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

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

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

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

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.

Novel mechanisms of epithelial ion transport: insights from the cryptonephridial system of lepidopteran larvae.

Lepidopterans are among the most widespread and easily recognized insects. Whereas adult lepidopterans are known for their beauty and ecological importance as pollinators and sources of food for other animals, larvae are economically important pests of forests and agricultural crops. In the larval body, rapid growth while feeding on plant-based diet is associated with extreme alkalinity (up to pH = 11) of the midgut lumen that helps digest plant proteins. Additionally, the presence of plant secondary metabolites which serve as anti-herbivory agents requires uninterrupted excretory function, accomplished primarily by the Malpighian tubules (MTs). The so-called cryptonephridial condition, along with extreme regional heterogeneity of the MTs, and the ability to rapidly and reversibly alter the direction of epithelial ion transport are features that allow uninterrupted MT functioning and recycling of base equivalents. Studies of MTs in lepidopteran larvae have revealed that rapid adjustments in epithelial ion transport include unexpected roles for voltage-gated, ligand-gated and mechanosensitive ion channels, as well as gap junctions. These molecular components are present in epithelia of a variety of vertebrates and invertebrates and thus are likely to constitute a universal epithelial toolkit for rapid autonomous regulation of epithelial function.

Voltage-gated calcium channels regulate K+ transport in the Malpighian tubules of the larval cabbage looper, Trichoplusia ni.

Transporting epithelia are tissues that specialize in the directional movements of ions and water and are typically either secretory or reabsorptive. Recent work on the Malpighian tubule of larval lepidopterans (caterpillars) demonstrated that the distal ileac plexus segment of this epithelium is capable of rapidly switching between ion secretion and reabsorption. Subsequent transcriptomic studies suggested expression of voltage-gated ion channels in the lepidopteran MTs (which are not contractile and not innervated). The present study shows that isolated MTs of larval Trichoplusia ni express α1, ß2, and α2δ4 subunits of voltage-gated Ca2+ channel CaV1 and that pan-CaVα immunoreactivity is present in the apical and basolateral membranes of the principal cells. Basolateral membrane potential (Vbl) in isolated MTs of larval Trichoplusia ni was influenced by CaV1 functioning; pharmacological inhibition of CaV1 reversed Vbl from inside-negative to inside-positive, and also reduced transepithelial potential (Vte), lowered [Ca2+]i and reversed the direction of K+ transport from secretion to reabsorption. Thus, our findings indicate that a functional CaV1 channel is necessary for constitutive K+ secretion observed in isolated preparations of lepidopteran MTs. Lastly, Vte and Vbl of isolated MTs were influenced by changes in bathing saline [K+]. Our findings suggest that epithelia may rely on CaV channels to enable robust ion secretion and downregulation of CaV channels, together with other transcriptional changes, enables ion reabsorption.

A unique Malpighian tubule architecture in Tribolium castaneum informs the evolutionary origins of systemic osmoregulation in beetles.

Maintaining internal salt and water balance in response to fluctuating external conditions is essential for animal survival. This is particularly true for insects as their high surface-to-volume ratio makes them highly susceptible to osmotic stress. However, the cellular and hormonal mechanisms that mediate the systemic control of osmotic homeostasis in beetles (Coleoptera), the largest group of insects, remain largely unidentified. Here, we demonstrate that eight neurons in the brain of the red flour beetle Tribolium castaneum respond to internal changes in osmolality by releasing diuretic hormone (DH) 37 and DH47-homologs of vertebrate corticotropin-releasing factor (CRF) hormones-to control systemic water balance. Knockdown of the gene encoding the two hormones (Urinate, Urn8) reduces Malpighian tubule secretion and restricts organismal fluid loss, whereas injection of DH37 or DH47 reverses these phenotypes. We further identify a CRF-like receptor, Urinate receptor (Urn8R), which is exclusively expressed in a functionally unique secondary cell in the beetle tubules, as underlying this response. Activation of Urn8R increases K+ secretion, creating a lumen-positive transepithelial potential that drives fluid secretion. Together, these data show that beetle Malpighian tubules operate by a fundamentally different mechanism than those of other insects. Finally, we adopt a fluorescent labeling strategy to identify the evolutionary origin of this unusual tubule architecture, revealing that it evolved in the last common ancestor of the higher beetle families. Our work thus uncovers an important homeostatic program that is key to maintaining osmotic control in beetles, which evolved parallel to the radiation of the "advanced" beetle lineages.

