Geoffrey M. Coast

Iain Kay

Jum-Sook Chung

Timothy K. Hayes

Journal of Experimental Biology

English

OAKTrust Digital Repository (Texas A&M Univ)

J. exp. Biol. 162, 331-338 (1992) 3 3 1Printed in Great Britain © The Company of Biologists Limited 1992EFFECT OF MANDUCA SEXTA DIURETIC HORMONE ANDRELATED PEPTIDES ON ISOLATED MALPIGfflAN TUBULESOF THE HOUSE CRICKET ACHETA DOMESTICUS (L.)BY GEOFFREY M. COAST1, TIMOTHY K. HAYES2, IAIN KAY1 ANDJUM-SOOK CHUNG11 Department of Biology, Blrkbeck College, London WC1E 7HX, UK and2Department of Entomology, Texas A & M University, College Station, TX77843, USAAccepted 5 September 1991SummaryPreviously, a corticotropin releasing factor (CRF)-like diuretic peptide (Man-duca-DH) has been isolated from Manduca sexta and shown to stimulate fluidexcretion in vivo in post-eclosion Pieris rapae adults and in pre-wandering post-feeding Manduca sexta larvae. However, Manduca-DH was reported to have noeffect on Malpighian tubules in vitro.Manduca-DH and [N\e2,u]-Manduca-DH were synthesized in Texas andassayed in London on isolated Malpighian tubules of Acheta domesticus. Man-duca-DH stimulated fluid secretion by about 60% of the maximum responseachievable with extracts of corpora cardiaca and increased the production of cyclicAMP. In combination with 10~4moirL 3-isobutyl-l-methyl xanthine (IBMX),Manduca-DH stimulated maximal secretion. A number of CRF-related peptidesalso stimulated fluid secretion and cyclic AMP production in cricket tubules, andthe CRF antagonist ff-helical-CRFp^i] blocked the stimulation of fluid secretionby Manduca-DH. [N\e2,ii\-Manduca-DH was more active than Manduca-DH inboth assays, suggesting that methionine residues in the natural peptide maybecome oxidized.Taken in conjunction with previous in vivo studies, the present findings suggestthat a Manduca-DH-Mke diuretic peptide is the hormone controlling post-eclosiondiuresis in butterflies, and Manduca-DH was shown to stimulate both fluidsecretion and cyclic AMP production in Malpighian tubules from 1-12 h post-eclosion Pieris rapae adults. The function of the peptide in Manduca sexta isdiscussed.IntroductionKataoka et al. (1989) identified a 41-residue diuretic peptide (Manduca-diuretichormone; Manduca-DH) from Manduca sexta, which resembles vertebrateKey words: diuretic hormone, Malpighian tubules, fluid secretion, cyclic AMP, Manduca sexta,Pieris rapae, Acheta domesticus.332 G. M. COAST AND OTHERSneuropeptides of the corticotropin releasing factor (CRF) family. SyntheticManduca-DH stimulated fluid excretion in vivo in head-ligated post-eclosionPieris rapae adults and a pronounced loss of fluid through the gut and theepidermis in pre-wandering post-feeding Manduca sexta larvae. Malpighiantubules are generally the primary target for diuretic hormones, but Kataoka et al.(1989) found that Manduca-DH did not have a direct effect on isolated tubules ofManduca sexta larvae, and suggested that the peptide may release a 'true' diuretichormone. This is an interesting suggestion, given that CRF stimulates the releaseof adrenocorticotropic hormone (ACTH) from the vertebrate pituitary, andRafaeli et al. (1986) found that ACTH stimulates cyclic AMP production and fluidsecretion by isolated Malpighian tubules of Locusta migratoria.Materials and methodsAnimalsMalpighian tubules from adult virgin female Acheta domesticus (Linnaeus) 1-2weeks old were used in the majority of experiments. Crickets were maintained andreared as previously described (Clifford et al. 1977; Coast, 1988) and were fed on adiet of turkey starter crumbs with water provided ad libitum. In one set ofexperiments, Malpighian tubules from 1-12 h post-eclosion Pieris rapae adultswere used. Pieris rapae pupae were obtained from Professor L. M. Schoonhoven(The Netherlands).Peptide synthesisManduca-DH and a chemically more stable analogue in which methionine atresidues 2 and 11 was replaced by norleucine ([Nle2,n]-Manduca-DH) weresynthesized in Texas with tBoc solid-phase peptide chemistry using methodssimilar to those employed by Kataoka et al. (1989). Final cleavage-deprotection ofManduca-DH was by low-high hydrofluoric acid (HF) cleavage (Tarn et al. 1983).The cleavage of both peptides was with the scavenger-HF mixture (1.5 ml ofanisole: 500/il of ethanedithiol: 10 ml of liquid HF per gram of resin) for 2h at-15°C. The temperature was raised to 0°C for 45min to ensure removal of tosylgroups from arginine side chains. Analytical HPLC immediately after preparativeHPLC showed the chosen fractions to be homogeneous. Peptides were quantifiedby PicoTag amino acid analysis (Cohen et al. 1984) and the amino acid compositionand mass, determined by fast atom bombardment mass spectrometry, wereconsistent with the target sequences. Samples were dried by vacuum centrifu-gation for transatlantic shipment.BioassaysIn London, the peptides were redissolved in methanol at 10~5 mol I"1 and storedat —20°C. Prior to assay, samples were transferred to polypropylene centrifugetubes containing 12.5 fi% of gamma globulin and taken to dryness in a vacuumEffect of Manduca-Z)H on cricket tubules 333centrifuge. Test material was resuspended in Acheta domesticus saline (Coast,1988) and assayed for its ability to stimulate fluid secretion and cyclic AMPproduction by isolated Malpighian tubules of Acheta domesticus. These bioassayshave been described in detail elsewhere (Coast, 1988; Coast et al. 1991; Kay et al.1991). Fluid secretion was measured over 40min periods before and after theaddition of assay material. Diuretic activity was determined as the increase in rateof secretion compared with controls (Ringer substitution) and expressed as apercentage of the response to a supramaximal amount of corpora cardiaca extract(5 gland pairs per 50/^1; Coast and Wheeler, 1990). Cyclic AMP production wasdetermined in groups of 4-5 tubules incubated at room temperature in Ringercontaining 10~4moir1 3-isobutyl-l-methyl xanthine (IBMX) to inhibit phospho-diesterase activity. Reactions were stopped after 30 min by the addition of ice-coldmethanol and the cyclic AMP released by sonication was measured using acompetitive protein binding assay (Coast et al. 1991). Additionally, Manduca-DKwas assayed for its ability to stimulate fluid secretion and cyclic AMP productionby isolated Malpighian tubules of Pieris rapae using identical methods to thoseemployed with cricket tubules, but with the saline of Nicolson (1976a).ChemicalsSynthetic bovine CRF, sucker fish urotensin-I, sauvagine, bovine gammaglobulin and IBMX were obtained from Sigma.StatisticsAll data are presented as mean±standard error (S.E.). Student's Mest was usedto determine significant differences in fluid secretion and cyclic AMP productioncaused by peptide addition. Values for t and degrees of freedom (d.f.) are given,with /'<0.05 accepted as significant. Dose-response curves were analysed usingthe Maximum Likelihood Program (Numerical Algorithms Group Ltd).Results and discussionWithin 2 min of the addition of 10~6moll~1 Manduca-DH there was asignificant increase in fluid secretion (^=3.011; d.f. = 13; F<0.01), and the responsepeaked after 15 min (Fig. 1A). The dose-response curve for the effect ofManduca-DH on fluid secretion (Fig. 2A) appeared truncated and saturated atabout 60% of the maximum response achievable with extracts of corporacardiaca. Analysis of the dose-response curve gave an EC50 of1.3xl0"6±0.2xl0"6moir1 . In Manduca sexta, an EG50 of 0.02pmol of injectedpeptide was cited by Kataoka et al. (1989) which, with a haemolymph volume ofapproximately lml (S. E. Reynolds, personal communication), is equivalent toabout 2xlO~11 moll"1. Thus, the peptide is about five orders of magnitude morepotent in Manduca sexta than in Acheta domesticus. Addition of 10~4moll"1IBMX displaced the dose-response curve to the left (Fig. 2A; ECso=5.4xl0" 7 ±0.6xl0" 7 moir 1 ) , and the diuretic activity of 10" 6 moi r 1 Manduca-334 G. M. COAST AND OTHERS40CM300-10040 50 60Time (min)7010050 60 90Time (min)100Fig. 1. Stimulation of tubule fluid secretion by (A) 10 6moll ' Manduca-DH and (B)5xlO~6moir' urotensin-I. Points represent the means of 6-8 determinations andvertical lines are ±1 S.E. The time of addition of the peptides is shown by the arrows.Note the difference in scale of the _y-axis in the two graphs.DH did not differ significantly (t=0.799; d.f. = 15; P=0.442) from the maximum(IBMX alone increased fluid secretion by 16±2.4 % of maximum). This suggestedthat the peptide stimulated adenylyl cyclase. 