/H+ exchanger isoform 2 (NHE2) and with moesin in stellate cells. Knockdown of NHE2 specifically to the stellate cells also increased renal uric acid stone formation, and so a model was developed in which SIP1 normally regulates NHE2 activity and luminal pH, ultimately leading to uric acid stone formation. Drosophila renal tubules may thus offer a useful model for urate nephrolithiasis.

Novel roles for GATAe in growth, maintenance and proliferation of cell populations in the Drosophila renal tubule.

The GATA family of transcription factors is implicated in numerous developmental and physiological processes in metazoans. In Drosophila melanogaster, five different GATA factor genes (pannier, serpent, grain, GATAd and GATAe) have been reported as essential in the development and identity of multiple tissues, including the midgut, heart and brain. Here, we present a novel role for GATAe in the function and homeostasis of the Drosophila renal (Malpighian) tubule. We demonstrate that reduced levels of GATAe gene expression in tubule principal cells induce uncontrolled cell proliferation, resulting in tumorous growth with associated altered expression of apoptotic and carcinogenic key genes. Furthermore, we uncover the involvement of GATAe in the maintenance of stellate cells and migration of renal and nephritic stem cells into the tubule. Our findings of GATAe as a potential master regulator in the events of growth control and cell survival required for the maintenance of the Drosophila renal tubule could provide new insights into the molecular pathways involved in the formation and maintenance of a functional tissue and kidney disease.

Renal neuroendocrine control of desiccation and cold tolerance by Drosophila suzukii.

BACKGROUND: Neuropeptides are central to the regulation of physiological and behavioural processes in insects, directly impacting cold and desiccation survival. However, little is known about the control mechanisms governing these responses in Drosophila suzukii. The close phylogenetic relationship of D. suzukii with Drosophila melanogaster allows, through genomic and functional studies, an insight into the mechanisms directing stress tolerance in D. suzukii. RESULTS: Capability (Capa), leucokinin (LK), diuretic hormone 44 (DH44 ) and DH31 neuropeptides demonstrated a high level of conservation between D. suzukii and D. melanogaster with respect to peptide sequences, neuronal expression, receptor localisation, and diuretic function in the Malpighian tubules. Despite D. suzukii's ability to populate cold environments, it proved sensitive to both cold and desiccation. Furthermore, in D. suzukii, Capa acts as a desiccation- and cold stress-responsive gene, while DH44 gene expression is increased only after desiccation exposure, and the LK gene after nonlethal cold stress recovery. CONCLUSION: This study provides a comparative investigation into stress tolerance mediation by neuroendocrine signalling in two Drosophila species, providing evidence that similar signalling pathways control fluid secretion in the Malpighian tubules. Identifying processes governing specific environmental stresses affecting D. suzukii could lead to the development of targeted integrated management strategies to control insect pest populations. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The corticotropin-releasing factor-like diuretic hormone 44 (DH44) and kinin neuropeptides modulate desiccation and starvation tolerance in Drosophila melanogaster.

Malpighian tubules are critical organs for epithelial fluid transport and stress tolerance in insects, and are under neuroendocrine control by multiple neuropeptides secreted by identified neurons. Here, we demonstrate roles for CRF-like diuretic hormone 44 (DH44) and Drosophila melanogaster kinin (Drome-kinin, DK) in desiccation and starvation tolerance. Gene expression and labelled DH44 ligand binding data, as well as highly selective knockdowns and/or neuronal ablations of DH44 in neurons of the pars intercerebralis and DH44 receptor (DH44-R2) in Malpighian tubule principal cells, indicate that suppression of DH44 signalling improves desiccation tolerance of the intact fly. Drome-kinin receptor, encoded by the leucokinin receptor gene, LKR, is expressed in DH44 neurons as well as in stellate cells of the Malpighian tubules. LKR knockdown in DH44-expressing neurons reduces Malpighian tubule-specific LKR, suggesting interactions between DH44 and LK signalling pathways. Finally, although a role for DK in desiccation tolerance was not defined, we demonstrate a novel role for Malpighian tubule cell-specific LKR in starvation tolerance. Starvation increases gene expression of epithelial LKR. Also, Malpighian tubule stellate cell-specific knockdown of LKR significantly reduced starvation tolerance, demonstrating a role for neuropeptide signalling during starvation stress.

A novel role of Drosophila cytochrome P450-4e3 in permethrin insecticide tolerance.

