Proc. Nati. Acad. Sci. USAVol. 86, pp. 2976-2980, April 1989Physiological Sciences

Isolation and identification of a diuretic hormone from the tobaccohornworm, Manduca sexta

(osmoregulation/amino acid sequence/corticotropin-releasing factor/sauvagine/urotensin I)

HIROSHI KATAOKA*, RUTH G. TROETSCHLER, JORGE P. Li, STEVEN J. KRAMER, ROBERT L. CARNEY,AND DAVID A. SCHOOLEYtZoecon Research Institute, Sandoz Crop Protection, P. 0. Box 10975, Palo Alto, CA 94303-1104

Communicated by Wendell L. Roelofs, January 9, 1989 (received for review October 24, 1988)

ABSTRACT A diuretic hormone (DH) has been isolatedfrom pharate adult heads of Manduca sexta by a nine-steppurification procedure. The primary structure of the amino-terminal 40 residues was determined by sequence analysis ofintact DH. The structure of an amidated carboxyl-terminaltryptic hexapeptide was characterized by sequence analysis ofthe peptide, and this hexapeptide was later compared byreversed-phase liquid chromatography with two synthetichexapeptides with the free acid or amide at the carboxylterminus. The complete structure of M. sexta DH was estab-lished as a 41-residue peptide without disulfide bonds: H-Arg-Met-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Pro-Met-Ser-Val-Leu-Arg-Gln-Lys-Leu-Ser-Leu-Glu-Lys-Glu-Arg-Lys-Val-His-Ala-Leu-Arg-Ala-Ala-Ala-Asn-Arg-Asn-Phe-Leu-Asn-Asp-Ile-NH2. M. sexta DH was synthesized and shown to have chro-matographic and biological properties identical with those ofthe native material. Synthetic DH stimulated fluid excretion invivo upon injection into larval M. sexta and newly emergedadult Pieris rapae. M. sexta DH has considerable sequencehomology with corticotropin-releasing factor, urotensin I, andsauvagine.

Insects regulate the osmotic composition of their bloodwithin relatively narrow limits in spite of an unfavorablesurface-to-volume ratio (1). The major organs responsible forfluid and ion secretion are the Malpighian tubules (Mt).Primary urine from the Mt moves into the gut and eventuallyto the rectum, where selective resorption of essential metab-olites and water typically occurs (for reviews see refs. 1-3).

It is generally believed that regulation of fluid secretion ininsects is controlled by one or more peptidic diuretic hor-mones (DHs), while resorption may be regulated by antidi-uretic hormones (1, 2, 4, 5). By using in vitro Mt assays,substances with diuretic activity have been found in gangliafrom the head, thorax, and abdomen, and in a glandulartissue, the corpora cardiaca (CC) (4-8). Partial purificationsofDH from several species indicate that there may be severalDHs which differ in size and possibly in modes of action (3,5, 8-12).To date, isolation of two insect DHs, both from Locusta

migratoria, has been reported (13, 14). One of these wasisolated from subesophageal and thoracic ganglia, using itsimmunological cross-reactivity with an antibody to [Arg8]-vasopressin (AVP) as an assay (14). This DH was identifiedas an antiparallel dimer of [Leu2,Thr4,Arg8]vasotocin (15),and it occurs in tissue with the corresponding monomer,which has no diuretic activity (15). The otherDH was isolatedfrom CC of this locust by monitoring in vitro stimulation ofcAMP in Mt during purification, but only a partial sequencewas determined (13).

Several species of butterflies exhibit significant diuresissoon after adult eclosion. This phenomenon allowed devel-opment of a facile in vivo DH assay (16). Though thewell-studied tobacco hornworm moth, Manduca sexta, doesnot show diuresis at this stage, we were able to utilize thebutterfly Pieris rapae in a similar bioassay and to purify a DHfrom trimmed head tissue of pharate adult M. sexta. We herereport the isolation and structure identification of a M. sextaDH, which has homology with sauvagine, corticotropin-releasing factor (CRF), and urotensin I.

