Elsevier

Journal of Insect Physiology

Volume 112, January 2019, Pages 73-89
Journal of Insect Physiology

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

https://doi.org/10.1016/j.jinsphys.2018.12.005Get rights and content

Highlights

  • Previously uncharacterized ion transport mechanisms targeted in the DIP with RNAseq.

  • Several voltage-gated, ligand-gated and mechanosensitive channels were detected.

  • Na+- and H+-dependent reabsorption of solutes “flushed” during fluid secretion.

  • Cl secretion mechanisms may be correlated with base recovery.

  • Response to, processing, and sometimes production of endocrine ligands.

Abstract

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.

Introduction

Insects represent more than three-quarters of terrestrial animals and are of extreme importance as pollinators, disease vectors, invasive species and agricultural pests. Butterflies and moths belong to a group of insects that contains many agricultural pests, 28 species of which have warranted government-level management programs in the last three decades (Gullan and Cranston, 2014, Suckling et al., 2017). Lepidopteran larvae (caterpillars), are voracious feeders that are capable of 1000-fold increases in mass during their development (Grunert et al., 2015). Caterpillars, (e.g., T. ni) consume three times their own weight daily, feeding on a wide variety of cultivated plants and weeds (McEwen and Hervey, 1960). Physiological consequences of ingesting such large amounts of food include a constant need for active excretion of metabolites and the need to maintain ion balance as hemolymph volume increases during rapid growth. A deep understanding of the complement of transporters and endocrine receptors expressed in an insect renal system is a necessary first step toward developing new and highly specific methods of insect control targeting this system. The current paper will utilise RNAseq methodology to uncover the mechanisms and control of ion transport, and excretion present in the renal system of lepidopteran larvae.

Excretion in insects is accomplished by the combined actions of the Malpighian tubules (MT) and hindgut, which together form the functional ‘kidney’. Active transport of primary osmolytes (e.g., K+ and Cl) from haemolymph into the MT drives fluid secretion by osmosis. Ions, metabolic wastes and toxins can also be added to the secreted fluid by passive paracellular leakage (see Jonusaite et al., 2016, Furuse and Izumi, 2017 for review) or secondary active transport mechanisms (O’Donnell, 2008). Beyenbach and colleagues have convincingly demonstrated that MTs of Aedes aegypti can alter paracellular permeability selectively for Cl (Beyenbach and Piermarini, 2011), while permeability to large macromolecules has been demonstrated in the MTs of Rhodnius prolixus (O’Donnell et al., 1984) and A. aegypti (Jonusaite et al., 2017). The primary urine is then modified in downstream segments of the tubule or the hindgut (Wigglesworth, 1961, Bradley, 1985). For instance, in MTs of dipteran Drosophila melanogaster (O’Donnell and Maddrell, 1995), hemipteran R. prolixus (Haley and Donnell, 1997) and orthopteran Acheta domesticus (Spring and Hazelton, 1987), much of the water and ions are reabsorbed from the lumen before the excreta leave the body. In general, the ionomotive enzymes used to drive fluid secretion employ ions supplied in abundance through the diet. Current models of ion transport in dipterans (e.g., Piermarini and Gillen, 2015) and lepidopterans (e.g., Kolosov et al., 2018b) propose uptake of ions across the basolateral membrane by Na+/K+-ATPase, Na+:K+:2Cl (NKCC) cotransporters and inward rectifying K+ channels (Kir). Across the apical membrane, a vacuolar-type H+-ATPase coupled to Na+/H+ or K+/H+ exchangers drives K+ and Na+ from cell to tubule lumen (Wieczorek et al., 2009). One advantage of this arrangement is that phytophagous insects feeding on K+-rich diets can secrete K+-rich fluids.

Several classical studies have highlighted the anatomical complexity of the lepidopteran MT system and highlighted their juxtaposition to the regions of the gut (Irvine, 1969, Ramsay, 1976, Moffett, 1994). Detailed pictorial descriptions of MT regions in the larval T. ni can be found in O’Donnell and Ruiz-Sanchez, 2015, Kolosov et al., 2018a. Briefly, six MTs present in the larvae are divided into the cryptonephridial MT (embedded into rectal complex adjacent to the rectum), the ileac plexus (IP) (highly convoluted and juxtaposed to the ileum), and the yellow and white regions (adjacent to the midgut). Recent studies have described several unusual aspects of ion transport in the DIP. Of special interest was the reversal from ion secretion to ion reabsorption in the DIP of caterpillars acutely exposed to High-K+ or High-Na+ diets (Kolosov et al., 2018a). Therefore, transcriptomic data were obtained from the DIP (see Fig. 1) in this study with the intention of identifying differentially expressed transcripts during this reversal in the direction of ion transport.