The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response necessary for survival. This response includes the induction of genes that encode key Cytochrome P450 monooxygenase detoxification enzymes. Drosophila melanogaster Malpighian (renal) tubules are critical organs in the detoxification and elimination of these foreign compounds, so the tubule response induced by dietary exposure to the insecticide permethrin was examined. We found that expression of the gene encoding Cytochrome P450-4e3 (Cyp4e3) is significantly up-regulated by Drosophila fed on permethrin and that manipulation of Cyp4e3 levels, specifically in the principal cells of the Malpighian tubules, impacts significantly on the survival of permethrin-fed flies. Both dietary exposure to permethrin and Cyp4e3 knockdown cause a significant elevation of oxidative stress-associated markers in the tubules, including H2O2 and lipid peroxidation byproduct, HNE (4-hydroxynonenal). Thus, Cyp4e3 may play an important role in regulating H2O2 levels in the endoplasmic reticulum (ER) where it resides, and its absence triggers a JAK/STAT and NF-κB-mediated stress response, similar to that observed in cells under ER stress. This work increases our understanding of the molecular mechanisms of insecticide detoxification and provides further evidence of the oxidative stress responses induced by permethrin metabolism.

Tracing the evolutionary origins of insect renal function.

Knowledge on neuropeptide receptor systems is integral to understanding animal physiology. Yet, obtaining general insight into neuropeptide signalling in a clade as biodiverse as the insects is problematic. Here we apply fluorescent analogues of three key insect neuropeptides to map renal tissue architecture across systematically chosen representatives of the major insect Orders, to provide an unprecedented overview of insect renal function and control. In endopterygote insects, such as Drosophila, two distinct transporting cell types receive separate neuropeptide signals, whereas in the ancestral exopterygotes, a single, general cell type mediates all signals. Intriguingly, the largest insect Order Coleoptera (beetles) has evolved a unique approach, in which only a small fraction of cells are targets for neuropeptide action. In addition to demonstrating a universal utility of this technology, our results reveal not only a generality of signalling by the evolutionarily ancient neuropeptide families but also a clear functional separation of the types of cells that mediate the signal.

Insect capa neuropeptides impact desiccation and cold tolerance.

The success of insects is linked to their impressive tolerance to environmental stress, but little is known about how such responses are mediated by the neuroendocrine system. Here we show that the capability (capa) neuropeptide gene is a desiccation- and cold stress-responsive gene in diverse dipteran species. Using targeted in vivo gene silencing, physiological manipulations, stress-tolerance assays, and rationally designed neuropeptide analogs, we demonstrate that the Drosophila melanogaster capa neuropeptide gene and its encoded peptides alter desiccation and cold tolerance. Knockdown of the capa gene increases desiccation tolerance but lengthens chill coma recovery time, and injection of capa peptide analogs can reverse both phenotypes. Immunohistochemical staining suggests that capa accumulates in the capa-expressing Va neurons during desiccation and nonlethal cold stress but is not released until recovery from each stress. Our results also suggest that regulation of cellular ion and water homeostasis mediated by capa peptide signaling in the insect Malpighian (renal) tubules is a key physiological mechanism during recovery from desiccation and cold stress. This work augments our understanding of how stress tolerance is mediated by neuroendocrine signaling and illustrates the use of rationally designed peptide analogs as agents for disrupting protective stress tolerance.

Chloride channels in stellate cells are essential for uniquely high secretion rates in neuropeptide-stimulated Drosophila diuresis.

Epithelia frequently segregate transport processes to specific cell types, presumably for improved efficiency and control. The molecular players underlying this functional specialization are of particular interest. In Drosophila, the renal (Malpighian) tubule displays the highest per-cell transport rates known and has two main secretory cell types, principal and stellate. Electrogenic cation transport is known to reside in the principal cells, whereas stellate cells control the anion conductance, but by an as-yet-undefined route. Here, we resolve this issue by showing that a plasma membrane chloride channel, encoded by ClC-a, is exclusively expressed in the stellate cell and is required for Drosophila kinin-mediated induction of diuresis and chloride shunt conductance, evidenced by chloride ion movement through the stellate cells, leading to depolarization of the transepithelial potential. By contrast, ClC-a knockdown had no impact on resting secretion levels. Knockdown of a second CLC gene showing highly abundant expression in adult Malpighian tubules, ClC-c, did not impact depolarization of transepithelial potential after kinin stimulation. Therefore, the diuretic action of kinin in Drosophila can be explained by an increase in ClC-a-mediated chloride conductance, over and above a resting fluid transport level that relies on other (ClC-a-independent) mechanisms or routes. This key segregation of cation and anion transport could explain the extraordinary fluid transport rates displayed by some epithelia.

The D. melanogaster capa-1 neuropeptide activates renal NF-kB signaling.

