Efficacy and biosafety assessment of neuropeptide CAPA analogues against the peach-potato aphid (Myzus persicae).

Insect CAPA neuropeptidesare considered to affect water and ion balance by mediating the physiological metabolism activities of the Malpighian tubules. In previous studies, the CAPA-PK analogue 1895 (2Abf-Suc-FGPRLamide) was reported to decrease aphid fitness when administered through microinjection or via topical application. However, a further statistically significant decrease in the fitness of aphids and an increased mortality could not be established with pairwise combinations of 1895 with other CAPA analogue. In this study, we assessed the topical application of new combinations of 1895 with five CAPA-PVK analogues on the fitness of aphids. We found that 1895 and CAPA-PVK analogue 2315 (ASG-[ß3 L]-VAFPRVamide) was statistically the most effective combination to control the peach potato aphid Myzus persicae nymphs via topical application, leading to 72% mortality. Additionally, the combination (1895+2315) was evaluated against a selection of beneficial insects, that is, a pollinator (Bombus terrestris) and three natural enemies (Chrysoperla carnea, Nasonia vitripennis, and Adalia bipunctata). We found no significant influence on food intake, weight increase, and survival for the pollinator and the three representative natural enemies. These results could facilitate to further establish and generate CAPA analogues as alternatives to broad spectrum and less friendly insecticides.

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.

Assessment of neuropeptide binding sites and the impact of biostable kinin and CAP2b analogue treatment on aphid (Myzus persicae and Macrosiphum rosae) stress tolerance.

BACKGROUND: Neuropeptides are regulators of critical life processes in insects and, due to their high specificity, represent potential targets in the development of greener insecticidal agents. Fundamental to this drive is understanding neuroendocrine pathways that control key physiological processes in pest insects and the screening of potential analogues. The current study investigated neuropeptide binding sites of kinin and CAPA (CAPA-1) in the aphids Myzus persicae and Macrosiphum rosae and the effect of biostable analogues on aphid fitness under conditions of desiccation, starvation and thermal (cold) stress. RESULTS: M. persicae and M. rosae displayed identical patterns of neuropeptide receptor mapping along the gut, with the gut musculature representing the main target for kinin and CAPA-1 action. While kinin receptor binding was observed in the brain and VNC of M. persicae, this was not observed in M. rosae. Furthermore, no CAPA-1 receptor binding was observed in the brain and VNC of either species. CAP2b/PK analogues (with CAPA receptor cross-activity) were most effective in reducing aphid fitness under conditions of desiccation and starvation stress, particularly analogues 1895 (2Abf-Suc-FGPRLa) and 2129 (2Abf-Suc-ATPRIa), which expedited aphid mortality. All analogues, with the exception of 2139-Ac, were efficient at reducing aphid survival under cold stress, although were equivalent in the strength of their effect. CONCLUSION: In demonstrating the effects of analogues belonging to the CAP2b neuropeptide family and key analogue structures that reduce aphid fitness under stress conditions, this research will feed into the development of second generation analogues and ultimately the development of neuropeptidomimetic-based insecticidal agents. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Epithelial Function in the Drosophila Malpighian Tubule: An In Vivo Renal Model.

The insect renal (Malpighian) tubule has long been a model system for the study of fluid secretion and its neurohormonal control, as well as studies on ion transport mechanisms. To extend these studies beyond the boundaries of classical physiology, a molecular genetic approach together with the 'omics technologies is required. To achieve this in any vertebrate transporting epithelium remains a daunting task, as the genetic tools available are still relatively unsophisticated. Drosophila melanogaster, however, is an outstanding model organism for molecular genetics. Here we describe a technique for fluid secretion assays in the D. melanogaster equivalent of the kidney nephron. The development of this first physiological assay for a Drosophila epithelium, allowing combined approaches of integrative physiology and functional genomics, has now provided biologists with an entirely new model system, the Drosophila Malpighian tubule, which is utilized in multiple fields as diverse as kidney disease research and development of new modes of pest insect control.

New views on the Malpighian tubule from post-genomic technologies.

Successful insect diversification depends at least in part on the ability to osmoregulate successfully across a broad range of ecological niches. First described in the 17th Century, and Malpighian tubules have been studied physiologically for 70 years. However, our understanding has been revolutionized by the advent of genomics, transcriptomics, proteomics and metabolomics. Such technologies are natural partners with (though do not obligatorily require) model organisms and transgenic technologies. This review describes the recent impact of multi-omic technologies on our understanding or renal function and control in insects.

FlyAtlas 2: a new version of the Drosophila melanogaster expression atlas with RNA-Seq, miRNA-Seq and sex-specific data.

