DEVELOP~NTAL AND COMPARATIVE IMMUNOLOGY, Vol. I0, pp. 167-179, 1986. O145-305X/86 $3.00 + .OO Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.

THE HUMORAL ANTIBACTERIAL RESPONSE OF DROSOPHILA ADULTS

Mark Robertson and John H. Post le thwai t Department of Bio logy, Un ive rs i t y of Oregon, Eugene, OR 97403

ABSTRACT: Hemolymph from a normal adu l t Drosophila melanogaster lacks fac tors that block the growth of Escherichia c o l i , but hemolymph from a f l y p rev ious ly inoculated with Enterobacter cloacae i n h i b i t s bac ter ia l growth. An t i bac te r i a l a c t i v i t y appears w i th in two hours a f t e r i nocu la t i on , and is s t i l l detectable s i x t y days l a t e r . A c t i v i t y is potent, and can be detected in as l i t t l e as a quar ter of the hemolymph from a s ing le inoculated male f l y . A f te r i nocu la t i on , at least e ight new polypept ides not of bac ter ia l o r i g i n appear in hemolymph wi th a time course s im i l a r to the appearance of an t i bac te r i a l a c t i v i t y ; these are ca l led An t i bac te r i a l Response Polypept ides, or ARs. The most prominent polypept ides are AR24, AR22, and ARI9 wi th molecular weights of about 24, 22, and 19 k i loda l tons (kd). Other bands wi th as much as 75kd and as l i t t l e as 5kd were also found. Electrophoresis of ac t ive hemolymph under non-denatur ing cond i t i ons , and i s o e l e c t r i c focusing separate several p ro te in species that block bac ter ia l growth (An t i bac te r i a l Prote ins, or ABs); one AB is neutra l (AB7.1) and three are basic (AB8.7, AB9.0 and AB9.2). Two dimensional gels show that AR24, AR22 and ARI9 have pls i den t i ca l to the basic a n t i b a c t e r i a l p ro te ins . Rad io labe l l ing experiments proved that the ARs were synthesized de novo a f t e r bac ter ia l i nocu la t i on . ARs in s ix species of Drosophila showed fundamental ly s i m i l a r e lec t rophore t i c pat terns.

INTRODUCTION

Many species of insects possess an a n t i b a c t e r i a l response that is induced by exposure to l i v e bacter ia or ce l l wall cons t i t uen ts , and that provides the insect wi th immunity to bac ter ia l i n f ec t i on ( I ) . This immune system involves the synthesis of about a dozen new polypept ides that can be ca l led An t i bac te r i a l Response Polypept ides, or ARs. A subset of the ARs, ca l led An t i bac te r i a l Proteins (ABs), d i r e c t l y block bac te r ia l growth. In diapausing pupae of the s i lkmoth Hyalophora cecropia, there are three classes of ABs: lysozyme (2) (14 k i l oda l t on (kd ) ) , whose a c t i v i t y is l im i ted to a few Gram pos i t i ve organisms; a t t ac i ns , (3) (20kd) wi th a c t i v i t y only

167

168 ~NTIBACTERIAI. PROTEINS IN FRUIT FLIES Vol . 10, No. 2

against a few Gram-negative bacter ia; and cecropins (4), which are small basic proteins (4kd) e f fec t i ve against many Gram-positive and -negative bacter ia. These polypeptides are produced by the insect 's fa t body (5), although blood cel ls are also necessary for induction of immunity (6). Recent work (7) has suggested that henlocytes phagocytose and p a r t i a l l y degrade bacter ia, releasing peptidoglycans that then cause the fa t body to synthesize ant ibac ter ia l proteins. In order to use mutations to help unravel the mechanisms of immunity in insects, we have begun to invest igate the an t ibac ter ia l response of Drosophila melanogaster.