The capa peptide family exists in a very wide range of insects including species of medical, veterinary and agricultural importance. Capa peptides act via a cognate G-protein coupled receptor (capaR) and have a diuretic action on the Malpighian tubules of Dipteran and Lepidopteran species. Capa signaling is critical for fluid homeostasis and has been associated with desiccation tolerance in the fly, Drosophila melanogaster. The mode of capa signaling is highly complex, affecting calcium, nitric oxide and cyclic GMP pathways. Such complex physiological regulation by cell signaling pathways may occur ultimately for optimal organismal stress tolerance to multiple stressors. Here we show that D. melanogaster capa-1 (Drome-capa-1) acts via the Nuclear Factor kappa B (NF-kB) stress signaling network. Human PCR gene arrays of capaR-transfected Human Embryonic Kidney (HEK) 293 cells showed that Drome-capa-1 increases expression of NF-kB, NF-kB regulated genes including IL8, TNF and PTGS2, and NF-kB pathway-associated transcription factors i.e. EGR1, FOS, cJUN. Furthermore, desiccated HEK293 cells show increased EGR1, EGR3 and PTGS2 - but not IL8, expression. CapaR-transfected NF-kB reporter cells showed that Drome-capa-1 increased NF-kB promoter activity via increased calcium. In Malpighian tubules, both Drome-capa-1 stimulation and desiccation result in increased gene expression of the D. melanogaster NF-kB orthologue, Relish; as well as EGR-like stripe and klumpfuss. Drome-capa-1 also induces Relish translocation in tubule principal cells. Targeted knockdown of Relish in only tubule principal cells reduces desiccation stress tolerance of adult flies. Together, these data suggest that Drome-capa-1 acts in desiccation stress tolerance, by activating NF-kB signaling.

Cell signalling mechanisms for insect stress tolerance.

Insects successfully occupy most environmental niches and this success depends on surviving a broad range of environmental stressors including temperature, desiccation, xenobiotic, osmotic and infection stress. Epithelial tissues play key roles as barriers between the external and internal environments and therefore maintain homeostasis and organismal tolerance to multiple stressors. As such, the crucial role of epithelia in organismal stress tolerance cannot be underestimated. At a molecular level, multiple cell-specific signalling pathways including cyclic cAMP, cyclic cGMP and calcium modulate tissue, and hence, organismal responses to stress. Thus, epithelial cell-specific signal transduction can be usefully studied to determine the molecular mechanisms of organismal stress tolerance in vivo. This review will explore cell signalling modulation of stress tolerance in insects by focusing on cell signalling in a fluid transporting epithelium--the Malpighian tubule. Manipulation of specific genes and signalling pathways in only defined tubule cell types can influence the survival outcome in response to multiple environmental stressors including desiccation, immune, salt (ionic) and oxidative stress, suggesting that studies in the genetic model Drosophila melanogaster may reveal novel pathways required for stress tolerance.

Signaling by Drosophila capa neuropeptides.

The capa peptide family, originally identified in the tobacco hawk moth, Manduca sexta, is now known to be present in many insect families, with increasing publications on capa neuropeptides each year. The physiological actions of capa peptides vary depending on the insect species but capa peptides have key myomodulatory and osmoregulatory functions, depending on insect lifestyle, and life stage. Capa peptide signaling is thus critical for fluid homeostasis and survival, making study of this neuropeptide family attractive for novel routes for insect control. In Dipteran species, including the genetically tractable Drosophila melanogaster, capa peptide action is diuretic; via elevation of nitric oxide, cGMP and calcium in the principal cells of the Malpighian tubules. The identification of the capa receptor (capaR) in several insect species has shown this to be a canonical GPCR. In D. melanogaster, ligand-activated capaR activity occurs in a dose-dependent manner between 10(-6) and 10(-12)M. Lower concentrations of capa peptide do not activate capaR, either in adult or larval Malpighian tubules. Use of transgenic flies in which capaR is knocked-down in only Malpighian tubule principal cells demonstrates that capaR modulates tubule fluid secretion rates and in doing so, sets the organismal response to desiccation. Thus, capa regulates a desiccation-responsive pathway in D. melanogaster, linking its role in osmoregulation and fluid homeostasis to environmental response and survival. The conservation of capa action between some Dipteran species suggests that capa's role in desiccation tolerance may not be confined to D. melanogaster.

Phosphodiesterase-8A binds to and regulates Raf-1 kinase.

