Proton-driven sodium secretion in a saline water animal.

Aquatic animals residing in saline habitats either allow extracellular sodium concentration to conform to environmental values or regulate sodium to lower levels. The latter strategy requires an energy-driven process to move sodium against a large concentration gradient to eliminate excess sodium that diffuses into the animal. Previous studies of invertebrate and vertebrate species indicate a sodium pump, Na+/K+ ATPase, powers sodium secretion. We provide the first functional evidence of a saline-water animal, Aedes taeniorhynchus mosquito larva, utilizing a proton pump to power this process. Vacuolar-type H+ ATPase (VHA) protein is highly expressed on the apical membrane of the posterior rectal cells, and in situ sodium flux across this epithelium increases significantly in larvae held in higher salinity and is sensitive to Bafilomycin A1, an inhibitor of VHA. We also report the first evidence of splice variants of the sodium/proton exchanger, NHE3, with both high and low molecular weight variants highly expressed on the apical membrane of the posterior rectal cells. Evidence of NHE3 function was indicated with in situ sodium transport significantly inhibited by a NHE3 antagonist, S3226. We propose that the outward proton pumping by VHA establishes a favourable electromotive gradient to drive sodium secretion via NHE3 thus producing a hyperosmotic, sodium-rich urine. This H+- driven Na+ secretion process is the primary mechanism of ion regulation in salt-tolerant culicine mosquito species and was first investigated over 80 years ago.

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

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

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.

Osmoregulatory Challenges Faced by Mosquitoes and Techniques for Studying the Transporters Involved in Their Adaptations.

Mosquitoes are the most important disease vector in the world, and gaining knowledge of their physiology to develop novel population control strategies has been a focus of research for some time. Both aquatic larvae and terrestrial adults face harsh environmental factors that severely challenge their salt and water balance, which are regulated by the function of epithelia of various organs. The regulated passage of water and solutes across epithelia occurs, in part, through transporters expressed in epithelial cell membranes. Identifying these transporters and their localization is necessary to understand how mosquitoes regulate salt and water balance. Here, we review environmental challenges faced by mosquitoes and how they cope with them, in addition to introducing techniques used to identify organ epithelial transporters.

Localizing Protein Expression of Transporters in Epithelial Cell Membranes of Mosquitoes.

Identifying and localizing ion transport proteins in epithelia is important for understanding how these tissues can regulate salt and water balance in animals. Mosquitoes face distinct challenges regarding salt and water balance as larvae live in water of varying ionic composition while adult female mosquitoes must deal with periodic large blood meals. This protocol will explain how to localize ion transporters in epithelia of mosquitoes. Antibodies raised against specific proteins can be used to carry out immunohistochemistry to tag and visualize native proteins in tissues and cells. This technique may be used for whole tissues and organs or can also be used on histological thin sections of fixed tissue. This protocol will detail the use of immunohistochemistry to localize membrane proteins in mosquito organs.

Hiat1 as a new transporter involved in ammonia regulation.

The orphan transporter hippocampus-abundant transcript 1 (Hiat1) was first identified in the mammalian brain. Its specific substrate specificity, however, has not been investigated to date. Here, we identified and analyzed Hiat1 in a crustacean, the green crab Carcinus maenas. Our phylogenetic analysis showed that Hiat1 protein is conserved at a considerable level between mammals and this invertebrate (ca. 78% identical and conserved amino acids). Functional expression of Carcinus maenas Hiat1 in Xenopus laevis oocytes demonstrated the capability to transport ammonia (likely NH4+) in a sodium-dependent manner. Furthermore, applying quantitative polymerase chain reaction, our results indicated a physiological role for Carcinus maenas Hiat1 in ammonia homeostasis, as mRNA abundance increased in posterior gills in response to elevated circulating hemolymph ammonia upon exposure to high environmental ammonia. Its ubiquitous mRNA expression pattern also suggests an essential role in general cellular detoxification of ammonia. Overall, our results introduce a new ubiquitously expressed ammonia transporter, consequently demanding revision of our understanding of ammonia handling in key model systems from mammalian kidneys to crustacean and fish gills.

Physiological responses of freshwater insects to salinity: molecular-, cellular- and organ-level studies.

