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1 Developmental and Comparative Immunology, Vol. 20, No. 3, pp. 175-181, 1996 Copyright 0 1996 Elsevier Science Ltd. All rights reserved Pergamon Printed in Great Britain 0145-305x/96 $15.00+0.00 PIL s0145-305X(%)00017-1 EXPRESSION OF ANTIMICROBIAL PEPTIDE GENES AFTER INFECTION BY PARASITOID WASPS IN Drosophila Emmanuelle Nicolas,* Anthony J. Nappit and Bruno Lemaitre* *Institut de Biologie Molkulaire et Cellulaire, UPR 9022 du CNRS, 15 rue Rene Descartes, 67084 Strasbourg, Cedex, France TDepartment of Biology, Loyola University of Chicago, 6525 North Sheridan Road, IL 60626, Chicago, U.S.A. {~~b~it~~d 1 Febrwry 1996; Accecpted I April 1996) IJAbstract-We report here the use of a spe- loxidase, and lectins (l-3). The cellular cific P-galactosidase staining assay and immune reactions consist essentially of Northern blotting technique to examine the phagocytosis and encapsulation by circu- expression of three genes encoding either lating blood cells which are frequently antibacterial peptides (diptericin, cecropin mobilized to form melanotic capsules A) or an antifungal peptide (drosomycin) in around foreign entities that are too large Drosophila following infection by larval and pupal parasitoids. The results show that the to be phagocytosed (4-6). The mechanism genes encoding these peptides are either not leading to the cellular encapsulation of induced or minimally induced in wasp- parasites has been well documented at the infected hosts, but remain responsive and morphological level (6) but is still poorly are induced upon microbial challenge. As understood at the molecular level (7). The the parasitoids elicit a cellular response, our inducible antimicrobial peptides that data suggest that the antimicrobial responses appear in the hemolymph of insects in are activated and/or regulated by mechan- response to bacterial or fungal challenge isms that are independent of those mediating are synthesized primarily by the fat body cellular encapsulation. Copyright Q 1996 and hemocytes. In Drosophila, the genes Elsevier Science Ltd. or cDNAs encoding some of these indu- qKeywords-Antibacterial peptides; Insect cible molecules have been cloned (i.e. immunity; Parasitoid wasps. cecropins f&9); diptericin (10); defensin (11); drosocin (12); attacin (13); droso- mycin (14)). The mechanism(s) control- ling the expression of these antimicrobial Introduction peptides after immune challenge is an important questions in the field of insect The humoral immune molecules in insects immunology. include antibacterial and antifungal pep- The objective of this investigation was tides, proteases, prophenoloxidase/pheno- to examine the extent to which mechan- isms regulating the production of anti- bacterial peptides and cellular immune Address correspondence to Dr Bruno components in Drosophila ~la~5g~~~r Lemaitre, UPR 9022, Institut de Biologie are co-replated. To study the possible Molhlaire et Cellulaire, 15 rue Renk Des- involvement of antibacterial genes in cartes, 67084 Strasbourg Cedex, France. cellular encapsulation we monitored the 175

2 176 E. Nicolas et al. inducible expression of two antibacterial oviposition period on standard medium. peptides and one antifungal peptide in Second stage Drosophila larvae (approxi- Drosophila melanogaster infected by the mately 50 h) were exposed for 8 h to wasp parasitoids Leptopilina boulardi, Leptopilina females that had not pre- Leptopilina heterotoma, or Trichopria sp., viously oviposited (4). To ensure adequate which oviposit a single egg into the body numbers of parasitized larvae and to cavity of Drosophila larvae or pupae, minimize multiparasitism, five female respectively. parasitoids were used per 200-300 larvae. Under these conditions, 80% of the larvae were infected. From 48 to 72 h Materials and Methods after parasitization, larvae were collected and divided into two batches. The first Drosophila Stocks group was dissected to determine the percentage of parasitization, and the Oregon R flies were used as a standard second group was used to evaluate gene wild-type strain. The transgenic strain, expression. A similar procedure was Dipt2.2~la&l is a ry506 C.S. line carrying followed when 24day-old pupae were a diptericin reporter gene on the X exposed to Trichopria. chromosome (10). The fusion gene con- taining 2.2 kb of diptericin upstream sequences fused to the bacterial 1acZ Injury Experiments coding region was inserted into the