Claudins of sea lamprey (Petromyzon marinus) - organ-specific expression and transcriptional responses to water of varying ion content.

The role of lamprey epithelium tight junctions (TJs) in the regulation of salt and water balance is poorly understood. This study reported on claudin (Cldn) TJ protein transcripts of pre-metamorphic larval and post-metamorphic juvenile sea lamprey (Petromyzon marinus) and the transcriptional response of genes encoding Cldns to changed environmental ion levels. Transcripts encoding Cldn-3b, -4, -5, -10, -14, -18 and -19 were identified, and mRNA expression profiles revealed the organ-specific presence of cldn-5 and -14, broad expression of cldn-3b, -4, -10, -18 and -19 and spatial differences in the mRNA abundance of cldn-4, -3b and -14 along the ammocoete intestine. Expression profiles were qualitatively similar in ammocoetes and juvenile fishes. Transcript abundance of genes encoding Cldns in osmoregulatory organs (gill, kidney, intestine and skin) was subsequently investigated after exposure of ammocoetes to ion-poor water (IPW) and juveniles to hyperosmotic conditions [60% sea water (SW)]. IPW-acclimated ammocoetes increased mRNA abundance of nearly all cldns in the gill. Simultaneously, cldn-10 abundance increased in the skin, whereas cldn-4, -14 and -18 decreased in the kidney. Ammocoete cldn mRNA abundance in the intestine was altered in a region-specific manner. In contrast, cldn transcript abundance was mostly downregulated in osmoregulatory organs of juvenile fish acclimated to SW - cldn-3b, -10 and -19 in the gill; cldn-3b, -4, -10 and -19 in the skin; cldn-3b in the kidney; and cldn-3b and -14 in the intestine. Data support the idea that Cldn TJ proteins play an important role in the osmoregulatory physiology of pre- and post-metamorphic sea lamprey and that Cldn participation can occur across organs, in an organ-specific manner, as well as differ spatially within organs, which contributes to the regulation of salt and water balance in these fishes.

Mechanisms and regulation of chloride transport in the Malpighian tubules of the larval cabbage looper Trichoplusia ni.

Malpighian tubules (MTs) and the hindgut together constitute the excretory system of insects. Larvae of lepidopterans (butterflies and moths) demonstrate the so-called cryptonephric arrangement, where the distal blind end of each MT is embedded into the rectal complex. The rest of the free tubule is modified into several distinct regions that differ greatly in the transport of cations and water. However, relatively little is known about the transport of counter-anions (e.g., Cl- and HCO3-) by the MTs of lepidopteran larvae. In the current study we used ion-selective microelectrodes to characterize Cl- transport in the distinct regions of the free MT of the larval Trichoplusia ni. Firstly, we note that Cl- transport in the MTs is sensitive to the Cl- concentration of the bathing saline, and several regions of the MTs are capable of either secreting or reabsorbing Cl-. In the distal ileac plexus (DIP), a region previously characterized by cellular heterogeneity and its ability to switch between cation secretion and reabsorption, principal cells (PCs) toggled between Cl- reabsorption (in high-Cl- saline) and Cl- secretion (in low-Cl- saline). In contrast, secondary cells (SCs) in the DIP secreted Cl- regardless of saline Cl- concentration. Mechanistically, we have detected a number of 'leak' and ligand-gated Cl- channels (ClC) and demonstrated that Cl- channels are involved in Cl- secretion. Additionally, we demonstrated that the lumen-positive transepithelial potential increased in response to glycine. Using the scanning ion-selective electrode technique we demonstrated that glycine stimulated Cl- secretion by SCs, but not by PCs. In contrast, when MTs were deprived of glycine, a decrease in Cl- secretion, coupled with a decrease in the TEP, was observed. In contrast to the effects of glycine, an active dose of helicokinin reduced Cl- secretion by PCs, but not by SCs. Lastly, we detected expression of chloride-bicarbonate exchangers (CBE) in all regions of the free tubule. Scans of H+ transport across the tubule indicated that base equivalents are likely reabsorbed across the ileac plexus. Blocking ClC or CBE led to secretion of a more basic fluid, indicating lack of base reabsorption. We suggest that the transport of Cl- in the MTs of larval lepidopterans (i) may be correlated with the reabsorption of base, (ii) may be sensitive to Cl- concentration in the haemolymph, and (iii) could be regulated by helicokinin and glycine.