Salinization of freshwater is occurring throughout the world, affecting freshwater biota that inhabit rivers, streams, ponds, marshes and lakes. There are many freshwater insects, and these animals are important for ecosystem health. These insects have evolved physiological mechanisms to maintain their internal salt and water balance based on a freshwater environment that has comparatively little salt. In these habitats, insects must counter the loss of salts and dilution of their internal body fluids by sequestering salts and excreting water. Most of these insects can tolerate salinization of their habitats to a certain level; however, when exposed to salinization they often exhibit markers of stress and impaired development. An understanding of the physiological mechanisms for controlling salt and water balance in freshwater insects, and how these are affected by salinization, is needed to predict the consequences of salinization for freshwater ecosystems. Recent research in this area has addressed the whole-organism response, but the purpose of this Review is to summarize the effects of salinization on the osmoregulatory physiology of freshwater insects at the molecular to organ level. Research of this type is limited, and pursuing such lines of inquiry will improve our understanding of the effects of salinization on freshwater insects and the ecosystems they inhabit.

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito.

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the foundation to develop novel strategies for pest control. Here, a series of methods are described that are routinely utilized to determine the functional roles of neuropeptides and other neuronal factors (i.e., biogenic amines) on the excretory system of the mosquito, Aedes aegypti. The Malpighian tubules (MTs), responsible for primary urine formation, can continue functioning for hours when removed from the mosquito, allowing for fluid secretion measurements following hormone treatments. As such, the Ramsay assay is a useful technique to measure secretion rates from isolated MTs. Ion-selective microelectrodes (ISME) can sequentially be used to measure ion concentrations (i.e., Na+ and K+) in the secreted fluid. This assay allows for the measurement of several MTs at a given time, determining the effects of various hormones and drugs. The Scanning Ion-selective Electrode Technique uses ISME to measure voltage representative of ionic activity in the unstirred layer adjacent to the surface of ion transporting organs to determine transepithelial transport of ions in near real time. This method can be used to understand the role of hormones and other regulators on ion absorption or secretion across epithelia. Hindgut contraction assays are also a useful tool to characterize myoactive neuropeptides, that may enhance or reduce the ability of this organ to remove excess fluid and waste. Collectively, these methods provide insight into how the excretory system is regulated in adult mosquitoes. This is important because functional coordination of the excretory organs is crucial in overcoming challenges such as desiccation stress after eclosion and before finding a suitable vertebrate host to obtain a bloodmeal.

The transcriptome of anal papillae of Aedes aegypti reveals their importance in xenobiotic detoxification and adds significant knowledge on ion, water and ammonia transport mechanisms.

The anal papillae of mosquito larvae are osmoregulatory organs in direct contact with the external aquatic environment that actively sequester ions and take up water in dilute freshwater. In the disease vector Aedes aegypti mechanisms of ion, water and ammonia transport have only been partially resolved. Furthermore, A. aegypti larvae are known to reside in high ammonia sewage and high salt brackish waters, and understanding of anal papillae function in these conditions is in its infancy. The objective of this study was to identify the complement of ion and water transport genes expressed by the anal papillae of freshwater larvae by sequencing their transcriptome, and comparing their expression in anal papillae of larvae abruptly transferred to brackish water for 24 h. Results identified a number of ion and water transport proteins, ammonia detoxifying enzymes, a full suite of xenobiotic detoxifying enzymes and transporters, and G-protein coupled receptors of specific hormones. We identified a marked increase in transcript and protein abundance of aquaporin AaAQP2 in the anal papillae with abrupt transfer to brackish water. We present an updated and more comprehensive model for ion and water transport with additional putative transporters for Na+ and Cl- uptake in the anal papillae. These are organs which are actively engaged in Na+, Cl- and water uptake and regulation when the aquatic larvae encounter fluctuating salinities over the course of their development. Furthermore the transcriptome of the anal papillae includes a full set of xenobiotic detoxification genes suggesting that these are important detoxification organs which is particularly important when larvae reside in polluted water.

Ammonium transporter expression in sperm of the disease vector Aedes aegypti mosquito influences male fertility.