FlyAtlas 2 (www.flyatlas2.org) is part successor, part complement to the FlyAtlas database and web application for studying the expression of the genes of Drosophila melanogaster in different tissues of adults and larvae. Although generated in the same lab with the same fly line raised on the same diet as FlyAtlas, the FlyAtlas2 resource employs a completely new set of expression data based on RNA-Seq, rather than microarray analysis, and so it allows the user to obtain information for the expression of different transcripts of a gene. Furthermore, the data for somatic tissues are now available for both male and female adult flies, allowing studies of sexual dimorphism. Gene coverage has been extended by the inclusion of microRNAs and many of the RNA genes included in Release 6 of the Drosophila reference genome. The web interface has been modified to accommodate the extra data, but at the same time has been adapted for viewing on small mobile devices. Users also have access to the RNA-Seq reads displayed alongside the annotated Drosophila genome in the (external) UCSC browser, and are able to link out to the previous FlyAtlas resource to compare the data obtained by RNA-Seq with that obtained using microarrays.

Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila.

Multiple neuropeptides are known to regulate water and ion balance in Drosophila melanogaster. Several of these peptides also have other functions in physiology and behavior. Examples are corticotropin-releasing factor-like diuretic hormone (diuretic hormone 44; DH44) and leucokinin (LK), both of which induce fluid secretion by Malpighian tubules (MTs), but also regulate stress responses, feeding, circadian activity and other behaviors. Here, we investigated the functional relations between the LK and DH44 signaling systems. DH44 and LK peptides are only colocalized in a set of abdominal neurosecretory cells (ABLKs). Targeted knockdown of each of these peptides in ABLKs leads to increased resistance to desiccation, starvation and ionic stress. Food ingestion is diminished by knockdown of DH44, but not LK, and water retention is increased by LK knockdown only. Thus, the two colocalized peptides display similar systemic actions, but differ with respect to regulation of feeding and body water retention. We also demonstrated that DH44 and LK have additive effects on fluid secretion by MTs. It is likely that the colocalized peptides are coreleased from ABLKs into the circulation and act on the tubules where they target different cell types and signaling systems to regulate diuresis and stress tolerance. Additional targets seem to be specific for each of the two peptides and subserve regulation of feeding and water retention. Our data suggest that the ABLKs and hormonal actions are sufficient for many of the known DH44 and LK functions, and that the remaining neurons in the CNS play other functional roles.

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.

Molecular mechanism and functional significance of acid generation in the Drosophila midgut.

The gut of Drosophila melanogaster includes a proximal acidic region (~pH 2), however the genome lacks the H(+)/K(+) ATPase characteristic of the mammalian gastric parietal cell, and the molecular mechanisms of acid generation are poorly understood. Here, we show that maintenance of the low pH of the acidic region is dependent on H(+) V-ATPase, together with carbonic anhydrase and five further transporters or channels that mediate K(+), Cl(-) and HCO3(-) transport. Abrogation of the low pH did not influence larval survival under standard laboratory conditions, but was deleterious for insects subjected to high Na(+) or K(+) load. Insects with elevated pH in the acidic region displayed increased susceptibility to Pseudomonas pathogens and increased abundance of key members of the gut microbiota (Acetobacter and Lactobacillus), suggesting that the acidic region has bacteriostatic or bacteriocidal activity. Conversely, the pH of the acidic region was significantly reduced in germ-free Drosophila, indicative of a role of the gut bacteria in shaping the pH conditions of the gut. These results demonstrate that the acidic gut region protects the insect and gut microbiome from pathological disruption, and shed light on the mechanisms by which low pH can be maintained in the absence of H(+), K(+) ATPase.

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.

The capacity to maintain ion and water homeostasis underlies interspecific variation in Drosophila cold tolerance.

Many insects, including Drosophila, succumb to the physiological effects of chilling at temperatures well above those causing freezing. Low temperature causes a loss of extracellular ion and water homeostasis in such insects, and chill injuries accumulate. Using an integrative and comparative approach, we examined the role of ion and water balance in insect chilling susceptibility/ tolerance. The Malpighian tubules (MT), of chill susceptible Drosophila species lost [Na(+)] and [K(+)] selectivity at low temperatures, which contributed to a loss of Na(+) and water balance and a deleterious increase in extracellular [K(+)]. By contrast, the tubules of chill tolerant Drosophila species maintained their MT ion selectivity, maintained stable extracellular ion concentrations, and thereby avoided injury. The most tolerant species were able to modulate ion balance while in a cold-induced coma and this ongoing physiological acclimation process allowed some individuals of the tolerant species to recover from chill coma during low temperature exposure. Accordingly, differences in the ability to maintain homeostatic control of water and ion balance at low temperature may explain large parts of the wide intra- and interspecific variation in insect chilling tolerance.