MATERIALS AND METHODSInsects. M. sexta were reared on an artificial diet (17).

Pharate adult M. sexta were beheaded 24-48 hr before adulteclosion, and the heads were frozen. A posterior section ofthese frozen heads containing the brain and the CC/corporaallata complex was punched out with a 5-mm-diameter corkborer and stored at -800C until extraction.Cabbageworms (P. rapae larvae) were reared on cabbage

plants until about the third stadium, when they were trans-ferred to an artificial diet (17), incorporating fresh cabbageleaves as a phagostimulant. Leaves of one immature cabbageplant were heated (15 min, 1630C) before grinding in a blenderwith each 3-liter batch of diet. P. rapae pupae were held at280C during the day, 270C during the night on a 16:8 light:darkcycle until the morning of emergence.In Vivo Bioassay. DH activity in fractions from each

purification step was detected by using newly emerged adultP. rapae, in an assay modified from one previously reportedfor Danaus plexippus (16). In addition to utilizing an increasein temperature just after lights-on (16) to synchronize adulteclosion, we placed pharate adults in a cardboard containerseated on a warm water bath (s450C) to trigger almostsimultaneous emergence of large numbers of young adults.These young butterflies were anesthetized with CO2 within 1min of eclosion. They were neck ligated within 15 min, thenbeheaded, and the wound was sealed with melted wax. Eachanimal was suspended from the side of a 30-ml plastic cupuntil the wings expanded. Beheaded butterflies were weighedwithin 1 hr of the molt. Untreated controls were weighedabout 15 min after eclosion.To reduce possible adsorption ofDH to containers, bovine

serum albumin (50 gg) was added to test fractions beforeevaporating the solvent. Treated butterflies were injected inthe ventral thorax with the test fraction dissolved in 5 dul of

Abbreviations: DH, diuretic hormone; AVP, [Arg8lvasopressin; LC,liquid chromatography; TFA, trifluoroacetic acid; Boc, t-butoxycar-bonyl; Mt, Malpighian tubule(s); CC, corpora cardiaca; CRF, cor-ticotropin-releasing factor; oCRF, ovine CRF; ACTH, adrenocorti-cotropic hormone.*Present address: Department of Agricultural Chemistry, Universityof Tokyo, Tokyo 113, Japan.tTo whom reprint requests should be addressed at: BiochemistryDepartment, University of Nevada, Reno, NV 89557.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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"Manduca saline" (18) (4 mM NaCl/40 mM KCI/3 mMCaCl2/18 mM MgCl2); beheaded controls received 5 1.L ofManduca saline. The plastic cups were capped to reduceevaporation. The butterflies were weighed again 3 hr afterinjection; weight loss during this period was considered to befluid excretion.To determine net normal diuresis, the mean weight loss of

beheaded controls was subtracted from the mean weight lossof intact controls. The mean weight loss of a typical 114-mguntreated intact control animal in the 3-hr assay period was23 mg (20% of mean body weight), that of a typical 118mg-beheaded control animal injected with 5 pJ of saline was11 mg (9% of mean body weight), and that of a typical 118-mgbeheaded animal injected with 1 head equivalent of extract in5 1Ld of saline was 24 mg (20% of mean body weight). Theweight loss of treated, beheaded animals was expressed as afraction of net normal diuresis.M. sexta in Vivo DH Bioassay. Although P. rapae retain

most of their fluids during the postfeeding prepupal period,M. sexta wandering fifth-stadium larvae lose =30% of theirbody weight of -9 g (19) during the 48 hr that precedepupation. We used prewandering postfeeding M. sexta justafter lights-out (12:12 photoperiod) to assay for increaseddiuresis triggered by DH. These larvae were weighed, theninjected with 25 ,u1 of Manduca saline or 25 ,ul of this salinecontaining 0.1 ,ug of synthetic DH. After 4 hr, each larva wasagain weighed and dry weight of frass was subtracted todetermine net weight of water lost.