Except for a b r ie f period during pupation, Drosophila has l i t t l e natura l ly occuring an t ibac ter ia l a c t i v i t y (8), although ant ibac ter ia l a c t i v i t y can be induced by in jec t ing bacteria during the adult stage (9). Despite advances in the ce l l u la r immune responses of Drosophila (10,11), and the use of Drosophila to test the virulence of d i f f e ren t bacteria (12, 13), there has been no d i rec t work on Drosophila's humoral an t ibac ter ia l response since 1972. In the Drosophila experiments reported here, we have sought to ( I ) i den t i f y proteins that are responsible for an t ibac ter ia l a c t i v i t y ; (2) inventory hemolymph polypeptides that appear a f ter inoculat ion; (3) tes t whether ant ibac ter ia l response polypeptides ar ise due to de novo synthesis; and (4) begin to assess the genetic v a r i a b i l i t y in the an t ibac ter ia l response. We report that in Drosophila, proteins with some s i m i l a r i t i e s to the bacter ic idal proteins of H__ L cecropia begin to be synthesized de novo wi th in a few hours a f ter exposure to bacter ia.

MATERIALS AND METHODS

Stocks: Most experiments were conducted with the Oregon R wi ld type stock of Drosophila melanogaster. Wild type stocks of the fol lowing species were also used: D. simulans, D. mauri tania, D. erecta, D. t e i s s i e r i , and D. yakuba, Fl ies were cultured on ~tandard medium at 22-25 C. Bacteria stocks were Enterobacter cloacae, s t ra in beta-12, na lad ix ic acid res is tan t (14), and Escherichia co l i , s t ra in D31 {15), streptomycin res is tan t .

Surgical procedures: Fl ies were injected in the abdomen with O.lul of a suspension of s ta t ionary phase E. cloacae cel ls grown in LB broth (16) to 2 x 109/ml and d i lu ted 1:5 with s ~ r i l e 0.9% NaCI. Hemolymph was col lected by in jec t ing 0.3 ul of s t e r i l e O.9L NaCI containing phenylthiourea (10% saturated) and aprot in in (2.3 TIU/ml, Sigma) into the abdominal body cav i t y , and recovering the solut ion in a glass cap i l l a ry . Samples used in electrophoresis and i soe lec t r i c focusing were centr i fuged in a cap i l l a ry centr i fuge at 4 C for 10 minutes to remove ce l ls . In some cases, hemolymph was frozen in l i qu id nitrogen before tes t ing , a treatment that was not found to a f fec t an t ibac ter ia l a c t i v i t y . A mixture of 14C-amino acids (55mCi/m atom carbon, NEN) was d i lu ted to 0 . i uCi/ul in 0.9~i NaCI, and injected into the abdominal body cav i ty of male f l i e s .

Gels: Electrophoresis at pH 6.8 (neutral gels) in 151~ acrylamide gels used a discontinuous (potassium-MOPS/histidine-MOPS) buffer system (17). I soe lec t r i c focusing (IEF) was performed on LKB PAG plates at pH 3.5-9.5 according to manufacturers ins t ruc t ions . Electrophoresis under denaturing condit ions in sodium.dodecyl su l fa te-conta in ing gels of polyacrylamide (SDS- PAGE) employed a 9-18~ acrylamide gradient (acrylamide/bis-acrylamide ra t io = 20:1) (18). For two dimensional e lectrophoresis, lanes from IEF were f ixed in t r i ch lo roace t i c ac id -su l fosa lacy l i c acid for one hour, washed ten minutes in iOmM d i t h i o t h r e i t o l , 8';i g lycero l , 2.3% SDS in stacking buf fer , and placing on top of an SDS-PAGE gel. Gels were f ixed in 5% formaldehyde, 25%

V o l . 10, No. 2 ANTIBACTERIAL PROTELNS IN FRUIT FLIES 169

EtOH, i0 . acetic acid and stained with s i l ve r (19). The Pharmacia low molecular weight standards was used as a size marker.

Ant ibactcr ia l a c t i v i t y : Ant ibacter ia l a c t i v i t y was tested by the inh ib i t i on zone assay (4), or by "bug blots" prepared by overlaying electrophoret ic gels with phosphate buffered LB agar seeded with E. col i D31 to reveal bands of a c t i v i t y (20).

RESULTS

Potency and time course of the humoral ant ibacter ia l response.