10~6moll~1 Manduca-DH signifi-cantly (f=19.117; d.f. = 10; P<s0.001) increased the production of cyclic AMP byisolated Malpighian tubules (Fig. 3).Some CRF-related peptides (Table 1) were also assayed. Kataoka et al. (1989)showed that injection of 500pmol of CRF, urotensin-I or sauvagine had no effectTable 1. Amino acid sequences of Manduca-D// and the CRF-related peptidesused in this studyManduca-DHR M P S L S I D L P M S V L R Q K L S L E K E R K V H A L R A A A N R N F L N D T-NH2Sucker fish urotensin-1N D D P P I S I D L T F H L L R N M I E M A R I E N E Q A G L N R K Y L D E V-NH2Sauvaginep Q G P P I S L D L T F H L L R E V L E M T K A D Q L A Q Q A H N N R K L L D I A-NH2Bovine-CRFS Q E P P I S L D L T F H L L R E V L E M T K A D Q L A Q Q A H N N R K L L D I A-NH2cr-helicalD L T F H L L R E M L E M A K A E Q E A E Q A A L N R L L L E E A-NH2Effect of Manduca-D// on cricket tubules 335100-iE4 - 1oage8060I402°100-80-IBMX-free10" 10" 10" 10_S 1 0 -7 1 0 -6 1 Q -Dose (molP1)Fig. 2. Dose-response curves for the diuretic activities of (A) Manduca-DH alone (•)and in the presence of 10~4moir1 IBMX (•) , and (B) [N\e2M]-Manduca-DH.Diuretic activity is expressed as a percentage of the response to an extract of corporacardiaca that elicits a maximal stimulation. Points represent the means of 5-10determinations, and vertical lines are ±1S.E.on fluid excretion by head-ligated post-eclosion Pieris rapae adults. At5xlO~6moll~1, urotensin-I brought about a significant stimulation of fluidsecretion by Acheta domesticus tubules after 2min (f=2.550; d.f. = l l ; P<0.05),but this was not sustained (Fig. IB). Urotensin-I, sauvagine and CRF allstimulated fluid secretion in a dose-dependent fashion (results not shown), but theresponses did not saturate. At 10~5moll~1, all three peptides increased fluidsecretion by about 20 % of the maximum. Ovine CRF stimulates adenylyl cyclasein the pituitary (Labrie et al. 1983) and 10~6moll~1 urotensin-I, sauvagine andCRF all elicited small increases in cyclic AMP production in cricket tubules(Fig. 3). The CRF antagonist a-helical-CRF[9_4i] (Rivier etal. 1984) had nosignificant effect either on cyclic AMP production (Fig. 3) or on fluid secretion(f=0.241; d.f. = l l ; P=0.192). Manduca-DU (10"6moH"1) was assayed alone andtogether with CRF antagonist. Manduca-DH alone increased fluid secretion by114±19.2 p lmm^min" 1 (N=7). At 10~ 4 mo i r \ a-helical-CRF[9_41] blocked theresponse, the difference between the control and experimental rates being-47±53.8 plmm~1min~1 (N=6). Lower concentrations of the antagonist werewithout effect.The [Nle2.11]-Manduca-DH analogue stimulated cyclic AMP production to agreater extent than did Manduca-DH (Fig. 3) and was more active in the fluidsecretion assay, giving a maximum response without the addition of IBMX336 G. M. COAST AND OTHERSX)-18.ooOa1.4-1.2-1.0-0.80.60.40.20,0(4)(8)n(4)(4)JLTreatment &Fig. 3. The effect of 10 6moll l Manduca-DH and related peptides on cyclic AMPproduction by isolated Malpighian tubules. Results are expressed as pmol of cyclicAMP produced per tubule over a 30min incubation. Bars represent the means of thenumber of determinations given in parentheses and vertical lines +1S.E. Student's/-test was used to compare results with those from control incubations (Ringeraddition). Asterisks denote P<0.001; NS, not significant. Manduca-DH, Mas-DH;[Nle2,n]-Manduca-DH, Nle-Mas-DH; CRF, corticotropin releasing factor.