The endocrine control of salt balance in insects

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General and Comparative Endocrinology 152 (2007) 332–338

The endocrine control of salt balance in insects

Geoffrey Coast *

School of Biological & Chemical Sciences, Birkbeck (University of London), Malet Street, London WC1E 7HX, UK

Received 3 November 2006; revised 12 February 2007; accepted 17 February 2007Available online 27 February 2007

Abstract

An overview is given of the role of Malpighian (renal) tubules and the hindgut (ileum and rectum) in the excretory process of insects.The review focuses on the mechanism of primary urine production by Malpighian tubules and its control by neurohormones, whichincludes serotonin and neuropeptides resembling mammalian corticotropin-releasing factor (CRF) and calcitonin. Particular emphasisis given to in vitro studies of the effect of neurohormones on Malpighian tubule ion transport and a consideration of their likely role in theregulation of salt balance in vivo.� 2007 Elsevier Inc. All rights reserved.

Keywords: Malpighian tubules; Hindgut; Diuresis; Natriuresis; Diuretic hormones; Antidiuretic factors

1. Introduction

The insect excretory system comprises Malpighian(renal) tubules and the hindgut, notably the ileum and rec-tum. Malpighian tubules (MTs) generate a flow of primaryurine approximately isosmotic to haemolymph (blood),rich in KCl and/or NaCl, and containing low molecularweight solutes that enter diffusively and organic soluteswhich are actively transported into the lumen. The primaryurine drains into the hindgut where it may be substantiallymodified by the absorption of ions, water and essentialmetabolites before being voided from the anus. The finalexcreta can be hypo- or hyperosmotic to haemolymph,and is enriched in nitrogenous and toxic wastes. The roleof MTs in the excretory process is therefore functionallyequivalent to that of the glomerulus of the vertebratekidney, whereas the ileum and rectum perform roles thatare analogous to those performed by the proximal andmore distal tubular segments, respectively.

Malpighian tubule and hindgut functions are indepen-dently regulated by neurohormones with either diuretic

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or antidiuretic activity (Coast et al., 2002; Schooley et al.,2005). These hormones are the products of neurosecretorycells in the brain and/or the ventral nerve cord. They arereleased into the circulation from neurohaemal areas suchas the corpora cardiaca (the insect equivalent of the verte-brate pituitary), perivisceral organs, and from sites alongabdominal nerves. In general, diuretics stimulate MT secre-tion whereas antidiuretics promote fluid reabsorption fromthe ileum and rectum. Recently, however, antidiureticshave been identified that reduce MT secretion.

MT secretion is readily studied in vitro with the Ramsayassay (Ramsay, 1954). For this assay, MTs are severedfrom the gut and transferred individually to small dropsof bathing fluid (5–50 lL) held beneath paraffin oil. Thecut end of each MT is withdrawn from the bathing fluidand secreted urine collects as a discrete droplet in thesurrounding paraffin oil. Measurements of the volumeand composition of urine droplets collected over set timeintervals allow rates of MT secretion and transepithelialion transport to be calculated. The assay has been usedextensively to investigate the effect of endocrine factorson primary urine formation in a number of species, includ-ing fruit flies and mosquitoes. Such endocrine factorsinclude serotonin and members of several different neuro-peptide families (Table 1). In contrast, the only detailed

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Table 1Structures of selected diuretic and antidiuretic peptides in insects

Family/ peptide Sequence Reference

CRF-DH

Manse-DH RMPSLSIDLPMSVLRQKLSLEKERKVHALRAAANRNFLNDI-NH2 Kataoka et al., 1989 #496

CT-DH

Dippu-DH31 GLDLGLSRGFSGSQAAKHLMGLAAANYAGGP-NH2 Furuya et al., 2000 #5221

Kinin

Locmi-TK-I DPAFNSWG-NH2 Holman et al., 1986 #258

CAP2b (Capa)

Manse-CAP2b pQLYAFPRV-NH2 Huesmann et al., 1995 #1956

ADFs

Tenmo-ADFa VVNTPGHAVSYHVY-COOH Eigenheer et al., 2002 #6046Tenmo-ADFb YDDGSYKPHIYGF-COOH Eigenheer et al., 2003 #6529

ITP

Schgr-ITP SFFDIQCKGVYDKSIFARLDRICEDCYNLFREPQLHSLC Meredith et al., 1996 #2614RSDCFKSPYFKGCLQALLLIDEEEKFNQMVEIL-NH2

The structures shown are those of the first identified member of each peptide family.