Hemolymph removed from normal 2-4 day old male f l i e s did not i nh i b i t the growth of test bacteria suspended in nutr ient agar in the inh ib i t i on zone assay (Fig. I ) , showing that naive f l i es lack detectable humoral ant ibacter ia l a c t i v i t y . Likewise, f l i e s injected with s t e r i l e sal ine or f l i es injured by pricking showed no ant ibacter ia l a c t i v i t y . However, when male f l i es were inoculated with E. cloacae, and exsanguinated 24 hours la te r , the i r hemolymph blocked the growth of E. col i in a plaque surrounding the test well (Fig. i ) . This resul t confirms that Drosophila adults possess an inducible ant ibacter ia l system (9). Ant ibacter ia l a c t i v i t y is quite potent, since i t was detected from as l i t t l e as one-quarter of the hemolymph drawn from a single inoculated f l y (about 0.05 u l ) . Females as well as males have an inducible humoral ant ibacter ia l response, but males were used rout ine ly to avoid complications related to oogenesis, such as the rapid synthesis of yolk protein by female fat bodies.

~_.~: ~ • 1; 4 ..'~ . . . . . . , - , ~ , ~

T. ~

, ,

FIG.I Drosophila possesses a potent inducible humoral ant ibacter ia l response. Two day old adult male f l i es were injected with 0 . i ul of saline or 0 . i ul of a suspension of streptomycin-sensi t ive E. cloacae. Af ter 24 hours, hemolymph was col lected, centr i fuged, and the supernatant solut ion tested by the inh ib i t i on zone assay. Numbered wells contained immune hemolymph from i0, 5, 3, 2, I or half of an inoculated f l y . Hemolymph from ten sa l ine- in jected f l i es f i l l e d the well labeled "buffer"

Induction kinet ics and persistence of ant ibacter ia l ~ c t i v i t y was tested by inoculat ing two day old males with bacteria and withdrawing hemolymph at in terva ls from i hour to 65 days la ter . Fig. 2 shows that ant ibacter ia l a c t i v i t y had already appeared within two hours a f ter inoculat ion, and that i t continued to increase unt i l about 48 hours, when i t leveled o f f . A high level of a c t i v i t y persisted for at least 65 days af ter inoculat ion. We

170 ANTIBACTERIAL PROTEINS IN FRUIT FLIES Vol. IO, No. 2

conclude that the a n t i b a c t e r i a l system is rap id l y induc ib le and long l as t i ng .

L E I I I I . i [ _ l L L I

O

tOO- ~-

• T. 80- 0 u • I • _ 6 0 -

4 0 - • • I- .L ~ 2o

'~ , I i , I/ /--IEI3TO ; CbF-- 0 4 8 12 16 20 24 2 I0 20 30 40 50 60

Hours DGys Time After T_noculolion

FIG 2 The a n t i b a c t e r i a l response is rap id l y induced and long l as t i ng . Male f l i e s were inoculated wi th bacter ia and hemolymph was withdrawn from ten of them at i n t e r v a l s the rea f te r and tested in the i n h i b i t i o n zone assay. The area of the c lear plaque was p lo t ted against time a f t e r i nocu la t i on . . . . three d i f f e r e n t ser ies of inoculated f l i e s ; , un in jected con t ro l s .

The a n t i b a c t e r i a l response involves several components.

To determine i f the humoral a n t i b a c t e r i a l a c t i v i t y has mu l t i p l e components, we separated ac t i ve and contro l hemolymph e l e c t r o p h o r e t i c a l l y in nat ive gels at neutra l pH, and by i s o e l e c t r i c focusing. To detect a n t i b a c t e r i a l a c t i v i t y , the gels were over la id wi th bacter ia-seeded agar, in to which prote ins from the gel d i f fused (20). The resu l t s (Fig. 3) showed that ac t i ve hemolymph contained several a n t i b a c t e r i a l fac tors wi th d i s t i n c t migrat ion pat terns. In no case did unin jected contro l hemolymph show a c t i v i t y . Neutral gels (Fig. 3) revealed three regions of bac ter ia l growth- blocking a c t i v i t y moving towards the cathode: the most r ap id l y migrat ing band was the s t rongest , fol lowed by a weaker, narrow band, and f i n a l l y , a ser ies of three c lose ly grouped bands. I s o e l e c t r i c focusing (Fig. 3) showed at leas t four a n t i b a c t e r i a l components (AB), one near ly neutra l (AB7.1), and three basic (AB8.7, AB9.0, and AB9.2). These experiments suggest that there are at least four major molecular species capable of b locking bac te r ia l growth that appear in the hemolymph of inoculated Drosophi la adu l ts .

New polypept ides appear in the hemolymph of inoculated f l i e s .