(EC5 0=2.8xl0~7±0.8xl0~7moir1; Fig. 2B). Methionine residues in Manduca-DH may, therefore, be prone to oxidation. A sample of synthetic Manduca-DHfrom Peninsula Laboratories Inc. gave a truncated dose-response curve in thefluid secretion assay and saturated at 69% of maximum, somewhat higher thanthat for the peptide synthesized by us (60 % of maximum), but lower than that for[N\&2tn]-Manduca-DH. The material from Peninsula Laboratories was also about10 times more potent (EC5 0=1.6xl0"7±0.4xl0~7moir1) . Such differences mayreflect the extent to which oxidation had occurred.Because Manduca-DH acts directly on cricket tubules it is unlikely to be areleasing factor in the host species. In Pieris brassicae, post-eclosion diuresis isstimulated by a peptide from the brain and the retrocerebral complex working viacyclic AMP to increase tubule fluid secretion (Nicolson, 1976a,b). Manduca-DHstimulates fluid excretion in post-eclosion Pieris rapae adults and, when assayed at10~6moll~1 on Malpighian tubules taken from adult butterflies within 1-12 h ofeclosion, it increased fluid secretion by 196±26.1% 0=5.793; d.f. = l l ; P<0.001)and cyclic AMP production from 1.6±0.22 to 4.3±0.37pmol per tubule (f=6.309;d.f.=8; P<0.001). Thus, a Manduca-hke peptide is probably the hormonecontrolling post-eclosion diuresis in butterflies. In Manduca sexta, weight lossoccurs largely during the 48 h preceding pupation (Baker et al. 1987). However, atEffect of Manduca-Z)// on cricket tubules 337this time, Manduca sexta tubules lose their ability to excrete dyes (Nijhout, 1975),and fluid secretion may be switched off prior to the end of the stadium (Nicolson,1976b; Ryerse, 1978; Chung and Keeley, 1989). Reabsorption of fluid from therectum of Manduca sexta larvae might be subject to neural or hormonal control(Reynolds and Bellward, 1989). Thus, Manduca-DH could act in vivo to inhibitfluid uptake from the rectum in the period before tubule secretion is switched off,so increasing faecal water loss.Manduca-DH belongs to a family of insect diuretic peptides that are themselvesmembers of a superfamily of peptides including CRF, sauvagine and urotensin-I(G. M. Coast, G. J. Goldsworthy, M. S. Johnson, I. Kay and C. H. Wheeler, inpreparation). These peptides are involved, either directly or indirectly, in theregulation of hydromineral balance, and all appear to act via cyclic AMP. They aresufficiently similar for there to be discernible cross-reactivity in Acheta domesticustubules. As in vertebrates, a^helical CRF[9_4i] appears to be an antagonist, and theamino terminal region of the peptides may be important for signal transduction.This could explain differences in activity between Manduca-DH and [Nle2,n]-Manduca-DH.The work at Birkbeck was supported by a grant from the AFRC, and at Texas A& M by an NSF grant (DCB-8918438) and the Tamu Biotechnology SupportLaboratory which provided the instrumentation. We thank Alan Tyler for histechnical assistance and Graham Goldsworthy for comments on a final draft of thismanuscript.ReferencesBAKER, F. C , TSAI, L. W., REUTER, C. C. AND SCHOOLEY, D. A. (1987). In vivo fluctuation ofJH, JH acid, and ecdysteroid titer, and JH esterase activity, during development of fifthstadium Manduca sexta. Insect Biochem. 17, 989-996.CHUNG, J.-S. AND KEELEY, L. L. (1989). Evidence and bioassay for diuretic factors in thenervous system of larval Heliothis virescens. J. comp. Physiol. B 159, 359-370.CLIFFORD, C. W., ROE, R. M. AND WOODRING, J. P. (1977). Rearing methods for obtaininghouse crickets, Acheta domesticus, of known age, sex and instar. Ann. ent. Soc. Am. 70,69-74.COAST, G. M. (1988). Fluid secretion by single isolated Malpighian tubules of the house cricket,Acheta domesticus, and their response to diuretic hormone. Physiol. Ent. 13, 381-391.COAST, G. M., CUSINATO, O., KAY, I. AND GOLDSWORTHY, G. J. (1991). An evaluation of therole of cyclic AMP as an intracellular messenger in Malpighian tubules of the house cricket,Acheta domesticus. J. Insect Physiol. (in press).COAST, G. M. AND WHEELER, C. H. (1990). The distribution and relative potency of diureticpeptides in the house cricket, Acheta domesticus. Physiol. Ent. 15, 13—21.COHEN, S. A., TARVIN, T. L. AND BIDLINGMEYER, B. A. (1984). Analysis of amino acids usingprecolumn derivitization with phenylthioisocyanate. Am. Lab. 16, 48-59.KATAOKA, H., TROETSCHLER, R. G., LI , J. P., KRAMER, S. J., CARNEY, R. L. AND SCHOOLEY,D. A. (1989). Isolation and identification of a diuretic hormone from the tobacco hornworm,Manduca sexta. Proc. natn. Acad. Sci. U.S.A. 86, 2976-2980.KAY, I., COAST, G. M., CUSINATO, O., WHEELER, C. H., TOTTY, N. F. AND GOLDSWORTHY, G. J.