The ammonium transporter (AMT)/methylammonium permease (MEP)/Rhesus glycoprotein (Rh) family of ammonia (NH3/NH4+) transporters has been identified in organisms from all domains of life. In animals, fundamental roles for AMT and Rh proteins in the specific transport of ammonia across biological membranes to mitigate ammonia toxicity and aid in osmoregulation, acid-base balance, and excretion have been well documented. Here, we observed enriched Amt (AeAmt1) mRNA levels within reproductive organs of the arboviral vector mosquito, Aedes aegypti, prompting us to explore the role of AMTs in reproduction. We show that AeAmt1 is localized to sperm flagella during all stages of spermiogenesis and spermatogenesis in male testes. AeAmt1 expression in sperm flagella persists in spermatozoa that navigate the female reproductive tract following insemination and are stored within the spermathecae, as well as throughout sperm migration along the spermathecal ducts during ovulation to fertilize the descending egg. We demonstrate that RNA interference (RNAi)-mediated AeAmt1 protein knockdown leads to significant reductions (∼40%) of spermatozoa stored in seminal vesicles of males, resulting in decreased egg viability when these males inseminate nonmated females. We suggest that AeAmt1 function in spermatozoa is to protect against ammonia toxicity based on our observations of high NH4+ levels in the densely packed spermathecae of mated females. The presence of AMT proteins, in addition to Rh proteins, across insect taxa may indicate a conserved function for AMTs in sperm viability and reproduction in general.

A comparison of aquaporin expression in mosquito larvae (Aedes aegypti) that develop in hypo-osmotic freshwater and iso-osmotic brackish water.

The mosquito Aedes aegypti vectors the arboviral diseases yellow fever, dengue, Zika and chikungunya. Larvae are usually found developing in freshwater; however, more recently they have been increasingly found in brackish water, potential habitats which are traditionally ignored by mosquito control programs. Aedes aegypti larvae are osmo-regulators maintaining their hemolymph osmolarity in a range of ~ 250 to 300 mOsmol l-1. In freshwater, the larvae must excrete excess water while conserving ions while in brackish water, they must alleviate an accumulation of salts. The compensatory physiological mechanisms must involve the transport of ions and water but little is known about the water transport mechanisms in the osmoregulatory organs of these larvae. Water traverses cellular membranes predominantly through transmembrane proteins named aquaporins (AQPs) and Aedes aegypti possesses 6 AQP homologues (AaAQP1 to 6). The objective of this study was to determine if larvae that develop in freshwater or brackish water have differential aquaporin expression in osmoregulatory organs, which could inform us about the relative importance and function of aquaporins to mosquito survival under these different osmotic conditions. We found that AaAQP transcript abundance was similar in organs of freshwater and brackish water mosquito larvae. Furthermore, in the Malpighian tubules and hindgut AaAQP protein abundance was unaffected by the rearing conditions, but in the gastric caeca the protein level of one aquaporin, AaAQP1 was elevated in brackish water. We found that AaAQP1 was expressed apically while AaAQP4 and AaAQP5 were found to be apical and/or basal in the epithelia of osmoregulatory organs. Overall, the results suggest that aquaporin expression in the osmoregulatory organs is mostly consistent between larvae that are developing in freshwater and brackish water. This suggests that aquaporins may not have major roles in adapting to longterm survival in brackish water or that aquaporin function may be regulated by other mechanisms like post-translational modifications.

Active transport of brilliant blue FCF across the Drosophila midgut and Malpighian tubule epithelia.

Under conditions of stress, many animals suffer from epithelial barrier disruption that can cause molecules to leak down their concentration gradients, potentially causing a loss of organismal homeostasis, further injury or death. Drosophila is a common insect model, used to study barrier disruption related to aging, traumatic injury, or environmental stress. Net leak of a non-toxic dye (Brilliant blue FCF) from the gut lumen to the hemolymph is often used to identify barrier failure under these conditions, but Drosophila are capable of actively transporting structurally-similar compounds. Here, we examined whether cold stress (like other stresses) causes Brilliant blue FCF (BB-FCF) to appear in the hemolymph of flies fed the dye, and if so whether Drosophila are capable of clearing this dye from their body following chilling. Using in situ midgut leak and transport assays as well as Ramsay assays of Malpighian tubule transport, we tested whether these ionoregulatory epithelia can actively transport BB-FCF. In doing so, we found that the Drosophila midgut and Malpighian tubules can mobilize BB-FCF via an active transcellular pathway, suggesting that elevated concentrations of the dye in the hemolymph may occur from increased paracellular permeability, reduced transcellular clearance, or both. SUMMARY STATEMENT: Drosophila are able to actively secrete Brilliant blue FCF, a commonly used marker of barrier dysfunction.