To i d e n t i f y hemolymph polypept ides that might be responsib le for induc ib le a n t i b a c t e r i a l a c t i v i t y , we inoculated males, withdrew t h e i r hemolymph at i n t e r v a l s , and subjected i t to SDS-PAGE. Fig. 4 shows that

Vol. IO, No. 2 ANTIBACTERIAL PROTEINS IN FRUIT FLIES 171

FIG.3 The a n t i b a c t e r i a l response involves several a n t i b a c t e r i a l prote ins (ABs). Hemolymph from bac te r ia - inocu la ted f l i e s ( I ) or s a l i n e - i n j e c t e d cont ro ls (C) were separated by e lec t rophores is . The gels were ove r la id wi th n u t r i e n t agar seeded wi th bacter ia and incubated overn ight . Lanes A-C, neutra l gel ; lanes D-E, i s o e l e c t r i c focusing. AB7.1, AB8.7, AB9.0, AB9.2, an t i bac te r i a l prote ins wi th the ind icated i s o e l e c t r i c po in ts .

About e igh t new polypept ides appeared in the hemolymph in the f i r s t two days a f t e r i nocu la t i on . Fly-produced polypept ides that appear a f te r i nocu la t i on wi th bacter ia are ca l led An t i bac te r i a l Response Polypeptides (ARs). The ARs ranged in size from about 5kd up to about 75kd. The most prominent bands were ARIO, which was already detectable four hours a f t e r i nocu la t i on , ARt9, which stained b r i gh t golden, and AR24, one of the most abundant hemolymph polypept ides a f t e r i nocu la t i on . AR75 was negat ive ly stained by the s i l v e r . Since the ARs increase wi th a time course cons is ten t wi th the increase in a n t i b a c t e r i a l a c t i v i t y , they are candidates fo r prote ins wi th a n t i b a c t e r i a l a c t i v i t y (ABs).

While ARs were increasing in concentrat ion in the hemolymph, polypept ide bands c h a r a c t e r i s t i c of the inoculated bacter ia were decreasing ( i . e . , B34 and B20 in Fig. 4) , and by twelve hours a f t e r i nocu la t i on , they were gone. We conclude that bacter ia are cleared from the hemolymph w i th in a few hours a f t e r i nocu la t i on .

To determine whether any of the ARs defined by SDS-PAGE in Fig. 4 are responsib le for the a n t i b a c t e r i a l a c t i v i t y detected on nat ive gels shown in Fig. 3, we analyzed hemolymph from uninoculated con t ro ls and from bac ter ia - in jec ted male f l i e s on two dimensional gels, using i s o e l e c t r i c focusing in the f i r s t dimension and SDS-PAGE in the second. The resu l t s (Fig. 5) ind icated that AR24, AR23, and AR21 had i s o e l e c t r i c points the same as AB8.7 and AB9.0, and AR 19 had an i s o e l e c t r i c point the same as AB9.2. No AR could be i d e n t i f i e d on these two dimensional gels that had an i s o e l e c t r i c po in t the same as AB7.1. AR24 and AR22 are both present in d i f f e r e n t l y charged forms, suggesting some type of pos t - t r ans l a t i ona l mod i f i ca t ion . These experiments suggested that the most prominent ARs of about 19-24kd form prote ins wi th a n t i b a c t e r i a l a c t i v i t y .

£72 ANTIBACTERIAL PROTEINS IN FRUIT FLIES Vol. lO, No. 2

BACTERIA HEMC~YMPH C ON TR¢~. INOCULATED

IOmin Ih 2k 4k 12h 24h 4Sh

FIG 4 Eight ant ibacter ia l response polypeptides (ARs) are secreted into the hemolymph of f l i es inoculated with bacteria. Flies were injected with bacteria and hemolymph was col lected at I0 minutes, and i , 2, 4, 12, 24, and 48 hours af ter inoculat ion. Proteins were separated by SDS-PAGE and stained with s i l ve r . Positions of bacter ial polypeptides are indicated on the l e f t of the f igure, and locations of ARs are shown on the r igh t .

The basic ant ibacter ia l proteins are heat and acid stable.