C-type natriuretic peptide regulates the molecular components of the rainbow trout gill epithelium tight junction complex.

Freshwater (FW) fish experience passive paracellular loss of ions into the surrounding environment across water-exposed epithelia such as the gill. The mitigation of paracellular ion loss is thought to be regulated by proteins of the tight junction (TJ) complex and in particular, the large superfamily of claudin (cldn) TJ proteins plays an important role. Transcript and protein levels of TJ proteins in teleosts are known to be under endocrine control of several important osmoregulatory hormones and the current study was aimed at determining whether the osmoregulatory hormone, C-type natriuretic peptide (CNP), can alter paracellular permeability and TJ protein abundance in a primary cultured gill epithelium derived from rainbow trout. Natriuretic peptide receptors were detected in the cultured trout gill epithelium. It was found that (i) developing cultured gill epithelia "grown" in the presence of 10 nM CNP, and (ii) mature cultured gill epithelia exposed to 10 nM CNP for 48 h, exhibited augmented barrier properties. This occurred in association with reduced flux rates of a paracellular permeability marker (polyethylene glycol, molecular mass 400; PEG-400) and, reduced ion efflux (i.e. ion loss) when preparations were exposed to apical FW. Exposure to CNP altered mRNA abundance of cldn-3a, -5a, -6, - 8c, -20a, -25b, -28a, -32a and cgn, but differences in the transcriptional response were observed between chronic and acute CNP exposure. In contrast, chronic and acute exposure to CNP resulted in reduced cldn-10e/Cldn-10e abundance. Data suggest that CNP may play a role in regulating the molecular physiology of the TJ complex in the fish gill epithelium and contribute to the regulation of salt and water balance by influencing the paracellular permeability properties of this tissue.

Malpighian tubules of caterpillars: blending RNAseq and physiology to reveal regional functional diversity and novel epithelial ion transport control mechanisms.

The Malpighian tubules (MTs) and hindgut constitute the functional kidney of insects. MTs are outpouchings of the gut and in most insects demonstrate proximodistal heterogeneity in function. In most insects, such heterogeneity is confined to ion/fluid secretion in the distal portion and ion/fluid reabsorption in the proximal portion. In contrast, MTs of larval Lepidoptera (caterpillars of butterflies and moths) are composed of five regions that differ in their association with the gut, their structure and ion/fluid transport function. Recent studies have shown that several regions can rapidly and reversibly switch between ion secretion and reabsorption. The present study employed RNAseq, pharmacology and electrophysiology to characterize four distinct regions of the MT in larval Trichoplusia ni Luminal microelectrode measurements indicate changes in [K+], [Na+] and pH as fluid passes through different regions of the tubule. In addition, the regions examined differ in gene ontology enrichment, and demonstrate robust gradients in expression of ion transporters and endocrine ligand receptors. Lastly, the study provides evidence for direct involvement of voltage- and ligand-gated ion channels in epithelial ion transport of insect MTs.