V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) is a key activator of the ERK pathway and is a target for cross-regulation of this pathway by the cAMP signaling system. The cAMP-activated protein kinase, PKA, inhibits Raf-1 by phosphorylation on S259. Here, we show that the cAMP-degrading phosphodiesterase-8A (PDE8A) associates with Raf-1 to protect it from inhibitory phosphorylation by PKA, thereby enhancing Raf-1's ability to stimulate ERK signaling. PDE8A binds to Raf-1 with high (picomolar) affinity. Mapping of the interaction domain on PDE8A using peptide array technology identified amino acids 454-465 as the main binding site, which could be disrupted by mutation. A cell-permeable peptide corresponding to this region disrupted the PDE8A/Raf-1 interaction in cells, thereby reducing ERK activation and the cellular response to EGF. Overexpression of a catalytically inactive PDE8A in cells displayed a dominant negative phenotype on ERK activation. These effects were recapitulated at the organism level in genetically modified (PDE8A(-/-)) mice. Similarly, PDE8 deletion in Drosophila melanogaster reduced basal ERK activation and sensitized flies to stress-induced death. We propose that PDE8A is a physiological regulator of Raf-1 signaling in some cells.

Functional correlates of positional and gender-specific renal asymmetry in Drosophila.

BACKGROUND: In humans and other animals, the internal organs are positioned asymmetrically in the body cavity, and disruption of this body plan can be fatal in humans. The mechanisms by which internal asymmetry are established are presently the subject of intense study; however, the functional significance of internal asymmetry (outside the brain) is largely unexplored. Is internal asymmetry functionally significant, or merely an expedient way of packing organs into a cavity? METHODOLOGY/PRINCIPAL FINDINGS: Like humans, Drosophila shows internal asymmetry, with the gut thrown into stereotyped folds. There is also renal asymmetry, with the rightmost pair of renal (Malpighian) tubules always ramifying anteriorly, and the leftmost pair always sitting posteriorly in the body cavity. Accordingly, transcriptomes of anterior-directed (right-side) and posterior-directed (left-side) Malpighian (renal) tubules were compared in both adult male and female Drosophila. Although genes encoding the basic functions of the tubules (transport, signalling) were uniformly expressed, some functions (like innate immunity) showed positional or gender differences in emphasis; others, like calcium handling or the generation of potentially toxic ammonia, were reserved for just the right-side or left-side tubules, respectively. These findings correlated with the distinct locations of each tubule pair within the body cavity. Well known developmental genes (like dorsocross, dachshund and doublesex) showed continuing, patterned expression in adult tubules, implying that somatic tissues maintain both left-right and gender identities throughout life. Gender asymmetry was also noted, both in defence and in male-specific expression of receptors for neuropeptide F and sex-peptide: NPF elevated calcium only in male tubules. CONCLUSIONS/SIGNIFICANCE: Accordingly, the physical asymmetry of the tubules in the body cavity is directly adaptive. Now that the detailed machinery underlying internal asymmetry is starting to be delineated, our work invites the investigation, not just of tissues in isolation, but in the context of their unique physical locations and milieux.

Immune and stress response 'cross-talk' in the Drosophila Malpighian tubule.

The success of insects is in large part due to their ability to survive environmental stress, including heat, cold, and dehydration. Insects are also exposed to infection, osmotic or oxidative stress, and to xenobiotics or toxins. The molecular mechanisms of stress sensing and response have been widely investigated in mammalian cell lines, and the area of stress research is now so vast to be beyond the scope of a single review article. However, the mechanisms by which stress inputs to the organism are sensed and integrated at the tissue and cellular level are less well understood. Increasingly, common molecular events between immune and other stress responses are observed in vivo; and much of this work stems of efforts in insect molecular science and physiology. We describe here the current knowledge in the area of immune and stress signalling and response at the level of the organism, tissue and cell, focussing on a key epithelial tissue in insects, the Malpighian tubule, and drawing together the known pathways that modulate responses to different stress insults. The tubules are critical for insect survival and are increasingly implicated in responses to multiple and distinct stress inputs. Importantly, as tubule function is central to survival, they are potentially key targets for insect control, which will be facilitated by increased understanding of the complexities of stress signalling in the organism.

Mechanism and function of Drosophila capa GPCR: a desiccation stress-responsive receptor with functional homology to human neuromedinU receptor.