To test the s t a b i l i t y of the ant ibacter ia l factors, we treated active and control hemolymph to e i ther 100 C for 5 minutes in 0.2 M acetic acid, or 25 C for 5 minutes in 0.2 M acetic acid. Samples were then centrifuged to remove denatured proteins, and the supernatant analyzed for a c t i v i t y and protein. Since the inh ib i t i on zone assay showed that substantial a c t i v i t y remained af ter the acid/heat treatment, the supernatant solut ion from acid/heat treated hemolymph was resolved by i soe lec t r i c focusing (Fig. 6). We found that the highly basic ABs retained the i r a c t i v i t y a f ter the acid/heat treatment, but that AB7.1 had disappeared. When the supernatant solut ions from acid/heat treated hemolymph were displayed on SDS-PAGE, many of the ARs had remained in so lut ion, including AR24, AR22, ARI7, and the b r igh t l y negatively stained pair of bands ARI3 and ARIO. This experiment shows that a major f ract ion of the ant ibacter ia l a c t i v i t y is stable to heat and to acid, and that th is stable f ract ion contains several ARs.

Ant ibacter ia l response polypeptides are due to de nov0 synthesis.

Do the ARs resu l t from the synthesis of new proteins that are not being made by normal f l i e s , or do they appear from post - t rans la t iona l modif icat ion

Vol. IO, No. 2 ~NTIBACTERIAL PROTEINS IN FRUIT FLIES 173

94

67

43

30

I NOCULATE D

p~ $ Zl UI~

-=2, ~ ~ N m l E , L

CON TROu

S

t P

FIG 5 Four hemolymph ARs have the same i s o e l e c t r i c points as prote ins wi th a n t i b a c t e r i a l a c t i v i t y . Immune ( l e f t ) and contro l ( r i g h t ) hemolymph samples were placed at the pos i t ion marked S and separated f i r s t by i s o e l e c t r i c focusing, and then in the second dimension by SDS-PAGE and stained with s i l v e r . The lanes labeled IH (immune hemolymph) and CH (contro l hemolymph) were run d i r e c t l y in SDS-PAGE. I s o e l e c t r i c points of a n t i b a c t e r i a l prote ins are ind icated on top of the gel , and the migrat ion of molecular weight standards on the l e f t s ide. The loca t ion of ARI9, AR22, AR23, and AR24 are ind icated by numbers and arrow heads.

of p re -ex i s t i ng polypept ides? I f ARs resu l t from de novo synthes is , then they should become rap id l y labeled with rad ioac t ive amino acids a f te r i nocu la t i on . To tes t th i s p red i c t i on , two day old male f l i e s were inocu la ted, and 24 hours l a t e r they were in jec ted wi th a mixture of 14C- labeled amino acids. A f te r an add i t iona l incubat ion of one hour in the presence of labe l , hemolymph was co l lec ted , separated by SDS-PAGE, and the gel was stained wi th s i l v e r and autoradiographed. When the autoradiograph was superimposed on the stained gel (Fig. 7), i t became apparant that a l l e igh t ARs become labeled and stand out in cont rast to the con t ro ls . The synthesis of AR75, AR24, and AR21 were not detected in the hemolymph of contro l f l i e s , but became three of the four most h igh ly labeled bands in the hemolymph of immunized f l i e s . Polypeptides that were being synthesized before i nocu la t i on cont inue to be made the rea f te r . Since Fig. 4 shows that by 24 hours bac ter ia l prote ins had become undetectable, i t is very u n l i k e l y that the labeled bands were made by bacter ia . We conclude that ARs ar ise by the synthesis of a new set of p ro te ins .

Several c lose ly re la ted species o f Drosophila make the same ARs.

To determine whether s i g n i f i c a n t genetic v a r i a b i l i t y ex i s t s in the a n t i b a c t e r i a l response, we examined the size of humoral ARs from s ix c lose ly re la ted Drosophila species of the melanogaster group. Two day old male f l i e s of each species were in jec ted with E. cloacae, and uninoculated f l i e s served

174 ANTIBACTERIAL PROTEINS IN FRUIT FLIES Vol. iO, No. 2

I [ P ~ ) S - m , G E

U n t r m a l ~ Acid/H41al c . ~ j

i ¢ i c ~ d e ~ A A H

,mmIII-"

FIG 6 Basic ant ibacter ia l proteins are heat and acid stable. Active and control hemolymph samples were e i ther l e f t untreated or brought to 0.2 M acetic acid and treated for f ive minutes at 100 C or 25 C and centr i fuged. The supernatant solut ions were analyzed by i soe lec t r i c focusing (IEF) and SDS- PAGE. i , hemolymph from inoculated f l i e s ; c, hemolymph from uninjected control f l i e s ; A/H, acid/heated hemolymph; A, acid treated hemolymph.