Section-specific H+ flux in renal tubules of fasted and fed goldfish.

A recent study demonstrated that in response to a feeding-induced metabolic acidosis, goldfish (Carassius auratus) adjust epithelial protein and/or mRNA expression in their kidney tubules for multiple transporters known to be relevant for acid-base regulation. These include Na+/H+ exchanger (NHE), V-type H+-ATPase (V-ATPase), cytoplasmic carbonic anhydrase, HCO3- transporters and Rhesus proteins. Consequently, renal acid output in the form of protons and NH4+ increases. However, little is known about the mechanistic details of renal acid-base regulation in C. auratus and teleost fishes in general. The present study applied the scanning ion-selective electrode technique (SIET) to measure proton flux in proximal, distal and connecting tubules of goldfish. We detected increased H+ efflux into the extracellular fluid from the tubule in fed animals, resulting from paracellular back-flux of H+ through the tight junction. By applying inhibitors for selected acid-base regulatory epithelial transporters, we found that cytosolic carbonic anhydrase and HCO3- transporters were important in mediating H+ flux in all three tubule segments of fed goldfish. Contrastingly, V-ATPase seemed to play a role in H+ flux only in proximal and distal tubules, and NHE in proximal and connecting tubules. We developed working models for transport of acid-base relevant equivalents (H+, HCO3-, NH3/NH4+) for each tubule segment in C. auratus kidney. While the proximal tubule appears to play a major role in both H+ secretion and HCO3- reabsorption, the distal and connecting tubules seem to mainly serve for HCO3- reabsorption and NH3/NH4+ secretion.

The mineralocorticoid receptor contributes to barrier function of a model fish gill epithelium.

Cortisol-induced epithelial tightening of a primary cultured rainbow trout gill epithelium model occurs in association with reduced paracellular permeability and increased abundance of select barrier-forming tight junction (TJ) proteins. Corticosteroid receptor (CR) pharmacological blocker studies have suggested that to produce this tightening effect, cortisol acts on the mineralocorticoid receptor (MR) as well as glucocorticoid receptors (GRs). This study considered how cortisol influences model gill epithelium permeability and TJ properties by transcriptional knockdown of the gene encoding the MR (mr-KD) using double-stranded RNA. Following mr-KD, a significant reduction in MR protein abundance was observed in the epithelium. The mr-KD epithelium demonstrated reduced transepithelial resistance (TER) and an increase in the paracellular flux of [3H]polyethylene glycol (MW 400 kDa, PEG-400). Concurrently, mRNA abundance of gr2 and 11ßhsd increased, indicating a possible compensatory response to mr-KD. Transcript abundance of claudin (cldn)-6, -8d, -23a and -28b decreased while that of cldn-20a increased in mr-KD preparations. Cortisol-induced epithelial tightening was enhanced in mr-KD preparations, suggesting that alterations in CRs and TJ composition augmented model epithelium barrier function in response to lowered MR abundance. Cortisol treatment significantly increased the transcript and protein abundance of TJ proteins such as Cldn-8d and -28b. However, in mr-KD preparations, Cldn-28b protein abundance did not significantly alter in response to cortisol treatment, while Cldn-8d abundance was significantly elevated. Data suggest that mr-KD compromises normal barrier function of a primary cultured rainbow trout gill epithelium in both the presence and absence of cortisol and that Cldn-28b protein abundance may be modulated by cortisol via the MR only.

Water and ion transport across the eversible vesicles in the collophore of the springtail Orchesella cincta.