Extraction and Preliminary Purification (Steps 1-4). Tenthousand trimmed heads of M. sexta (fresh weight 420 g)were homogenized in 1500 ml of cold acetone and filtered.Residues were extracted with 1500 ml of 1 M HOAc/20 mMHCI (containing 0.1 mM phenylmethylsulfonyl fluoride and0.01 mM pepstatin A, prepared freshly) and centrifuged at10,000 X g for 20 min. After reextraction of the pellet with1200 ml of the same solution, and recentrifugation, thecombined supernatants were applied to an SP-Sephadex C-25column (Pharmacia; 25 mm inner diameter x 750 mm,containing 300 ml of packing) equilibrated with 1 M HOAc.The column was eluted with 1000 ml each of 1 M HOAc, 0.05M NH4OAc (pH 4.0), and 0.05 M, 0.1 M, 0.4 M, and 0.8 MNH4OAc (pH 7.0). The 0.4 M and 0.8 M NH4OAc fractions,which both had DH activity, were applied directly to 10 g ofreversed-phase Vydac C4 packing material (20- to 30-,umpacking contained in a 75-ml polypropylene syringe barrelwith polyethylene frit) equilibrated with 0.1% trifluoroaceticacid (TFA). The cartridge was eluted with 100 ml each of0.1% TFA and 20%, 35%, and 50% (vol/vol) CH3CN in 0.1%TFA. DH was recovered in the 35% CH3CN/0.1% TFAfraction.Vydac C4 Semipreparative Liquid Chromatography (LC)

(Steps 5 and 6). This and subsequent purifications by LC wereperformed with a Perkin-Elmer Model 410 Bio pump, aRheodyne loop injector, and a Kratos model 783 variable-wavelength detector, usually set at 220 nm. The pump wasmodified with a Rheodyne model 5302 valve installed in the"D" solvent tube before the solvent proportioning valve, sothat water-diluted fractions could be pumped into the col-umn. The active fraction from step 4 was diluted with 200 mlof water and pumped onto a Vydac C4 semipreparativecolumn (10-,tm packing, 10 x 250 mm) previously equili-brated with 20% CH3CN in 0.1% TFA. The column waseluted with an 80-min linear gradient of 20-40% CH3CN in0.1% TFA at a flow rate of 5 ml/min; 10-ml fractions werecollected. DH activity was found in fractions which eluted at34-38 min. These were combined and diluted with 40 ml ofwater, and again pumped onto the same column, but nowequilibrated with 10% (vol/vol) 1-propanol/0.1% TFA. Theretained materials were eluted with an 80-min linear gradientof 10-30% 1-propanol in 0.1% TFA at a flow rate of 5ml/min,

with 10-ml fractions collected. DH was recovered in the 48-to 52-min fractions.TSK SP-5PW LC (Step 7). After addition of 2 ml of 0.2 M

sodium phosphate buffer (pH 6.25), the active fractions fromstep 6 were applied to a TSK SP-5PW ion-exchange column(Beckman; 7.5 x 75 mm) equilibrated with 0.02 M sodiumphosphate buffer (pH 6.25) containing 10% CH3CN. Thecolumn was eluted with two successive gradients at 1ml/min: 10 min of0-0.1 M NaCl in 0.02M sodium phosphatebuffer (pH 6.25) containing 10% CH3CN and 60 min of0.1-0.4M NaCl in the same buffer. Two-milliliter fractions werecollected. DH was recovered in the 48- to 52-min fractions.Vydac C4 Analytical LC (Step 8). The active fractions from

step 7, containing buffer salts and =10% CH3CN, werepooled and applied by means of a 5-ml loop injector to aVydac C4 column (5-pum packing, 4.6 x 150 mm) equilibratedwith 30% CH3CN in 0.1% heptafluorobutyric acid (HFBA).The column was eluted with a 75-min linear gradient of 30-45% CH3CN in 0.1% HFBA at aflow rate of 1.5 ml/min, with3-ml fractions collected. DH was recovered only in the 36- to38-min fraction.Vydac C4 Microbore LC (Step 9). Finally, the active