(1991). Isolation and characterization of a diuretic peptide from Acheta domesticus: evidencefor a family of insect diuretic peptides. Biol. Chem. Hoppe-Seyler 372, 505-512.338 G. M. COAST AND OTHERSLABRIE, F., GAGNE, B.,LEFEVRE, G. ANDMEUNIER, H. (1983). CRFstimulates adenylate cyclaseactivity in the intermediate lobe of the pituitary gland. Molec. cell. Endocr. 30, 347-351.NICOLSON, S. W. (1976a). Diuresis in the cabbage white butterfly, Pieris brassicae: fluidsecretion by the Malpighian tubules. J. Insect Physiol. 22,1347-1356.NICOLSON, S. W. (1976f>). The hormonal control of diuresis in the cabbage white butterfly, Pierisbrassicae. J. exp. Biol. 65, 565-575.NUHOUT, H. F. (1975). Excretory role of the midgut in larvae of the tobacco hornworm,Manduca sexta (L.). J. exp. Biol. 62, 221-230.RAFAELI, A., APPLEBAUM, S. W. AND MOSHITZKY, P. (1986). An ACTH-like diuretic hormone inlocusts. In Insect Neurochemistry and Neurophysiology, 1986 (ed. A. B. Borkovec and D. B.Gelman), pp. 355-357. New York: Humana Press.REYNOLDS, S. E. AND BELLWARD, K. (1989). Water balance in Manduca sexta caterpillars: waterrecycling from the rectum. J. exp. Biol. 141, 33-45.RIVIER, J., RIVIER, C. AND VALE, W. (1984). Synthetic competitive antagonists of corticotropinreleasing factor: effect on ACTH secretion in rat. Science 224, 889-891.RYERSE, J. S. (1978). Developmental changes in Malpighian tubule fluid transport. J. InsectPhysiol. 24, 315-319.TAM, J. P., HEALTH, W. F. AND MERRIFIELD, R. B. (1983). S^2 deprotection of syntheticpeptides with a low concentration of HF in dimethyl sulfide: evidence and application inpeptide synthesis. J. Am. chem. Soc. 105, 6442-6455.

Effect of Manduca Sexta Diuretic Hormone and related Peptides on isolated Malpigfflan Tubules of the House Cricket Acheta Domesticus (L.)

Texas A&M University

J. exp. Biol. 162, 331-338 (1992) 3 3 1
Printed in Great Britain © The Company of Biologists Limited 1992
EFFECT OF MANDUCA SEXTA DIURETIC HORMONE AND
RELATED PEPTIDES ON ISOLATED MALPIGfflAN TUBULES
OF THE HOUSE CRICKET ACHETA DOMESTICUS (L.)
BY GEOFFREY M. COAST1, TIMOTHY K. HAYES2, IAIN KAY1 AND
JUM-SOOK CHUNG1
1
 Department of Biology, Blrkbeck College, London WC1E 7HX, UK and
2Department of Entomology, Texas A & M University, College Station, TX
77843, USA
Accepted 5 September 1991
Summary
Previously, a corticotropin releasing factor (CRF)-like diuretic peptide (Man-
duca-DH) has been isolated from Manduca sexta and shown to stimulate fluid
excretion in vivo in post-eclosion Pieris rapae adults and in pre-wandering post-
feeding Manduca sexta larvae. However, Manduca-DH was reported to have no
effect on Malpighian tubules in vitro.
Manduca-DH and [N\e2,u]-Manduca-DH were synthesized in Texas and
assayed in London on isolated Malpighian tubules of Acheta domesticus. Man-
duca-DH stimulated fluid secretion by about 60% of the maximum response
achievable with extracts of corpora cardiaca and increased the production of cyclic
AMP. In combination with 10~4moirL 3-isobutyl-l-methyl xanthine (IBMX),
Manduca-DH stimulated maximal secretion. A number of CRF-related peptides
also stimulated fluid secretion and cyclic AMP production in cricket tubules, and
the CRF antagonist ff-helical-CRFp^i] blocked the stimulation of fluid secretion
by Manduca-DH. [N\e2,ii\-Manduca-DH was more active than Manduca-DH in
both assays, suggesting that methionine residues in the natural peptide may
become oxidized.
Taken in conjunction with previous in vivo studies, the present findings suggest
that a Manduca-DH-Mke diuretic peptide is the hormone controlling post-eclosion
diuresis in butterflies, and Manduca-DH was shown to stimulate both fluid
secretion and cyclic AMP production in Malpighian tubules from 1-12 h post-
eclosion Pieris rapae adults. The function of the peptide in Manduca sexta is
discussed.
Introduction
Kataoka et al. (1989) identified a 41-residue diuretic peptide (Manduca-diuretic
hormone; Manduca-DH) from Manduca sexta, which resembles vertebrate
Key words: diuretic hormone, Malpighian tubules, fluid secretion, cyclic AMP, Manduca sexta,
Pieris rapae, Acheta domesticus.