Carnegie 20 vector (15). The develop- Injury experiments were performed by mental and inducible expression of the pricking flies with a needle that had been Dipt2.2-ZacZ transgene has been pre- previously dipped in a concentrated bac- viously described (10). The inducible terial culture of Escherichia coli and expression of the Dipt2.2-1acZ is super- Micrococcus luteus. The majority of bac- imposable to that of the resident dipter- terially challenged larvae died during the icin gene at the end of the third larval pupal stage. stage. All experiments were performed at 25C unless otherwise stated. Quantitative Measurement of P-Galactosidase Activity Origin of Wasp Strains and The procedure described by Lemaitre Infection Procedures and Coen (16) was applied to homoge- nates made from groups of five indivi- The parasitic wasps L. boulardi, L. duals of either third stage larvae or pupae. heterotoma, and Trichopria sp. used in Results are given in nanomoles of product this study were raised at 25C on a wild- formed/(min mg-) protein. type strain of D. melanogaster (Oregon R). Leptopilina species lay their eggs inside Drosophila larvae. The egg hatches P-Galactosidase Localization after approximately 48 h and the young wasp larva develops to the adult stage and The fat bodies were fixed for 5 min in emerges as an adult from the host PBS, pH 7.5, containing 1% glutaralde- puparium after 18-20 days. Trichopria hyde and 1 mM MgClz (17). Subse- lay eggs inside 2-day-old pupae and quently, tissues were washed in PBS and emerge as adults after 18 days. immersed in 0.2% 5-bromo-4-chloro-3- Drosophila females were provided a 6-h indolyl-8-o-galactopyranoside (X-gal),

3 Genetic regulation of Drosophila immunity 177 3.5 r&4 &Fe&N),, 3.5 IIIM K3Fe(CN)G, We have first investigated the expres- 1 mM MgCls, 150 mM N&l, 10 mM sion of the Dipt-la&Z reporter gene in NQHPO~, 10 mM NaHaPO4 and incu- larvae infected by Leptopi~~nabo~~ar~~ bated for 4 h at 37C. The tissues were and ~ptop~~ina heterotoma. For this, then mounted in glycerol. second stage Drosophila larvae carrying the transgene were parasitized and assayed for P-galactosidase activity 96 h post-infection (Table 1). In contrast to RNA Expression of Genes Encoding non-parasitized bacteria-challenged Antimicrobial Peptides larvae, no expression of the Dipt2.2lacZ reporter gene was observed in infected Total RNA extraction and Northern larvae. Thus, neither L. boulardi nor L. blotting experiments were performed with heteroto~a elicited a marked Dipt2.2- tissues removed from control, bacteria- la&: 1 expression. challenged, and parasitized Drosophila To exclude the possibility that the using methods described previously (18). absence of expression of the Dipt2.2- The following probes were used to detect IacZ: 1 transgene in wasp-infected second RNA expression: diptericin cDNA (19), instar larvae reflected the low level of drosomycin cDNA (14), rp49 cDNA, (a inducibility of the reporter gene at this PCR fragment of approximately 400 bp stage of development (lo), we have generated between two oligonucleotides parasitized older, third stage larvae designed after the rp49 coding sequence (approximately 96 h) with L. boulardi. (20)) and a 21-mer oligonucleotide (S- At 6-h post-infection, the fat body was GATTCCCAGTCCCTGGATTGT-3) removed from these hosts and stained for compl~en~~ to part of the coding ~-galactosidase activity (X-gal staining). sequence of cecropin Al which is identical Little or no P-galactosidase activity was for cecropin A2 (8). observed in parasitized larvae or in non- parasitized control larvae. In contrast, a deep blue coloration was apparent in fat Results body cells of parasitized larvae that were Analysis of the Expression of a table 1. Dipt-lacZ2.2 Activity in Wasp-infected Diptericin-1acZ Reporter Gene in Larvae Transgenic Parasitized Drosophila Number of Pgalactosidase Larvae measurements activity For our experiments we have used the transgenic DiptZZiacZ: 1 strain, which 1-I 7 1.6-11.4 has stably integrated a ZacZ reporter (+I 8 83.0 + 28.5 L.h.(--) 8 1.1 + 1.6 gene fused to the promoter (2.2 kb) of L.h.(+) 8 92.2k41.3 the gene encoding the antibacterial pep- L.b.(-) 8 1.3f0.8 tide diptericin. A previous study has L.b.(+) 8 107.6 f 58.5 shown that the induction of this reporter Results are the number of measurements, mean by immune challenge parallels that of the and the confidency interval (p