Metabolomic profiling of permethrin-treated Drosophila melanogaster identifies a role for tryptophan catabolism in insecticide survival.

Insecticides and associated synergists are rapidly losing efficacy in target insect pest populations making the discovery of alternatives a priority. To discover novel targets for permethrin synergists, metabolomics was performed on permethrin-treated Drosophila melanogaster. Changes were observed in several metabolic pathways including those for amino acids, glycogen, glycolysis, energy, nitrogen, NAD(+), purine, pyrimidine, lipids and carnitine. Markers for acidosis, ammonia stress, oxidative stress and detoxification responses were also observed. Many of these changes had not been previously characterized after permethrin exposure. From the altered pathways, tryptophan catabolism was selected for further investigation. The knockdown of some tryptophan catabolism genes (vermilion, cinnabar and CG6950) in the whole fly and in specific tissues including fat body, midgut and Malpighian tubules using targeted RNAi resulted in altered survival phenotypes against acute topical permethrin exposure. The knockdown of vermilion, cinnabar and CG6950 in the whole fly also altered survival phenotypes against chronic oral permethrin, fenvalerate, DDT, chlorpyriphos and hydramethylnon exposure. Thus tryptophan catabolism has a previously uncharacterized role in defence against insecticides, and shows that metabolomics is a powerful tool for target identification in pesticide research.

Transport proteins NHA1 and NHA2 are essential for survival, but have distinct transport modalities.

The cation/proton antiporter (CPA) family includes the well-known sodium/proton exchanger (NHE; SLC9A) family of Na(+)/H(+) exchangers, and the more recently discovered and less well understood CPA2s (SLC9B), found widely in living organisms. In Drosophila, as in humans, they are represented by two genes, Nha1 (Slc9b1) and Nha2 (Slc9b2), which are enriched and functionally significant in renal tubules. The importance of their role in organismal survival has not been investigated in animals, however. Here we show that single RNAi knockdowns of either Nha1 or Nha2 reduce survival and in combination are lethal. Knockdown of either gene alone results in up-regulation of the other, suggesting functional complementation of the two genes. Under salt stress, knockdown of either gene decreases survival, demonstrating a key role for the CPA2 family in ion homeostasis. This is specific to Na(+) stress; survival on K(+) intoxication is not affected by sodium/hydrogen antiporter (NHA) knockdown. A direct functional assay in Xenopus oocytes shows that Nha2 acts as a Na(+)/H(+) exchanger. In contrast, Nha1 expressed in Xenopus oocytes shows strong Cl(-) conductance and acts as a H(+)-Cl(-) cotransporter. The activity of Nha1 is inhibited by chloride-binding competitors 4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene and 4,4'-dibenzamido-2,2'-stilbenedisulphonate. Salt stress induces a massive up-regulation of NHA gene expression not in the major osmoregulatory tissues of the alimentary canal, but in the crop, cuticle, and associated tissues. Thus, it is necessary to revise the classical view of the coordination of different tissues in the coordination of the response to osmoregulatory 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.

A comprehensive transcriptomic view of renal function in the malaria vector, Anopheles gambiae.

Renal function is essential to maintain homeostasis. This is particularly significant for insects that undergo complete metamorphosis; larval mosquitoes must survive a freshwater habitat whereas adults are terrestrial, and mature females must maintain ion and fluid homeostasis after blood feeding. To investigate the physiological adaptations required for successful development to adulthood, we studied the Malpighian tubule transcriptome of Anopheles gambiae using Affymetrix arrays. We assessed transcription under several conditions; as third instar larvae, as adult males fed on sugar, as adult females fed on sugar, and adult females after a blood meal. In addition to providing the most detailed transcriptomic data to date on the Anopheles Malpighian tubules, the data provide unique information on the renal adaptations required for the switch from freshwater to terrestrial habitats, on gender differences, and on the contrast between nectar-feeding and haematophagy. We found clear differences associated with ontogenetic change in lifestyle, gender and diet, particularly in the neuropeptide receptors that control fluid secretion, and the water and ion transporters that impact volume and composition. These data were also combined with transcriptomics from the Drosophila melanogaster tubule, allowing meta-analysis of the genes which underpin tubule function across Diptera. To further investigate renal conservation across species we selected four D. melanogaster genes with orthologues highly enriched in the Anopheles tubules, and generated RNAi knockdown flies. Three of these genes proved essential, showing conservation of critical functions across 150 million years of phylogenetic separation. This extensive data-set is available as an online resource, MozTubules.org, and could potentially be mined for novel insecticide targets that can impact this critical organ in this pest species.

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.