As in the tubules of many insect species (Halberg et al., 2015), the majority of the DIP epithelium is comprised of the principal cells (PC) with secondary cells (SC) embedded in-between (O’Donnell and Ruiz-Sanchez, 2015). In most insects studied to date, SCs, along with PCs, contribute to the secretion of ions, which, in turn, drives secretion of osmotically obliged water into the tubule lumen (Dow, 2012). The two cell types have been found to be regulated by separate endocrine factors in dipterans, where PC’s secrete primarily cations (O'Donnell et al., 1996, Sajadi et al., 2018). Anions, on the other hand, are secreted by both paracellular and transcellular pathways in MTs of mosquitoes (Aedes aegypti) (Beyenbach and Piermarini, 2011), but primarily by transcellular pathways in MTs of Drosophila melanogaster (O'Donnell et al., 1996, Cabrero et al., 2014).

Recent studies have demonstrated several unique aspects of the SC-containing region of the MT in lepidopteran larvae, termed the distal ileac plexus (DIP). Firstly, SCs of lepidopterans reabsorb cations (Na+ and K+) instead of secreting them (O’Donnell and Ruiz-Sanchez, 2015). This reabsorption of cations by the SCs (at least in part) relies, on the gap junctional coupling to the neighbouring PCs (Kolosov et al., 2018a). Moreover, PCs in the DIP of larvae fed ion-rich diets, reabsorb cations in situ, but secrete cations when the tubules are isolated in the Ramsay assay (Ruiz-Sanchez et al., 2015, Kolosov et al., 2018b). Lastly, members of the kinin neuropeptide superfamily are known for diuretic effects through stimulation of transport pathways (Halberg et al., 2015), often in SCs (O'Donnell et al., 1996, O’Donnell et al., 1998, Beyenbach, 2003). In contrast, a lepidopteran kinin, Helicokinin, targets both PCs and SCs in the DIP of lepidopteran larvae and is antidiuretic with a narrow active dose range in isolated DIP preparations (Kolosov and O’Donnell, 2018).

The genome of T. ni (together with a transcriptome and proteome) has been released recently, simplifying a Next Generation Sequencing approach in the search for the molecular mechanisms of ion transport in the MT of this animal (Fu et al., 2017). The findings on the DIP described above imply the occurrence of novel ion transport mechanisms in this segment of the MT. Our goal in this paper is to identify these novel ion transport mechanisms using the RNA-Seq, a method of transcriptome profiling that uses deep-sequencing technologies (Wang et al., 2009). Our experimental design consisted of feeding larvae on high-K+ or high-Na+ diets that have been reported to induce ion transport reversal in the DIP when compared to the standard McMorran diet. We hypothesised that transcripts encoding the molecular machinery involved in the previously-reported reversal of ion transport in the DIP of ion-loaded larvae would be expressed differentially (as determined by DESeq2 protocols) in the DIP of larvae fed high-K+ and high-Na+ diets.

Section snippets

Experimental animals

Eggs of T. ni (Hübner 1800) were purchased from the Great Lakes Forestry Centre (Sault St. Marie, ON). Larvae were maintained at 23–25 °C and 40–50% relative humidity while fed synthetic McMorran diet containing 59 mM [K+] and 18 mM [Na+]. McMorran diet ingredients were purchased from Insect Production Services (Great Lakes Forestry Centre, Sault Ste. Marie, ON, Canada). The diet contained (per 1-liter): 860 ml distilled water, 17.36 g agar, 35 g casein, 35 g sucrose, 30.69 g toasted wheat

RNAseq approach to identifying the molecular machinery of ion transport in the distal ileac plexus (DIP) of the larval T. ni

The RNAseq approach was used to detect transcripts related to water and solute transport, that are expressed in the DIP of the larval cabbage looper (Fig. 1). Transcripts encoding 8 ion-motive ATPases (holoenzyme assemblies) were detected, along with 71 ion channels, 117 non-channel ion transporters (co-transporters and exchangers), 4 aquaporins (water channels), 52 nutrient (sugar and amino acid) transporters, and 23 xenobiotic/toxin transporters. In addition, the DIP was found to express 43

Overview and significance of RNAseq data

The advantage of the RNAseq approach for identification of mechanisms of ion transport in the MTs of lepidopterans is that it allows simultaneous detection of all relevant gene transcripts in the tissue. This in turn allows data mining of transcripts of interest that could be used in future hypothesis-driven studies. Numerous genomic and transcriptomic studies on the MTs of insects have been performed to elucidate ion transport mechanisms and their endocrine regulatory networks (Hewes and

Acknowledgements and funding sources

This work was supported by the following funding agencies. DK is funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) Post-Doctoral Fellowship. CD, HM and MJO are supported by NSERC Discovery Grants. Additionally, MJO was awarded Discovery Accelerator Supplement. The authors would like to thank Dr. Andrew Donini (York University, Toronto, Canada) and Dr. Jean-Paul Paluzzi (York University, Toronto, Canada) for fruitful discussions.

Statement of contributions

DK and MJO conceived and designed the experiments; DK performed the experiments; MJO contributed reagents and materials; DK and CD analyzed the data; DK and HM visualized the data; DK and MJO wrote the manuscript; CD and HM edited the manuscript; all authors read and approved the final manuscript version.

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