Development of Aedes aegypti (Diptera: Culicidae) mosquito larvae in high ammonia sewage in septic tanks causes alterations in ammonia excretion, ammonia transporter expression, and osmoregulation.

Larvae of the disease vector mosquito, Aedes aegypti (L.) readily develop in ammonia rich sewage in the British Virgin Islands. To understand how the larvae survive in ammonia levels that are lethal to most animals, an examination of ammonia excretory physiology in larvae collected from septic-water and freshwater was carried out. A. aegypti larvae were found to be remarkably plastic in dealing with high external ammonia through the modulation of NH4+ excretion at the anal papillae, measured using the scanning ion-selective electrode technique (SIET), and NH4+ secretion in the primary urine by the Malpighian tubules when developing in septicwater. Ammonia transporters, Amt and Rh proteins, are expressed in ionoregulatory and excretory organs, with increases in Rh protein, Na+-K+-ATPase, and V-type-H+-ATPase expression observed in the Malpighian tubules, hindgut, and anal papillae in septic-water larvae. A comparative approach using laboratory A. aegypti larvae reared in high ammonia septic-water revealed similar responses to collected A. aegypti with regard to altered ammonia secretion and hemolymph ion composition. Results suggest that the observed alterations in excretory physiology of larvae developing in septic-water is a consequence of the high ammonia levels and that A. aegypti larvae may rely on ammonia transporting proteins coupled to active transport to survive in septic-water.

An impressive capacity for cold tolerance plasticity protects against ionoregulatory collapse in the disease vector Aedes aegypti.

The mosquito Aedes aegypti is largely confined to tropical and subtropical regions, but its range has recently been spreading to colder climates. As insect biogeography is tied to environmental temperature, understanding the limits of A. aegypti thermal tolerance and their capacity for phenotypic plasticity is important in predicting the spread of this species. In this study, we report on the chill coma onset (CCO) and recovery time (CCRT), as well as low-temperature survival phenotypes of larvae and adults of A. aegypti that developed or were acclimated to 15°C (cold) or 25°C (warm). Cold acclimation did not affect CCO temperatures of larvae but substantially reduced CCO in adults. Temperature and the duration of exposure both affected CCRT, and cold acclimation strongly mitigated these effects and increased rates of survival following prolonged chilling. Female adults were far less likely to take a blood meal when cold acclimated, and exposing females to blood (without feeding) attenuated some of the beneficial effects of cold acclimation on CCRT. Lastly, larvae suffered from haemolymph hyperkalaemia when chilled, but cold acclimation attenuated the imbalance. Our results demonstrate that A. aegypti larvae and adults have the capacity to acclimate to low temperatures, and do so at least in part by better maintaining ion balance in the cold. This ability for cold acclimation may facilitate the spread of this species to higher latitudes, particularly in an era of climate change.

Septate junction in the distal ileac plexus of larval lepidopteran Trichoplusia ni: alterations in paracellular permeability during ion transport reversal.

The Malpighian tubules (MTs) and hindgut together act as the functional kidney in insects. MTs of caterpillars are notably complex and consist of several regions that display prominent differences in ion transport. The distal ileac plexus (DIP) is a region of MT that is of particular interest because it switches from ion secretion to ion reabsorption in larvae fed on ion-rich diets. The pathways of solute transport in the DIP are not well understood, but one potential route is the paracellular pathway between epithelial cells. This pathway is regulated by the septate junctions (SJs) in invertebrates, and in this study, we found regional and cellular heterogeneity in the expression of several integral SJ proteins. DIP of larvae fed ion-rich diets demonstrated a reduction in paracellular permeability, coupled with alterations in both SJ morphology and the abundance of its molecular components. Similarly, treatment in vitro with helicokinin (HK), an antidiuretic hormone identified by previous studies, altered mRNA abundance of many SJ proteins and reduced paracellular permeability. HK was also shown to target a secondary cell-specific SJ protein, Tsp2A. Taken together, our data suggest that dietary ion loading, known to cause ion transport reversal in the DIP of larval Trichoplusiani, leads to alterations in paracellular permeability, SJ morphology and the abundance of its molecular components. The results suggest that HK is an important endocrine factor that co-regulates ion transport, water transport and paracellular permeability in MTs of larval lepidopterans. We propose that co-regulation of all three components of the MT function in larval lepidopterans allows for safe toggling between ion secretion and reabsorption in the DIP in response to variations in dietary ion availability.