fraction from step 8 was diluted with 6 ml ofwater and appliedto a Vydac C4 microbore column (5-,um packing, 2.1 x 250mm) by using two injections with a 5-ml loop injector. Thecolumn was equilibrated with 20% CH3CN in 0.1% TFA.After elution with a 100-min linear gradient of 20-40%CH3CN/0.1% TFA at a flow rate of0.3 ml/min, pure DH wasrecovered in a peak at 69.0-71.5 min.

Trypsin Digestion. Purified DH (-1.5 nmol) was dissolvedin 50 ,l of 0.1 M Tris HCI (pH 8.0)/0.01 M CaCl2 containing1 ,g of trypsin (TPCK treated, Sigma). After incubation at35°C for 2 hr, the reaction mixture was applied to a Vydac C18column (5-gm packing, 4.6 x 100 mm). The fragment pep-tides were eluted with an 80-min gradient of 0-40% CH3CNin 0.1% TFA at a flow rate of 0.5 ml/min.

Sequence Analysis. Purified DH and tryptic peptides weresequenced by using an Applied Biosystems model 477Apulsed liquid-phase protein sequencer equipped with a model120A on-line phenylthiohydantoin (PTH) amino acid ana-lyzer.Amino Acid Analyses. Purified DH and tryptic peptides

were hydrolyzed in vapor from 6 M HCI/1% phenol (110°Cfor 20 hr). Hydrolysates were analyzed after conversion tophenylthiocarbamoyl amino acid derivatives by reversed-phase LC using an Ultrasphere ODS column (Beckman,5-gm spheres, 4.6 x 150 mm) and buffers similar to those ofEbert (20).

Peptide Synthesis. The DH was synthesized by the solid-phase method (21) on a Biosearch 9600 peptide synthesizer,using a t-butoxycarbonyl (Boc) protocol and p-methylbenz-hydrylamine resin (0.35 meq/g, 0.50 g). Side chains of aminoacids were protected as follows: Boc-Arg(tosyl), Boc-Glu(benzyl), Boc-His(dinitrophenyl), Boc-Lys(o-chloro-benzyloxycarbonyl), Boc-Ser(benzyl), and Boc-Asp(cyclo-hexyl ester). Coupling was achieved by means of symmetricalanhydrides, except asparagine and glutamine, which wereintroduced as preformed hydroxybenzotriazole active esters(22). A yield of 2.06 g of the peptide-resin was obtained. Thedinitrophenyl protecting group on histidine was removed bythiolysis (23) (thiophenol), giving 1.8 g of the peptide-resin.HF cleavage of 858 mg of the peptide-resin in the presence of6% (vol/vol) anisole and 4% (vol/vol) ethyl methyl sulfide at0°C for 1 hr afforded 437 mg of the crude peptide (with acarboxyl-terminal amide), which was purified by reversed-phase LC, using a 2.2 x 25 cm Vydac C4 column eluted with30% CH3CN/0. 1% TFA at a flow rate of 9.9 ml/min. Pure DHwas recovered at 13-14 min. The identity of the major peakwas checked by sequence analysis and by amino acidcomposition analysis.

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0.2-40468

0~~~~~~~~~~~~~o ~~~~~~~~~~~~0

4)w h cblack bar indicates the DH-activ fraction.00

.0~~~~~~~~~~~~~a

20 40 60 80Retention Time (min)

FIG. 1. Vydac C4 microbore, LC (step 9). Chromatographicconditions are described in Materials and Methods. The broken lineshows the concentration of acetonitrile in 0.1% TFA in water. Ablack bar indicates the DH-active fraction.