Springtails (Collembola) are ancient close relatives of the insects. The eversible vesicles are their unique paired transporting organs, which consist of an epithelium located inside a tube-like structure called the collophore on the first abdominal segment. The vesicles can be protruded out of the collophore and several lines of evidence indicate that they have a vital function in water uptake and ion balance. However, the amount of water absorbed by the vesicles and which other ions apart from Na+ are transported remain unknown. Using Orchesella cincta as a model, we developed protocols for two assays that enabled us to study water and ion movement across the eversible vesicles in whole living springtails. Using an inverse Ramsay assay we demonstrate that the eversible vesicles absorb water from a droplet applied onto their surface. Using the scanning ion-selective electrode technique (SIET), we show that the vesicles absorb Na+ and Cl- from the bathing medium, secrete NH4+, and both absorb and secrete K+ H+ is secreted at a low level in the anterior part and absorbed at the posterior part. We did not detect transport of Ca2+ at significant levels. The highest flux was the absorption of Cl-, and the magnitude of ion fluxes was significantly lower in fully hydrated springtails. Our data demonstrate that the eversible vesicles are a transporting epithelium functioning in osmo- and ionoregulation, nitrogenous waste excretion and probably also acid-base balance.

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.

Helicokinin alters ion transport in the secondary cell-containing region of the Malpighian tubule of the larval cabbage looper Trichoplusia ni.

Excretion in insects is accomplished by the combined actions of the Malpighian tubules (MTs) and hindgut, which together form the functional kidney. MTs of many insect groups consist of principal cells (PC) and secondary cells (SC). In most insect groups SCs are reported to secrete ions from haemolymph into the tubule lumen. Paradoxically, SCs in the MTs of the lepidopteran cabbage looper T. ni are used to reabsorb Na+ and K+ back into haemolymph. The current study was designed to investigate the effects and mode of action of the lepidopteran kinin, Helicokinin (HK), on ion transport in the SC-containing region of MT of T. ni. We identified a HK receptor (HK-R) homologue in T. ni and detected its expression in the SC-containing region of the MTs. The mRNA abundance of hk-r altered in response to changes in dietary K+ and Na+ content. HK-R immunolocalized to both PCs and SCs. Ramsay assays of preparations of the isolated distal ileac plexus (DIP) indicated that [HK] = 10-8 M: (i) decreased fluid secretion rate in unstimulated and serotonin-stimulated preparations, and (ii) increased [Na+]/[K+] ratio in the secreted fluid. Scanning ion-selective electrode technique measurements revealed that HK reduced: (i) K+ secretion by the PCs, and (ii) Na+ reabsorption by the SCs in intact tubules. In vitro incubation of the DIP with HK resulted in reduced mRNA abundance of hk-r as well as Na+/K+-ATPase subunit α (NKAα), Na+/K+/Cl- co-transporter (nkcc), Na+/H+ exchangers (nhe) 7 and 8, and aquaporin (aqp) 1. Taken together, results of the current study suggest that HK is capable of altering fluid secretion rate and [Na+]/[K+] ratio of the fluid, and that HK targets both PCs and SCs in the DIP of T. ni.

Transcriptomic analysis of the Malpighian tubules of Trichoplusia ni: Clues to mechanisms for switching from ion secretion to ion reabsorption in the distal ileac plexus.