Impact of salt-contaminated freshwater on osmoregulation and tracheal gill function in nymphs of the mayfly Hexagenia rigida.

The impact of freshwater (FW) salinization on osmoregulation as well as tracheal gill morphology and function was examined in nymphs of the mayfly Hexagenia rigida following exposure to salt contaminated water (SCW, 7.25 g/l NaCl) for a 7-day period. Ionoregulatory homeostasis was perturbed in SCW exposed H. rigida nymphs as indicated by increased hemolymph Na+, K+ and Cl- levels as well as hemolymph pH and water content. Despite this, SCW did not alter gill Na+-K+-ATPase (NKA) or V-type H+-ATPase (VA) activity. In addition, NKA and VA immunolocalization in gill ionocytes did not show alterations in enzyme location or changes in ionocyte abundance. The latter observation was confirmed using scanning electron microscopy (SEM) to examine exposed tracheal gill ionocyte numbers. Ionocyte surface morphometrics also revealed that SCW did not change individual ionocyte surface area or ionocyte fractional surface area. Nevertheless, analysis of Na+ movement across the tracheal gill of mayfly nymphs using scanning ion-selective electrode technique indicated that FW nymphs acquired Na+ from surrounding water, while tracheal gills of SCW nymphs had the capacity to secrete Na+. Because Na+ secretion across the gill of SCW-exposed animals occurred in the absence of any change in (1) NKA and VA activity or (2) ionocyte numbers/surface exposure, it was reasoned that Na+ movement across the gill of SCW animals may be occurring, at least in part, through the paracellular pathway. The ultrastructure of tracheal gill septate junctions (SJs) supported this idea as they exhibited morphological alterations indicative of a leakier pathway. Data provide a first look at alterations in osmoregulatory mechanisms that allow H. rigida nymphs to tolerate sub-lethal salinization of their surroundings.

Evidence that Rh proteins in the anal papillae of the freshwater mosquito Aedes aegypti are involved in the regulation of acid-base balance in elevated salt and ammonia environments.

Aedes aegypti commonly inhabit ammonia-rich sewage effluents in tropical regions of the world where the adults are responsible for the spread of disease. Studies have shown the importance of the anal papillae of A. aegypti in ion uptake and ammonia excretion. The anal papillae express ammonia transporters and Rhesus (Rh) proteins which are involved in ammonia excretion and studies have primarily focused on understanding these mechanisms in freshwater. In this study, effects of rearing larvae in salt (5 mmol l-1 NaCl) or ammonia (5 mmol l-1 NH4Cl) on physiological endpoints of ammonia and ion regulation were assessed. In anal papillae of NaCl-reared larvae, Rh protein expression increased, NHE3 transcript abundance decreased and NH4+ excretion increased, and this coincided with decreased hemolymph [NH4+] and pH. We propose that under these conditions, larvae excrete more NH4+ through Rh proteins as a means of eliminating acid from the hemolymph. In anal papillae of NH4Cl-reared larvae, expression of an apical ammonia transporter and the Rh proteins decreased, the activities of NKA and VA decreased and increased, respectively, and this coincided with hemolymph acidification. The results present evidence for a role of Rh proteins in acid-base balance in response to elevated levels of salt, whereby ammonia is excreted as an acid equivalent.

Anti-diuretic activity of a CAPA neuropeptide can compromise Drosophila chill tolerance.