The carboxyl-terminal acid analogue of DH was synthe-sized in identical fashion, starting with Boc-L-Ile-O-resin(0.74 meq/g, 0.3 g). After deprotection and HF cleavage, thecrude peptide was analyzed by reversed-phase LC, using aVydac C4 0.46 x 15 cm column eluted with a linear gradientof 20-40o CH3CN/0.1% TFA over 20 min at 1.5 ml/min.The peptide eluting at 14.6 min was analyzed by sequenceanalysis and by hydrolysis and amino acid analysis.Hexapeptides with the sequence of the tryptic fragment

T-5, Asn-Phe-Leu-Asn-Asp-Ile, were synthesized as thecarboxyl-terminal free acid and the amide by Biosearch undercontract. The crude peptides were purified by reversed-phaseLC essentially as described for tryptic digests.

RESULTSIsolation Procedure. After lipids had been removed from

the trimmed heads by extraction with acetone, DH wasextracted by using an acidic solution containing two proteaseinhibitors. This acidic solution was applied directly to anSP-Sephadex column, which was eluted stepwise with dif-ferent ammonium acetate buffers of increasing pH and/orionic strength. The active fractions from SP-Sephadex wereapplied to a C4 reversed-phase "cartridge" similar to com-mercially available disposable cartridges, except containing10 g of adsorbent. The cartridge was eluted stepwise withCH3CN/0.1% aqueous TFA; DH-active material eluted inthe 35% CH3CN fraction. The latter solution was diluted withtwice its volume ofwater and then loaded onto a semiprepar-ative reversed-phase LC column by using the pump. Thistechnique avoided losses of biological activity that occurredwhen fractions were evaporated at each step. Two successivesemipreparative reversed-phase purifications were per-formed, using different organic modifiers. The active fractionfrom the second semipreparative reversed-phase LC wasfurther purified by ion-exchange LC, analytical reversed-phase LC, and finally microbore reversed-phase LC (Fig. 1).At the final step, DH activity coincided with a peak thatabsorbed UV at 220 nm, but this material had no UVabsorbance at 280 nm (not shown). The overall yield wasabout 5 nmol from 10,000 trimmed heads.

0205C4)

0~~~~~~~~~~~~.0~~~ ~ ~ ~ ~ ~ ~ ~~~2.

U10 20Retention Time (min)

FIG. 3. Comparative reversed-phase Vydac C4 LC of native T-5and synthetic peptides. Upper trace, native T-5; lower trace, mixtureof synthetic hexapeptides (standard one-letter symbols) with eithera free acid or an amidated carboxyl terminus.

Structural Analyses. Automated Edman degradation ofintact DH (0.5 nmol) yielded a single amino acid sequencewith :80% initial yield and =z95% repetitive yield. Residues1-40 were assigned after use of 10% of the isolated sample(Fig. 2). Trypsin digestion of DH (1.5 nmol) gave sixfragments (T-1 to T-6). Their amino acid composition andsequences are shown in Table 1 and Fig. 2, These resultssuggest that T-5 is a carboxyl-terminal fragment of DH, asonly T-5 has neither arginine nor lysine at its carboxylterminus. Therefore, DH was regarded as a 41-residuepeptide. The nature of the carboxyl terminus was establishedby comparison of reversed-phase LC retention times of T-5and the synthetic hexapeptide Asn-Phe-Leu-Asn-Asp-Ileprepared in the amidated and free acid forms. The data showclearly that the carboxyl terminus of T-5 is amidated (Fig. 3).Hence, the complete structure of DH was established asshown in Fig. 2. This structure is also supported by aminoacid analysis of intact DH (Table 1).