4 178 E. Nicolas et al. subsequently challenged with bacteria addition, following bacterial challenge, (data not shown). In separate experi- the Dipt2.ZlacZ:l reporter gene was ments, quantitative measurements of j3- found to be fully inducible in pupae galactosidase activity were made of infected by Trichopria (Table 2). homogenates of five third instar parasi- tized and bacteria-challenged larvae. These experiments showed levels of RNA Expression of Genes Encoding reporter gene expression that were similar Antimicrobial Peptides in Parasitized to those obtained from non-parasitized, Drosophila bacteria-challenged larvae (see Table 1). These observations indicate that parasi- Northern blot analysis was used to tized larvae are not physiologically com- monitor the expression of various genes promised and are fully capable of eliciting encoding antimicrobial peptides after a wild-type induction of the diptericin infection by wasp parasitoids. RNAs reporter gene in response to bacterial from control, parasitized, and both para- challenge. sitized and bacteria-challenged larvae or We next studied the expression of the pupae were extracted. The Northern blot diptericin reporter gene in Drosophila filter was successively probed for the pupae parasitized by Trichopria. For diptericin, cecropin A and drosomycin these investigations, 2-day-old pupae genes. A probe corresponding to the rp49 from the Dipt2.2-lacZ:l stock were used gene which encodes a ribosomal protein, as hosts and exposed to parasitoids for was also used as a control for the amount 8 h. A low but significant level of reporter of RNA. gene expression was observed 24 h after The genes encoding the antibacterial Trichopria infection (Table 2). However, peptides diptericin or cecropin A were not the level of Dipt2.2-facZ: 1 expression was expressed in unchallenged larvae and significantly lower and induction kinetics pupae (Fig. l), but a detectable level of slower than those observed in non-para- expression was monitored for the anti- sitized, bacteria-challenged pupae. In fungal peptide drosomycin, corroborating previous published data in the literature Table 2. Dipt2.2~la&l Activity in Trchoprfa- (8,10,14). High levels of expression were infected Pupae observed for all three immune genes in bacteria-challenged insects (Fig. 1). In Number of &galactosidase agreement with the results obtained Pupae measurements activity above with the transgenic strain, there (-) 13 2.6 + 2.6 was little or no expression of these (+) a 51.5+ la.9 immune genes in wasp-infected indivi- 6 h P.l.(-) a 15.5 f 6.4 duals. In parasite-infected larvae and 14 h P.I. (-) a 11.5 * 5.5 14 h P.l.(+) a 57.8 + 20.0 pupae, there was considerable variation 24 h P.l.( -) a 19.7 zt 6.7 in gene expression. Cecropin A activity 48 h P.l.(-) a 13.6 f 6.6 was not induced by any parasitoid. The Results are the number of measurements, diptericin gene was expressed in indivi- mean and the confidency interval (p

5 Genetic regulation of Drosophilaimmunity 179 Dipt Ccc Dram Figure 1. Transcriptional profiles of diptericin, cecropin and drosomycin in wild-type, parasitized and parasitized plus challenged larvae and pupae. Total RNA was extracted at different time intervals (as indicated) after parasitization from wild-type [email protected] larvae and pupae. Twenty microgram samples were fractionated by denaturing 1% agarose/formaldehyde gel electrophoresis, transferred onto a nylon membrane and successively hybridized with a nick-translated diptericin (Dipt) and drosomycin (Drom) cDNA probe; an end-labeled oligonucleotide probe complementary to cecropins Al and A2 (CecA) transcripts and a nick-translated rp49 cDNA probe. -: unchallenged animals; +: 6 h chal- lenged animals; L.b.: larvae parasitized by L. boulardi; L.h.: larvae parasitized by L. heterotoma; T: pupae parasitized by Trichopria; L3, wandering third instar larva: P.I.: post-infection. type levels of induction of all three ovoposited in the larvae do not induce a immune genes were exhibited in wasp- full humoral antimicrobial response. infected larvae after bacterial challenge. Despite the low level of antimicrobial gene expression in wasp-infected Droso- phila, the three antimicrobial genes Discussion remained fully inducible and were expressed upon subsequent bacterial chal- Drosophila susceptible hosts were lenge. These observations strongly suggest examined for evidence of parasite- that the eggs ovoposited in the larvae are mediated induction of three genes not recognized by the hosts humoral encoding antimicrobial