332 G. M. COAST AND OTHERS
neuropeptides of the corticotropin releasing factor (CRF) family. Synthetic
Manduca-DH stimulated fluid excretion in vivo in head-ligated post-eclosion
Pieris rapae adults and a pronounced loss of fluid through the gut and the
epidermis in pre-wandering post-feeding Manduca sexta larvae. Malpighian
tubules are generally the primary target for diuretic hormones, but Kataoka et al.
(1989) found that Manduca-DH did not have a direct effect on isolated tubules of
Manduca sexta larvae, and suggested that the peptide may release a 'true' diuretic
hormone. This is an interesting suggestion, given that CRF stimulates the release
of adrenocorticotropic hormone (ACTH) from the vertebrate pituitary, and
Rafaeli et al. (1986) found that ACTH stimulates cyclic AMP production and fluid
secretion by isolated Malpighian tubules of Locusta migratoria.
Materials and methods
Animals
Malpighian tubules from adult virgin female Acheta domesticus (Linnaeus) 1-2
weeks old were used in the majority of experiments. Crickets were maintained and
reared as previously described (Clifford et al. 1977; Coast, 1988) and were fed on a
diet of turkey starter crumbs with water provided ad libitum. In one set of
experiments, Malpighian tubules from 1-12 h post-eclosion Pieris rapae adults
were used. Pieris rapae pupae were obtained from Professor L. M. Schoonhoven
(The Netherlands).
Peptide synthesis
Manduca-DH and a chemically more stable analogue in which methionine at
residues 2 and 11 was replaced by norleucine ([Nle2,n]-Manduca-DH) were
synthesized in Texas with tBoc solid-phase peptide chemistry using methods
similar to those employed by Kataoka et al. (1989). Final cleavage-deprotection of
Manduca-DH was by low-high hydrofluoric acid (HF) cleavage (Tarn et al. 1983).
The cleavage of both peptides was with the scavenger-HF mixture (1.5 ml of
anisole: 500/il of ethanedithiol: 10 ml of liquid HF per gram of resin) for 2h at
-15°C. The temperature was raised to 0°C for 45min to ensure removal of tosyl
groups from arginine side chains. Analytical HPLC immediately after preparative
HPLC showed the chosen fractions to be homogeneous. Peptides were quantified
by PicoTag amino acid analysis (Cohen et al. 1984) and the amino acid composition
and mass, determined by fast atom bombardment mass spectrometry, were
consistent with the target sequences. Samples were dried by vacuum centrifu-
gation for transatlantic shipment.
Bioassays
In London, the peptides were redissolved in methanol at 10~5 mol I"1 and stored
at —20°C. Prior to assay, samples were transferred to polypropylene centrifuge
tubes containing 12.5 fi% of gamma globulin and taken to dryness in a vacuum
Effect of Manduca-Z)H on cricket tubules 333
centrifuge. Test material was resuspended in Acheta domesticus saline (Coast,
1988) and assayed for its ability to stimulate fluid secretion and cyclic AMP
production by isolated Malpighian tubules of Acheta domesticus. These bioassays
have been described in detail elsewhere (Coast, 1988; Coast et al. 1991; Kay et al.
1991). Fluid secretion was measured over 40min periods before and after the
addition of assay material. Diuretic activity was determined as the increase in rate
of secretion compared with controls (Ringer substitution) and expressed as a
percentage of the response to a supramaximal amount of corpora cardiaca extract
(5 gland pairs per 50/^1; Coast and Wheeler, 1990). Cyclic AMP production was
determined in groups of 4-5 tubules incubated at room temperature in Ringer
containing 10~4moir1 3-isobutyl-l-methyl xanthine (IBMX) to inhibit phospho-
diesterase activity. Reactions were stopped after 30 min by the addition of ice-cold
methanol and the cyclic AMP released by sonication was measured using a
competitive protein binding assay (Coast et al. 1991). Additionally, Manduca-DK
was assayed for its ability to stimulate fluid secretion and cyclic AMP production
by isolated Malpighian tubules of Pieris rapae using identical methods to those
employed with cricket tubules, but with the saline of Nicolson (1976a).
Chemicals
Synthetic bovine CRF, sucker fish urotensin-I, sauvagine, bovine gamma
globulin and IBMX were obtained from Sigma.
Statistics
All data are presented as mean±standard error (S.E.). Student's Mest was used
to determine significant differences in fluid secretion and cyclic AMP production
caused by peptide addition. Values for t and degrees of freedom (d.f.) are given,
with /'<0.05 accepted as significant. Dose-response curves were analysed using
the Maximum Likelihood Program (Numerical Algorithms Group Ltd).