Synthesis and Biological Properties of M. sexta DH. Wesynthesized M. sexta DH, as well as an analogue with an acidat the carboxyl terminus, by using conventional automatedsolid-phase methods. The synthetic amidated and acidicforms were compared to native DH by reversed-phase LCanalysis, which again established the identity of native DHwith the synthetic amidated form. Synthetic DH and its acidanalogue were tested in the Pieris assay. The approximateED50 for the amide form was 0.1 ng per animal, that for theacid was 0.1 Ag per animal.We investigated the effects of synthetic DH on M. sexta

larvae in vivo. When 0.1 ,ug of synthetic DH was injected intopostfeeding, prewandering larvae, a mean of 226 mg of fluidwas lost over a 4-hr period from those injected with syntheticDH, while controls excreted a mean of 98 mg. This is a highlysignificant difference (P < 0.001).

DISCUSSIONControl of water balance is vital to the survival of insects,although the requirements for each species vary greatly. Forinsects living in dry environments water retention is critical,while other species may consume their body weight in foliage

H-Arg-Met-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Pro-Met-Ser-Val-Leu-Arg-Gln-Lys-Leu-Ser-Leu-f T-6

t I T-4

Glu-Lys-Glu-Arg-Lys-Val-His-Ala-Leu-Arg-Ala-Ala-Ala-Asn-Arg-Asfn-Phe-Leu-Asn-Asp-Ile-NH2

T T-2 T-5I T-l1

IT-3

FIG. 2. Amino acid sequence of M. sexta DH. T-1 to T-6 indicate sequences determined for tryptic peptides.

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Table 1. Amino acid composition of DH and its tryptic peptidesResidues per molecule

Residue Intact DH T-1 T-2 T-3 T-4 T-5 T-6Asx 4.92 (5) 0.73 (1) 3.14 (3) 0.79 (1)Glx 2.63 (3) 0.71 (1)Ser 3.69(4) 1.05 (1) 3.14 (3)Gly 0.24 (0)His 0.63 (1) 0.38 (1) 1.03 (1)Thr 0.41 (0)Ala 4.00 (4) 3.00 (3) 1.09 (1) 1.00 (1)Arg 5.43 (5) 1.20 (1) 1.04 (1) 1.09 (1) 1.14 (1)Pro 2.00 (2) 2.28 (2)Tyr 0.05 (0)Val 1.52 (2) 0.57 (1) 0.77 (1) 0.% (1)Met 1.56 (2) 1.59 (2)Ile 1.93 (2) 0.97 (1) 1.00 (1)Leu 6.72 (7) 1.00 (1) 1.04 (1) 2.00 (2) 1.06 (1) 3.00 (3)Phe 1.03 (1) 0.99 (1)Lys 3.38 (3) 0.89 (1) 0.95 (1)

Position 1-41 31-35 26-30 25-30 18-22 36-41 2-15Numbers in parentheses are from sequence analyses. Trp and Cys were not determined.

each day, requiring elimination ofmost of the ingested water.Diuresis is especially striking in blood-feeding insects whichrapidly excrete the fluids obtained from a blood meal of 2-12times the unfed body weight (1-3). Most aquatic insects mustrid themselves of excess water acquired by osmosis (3, 6).Certain insects excrete water during flight as a result of theintense metabolism required (2). The most difficult osmoreg-ulatory problems may occur in insect larvae that dwell in saltwater (3, 24).Most attempts to characterize DH structurally have been

based on difficult and tedious in vitro Mt bioassays (8). Otherefforts have been based on an in vivo amaranth dye excretiontest (9), and a semi-isolated Mt assay (10). Recently, moreindirect, but rapid in vitro bioassays have been adopted.Isolated Mt have been monitored for transepithelial voltagechanges after DH stimulation (11). RIA techniques have beenused to detect an increase in cAMP in DH-stimulated Mt (10,12).Our isolation was guided by an in vivo bioassay on P.

rapae. Newly emerged adult butterflies of this species wereneck-ligated and beheaded, depriving the abdomen of a pulseof DH, which ordinarily stimulates substantial loss of a clearfluid from the rectum. Neck-ligated animals excrete only adark meconium (waste products of pupal metabolism). In-jection of DH-active fractions into ligated butterflies causeda typical normal urine excretion.We isolated M. sexta DH in parallel with eclosion hormone

(EH), which we recently purified and sequenced (25). Bothhormones are extracted together, but they are separated bySP-Sephadex ion-exchange chromatography. The strategy ofsubsequent purification steps is nearly identical for EH and

DH, although the chromatographic conditions differ. About5 nmol of diuretic hormone was isolated from 10,000 trimmedpharate adult heads. Proving that the carboxyl terminus wasamidated required chemical synthesis of the tryptic fragmentT-5 in both the free acid and amidated forms.

