The Malpighian tubules (MTs) play a dominant role in elimination of potentially toxic compounds, including endogenous metabolites and xenobiotics (O'Donnell, 2009). Among these compounds are organic cations, a structurally diverse group of primary, secondary, tertiary or quaternary amines that carry a net positive charge on the amine nitrogen at physiological pH and may include both endogenous compounds, xenobiotics and xenobiotic metabolites. Endogenous organic cations include catecholamines, choline and N-methylnicotinamide (NMN), while xenobiotics include environmental pollutants, animal toxins, plant alkaloids and drugs such as tetraethylammonium (TEA). Previous studies of organic cations by renal epithelial in vertebrates and invertebrates have made extensive use of the prototypical organic cation tetraethylammonium, as reviewed previously (O'Donnell, 2009, Wright and Dantzler, 2004). TEA has long been used as a proxy for other endogenous cations because transport of TEA can be quantified using radiolabelled 14C-TEA or through use of ion exchanger-based microelectrodes which are highly selective for TEA relative to other cations.

Organic cations transported by insect MTs of 9 species from 6 orders examined to date include endogenous compounds such as choline and N-methylnicotinamide, plant secondary metabolites such as nicotine and quinine, and drugs such as tetraethylammonium (TEA) (O’Donnell, 2008, Rheault et al., 2006). Secretion of TEA is competitively inhibited by other organic cations such as cimetidine (Rheault et al., 2006). There is also a pronounced regionalization of TEA transport by Drosophila melanogaster tubules: rates of transport per unit area are highest in the lower tubule and ureter, relative to the fluid-secreting main segment (Rheault and O'Donnell, 2004). MTs isolated from larvae of D. melanogaster that have been exposed to dietary TEA excrete TEA at higher rates than tubules from controls fed a TEA-free diet (Bijelic et al., 2005). In addition to modulation of TEA secretion rates by dietary exposure to TEA, there is evidence that the rates change during the moult cycle. MTs of Rhodnius prolixus undergoing moulting secrete TEA at 6 times the rate measured in tubules of pre-moult insects (Rheault et al., 2006).

This study examines TEA transport by the MTs of the cabbage looper caterpillar, Trichoplusia ni. As in other lepidopteran larvae, the MTs of T. ni form a close association with the gut (Figure 1. The distal end of each tubule is termed the cryptonephridial Malpighian tubule (cMT) because it is applied to the surface of the rectum and is enveloped by the perinephric membrane (Kolosov and O'Donnell, 2019). The cMT is thus enclosed within the perinephric space (PNS). The cMTs, rectal epithelium and perinephric membrane together form the rectal complex. The rectal lead connects the cMT to the ‘free’ tubule (i.e., outside of the rectal complex). The first segment downstream of the rectal lead is the ileac plexus, which consists of tightly coiled and intertwined distal, middle and proximal regions that lie close to the ileum of the gut. The yellow MT (YMT) is a straight region that lies anterior to the ileac plexus and overlays the posterior midgut and the white MT (WMT) is a second straight region running posteriorly along the midgut. The white regions of three MTs on each side of the midgut join to form a common ureter which leads into the urinary bladder.

Recent studies of inorganic ion (Na+, K+, Cl-) secretion by MTs of the cabbage looper have revealed pronounced differences in ion transport rates and direction along the length of the tubule and by different cell types, as well as changes in ion transport in feeding larvae relative to those with empty guts (Kolosov et al., 2018b; O'Donnell and Ruiz-Sanchez, 2015). Caterpillar larvae are characterized by extremely alkaline midguts, in which high pH is thought to dissociate tannin-protein complexes and thus enhance digestion of plant material (Berenbaum, 1980). Reabsorption of K+ by the YMT and WMT is associated with base (HCO3-) recovery, allowing recycling of base equivalents for midgut alkalinization (Irvine, 1969, Moffett, 1994). Within the ileac plexus, transport function varies with cell type. The more numerous principal cells (PCs) are yellow and opaque and secrete K+, whereas the large translucent secondary cells (SCs) reabsorb K+. Transport may also differ in feeding larvae versus those in which the gut is empty. The distal rectal lead of larvae with empty guts, found in recently moulted larvae, reabsorbs K+, whereas the same region secretes K+ in tubules of larvae with full guts (O'Donnell and Ruiz-Sanchez, 2015). The distal ileac plexus of tubules in larvae with full guts also secretes K+, whereas the middle and proximal regions of the ileac plexus reabsorb K+ (O'Donnell and Ruiz-Sanchez, 2015). In animals fed K+-rich diet, however, the PCs of the distal ileac plexus switch from Na+/K+ secretion to reabsorption (Kolosov et al., 2018a). Surprisingly, when the ileac plexus is isolated from the gut of larvae fed K+-rich diet so that it no longer receives fluid and ions from the upstream rectal complex, PCs switch from reabsorption to K+ secretion within ∼ 10 minutes. Secretion of K+ by isolated tubules is dependent upon the functioning of a voltage-dependent Ca2+ channel (CaV1) in the cells of the ileac plexus. Pharmacological inhibition of CaV1 results in a lowering of intracellular Ca2+ levels and reverses the direction of K+ transport in isolated tubules from secretion to reabsorption (Kolosov et al., 2021).

Insect Malpighian tubules are characterized by a lumen-positive transepithelial potential (TEP) (Beyenbach et al., 2000), so secretion of fluid containing the cation TEA at concentrations above those in the bathing saline indicates active transport against opposing electrical and chemical gradients. Active secretion of organic cations, such as TEA, by an epithelium requires transport across the basolateral membrane, an intracellular compartment, and an apical membrane in series. Transmembrane electrical potentials across the two membranes can be calculated from measurements of TEP and basolateral membrane potential (Vbl). TEP is equal to the sum of Vbl and the apical membrane potential (Vap). The Vap can thus be calculated by the difference TEP – Vbl. Previous studies have shown that uptake of organic cations across the basolateral membrane of MTs of Drosophila melanogaster is carrier-mediated and potential-dependent (Rheault et al., 2005).

Most studies of caterpillar tubules to date have examined transport of inorganic ions (Na+, K+, Cl-) and bicarbonate (Kolosov and O'Donnell, 2019, Kolosov and O'Donnell, 2020). A previous study reported that MTs of the cabbage looper T. ni secrete the prototypical organic cation TEA (Rheault et al., 2006), but the regions involved and the influence of full versus empty guts on TEA secretion was not examined. This paper examines transport of the organic cation TEA by different segments of the MTs as well as the influence of TEA-enriched diets and the gut condition (full versus empty) on transport of TEA by in situ tubules, which retain their connection to the gut, and by isolated tubules. In addition, TEP and Vbl have been measured in regions of the ileac plexus to assess the influence of transmembrane electrical gradients on TEA transport.