Results and discussion
Within 2 min of the addition of 10~6moll~1 Manduca-DH there was a
significant increase in fluid secretion (^=3.011; d.f. = 13; F<0.01), and the response
peaked after 15 min (Fig. 1A). The dose-response curve for the effect of
Manduca-DH on fluid secretion (Fig. 2A) appeared truncated and saturated at
about 60% of the maximum response achievable with extracts of corpora
cardiaca. Analysis of the dose-response curve gave an EC50 of
1.3xl0"6±0.2xl0"6moir1 . In Manduca sexta, an EG50 of 0.02pmol of injected
peptide was cited by Kataoka et al. (1989) which, with a haemolymph volume of
approximately lml (S. E. Reynolds, personal communication), is equivalent to
about 2xlO~11 moll"1. Thus, the peptide is about five orders of magnitude more
potent in Manduca sexta than in Acheta domesticus. Addition of 10~4moll"1
IBMX displaced the dose-response curve to the left (Fig. 2A; ECso=
5.4xl0" 7 ±0.6xl0" 7 moir 1 ) , and the diuretic activity of 10" 6 moi r 1 Manduca-
334 G. M. COAST AND OTHERS
40CM
300-
100
40 50 60
Time (min)
70
100
50 60 90
Time (min)
100
Fig. 1. Stimulation of tubule fluid secretion by (A) 10 6moll ' Manduca-DH and (B)
5xlO~6moir' urotensin-I. Points represent the means of 6-8 determinations and
vertical lines are ±1 S.E. The time of addition of the peptides is shown by the arrows.
Note the difference in scale of the _y-axis in the two graphs.
DH did not differ significantly (t=0.799; d.f. = 15; P=0.442) from the maximum
(IBMX alone increased fluid secretion by 16±2.4 % of maximum). This suggested
that the peptide stimulated adenylyl cyclase. 10~6moll~1 Manduca-DH signifi-
cantly (f=19.117; d.f. = 10; P<s0.001) increased the production of cyclic AMP by
isolated Malpighian tubules (Fig. 3).
Some CRF-related peptides (Table 1) were also assayed. Kataoka et al. (1989)
showed that injection of 500pmol of CRF, urotensin-I or sauvagine had no effect
Table 1. Amino acid sequences of Manduca-D// and the CRF-related peptides
used in this study
Manduca-DH
R M P S L S I D L P M S V L R Q K L S L E K E R K V H A L R A A A N R N F L N D T-NH2
Sucker fish urotensin-1
N D D P P I S I D L T F H L L R N M I E M A R I E N E Q A G L N R K Y L D E V-NH2
Sauvagine
p Q G P P I S L D L T F H L L R E V L E M T K A D Q L A Q Q A H N N R K L L D I A-NH2
Bovine-CRF
S Q E P P I S L D L T F H L L R E V L E M T K A D Q L A Q Q A H N N R K L L D I A-NH2
cr-helical
D L T F H L L R E M L E M A K A E Q E A E Q A A L N R L L L E E A-NH2
Effect of Manduca-D// on cricket tubules 335
100-
i
E
4 - 1
o
ag
e
80
60
I
40
2
°
100-
80-
IBMX-free
10" 10" 10" 10_S 1 0 -7 1 0 -6 1 Q -
Dose (molP1)
Fig. 2. Dose-response curves for the diuretic activities of (A) Manduca-DH alone (•)
and in the presence of 10~4moir1 IBMX (•) , and (B) [N\e2M]-Manduca-DH.
Diuretic activity is expressed as a percentage of the response to an extract of corpora
cardiaca that elicits a maximal stimulation. Points represent the means of 5-10
determinations, and vertical lines are ±1S.E.
on fluid excretion by head-ligated post-eclosion Pieris rapae adults. At
5xlO~6moll~1, urotensin-I brought about a significant stimulation of fluid
secretion by Acheta domesticus tubules after 2min (f=2.550; d.f. = l l ; P<0.05),
but this was not sustained (Fig. IB). Urotensin-I, sauvagine and CRF all
stimulated fluid secretion in a dose-dependent fashion (results not shown), but the
responses did not saturate. At 10~5moll~1, all three peptides increased fluid
secretion by about 20 % of the maximum. Ovine CRF stimulates adenylyl cyclase
in the pituitary (Labrie et al. 1983) and 10~6moll~1 urotensin-I, sauvagine and
CRF all elicited small increases in cyclic AMP production in cricket tubules
(Fig. 3). The CRF antagonist a-helical-CRF[9_4i] (Rivier etal. 1984) had no
significant effect either on cyclic AMP production (Fig. 3) or on fluid secretion
(f=0.241; d.f. = l l ; P=0.192). Manduca-DU (10"6moH"1) was assayed alone and
together with CRF antagonist. Manduca-DH alone increased fluid secretion by
114±19.2 p lmm^min" 1 (N=7). At 10~ 4 mo i r \ a-helical-CRF[9_41] blocked the
response, the difference between the control and experimental rates being
-47±53.8 plmm~1min~1 (N=6). Lower concentrations of the antagonist were
without effect.