4 , ' -

I a I -

~ ' ' - _

• ~ ? . 4~ i . T - a ~

-. '.~'" i "

~iIi, i I

FIG 7 AR polypeptides are synthesized de novo. Flies were injected with stat ionary phase bacteria (INOC.) or sal ine (CONT.) and 23 hours la ter with 14C amino acids. After one hour, hemolymph was col lected and separated by SDS-PAGE. Stationary phase bacteria were cultured at 22 C with ~4C amino acids (BACT.). The gel was stained with s i l ve r ( l e f t ) , and autoradiographed ( r i gh t ) . Locations of AR polypeptides are shown on the r igh t of the f igure.

Vol. IO, No. 2 ANTIBACTERIAL PROTEINS IN FRUIT FLIES 175

as contro ls . One day a f te r immunization, hemolymph was withdrawn, separated by SDS-PAGE, and stained with s i l v e r . Figure 8 shows that the pattern of hemolymph polypeptide~ in th is group of species is general ly uniform, but a few di f ferences characterize each species. Superimposed on th is pattern are the AR polypeptides: in al l s ix species, most AR polypeptides are c lear ly d is t ingu ishab le , and are lacking from the respect ive contro ls. ARI9 to AR24 seem to migrate i den t i ca l l y in al l s ix species, but di f ferences ex is t in several species in the lower molecular weight ARs. ARI7 and AR75 are d i f f e ren t in D__~. yakuba. In D. erecta, AR21 was present in two forms with s l i g h t l y d i f f e ren t mob i l i t i es . We do not yet know i f the two bands are due to polymorphism in the D. erecta population or i f there are two AR21 genes with d i f f e ren t propert ies in th is species. We conclude that select ion has not allowed the size of the ARs to vary much during the rad iat ion of the melanogaster group of species.

I • I ¢ I • I I i • i ¢

"

~ m l mm v-q

• . -

FIG 8 Six Drosophila species c losely related to D. melanogaster a l l have very s im i la r AR polypeptides in the i r hemolymph. Male f l i e s of each species were inoculated with bacteria or l e f t uninjected as contro ls . One day la te r , hemolymph was col lected and separated by SDS-PAGE. i , inoculated; c, cont ro l ; yak, D__~. yakuba; mau, D. maurit iana; t e i , D. t e i s s i e r i ; ere, D. erecta; sim, D. simulans; mel, D. melanogaster. Dots indicate the locat ion of ARs in each species.

DISCUSSION

These experiments show that invading bacteria st imulate an immune mechanism in Drosophila adults to synthesize and secrete into the hemolymph proteins that block bacter ia l growth. This an t ibac ter ia l a c t i v i t y is heat and acid stable, and is due to several molecular forms with at least one neutral and several basic species. Other insects also have a multicomponent humoral an t ibac ter ia l system with neutral and basic fac tors , the best characterized being the three types of an t ibac ter ia l proteins found in moths (2, 7, 21).

SDS-PAGE stained with s i l v e r showed that hemolymph from immunized f l i e s contained at least e ight polypeptides in addi t ion to those found in cont ro ls , the most conspicuous of which were ARI9, AR22 and AR23. Two dimensional electrophoresis showed that these ARs migrated with proteins that had i soe lec t r i c points corresponding to an t ibac te r ia l proteins with pls of 8.7-9.2. Since these polypeptides have about the same size and charge as

176 ANTIBACTERIAL PROTEINS IN FRUIT FLIES V o l . 10, No. 2

some of the a t tac ins found in s i lkmoths (3) , we t e n t a t i v e l y suggest that Drosophi la may possess a t t a c i n - l i k e molecules. New polypept ides of about 5kd ( i . e . , the size of cecropins) also appear in immunized Drosophila hemolymph, and the humoral a n t i b a c t e r i a l a c t i v i t y in Drosophi la has thermal and pH s t a b i l i t y c h a r a c t e r i s t i c s of cecropins, but we were not able to confirm that the 5kd polypept ides have a n t i b a c t e r i a l a c t i v i t y . Since other higher f l i e s have been shown to contain cec rop in - l i ke substances (22-24), i t is l i k e l y that these low molecular weight a n t i b a c t e r i a l s are also found in Drosophi la. As in moths (25), a d i f f e r e n t set of AR polypept ides are found at d i f f e r e n t stages of the l i f e cycle in Drosophila (Kolstoe, Robertson, and Pos t le thwa i t , unpubl ished).