In early experiments, we fractionated M. sexta DH byusing Sephadex G-50. The mobility of bioactive fractionssuggested a molecular weight of 5000-6000. This is roughlyconsistent with the value of 4732 calculated for the sequenceshown in Fig. 2.

In an earlier attempt to isolate DH, we found that biologicalactivity was diminished or lost when we evaporated solventfrom either the reversed-phase cartridge step or the semi-preparative purifications, possibly due to association withproteins, adsorption, or oxidation. Accordingly, we modifiedthe pumping system so that water-diluted fractions contain-ing organic solvent could be loaded onto the column by thepump. M. sexta DH has two methionine residues (positions2 and 11), perhaps rendering it quite sensitive to oxidation.We checked for sequence similarity of M. sexta DH with

known peptides by using a computer program (Intelligenet-ics). This located a substantial similarity with sauvagine (ref.26, Fig. 4). Sauvagine was isolated from skins of the frogPhyllomedusa sauvagei while using a bioassay based on itspotent antidiuretic effect in rats (27). If allowance is made fora one-amino acid insertion of histidine in M. sexta DH(position 27), 40% of the residues are identical. An additional16 amino acids in M. sexta DH could result from single-basemutations in the sauvagine gene. The location of the muta-tions strengthens the supposition that histidine has beeninserted in a well-conserved sequence. Sauvagine is but one

N D D|P|P I|S I D LIT F H L

N D D

Q G

R M

P

P

P

S Q EIPI

P I

P I

S L

P I

S E EIPIP I

S I D LIS L E LIL R

S I D L

S L D L

S L D L

I.P M S VIL R

T F H LIL R

T F H LIL R

NMI EMARNENQ R E Q

R E Q

K M I E I E K Q E K E K Q Q

Q K L S L E K E R K V H A L R

E V L E M TKA D Q L A Q Q

EVLEMARAEQL AQQ

FIG. 4. Comparison of amino acid sequence ofManduca DH with sequences of urotensin I (carp and sucker), sauvagine, and CRF (caprine,ovine, human, and rat). One-letter amino acid symbols are used. Residues identical with those in M. sexta DH are enclosed by solid lines.

Carp Urotensin I

Sucker Urotensin I

Sauvagine

Manduca sexta DH

Caprine/ovine CRF

Human/Rat CRF

SI D LIT F H LIL RIN MI E M A RI EN E

N RIK Y

NR

NR

KY

L L

A G L

A G L

A A N

A A A

AH S

A H S

N RIN F

D E V NH2

D E V NH2

D T I-NH2

N D I-NH2

D I NH2

E I I

N RI

NR

K LL

K L N

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member of an important peptide family including CRF andurotensin I. CRF, originally isolated and identified fromsheep hypothalami (oCRF, ref. 28), has been sequencedsubsequently from the rat (rCRF, ref. 29), goat (cCRF, ref.30), and cow (bCRF, ref. 31). The structure of human CRFwas deduced by sequencing its gene isolated from a genomiclibrary (hCRF, ref. 32). The sequence identity ofM. sexta DHwith oCRF/cCRF is 32%; an additional 41% similarity wouldresult from single-base changes in the gene. Urotensin I,peptides biologically and chemically homologous to sau-vagine and CRF, were isolated and identified from fishurophysis (analogous to the hypothalamus). M. sexta DHshares 27% sequence identity with both white sucker (33) andcarp (34) urotensin I; an additional 44% identity with suckerurotensin I could come from single-base changes in the gene.Thus, M. sexta DH is the newest member of the sauvagine/