The capa peptide receptor, capaR (CG14575), is a G-protein coupled receptor (GPCR) for the D. melanogaster capa neuropeptides, Drm-capa-1 and -2 (capa-1 and -2). To date, the capa peptide family constitutes the only known nitridergic peptides in insects, so the mechanisms and physiological function of ligand-receptor signalling of this peptide family are of interest. Capa peptide induces calcium signaling via capaR with EC50 values for capa-1 = 3.06 nM and capa-2 = 4.32 nM. capaR undergoes rapid desensitization, with internalization via a b-arrestin-2 mediated mechanism but is rapidly re-sensitized in the absence of capa-1. Drosophila capa peptides have a C-terminal -FPRXamide motif and insect-PRXamide peptides are evolutionarily related to vertebrate peptide neuromedinU (NMU). Potential agonist effects of human NMU-25 and the insect -PRLamides [Drosophila pyrokinins Drm-PK-1 (capa-3), Drm-PK-2 and hugin-gamma [hugg]] against capaR were investigated. NMU-25, but not hugg nor Drm-PK-2, increases intracellular calcium ([Ca²âº]i) levels via capaR. In vivo, NMU-25 increases [Ca²âº]i and fluid transport by the Drosophila Malpighian (renal) tubule. Ectopic expression of human NMU receptor 2 in tubules of transgenic flies results in increased [Ca²âº]i and fluid transport. Finally, anti-porcine NMU-8 staining of larval CNS shows that the most highly immunoreactive cells are capa-producing neurons. These structural and functional data suggest that vertebrate NMU is a putative functional homolog of Drm-capa-1 and -2. capaR is almost exclusively expressed in tubule principal cells; cell-specific targeted capaR RNAi significantly reduces capa-1 stimulated [Ca²âº]i and fluid transport. Adult capaR RNAi transgenic flies also display resistance to desiccation. Thus, capaR acts in the key fluid-transporting tissue to regulate responses to desiccation stress in the fly.

Cell-specific inositol 1,4,5 trisphosphate 3-kinase mediates epithelial cell apoptosis in response to oxidative stress in Drosophila.

Organismal stress responses to oxidative stress are relevant to ageing and disease and involve key cell-/tissue-specific signal transduction mechanisms. Using Drosophila, an established in vivo model for stress studies, we show that cell-specific inositol phosphate signalling specifically via inositol 1,4,5 trisphosphate 3-kinase (InsP(3) 3-K, IP(3)K), negatively regulates organismal responses to oxidative stress. We demonstrate that the Drosophila Malpighian tubule (equivalent to vertebrate kidney and liver) is a key epithelial sensor for organismal oxidative stress responses: precise targeting of either gain-of-function constructs of Drosophila IP(3)Ks (IP(3)K-1 and IP(3)K-2), or loss-of-function (RNAi) constructs to only one cell type in tubule reversibly modulates survival of stress-challenged adult flies. In vivo, targeted IP(3)K-1 directly increases H(2)O(2) production, pro-apoptotic caspase-9 activity and mitochondrial membrane potential. The mitochondrial calcium load in tubule principal cells-assessed by luminescent and fluorescent genetically-encoded mitochondrial calcium reporters-is significantly increased by IP(3)K-1 under oxidative stress conditions, leading to apoptosis. The Drosophila orthologues of human apoptotic bcl-2 genes include debcl and buffy. Oxidative stress challenge does not modulate gene expression of either debcl or buffy in tubules; and altered debcl expression does not influence survival rates under oxidative stress challenge. Finally, targeted over-expression of either debcl or buffy to tubule principal cells does not impact on tubule caspase-9 activity. Thus, IP(3)K-1 modulates epithelial cell apoptosis without involvement of bcl-2-type proteins.

Mislocalization of mitochondria and compromised renal function and oxidative stress resistance in Drosophila SesB mutants.

Mitochondria accumulate at sites of intense metabolic activity within cells, but the adaptive value of this placement is not clear. In Drosophila, sesB encodes the ubiquitous isoform of adenine nucleotide translocase (ANT, the mitochondrial inner membrane ATP/ADP exchanger); null alleles are lethal, whereas hypomorphic alleles display sensitivity to a range of stressors. In the adult renal tubule, which is densely packed with mitochondria and hence enriched for sesB, both hypomorphic alleles and RNA interference knockdowns cause the mitochondria to lose their highly polarized distribution in the tissue and to become rounded. Basal cytoplasmic and mitochondrial calcium levels are both increased, and neuropeptide calcium response compromised, with concomitant defects in fluid secretion. The remaining mitochondria in sesB mutants are overactive and maintain depleted cellular ATP levels while generating higher levels of hydrogen peroxide than normal. When sesB expression is knocked down in just tubule principal cells, the survival of the whole organism upon oxidative stress is reduced, implying a limiting role for the tubule in homeostatic response to stressors. The physiological impacts of defective ANT expression are thus widespread and diverse.