For insects, chilling injuries that occur in the absence of freezing are often related to a systemic loss of ion and water balance that leads to extracellular hyperkalemia, cell depolarization and the triggering of apoptotic signalling cascades. The ability of insect ionoregulatory organs (e.g. the Malpighian tubules) to maintain ion balance in the cold has been linked to improved chill tolerance, and many neuroendocrine factors are known to influence ion transport rates of these organs. Injection of micromolar doses of CAPA (an insect neuropeptide) have been previously demonstrated to improve Drosophila cold tolerance, but the mechanisms through which it impacts chill tolerance are unclear, and low doses of CAPA have been previously demonstrated to cause anti-diuresis in insects, including dipterans. Here, we provide evidence that low (femtomolar) and high (micromolar) doses of CAPA impair and improve chill tolerance, respectively, via two different effects on Malpighian tubule ion and water transport. While low doses of CAPA are anti-diuretic, reduce tubule K+ clearance rates and reduce chill tolerance, high doses facilitate K+ clearance from the haemolymph and increase chill tolerance. By quantifying CAPA peptide levels in the central nervous system, we estimated the maximum achievable hormonal titres of CAPA and found further evidence that CAPA may function as an anti-diuretic hormone in Drosophila melanogaster We provide the first evidence of a neuropeptide that can negatively affect cold tolerance in an insect and further evidence of CAPA functioning as an anti-diuretic peptide in this ubiquitous insect model.

Molecular mechanisms of bi-directional ion transport in the Malpighian tubules of a lepidopteran crop pest, Trichoplusia ni.

Classical studies have described in detail the complex and regionalized morphology of the Malpighian tubule (MT) in larval Lepidoptera. Recent studies revealed unusual aspects of ion transport in the Malpighian tubules of the larva of the cabbage looper, Trichoplusia ni. These included: cation reabsorption via secondary cells (SC); coupling of SCs to neighbouring PCs via gap junctions to enable reabsorption; and a reversal from cation secretion to reabsorption by the principal cells in the distal ileac plexus region of the in situ tubule in response to dietary ion loading. The current paper aimed to identify molecular components of ion transport in the MTs of T. ni and to describe their role in the recently reported reversal of ion transport in ion-loaded animals. Using a combination of molecular, immunohistochemical and electrophysiological techniques, we assigned roles to Na+/K+-ATPase (NKA), V-type H+-ATPase (VA), Na+/K+/Cl- co-transporter (NKCC), K+/Cl- co-transporter (KCC), inward-rectifying K+ channel (Kir), and Na+/H+ exchangers (NHE)-7 and -8 in the transport of Na+ and K+ by the distal ileac plexus of T. ni. The yellow region of the tubule lacked all of the above except VA, and the white region lacked all of the above transporters but expressed an amiloride-sensitive Na+ channel (NaC). Overall, the ion transport machinery in the distal ileac plexus of the T. ni tubule shows remarkable similarity to that in tubules of other groups of insects, yet this region transports ions very differently. Shutdown of secretory ATPases and utilisation of the same molecular machinery in the face of changing ion gradients may enable ion transport reversal in lepidopteran MTs. We propose that gap junction-based coupling of the two cell types likely aids in toggling between ion secretion and ion reabsorption in this segment.

Galanin decreases spontaneous resting contractions and potentiates acetyl choline-induced contractions of goldfish gut.

Galanin (GAL) is a 29 amino acid peptide, first identified from the porcine intestine and widely distributed within the brain and peripheral tissues. Among GAL biological functions, its role as a potent appetite-stimulating peptide is probably the most studied. With galanin's established role in the modulation of food intake in fish, this study aims to evaluate the effects of GAL on the intestinal motility of the goldfish, Carassius auratus, using an organ bath system. Our results found that application of GAL to the organ bath causes a significant concentration-dependent decrease in the amplitude of spontaneous contractions of goldfish gut. Preincubations of intestinal strips with acetylcholine (ACh) and GAL showed that GAL increases the force of ACh-induced contractions of the goldfish gut. These results provide the first evidence for a role of GAL in gut motility in goldfish. This also suggests a crosstalk between the effects of GAL and ACh in such functions, thus pointing to a putative joint role between the two molecules. These findings offer novel information that strengthens the role of the galaninergic system in fish feeding.