CRF/urotensin I family of peptides, now represented in theclasses Insecta/Amphibia/Mammalia/Pisces. The moststudied physiological/pharmacological effects of this peptidefamily appear to be the stimulation of adrenocorticotropichormone (ACTH) release and hypotensive/vasodilatoryproperties; the antidiuretic effect of sauvagine has beenattributed to its potent hypotensive action (26). In addition,stimulation of f-endorphin release accompanies ACTH re-lease induced by oCRF (28) or sauvagine (26). The potencyand character of the effects elicited by each peptide vary withthe species in which it is tested. For example, both types ofurotensin I and sauvagine elicit a strong and selectivehypotensive/vasodilatory effect in mammals, actually morepotent than that caused by the mammalian form oCRF (34).In contrast, injection of 500 pmol of urotensin I, sauvagine,and oCRF did not stimulate fluid excretion in the P. rapaeassay (R.G.T., unpublished data). By comparison, the ED50of M. sexta DH is :0.02 pmol.Proux et al. (15) recently identified an antiparallel ho-

modimer of [Leu2,Thr4,Arg8]vasotocin as a DH of L. migra-toria. This result seems difficult to reconcile with ouridentifying a member of a structurally divergent peptidefamily as a DH of M. sexta. However, the AVP-like locustDH has no activity in the P. rapae assay (R.G.T., unpub-lished data), and conversely, immunological studies haveshown there is no AVP-like factor in M. sexta (S.J.K. and A.Toschi, unpublished data). It is interesting that AVP (whileless potent than CRF) also triggers release of ACTH frommammalian pituitary (28). It is also intriguing that urotensinI has been suspected of playing a role in osmoregulation (35).Thus, there may be some overlap in biological roles of thesetwo distinct peptide families.ACTH has been implicated in water balance in vertebrates

due to its control on aldosterone production. Accordingly,Rafaeli et al. (36) recently investigated the possible existenceof an ACTH-like material in locust CC. They found that theCC contain an ACTH-like material, and that ACTH at 10 ,AMstimulates liquid excretion from, and increases cAMP in,locust Mt. Extracts of CC produce similar effects in thetraditional (8) in vitro Mt assay (36). Morgan et al. (13),assaying the increase in cAMP in Mt induced by tissuefractions, reported a partial sequence for a diuretic peptidealso isolated from locust CC. This partial sequence has nosimilarity to M. sexta DH or to the AVP-like locust DH (15).Curiously, synthetic M. sexta DH does not increase cAMP inMt of M. sexta larvae (R.G.T., unpublished data).

In our P. rapae assay, injection of synthetic DH and its acidanalogue revealed that the acid form was about 1/1000th asbioactive as the amide form. Injection of DH into M. sextalarvae caused a profound loss of fluid, not only via the gut butthrough the epidermis as well. Our results do not rule out thepossibility, suggested by the work of Rafaeli et al. (36), thatthe CRF-like M. sexta "DH" could actually stimulate release

of another peptide, which would act as a DH on Mt. Thesedata do suggest that the concept of a unique "post-eclosionDH" (16) may be in error.

In summary, we have isolated a diuretic factor from M.sexta that exhibits diuretic effects in vivo, although we cannotsay at this time whether it acts directly on the Mt or triggersrelease of another peptide from a tissue outside the head. Thelatter result is suggested by the sequence similarity to a familyof releasing factors and by our inability to observe directeffects of M. sexta "DH" on the presumed target tissue.

We thank the staff of the insect culture group for providinganimals, Marilyn Bennett for technical assistance, Blake Cesarin fordetermining amino acid compositions, Dr. Alan Roter for a computersearch of peptide homology, and Dr. Herbert Roller for encourage-ment.

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Proc. Natl. Acad Sci. USA 86 (1989)

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