Select latest published work

Dr. Kolosov and his team have an appreciable track record of working with vertebrate (e.g., fish gill) and invertebrate (e.g., Malpighian tubules) epithelia. Links to several currently relevant publications are provided below. For a full list of publications please visit Dr. Kolosov's Google Scholar page

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

October 2021 

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.

1-s2.0-S0022191021000792-ga1_lrg.jpg

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 (a collaborative study with Dr. Andrew Donini's lab, York University, Canada)

June 20 2021 

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 hor- mones. 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.

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

March 17 2021 

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 chan- nels in the lepidopteran MTs (which are not contractile and not innervated). The present study shows that isolated MTs of larval Trichoplusia ni express α1, β2, 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 func- tioning; 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.

Screen Shot 2021-08-23 at 6.00.05 PM.png

A unique Malpighian tubule architecture in Tribolium castaneum informs the evolutionary origins of systemic osmoregulation in beetles (a collaborative study with Dr. Kenneth Halberg's lab, University of Copenhagen, Denmark)

February 22 2021 

Maintaining internal salt and water balance in response to fluctu- ating 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 con- trol of osmotic homeostasis in beetles (Coleoptera), the largest group of insects, remain largely unidentified. Here, we demon- strate that eight neurons in the brain of the red flour beetle Tri- bolium castaneum respond to internal changes in osmolality by releasing diuretic hormone (DH) 37 and DH47—homologs of ver- tebrate 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 recep- tor, Urinate receptor (Urn8R), which is exclusively expressed in a functionally unique secondary cell in the beetle tubules, as under- lying 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 tu- bules 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 architec- ture, revealing that it evolved in the last common ancestor of the higher beetle families. Our work thus uncovers an important ho- meostatic program that is key to maintaining osmotic control in beetles, which evolved parallel to the radiation of the “advanced” beetle lineages.

Screen Shot 2019-11-03 at 11.44.09 AM.pn

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

January 2020

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.

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

October 2019

Malpighian tubules (MTs) and hindgut constitute the functional kidney of insects. MTs are outpouches 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 comprised 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 current 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-gated and ligand-gated ion channels in epithelial ion transport of insect MTs.

SJ paper_edited.png

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

May 2019

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 Malpighian tubule 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 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 T. ni, leads to alterations in the paracellular permeability, SJ morphology and its molecular component abundance. 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.

Book chapter: The Malpighian tubules and cryptonephric complex in lepidopteran larvae

February 2019

Lepidopterans (butterflies and moths) are an ecologically and agriculturally important group of holometabolous insects. Their larvae and adults exhibit trophic partitioning, which is reflected by the various modifications of their digestive and excretory systems. Adults are capable of flight and feed mostly on the nectar of plants, acting as pollinators. Larvae are voracious leaf eaters whose extremely alkaline midgut(pH ≈ 11) is an adaptation to high tannin levels in the host plants. Morphologically and functionally regionalized Malpighian tubules of the larva modify fluid as it flows through them. The larvae also exhibit the so-called cryptonephric condition, where the distal end of the tubule is juxtaposed to the rectum and enveloped by the perinephric membrane. The distal part of the free tubule adjacent to the ileum, termed the distal ileac plexus, is characterized by a high density of secondary cells. Recent studies have identified several unusual aspects of ion transportphysiology of the distal ileac plexus: (i) gap junctional coupling of principal and secondary cells that allows them to transport ions in opposite directions, (ii) the ability to switch between K+ secretion and K+reabsorption depending on the input from the cryptonephric tubule, (iii) the presence of voltage-gated, ligand-gated and mechanosensitive ion channels and (iv) coordinated regulation of water and septate junctionpermeability during the reversal from secretion to reabsorption (presumably aimed at retention of water and solute content in the distal ileac plexus lumen, while Na+ and K+ are being reabsorbed). We describe recent advances in understanding ion-transporting and regulatory mechanisms in the Malpighian tubules of larval Lepidoptera with a special emphasis on the distal ileac plexus segment.

Water and ion transport across the eversible vesicles in the collophore of a springtail Orchesella cincta (Collaborative study with Dr. Barbora Konopova, University of Göttingen, Germany)

May 2019

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 on the first abdominal segment called the collophore. 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 sodium 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. We did not detect transport of Ca2+ at significant levels. The highest flux was the absorption of Cl-, and the magnitude of ion fluxes were 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 likely acid-base balance.

RNAseq study on Malpighian tubules

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

December 2018

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 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.

midgut pH.jpg

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

September 2018

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.

HK paper.jpg

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

July 2018

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.

mol mech paper.jpg

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

June 2018

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.

GJ paper.jpg

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.

January 2018

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 immunolocalised 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 ion supply for the SC function in this segment of the tubule.