immune peptides mechanism. In contrast, it has been (diptericin, cecropin A, and drosomycin) shown that in response to wasp infection, following infection by either the larval (L. larvae elicit a cellular response (reviewed boulardi and L. heterotoma) or the pupal in Ref. 21). Rizki and Rizki showed that stage (Trichopria) parasitoids. We found infection by Leptopilina wasps induces no evidence for strongly induced antimi- lamellocyte differentiation in larvae (22). crobial immune responses in parasitized Lamellocyte are large flattened hemocytes Drosophila using either specific P-galacto- derived from plasmatocytes which are sidase titration or Northern blotting involved in the formation of the capsule techniques. These data indicate that eggs (21). This last observation indicates that

6 180 E. Nicolas et al. the presence of the parasitoid is recog- existence of different recognition mechan- nixed by the ~rnun~ system of the host. isms does not preclude that the cellular This cellular response is, however, later and humoral responses share common selectively incapacited in susceptible regulatory elements. This was recently larvae (23). Indeed as shown for L. suggested by two reports indicating that heterotoma, the number of lamellocytes rel proteins and the Toll receptor may be from infected larvae decreased, probably involved in both the control of antimi- due to a destructive factor derived from crobial genes and lamellocyte differentia- the accessory gland of the female wasps tion (18,26). reproductive system (24). Taken together, As with other organisms, insects have the results of this study indicate that, evolved with different adaptative while altering the cellular response, infec- responses to various types of aggression tion of Drosophila by wasp parasitoids (i.e. infection by metazoan parasitoids, does not affect the hosts humoral bacteria, or fungi). The results of this immune mechanism. This last result investigation suggest that, in Drosophila, suggests that the humoral and cellular the humoral response elicited by micro- immune mechanisms may be activated by bial agents is regulated by mechanisms different mechanisms. that differ from those which control The observations made in this study cellular encapsulation. Additional evi- using two species of Leptopilina support dence to support this proposal comes in part the recent investigations of from recent studies showing the differen- Coustau et al. (25) who likewise were tial induction of antibacterial and anti- unable to detect the induction of anti- fungal genes (27). An important goal of bacterial peptides in immune-reactive current research in the field of insect (resistant) Drosophila infected with L. i~~ity is to decipher the specific bou~ardi. This result demonstrates that reposition m~hanism(s) for pathogens the presence of antimicrobial peptides is that trigger the immune defense. not required for successful encapsulation of the parasitoid (25). However, these authors detected an elevated antibacterial Acknowledgements-The authors are indebted activity in susceptible hosts infected by L. to Pr Michel Bouletreaux for sending para- boulurdi. (25). In contrast, with the three sitoid wasps and to Dr Marie Meister. The different parasitoid species tested here, technical assistance of Reine K.lock and only a low level of antimicrobial expres- Raymonde Syllas is gratefully acknowledged. sion in both parasitized Drosophila larvae Anthony Nappi is grateful for the assistance and pupae was observed. This was attrib- provided by the National Science Foundation @BN 950 4796), the Research Services of uted to bacterial con~mination which Loyola University Chicago, and to Emily occurred during parasitoid oviposition. Vass of the Department of Biology at Loyola The combined data suggest that infection University Chicago. Emmanuelie Nicolas and of Drosophila by wasp parasitoids does Bruno Lemaitre acknowledge support from not diminish the hosts antimicrobial the Centre National de la Recherche Scienti- immune response, and that this humoral fique, the University Louis Pasteur. They mechanism is regulated independently of thank Pr Jules Hoffmann for his interest in the hosts cellular immune system. The this study. References 1. Ashida, M.; Yamazaki, H. 1. Biochemistry of the Ishizaki, H., Eds Molting and metamo~ho~s. phenoloxidase system in insects: with special Tokyo/Berlin: Japan Sci. Sot. Pr~/Sp~nger; refereuce to its activation. In Oh&hi, E.; 1990: 239-265.