G. Coast / General and Comparative Endocrinology 152 (2007) 332–338 333

investigations of hindgut function have been in the desertlocust (Schistocerca gregaria) by the laboratory of JohnPhillips (University of British Columbia, Canada). Themajor transepithelial active transport process in both ileumand rectum is an apical membrane electrogenic Cl� pump,which generates a Cl�-dependent short-circuit current (Iscc)(Phillips et al., 1996). Ileal Iscc (i.e. Cl� transport) is stimu-lated 10-fold by Ion Transport Peptide (ITP), which is amember of the Crustacean Hyperglycaemic Hormone(CHH) family (see Table 1). ITP also stimulates Na+ andK+ uptake, and increases isosmotic fluid absorption 4-fold.The rectum can absorb fluid hypo-osmotic to the luminalcontents, resulting in the production of concentrated urine.Rectal Iscc and fluid uptake are stimulated by ChlorideTransport Stimulating Hormone (CTSH), which has notbeen characterised, but differs from ITP in being acid labile(Coast et al., 2002).

Dissecting the contribution made by the MTs and hind-gut to the excretory process in vivo is hampered by the lackof a convenient method to quantify primary urine produc-tion in the intact insect. The focus of this review is theendocrine control of MT ion transport, but ion and fluidabsorption in the hindgut will normally have a major influ-ence on excretory losses. Indeed, >90% of primary urineentering the hindgut of terrestrial insects may be reab-sorbed therein, except during brief periods of enhancedexcretory water loss after moulting (post-eclosion diuresis)and feeding (post-prandial diuresis).

2. Ion transport processes in Malpighian tubules

Primary urine production is driven by active cation(K+/Na+) transport into the lumen, which establishes atransepithelial voltage favouring passive entry of thecounterion (Cl�). Water enters the lumen osmoticallydown the transepithelial osmotic gradient created by thenet secretion of KCl/NaCl. Other ions, sugars and amino

acids cross the tubule epithelium by passive diffusion, butthis is augmented by the active transport of a wide rangeof organic solutes. Indeed, a transcriptome analysis of thefruit fly (Drosophila melanogaster) MT showed organicsolute transporters are more highly expressed than keycomponents of the ion transport pathways (Wang et al.,2004).

2.1. Transport processes at the apical membrane

Active cation transport takes place across MT principalcells, which are the dominant cell type in the tubule epithe-lium. They are characterised by long slender apical micro-villi, virtually all of which contain a mitochondrion.Transport is driven by a bafilomycin A1-sensitive V-typeH+ ATPase that is densely expressed in the apical brushborder of principal cells (Weng et al., 2003). The activityof this pump establishes a favourable gradient for thereturn of protons to the cell, which takes place via amilo-ride-sensitive cation (Na+/K+)/H+ antiports in the apicalmembrane. The cation/H+ antiport may be electroneutral,as in the yellow fever mosquito, Aedes aegypti, in whichcase the proton gradient provides the sole driving forcefor Na+/K+ transport (Weng et al., 2003). Alternatively,it may import more than one proton for each cation, asin the midgut of the tobacco hornworm, Manduca sexta(Wieczorek et al., 1991), in which case the voltage differ-ence (cell negative) will contribute to the driving force. Itis unclear whether there are separate antiports for K+

and Na+, or a single exchanger with a preference for oneion over the other. In the blood sucking bug, Rhodnius

prolixus, there is evidence for the antiports having a prefer-ence for Na+ over K+ (Maddrell and O’Donnell, 1993), butin the house cricket (Acheta domesticus), an omnivorousinsect, and the stable fly (Stomoxys calcitrans), a bloodfeeder, there appears to be no preference for either cation(G.M. Coast, unpublished observations).