Excretion of metabolic wastes and toxins in insect Malpighian tubules (MTs) is coupled to secretion of ions and fluid. Larval lepidopterans demonstrate a complex and regionalized MT morphology, and recent studies of larvae of the cabbage looper, Trichoplusia ni, have revealed several unusual aspects of ion transport in the MTs. Firstly, cations are reabsorbed via secondary cells (SCs) in T. ni, whereas in most insects SCs secrete ions. Secondly, SCs are coupled to neighbouring principal cells (PCs) via gap junctions to enable such ion reabsorption. Thirdly, PCs in the SC-containing distal ileac plexus (DIP) region of the tubule reverse from cation secretion to reabsorption in response to dietary ion loading. Lastly, antidiuresis is observed in response to a kinin neuropeptide, which targets both PCs and SCs, whereas in most insects kinins are diuretics that act exclusively via SCs. Recent studies have generated a basic model of ion transport in the DIP of the larval T. ni. RNAseq was used to elucidate previously uncharacterised aspects of ion transport and endocrine regulation in the DIP, with the aim of painting a composite picture of ion transport and identifying putative regulatory mechanisms of ion transport reversal in this tissue. Results indicated an overall expression of 9103 transcripts in the DIP, 993 and 382 of which were differentially expressed in the DIP of larvae fed high-K+ and high-Na+ diets respectively. Differentially expressed transcripts include ion-motive ATPases, ion channels and co-transporters, aquaporins, nutrient and xenobiotic transporters, cell adhesion and junction components, and endocrine receptors. Notably, several transcripts for voltage-gated ion channels and cell volume regulation-associated products were detected in the DIP and differentially expressed in larvae fed ion-rich diet. The study provides insights into the transport of solutes (sugars, amino acids, xenobiotics, phosphate and inorganic ions) by the DIP of lepidopterans. Our data suggest that this region of the MT in lepidopterans (as previously reported) transports cations, fluid, and xenobiotics/toxic metals. Besides this, the DIP expresses genes coding for the machinery involved in Na+- and H+-dependent reabsorption of solutes, chloride transport, and base recovery. Additionally, many of the transcripts expressed by the DIP a capacity of this region to respond to, process, and sometimes produce, neuropeptides, steroid hormones and neurotransmitters. Lastly, the DIP appears to possess an arsenal of septate junction components, differential expression of which may indicate junctional restructuring in the DIP of ion-loaded larvae.

The Effect of Diet on Midgut and Resulting Changes in Infectiousness of AcMNPV Baculovirus in the Cabbage Looper, Trichoplusia ni.

Insecticide resistance has been reported in many important agricultural pests, and alternative management methods are required. Baculoviruses qualify as an effective, yet environmentally benign, biocontrol agent but their efficacy against generalist herbivores may be influenced by diet. However, few studies have investigated the tritrophic interactions of plant, pest, and pathogen from both a gene expression and physiological perspective. Here we use microscopy and transcriptomics to examine how diet affects the structure of peritrophic matrix (PM) in Trichoplusia ni larvae and consequently their susceptibility to the baculovirus, AcMNPV. Larvae raised on potato leaves had lower transcript levels for chitinase and chitin deacetylase genes, and possessed a thicker and more multi-layered PM than those raised on cabbage or artificial diet, which could contribute to their significantly lower susceptibility to the baculovirus. The consequences of these changes underline the importance of considering dietary influences on pathogen susceptibility in pest management strategies.

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.

Tricellular tight junction-associated angulins in the gill epithelium of rainbow trout.

Molecular physiology of the tricellular tight junction (tTJ)-associated proteins lipolysis-stimulated lipoprotein receptor ( lsr, = angulin-1) and an immunoglobulin-like domain-containing receptor ( ildr2, ≈angulin-3) was examined in model trout gill epithelia. Transcripts encoding lsr and ildr2 are broadly expressed in trout organs. A reduction in lsr and ildr2 mRNA abundance was observed during and after confluence in flask-cultured gill cells. In contrast, as high-resistance and low-permeability characteristics developed in a model gill epithelium cultured on permeable polyethylene terephthalate membrane inserts, lsr and ildr2 transcript abundance increased. However, as epithelia entered the developmental plateau phase, lsr abundance returned to initial values, while ildr2 transcript abundance remained elevated. When mitochondrion-rich cells were introduced to model preparations, lsr mRNA abundance was unaltered and ildr2 mRNA abundance significantly increased. Transcript abundance of ildr2 was not altered in association with corticosteroid-induced tightening of the gill epithelium, while lsr mRNA abundance decreased. Transcriptional knockdown of the tTJ protein tricelluin (Tric) reduced Tric abundance, increased gill epithelium permeability, and increased lsr without significantly altering ildr2 transcript abundance. Data suggest that angulins contribute to fish gill epithelium barrier properties but that Lsr and Ildr2 seem likely to play different roles. This is because ildr2 typically exhibited increased abundance in association with decreased model permeability, while lsr abundance changed in a manner that suggested a role in Tric recruitment to the tTJ.

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

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