Time course experiments showed that a n t i b a c t e r i a l a c t i v i t y remained in the hemolymph of Drosophila adul ts for at least two months. This d i f f e r s s i g n i f i c a n t l y from si lkmoth pupae, where a c t i v i t y peaks on day seven and then fades to contro l leve ls by day 16 (26). We have not yet inves t iga ted whether ABs cont inue to be synthesized in Drosophila fo r two months, or whether the prote ins are maintained in the hemolymph wi thout new synthes is , or whether bac ter ia , e i t he r sequestered in some compartment in the f l y or in the cu l tu re medium, cont inue to "reinduce" synthes is .

Time course studies f u r t he r showed that a f t e r immunization, a n t i b a c t e r i a l a c t i v i t y appeared in the hemolymph w i th in two hours a f t e r i nocu la t i on . This rapid evo lu t ion of a n t i b a c t e r i a l prote ins is too fas t for a process requ i r i ng massive ce l l d i v i s i o n , subs tan t ia l ce l l d i f f e r e n t i a t i o n , or extensive ce l l hypertrophy, and hence the induct ion process must re ly on t r i g g e r i n g a r e l a t i v e l y rapid process in p re -ex i s t i ng c e l l s , such as t r a n s c r i p t i o n or t r a n s l a t i o n . This i n t e r p r e t a t i o n is bolstered by the f i nd ing that the ARs rap id l y incorporated rad ioac t i ve amino acid t racers in inocu la ted, but not in contro l f l i e s . Whether in Drosophi la, as in s i lkmoths (27), the induct ion of AR synthesis is due to increased amounts of t r ans la tab le AR mRNA is not yet known.

The cur rent model for the induct ion of the a n t i b a c t e r i a l response in insects is that hemocytes phagocytose bacter ia and release a ce l l wall component that can s t imu la te the fa t body to synthesize and secrete a n t i b a c t e r i a l response polypept ides. (7, 28, 29). I t is possib le that in Drosophila th i s is also t rue, since Drosophila hemocytes rap id l y phagocytose in jec ted bacter ia ( I 0 ) . I nves t iga t ions are underway using mutations that a f fec t hemocytes in Drosophila to tes t the ro le of blood ce l l s in the induct ion of the a n t i b a c t e r i a l response.

F i n a l l y , a survey of s ix c lose ly re la ted species of Drosophila showed that a fundamental pat tern of four conspicuous ARs, inc lud ing the suspected a t t a c i n - l i k e substances, are s t rong ly conserved in amount and size. Nevertheless, some genet ic v a r i a b i l i t y was detected, which suggests that genet ic var ian ts of the ARs may be found w i th in Drosophila melanogaster. This w i l l a l low the polypept ides to be mapped g e n e t i c a l l y , thus a id ing t h e i r c lon ing and the product ion of mutations that block t h e i r func t ions . Current work is d i rected towards a molecular i nves t i ga t i on of the a n t i b a c t e r i a l polypept ides and a mutat ional d isec t ion of the induct ion pathway.

ACKNOWLEDGEMENTS

We thank Professors J. Hoffmann and P. Chambon in Strasbourg for the h o s p i t a l i t y of t h e i r l abora to r ies dur ing the i n i t i a l phases of t h i s work, and Drs. D. Hoffmann, and D. Zachary for subs tan t ia l help and encouragement

Vol. IO, No. 2 ANTIBACTERIAL PROTEINS IN FRUIT FLIES 177

they gave us as we began these experiments. For energetic help with inoculations and hemolymph col lect ions we thank V. Bilan, D. Kolstoe, D. Kon, K. Lodmel, G. Silvey, and P. White. For research support we thank the USDA, and Monsanto Company. A t ra in ing grant from NIH supported M.R., and an NSF/CNRS exchange supported J.H.P in Strasbourg.

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Received: November, 1985 Accepted: February, 1986