334 G. Coast / General and Comparative Endocrinology 152 (2007) 332–338

2.2. Transport processes at the basal membrane

The permeability properties of the basal membrane aredominated by a K+ conductance and this has beenassumed to be the major route for K+ uptake intoprincipal cells. However, measurements of the electro-chemical gradient for K+ across the basal membrane ofR. prolixus and D. melanogaster MTs show that it favoursK+ movement from the cell into the bathing fluid, whichis in the opposite direction of that required to supportfluid secretion (Ianowski and O’Donnell, 2004; Ianowskiet al., 2002). Instead, K+ uptake occurs via bumetanide-sensitive Na+/K+/2Cl� (NKCC) cotransport driven bythe favourable electrochemical gradient for Na+ entry.The Na+ gradient is maintained by the activity of a basalmembrane Na+/K+ ATPase, although ouabain may havelittle effect on MT secretion because it is rapidly trans-ported by an organic anion transporter on the basalmembrane (Torrie et al., 2004). In cricket MTs, ouabainhas no effect on fluid secretion, but increases the urine[Na+]:[K+] ratio from �0.5 to �1.0 (G.M. Coast, unpub-lished observation), suggesting the Na+/K+ ATPase com-petes with apical cation/H+ antiports for Na+. The effectis more dramatic in R. prolixus, where ouabain increasesboth fluid secretion and the urine [Na+] (Maddrell andOverton, 1988), reflecting the preference of apicalantiports for Na+.

There is generally a favourable transepithelial electro-chemical gradient for Cl� to enter the lumen by passive dif-fusion via either a paracellular or transcellular (through asecond cell type, the stellate cell) conductance pathway.However, the transepithelial voltage of R. prolixus MTsis negative relative to the bathing fluid and favours Cl�

movement in the reverse direction. Here, Cl� entering viaNKCC cotransport exits to the lumen through apical Cl�

channels (Ianowski and O’Donnell, 2006).

3. Endocrine control of ion transport and fluid secretion

Diuretic hormones (DHs) stimulate net KCl/NaCltransport into the MT lumen and have no significant effecton the already high osmotic permeability even when fluidsecretion is increased by up to 1000-fold (O’Donnellet al., 1982). The ion transport processes that are targetedvary with the different DHs and with the species under con-sideration as outlined below.

3.1. The V-type H+ ATPase as a target for DHs

The primary motive force driving MT secretion is theapical membrane V-type H+ ATPase and in D. melanogas-

ter this is targeted by a corticotropin releasing factor(CRF)-related DH (Drome-DH44) and a calcitonin (CT)-like DH (Drome-DH31), both acting via cAMP, and bycardioacceleratory peptides (Drome-CAP2b; Capa-1 and 2),which elevate intracellular Ca2+ levels and cGMP (Coastet al., 2002). V-type H+ ATPase activity can be increased

by promoting the association of catalytic V1 head groupswith membrane spanning V0 subunits, as in blowflysalivary glands stimulated with serotonin (Dames et al.,2006), or by controlling the availability of ATP, as shownrecently in an elegant study of the mode of action ofDrome-CAP2b, which found that a rise in intracellularCa2+ activates apical mitochondria and hence elevatesATP levels in the vicinity of the proton pump (Terhzazet al., 2006). Whatever the mechanism, stimulation of theV-type H+ ATPase has little effect on the urine[Na+]:[K+] ratio, which is dependent on the rate of deliveryof cations to apical cation/H+ antiports. However, thisdoes not mean that DHs targeting the V-type H+ ATPasehave no role in regulating haemolymph [Na+] and [K+],because the increased flow of primary urine places an addi-tional load on ion uptake processes in the hindgut and socontributes to the excretion of excess ions (along with othersolutes).

3.2. Targeting Cl� permeability

The kinin family of DHs act via Ca2+ to open a trans-epithelial Cl� conductance pathway and hence increasediffusion of the counterion into the MT lumen. InD. melanogaster, kinins act on stellate cells to open a trans-cellular Cl� conductance pathway (O’Donnell et al., 1998;Radford et al., 2002), while in A. aegypti they act on prin-cipal cells to open a paracellular conductance (Beyenbach,2003). Whatever the location of the conductance pathway,the increased availability of Cl� allows more K+ and Na+

to be transported into the lumen along with osmoticallyobliged water. This has no effect on the urine [Na+]:[K+]ratio, but adds to the load placed on ion uptake mecha-nisms in the hindgut.