The [Nle2.11]-Manduca-DH analogue stimulated cyclic AMP production to a
greater extent than did Manduca-DH (Fig. 3) and was more active in the fluid
secretion assay, giving a maximum response without the addition of IBMX
336 G. M. COAST AND OTHERS
X)
-1
8.
o
o
O
a
1.4-
1.2-
1.0
-
0.8
0.6
0.4
0.2
0,0
(4)
(8)
n
(4)
(4)
JL
Treatment &
Fig. 3. The effect of 10 6moll l Manduca-DH and related peptides on cyclic AMP
production by isolated Malpighian tubules. Results are expressed as pmol of cyclic
AMP produced per tubule over a 30min incubation. Bars represent the means of the
number of determinations given in parentheses and vertical lines +1S.E. Student's
/-test was used to compare results with those from control incubations (Ringer
addition). Asterisks denote P<0.001; NS, not significant. Manduca-DH, Mas-DH;
[Nle2,n]-Manduca-DH, Nle-Mas-DH; CRF, corticotropin releasing factor.
(EC5 0=2.8xl0~7±0.8xl0~7moir1; Fig. 2B). Methionine residues in Manduca-
DH may, therefore, be prone to oxidation. A sample of synthetic Manduca-DH
from Peninsula Laboratories Inc. gave a truncated dose-response curve in the
fluid secretion assay and saturated at 69% of maximum, somewhat higher than
that for the peptide synthesized by us (60 % of maximum), but lower than that for
[N\&2tn]-Manduca-DH. The material from Peninsula Laboratories was also about
10 times more potent (EC5 0=1.6xl0"7±0.4xl0~7moir1) . Such differences may
reflect the extent to which oxidation had occurred.
Because Manduca-DH acts directly on cricket tubules it is unlikely to be a
releasing factor in the host species. In Pieris brassicae, post-eclosion diuresis is
stimulated by a peptide from the brain and the retrocerebral complex working via
cyclic AMP to increase tubule fluid secretion (Nicolson, 1976a,b). Manduca-DH
stimulates fluid excretion in post-eclosion Pieris rapae adults and, when assayed at
10~6moll~1 on Malpighian tubules taken from adult butterflies within 1-12 h of
eclosion, it increased fluid secretion by 196±26.1% 0=5.793; d.f. = l l ; P<0.001)
and cyclic AMP production from 1.6±0.22 to 4.3±0.37pmol per tubule (f=6.309;
d.f.=8; P<0.001). Thus, a Manduca-hke peptide is probably the hormone
controlling post-eclosion diuresis in butterflies. In Manduca sexta, weight loss
occurs largely during the 48 h preceding pupation (Baker et al. 1987). However, at
Effect of Manduca-Z)// on cricket tubules 337
this time, Manduca sexta tubules lose their ability to excrete dyes (Nijhout, 1975),
and fluid secretion may be switched off prior to the end of the stadium (Nicolson,
1976b; Ryerse, 1978; Chung and Keeley, 1989). Reabsorption of fluid from the
rectum of Manduca sexta larvae might be subject to neural or hormonal control
(Reynolds and Bellward, 1989). Thus, Manduca-DH could act in vivo to inhibit
fluid uptake from the rectum in the period before tubule secretion is switched off,
so increasing faecal water loss.
Manduca-DH belongs to a family of insect diuretic peptides that are themselves
members of a superfamily of peptides including CRF, sauvagine and urotensin-I
(G. M. Coast, G. J. Goldsworthy, M. S. Johnson, I. Kay and C. H. Wheeler, in
preparation). These peptides are involved, either directly or indirectly, in the
regulation of hydromineral balance, and all appear to act via cyclic AMP. They are
sufficiently similar for there to be discernible cross-reactivity in Acheta domesticus
tubules. As in vertebrates, a^helical CRF[9_4i] appears to be an antagonist, and the
amino terminal region of the peptides may be important for signal transduction.
This could explain differences in activity between Manduca-DH and [Nle2,n]-
Manduca-DH.
The work at Birkbeck was supported by a grant from the AFRC, and at Texas A
& M by an NSF grant (DCB-8918438) and the Tamu Biotechnology Support
Laboratory which provided the instrumentation. We thank Alan Tyler for his
technical assistance and Graham Goldsworthy for comments on a final draft of this
manuscript.
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Effect of Manduca Sexta Diuretic Hormone and related Peptides on isolated Malpigfflan Tubules of the House Cricket Acheta Domesticus (L.)

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