3.3. Targeting Na+/K+/2Cl� cotransport

The CRF-related DH (Achdo-DH) of A. domesticus actsvia cAMP to stimulate MT secretion and increases theurine [Na+]:[K+] ratio from 0.4 to 1.3. Its diuretic activityrequires Na+, K+ and Cl� in the bathing fluid, which isconsistent with activation of basal membrane NKCCcotransport (Coast et al., 2002). Sodium entry now exceedsextrusion by the Na+/K+ ATPase, making more Na+ avail-able to apical cation/H+ antiports. The significance of thenatriuretic response is unclear, because crickets are notfaced by high Na+ intake from their diet. Possibly the addi-tional Na+ is required for absorption of sugars and aminoacids by Na+-coupled cotransport in the hindgut whenprimary urine secretion is accelerated.

3.4. Targeting a basal membrane Na+ channel

The blood meal of haematophagous insects represents amassive salt (NaCl) and water challenge. In A. aegypti mos-quitoes, this is met by a natriuresis and diuresis stimulatedby release of a natriuretic peptide (Mosquito Natriuretic


Peptide; MNP) into the circulation (Beyenbach and Petzel,1987; Wheelock et al., 1988). MNP has been identified as aCT-related DH (Anoga-DH31) in the malaria mosquito(Anopheles gambiae) and in A. aegypti (Coast et al.,2005). MNP (=Anoga-DH31) acts via cAMP to open aNa+ conductance in the principal cell basal membraneand thus stimulates NaCl transport into the MT lumen,with water following osmotically. Interestingly, the CRF-related DH (Anoga-DH44) also acts via cAMP, but doesnot have natriuretic activity (Coast et al., 2005), and itseems likely that cAMP signals are compartmentalised withonly the Anoga-DH31 response occurring in the vicinity ofcAMP-activated Na+ channels.

Male mosquitoes are nectivorous and are not con-fronted by large salt loads. Nevertheless, the head of adultmale A. aegypti contains a natriuretic factor (=MNP?) thatacts via cAMP to stimulate NaCl and fluid secretion byMTs from both males and females (Plawner et al., 1991).In contrast, cAMP has no natriuretic effect on MTs of lar-val A. aegypti and Ochlerotatus taeniorhynchus mosquitoesthat have been reared in freshwater or 30% seawater, andhas kaliuretic activity (increased K+ secretion) on MTsfrom saltwater O. taeniorhynchus larvae reared in 100%seawater (Donini et al., 2006). The absence of a natriureticresponse in larval MTs challenged with cAMP suggeststhat the expression and/or insertion of cAMP-activatedNa+ channels in the principal cell basal membrane occursonly after metamorphosis, and may therefore be linked tothe different osmoregulatory challenges facing a freshwaterlarva, which must continuously void excess water whileconserving ions, and the adult mosquito, which must pri-marily conserve water except after feeding on nectar(a water load) or on blood (both a water and salt load).

3.5. Multiple targets

The blood sucking bug R. prolixus imbibes a blood mealequivalent to �10-times the body weight of the unfedinsect. The ensuing natriuresis and diuresis is triggered byrelease of serotonin into the circulation, which stimulatesprimary urine production by the upper (secretory) portionof R. prolixus MTs up to 1000-fold. The dramatic acceler-ation of fluid secretion results from the cAMP-dependentstimulation of several transport processes, which involvein chronological order, the opening of an apical membraneCl� conductance, activation of the V-type H+ ATPase, andincreased activity of the basal membrane NKCC (Ianowskiand O’Donnell, 2001). The net result is a large increase inNaCl and KCl transport into the MT lumen along withosmotically obliged water. The fluid secreted by the upperregion of serotonin-stimulated MTs contains largeamounts of K+ ([Na+]:[K+] ratio �1), which would depletethe haemolymph of K+ within a minute (Maddrell et al.,1993a,b). This is prevented by KCl absorption from thelower segment of the MT, which is also stimulated byserotonin. The low osmotic permeability of this segmentensures that little water accompanies the KCl, and Na+-rich

urine hypo-osmotic to haemolymph flows into the hindgut tobe voided, thereby dealing with the volumic, ionic (NaCl)and osmotic challenges of the blood meal (mammalianblood is hypo-osmotic to haemolymph). KCl reabsorp-tion from the lower tubule must be stimulated inadvance of fluid secretion by the upper tubule, which isdue largely to the lower tubule responding more rapidlyto the same concentration of DH (Maddrell et al.,1993b). Subsequently, the haemolymph [K+] is regulatedby autonomous responses of the two MT segments.Thus, when haemolymph [K+] falls, K+ secretion bythe upper segment decreases while K+ absorption fromthe lower segment increases. The opposite occurs if thehaemolymph [K+] increases.

3.6. Synergism

Serotonin stimulates both cation and anion transport byR. prolixus MTs, whereas other insects may use a combina-tion of two (or possibly more?) DHs for the same purpose.For example, L. migratoria uses a CRF-related DH (Loc-mi-DH) and a kinin (Locmi-K) acting through differentsecond messengers (cAMP and Ca2+) to stimulate cationand anion transport, respectively (Coast, 1995). Atsubmaximal concentrations they act cooperatively andtheir combined effect is greater than the sum of their indi-vidual activities. This allows primary urine production tobe maximally stimulated at physiological concentrationsof the DHs and permits more precise control of MT func-tion, because a small change in the concentration of eitherDH has a pronounced effect on fluid secretion. Interest-ingly, serotonin and an unidentified peptide DH act syner-gistically in stimulating R. prolixus MTs, even though bothact via cAMP (Maddrell et al., 1993a). The peptide DH isunlikely to be a CRF-related DH, a CT-like DH or a kinin(Te Brugge et al., 2002,2005).

3.7. Antidiuretic factors acting on MTs

The high rate of secretion by MTs of haematophagousinsects, equivalent to the total haemolymph volume everyfew minutes, requires it to be precisely balanced by fluiduptake from the midgut. Once fluid uptake ceases, diuresismust rapidly be halted, which was thought to result fromthe cessation of DH release and the breakdown/excretionof DH present in the circulation. However, this may notbe quick enough, particularly since any reduction in hae-molymph volume would elevate the titre of circulatingDHs. A CAP2b peptide (Manse-CAP2b) from M. sexta

reduces secretion by serotonin-stimulated R. prolixus MTsi.e. it has antidiuretic activity (Quinlan et al., 1997).Manse-CAP2b uses cGMP as a second messenger andappears to activate a cAMP-specific phosphodiesterase,which degrades cAMP and so lowers intracellular levels ofthe second messenger that stimulates diuresis (Quinlan andO’Donnell, 1998). High concentrations of cGMP cause theMT to return to an unstimulated state, secreting K+-rich


urine at very low rates (Maddrell, 1980), which is consistentwith cGMP antagonising the actions of cAMP, because Na+

entry into principal cells is largely via the cAMP-activatedNKCC cotransporter (Ianowski et al., 2002). A CAP2b

peptide is present in R. prolixus and is released into thehaemolymph towards the end of the post-prandial diuresis(Paluzzi and Orchard, 2006).

Two antidiuretic peptides (Tenmo-ADFa and ADFb)have been identified in the yellow mealworm beetle, Teneb-

rio molitor (Eigenheer et al., 2002,2003), which is a pest of

Fig. 1. Body weight (open bars) and Na+ content (shaded bars) of adult fema(B) 0.1 lL 9% NaCl. Bars represent the means of 10–12 determinations and veboth groups of insects, but 4 h later little of the Na+ load is eliminated by mo1 lL saline.

Fig. 2. Body weight (A) and Na+ content (B) of adult female An. gambiae befordeterminations and vertical lines + 1s.e.m. Body weight and Na+ content at 4containing a 1:50 dilution of anti-Dippu-DH31 antiserum than in insects injeindicate significant differences between the means (P < 0.01).

stored products and lives under very dry conditions.Although unrelated to CAP2b, the Tenmo-ADFs act viacGMP and reduce secretion by MTs stimulated withTenmo-DH37, a CRF-related DH, probably by activatinga cAMP phosphodiesterase (Wiehart et al., 2002).Tenmo-ADFa also reduces secretion by A. aegypti MTs,but it has no effect on urine composition (Massaro et al.,2004). However, when tested on stable fly (Stomoxys calci-

trans) MTs that were partially stimulated with Anoga-DH31, the antidiuretic activity of Tenmo-ADFa was

le An. gambiae before and 4 h after the injection of (A) 1 lL 0.9% NaCl andrtical lines + 1s.e.m. Whole body Na+ content is increased post-injection insquitoes injected with 0.1 lL saline compared with the group injected with

e and after injection of 1 lL 0.9% NaCl. Bars represent the means of 10–12h post-injection are significantly higher in mosquitoes injected with salinected with the same dilution of normal rabbit antiserum. Different letters


accompanied by a decrease in the urine [Na+]:[K+] ratio,which is consistent with it antagonising the natriureticactions of cAMP (G. M. Coast, unpublished observations).An ADFa-like peptide could therefore be important forterminating both natriuresis and diuresis.

4. Diuresis and natriuresis in mosquitoes

Relatively few studies have investigated the role(s) of DHsin vivo. Serotonin and Locmi-DH have an endocrine func-tion in regulating the post-prandial diuresis of R. prolixus

and L. migratoria, respectively (Audsley et al., 1997; Madd-rell et al., 1991; Patel et al., 1995), and in the housefly (Musca

domestica) a kinin (Musdo-K) has been implicated in thediuretic response to hypervolemia (Coast, 2001). The post-prandial natriuresis and diuresis of blood fed femaleA. aegypti has been linked to increased levels of MNP-likeactivity in the circulation (Wheelock et al., 1988), and thereis now evidence from An. gambiae of Anoga-DH31

(=MNP) having natriuretic and diuretic activity in vivo

(see below).Female An. gambiae void fluid from the anus while tak-

ing a blood meal. The fluid voided in this ‘pre-diuresis’comes from the midgut, where blood cells are retainedwhile plasma is filtered into the hindgut and excreted. Thisallows the insect to ingest more blood cells (the source ofprotein for egg development) in a single meal than wouldotherwise be possible. The [Na+] and [K+] of fluid voidedduring pre-diuresis resemble human plasma. Diuresis com-mences after completion of the blood meal and the urinevoided comes largely (if not entirely) from the MTs. Themaximum rate of Na+ excretion is 8.5 nmol/min duringthe peak phase of diuresis. In contrast, isolated MTs max-imally stimulated with Anoga-DH31 secrete 0.5 nmol Na+/min (Coast et al., 2005), and assuming the five MTs arefunctionally identical, as in A. aegypti (Beyenbach et al.,1993), this would equate to �2.5 Na+ nmol/min. However,only about 30% of each MT is immersed in bathing fluid inthe Ramsay assay. Taking account of this, Na+ secretionby the five MTs would be �8.3 nl/min, and similar to themaximum rate of Na+ excretion. Therefore little or noNa+ is absorbed in the hindgut, and drops of Na+-richurine are voided every few seconds.

Anoga-DH31 activity in vivo has been investigated infemale An. gambiae injected with 0.9% saline, which avoidsvariations in the volume of blood consumed and theamount of Na+ and water voided during pre-diuresis. Fourhours after injecting 1 lL 0.9% NaCl about 70% of the vol-ume load and 60% of the Na+ load are voided (Fig. 1A).The stimulus for natriuresis appears to be the volume loadsince when the same amount of Na+ was injected in 0.1 lLsaline only 4% was voided after 4 h (Fig. 1B), The elimina-tion of injected salt and water is significantly reduced whenAnoga-DH31 is immunoneutralized using antiserum raisedagainst the CT-like DH (Dippu-DH31) from the cockroach,Diploptera punctata (Fig. 2), which suggests it is the major(if not only) natriuretic hormone of An. gambiae.

5. Concluding remarks

The excretion of water, ions and other solutes is a func-tion of secretory and reabsorptive processes in the MTsand hindgut. A number of endocrine factors influenceprimary urine formation and thereby modulate the deliveryof ions and water to the hindgut, which can result in theexcretion of excess ions and the retention of those in shortsupply. Their activities can differ both quantitatively (urineflow rate) and qualitatively (urine composition) even withina single species. It is possible that different hormones arereleased to meet specific ionic challenges, such as therelease of MNP after a blood meal in mosquitoes. On theother hand, a cocktail of factors may be needed for theprecise control of urinary excretory mechanisms in the faceof varied volumic, ionic and osmotic challenges. Unfortu-nately, although much is known about the mechanism ofprimary urine formation and how it is influenced by diure-tic/antidiuretic factors in vitro, little is known of how MTfunction is controlled in vivo, and how it relates to reab-sorptive processes in the hindgut.

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The endocrine control of salt balance in insects