7 Genetic regulation of Drosophila immunity 181 2. Hultmark, D. immune reactions in Drosophila 15. Rubin, G. M.; Spradling, A. C. Genetic trans- and other insects: a model for innate immunity. formation of Drosophila with transposable ele- Trends Genet. 9:178-183; 1993. ment vectors. Science 218:348-353; 1982. 3. Hoffmann, J. A. Innate i~~ty of irmmts. 16. Lemaitre, B.; Coen, D. P. Regulatory products Curr. Opin. Immunol. 24-10; 1995. repress in vivo the P promoter activity in P- 4. Carton, Y.; Frey, F.; Nappi, A. J. Inheritance of La& fusion genes. Proc. Natl. Acad. Sci. cellular immune resistance in Drosophila U.S.A. 88:44194%23; 1991. melanogaster. Heredity 69393-399; 1992, 17. Hiromi, Y.; Kuroiwa, A.; Gehring, W. I. 5. Nappi, A. J.; Vass, E. Melanogenesis and the Control of the Drosophila segmentation gene generation of cytotoxic molecules during insect fushi tarazu. Cell 43:603-614; 1985. cellular immune reactions. Pigment Cell Res. 18. Lemaitre, B.; Meister, M.; Goviad, S.; Georgel, 6:117-126; 1993. P.; Steward, R.; Reichhart, J. M.; Hoffmann, J. 6. Vass, E.; Nappi, A. J.; Carton, Y. Comparative A. Functional analysis and regulation of nuclear study of immune competence and host suscept- import of dorsal during the immune response in Drkophila. EMBO J. 14:53(i-545; 1995: ibility in Drosophila melanogaster parasitized by Leptopilina boulardi and Asobara tabida. J. 19. Wicker. C.: Reichhart. J. M.: Hoffmann. D.: Parasitol. 79106-112; 1993. Huhmark, D.; Samakovlis, C.;Hoffmann, J. A: Insect i~u~ty. Characterization of a Dross- 7. Kobayashi. M.; Johansson, M. W.; Soderh&ll, phiZa cDNA encoding a novel member of the K. The 76 kD cell-adhesion factor from crayfish diptericin family of immune peptides. J. Bioi. haemocytes promotes encapsulation in vitro. Chem. 26522493-22498; 1990. Cell. Tissue Res. 260~13-18; 1990. 20 OConnell, P.; Rosbash, M. Sequence, structure 8. Kylsten, P.; Samakovlis, C.; Hultmark, D. The aad codoa preference of the Drosophila ribo- cecropin locus in Drosophila; a compact gene somal protein 49 gene. Nucleic Acid Res. cluster involved in the response to infection. 12:5495-5513; 1984. EMBO J. 9:217-224; 1990. 21 Rizki, T.M.; Rizki, R.M. Parasitoid-induced 9. Tryselius, Y.; Samakovlis, C.; Kimbrell, D. A.; cellular immune deficiency in Drosophila. In: Hultmark, D. CecC, a cecropin gene expressed Beck. G. Ed. Primordial immunitv. Foundations during metamorphosis in Drosophila pupae. for the vertebrate immune system. Conference, Eur. J. Biochem. 204395399; 1992. Woods Hole, Massachusetts, U.S.A. May 2-5 10. Reichbart, J. M.; Meister, M.; Dimarcq, J. L.; 1993. Zachary, D.; Hoffmann, D.; Ruiz, C.; Richards, 22. Rizki, T. M.; Rizki, R. M. Lamellocyte differ- G.; Hoffmann, J. A. Insect immunity: develop- entiation in Drosophila parasitized by mental and inducible activity of the Drosophila ~ptop~Zina. Dev. Comp. Immuaol. 16:103; diptericin promoter. EMBO J. 11:1469-1477; 1993-110. 1992. 23. Rizki, T. M.; Rizki, R. M.; Carton, Y. 11. Dimarcq, J. L.; Hoffmann, D.; Meister, M.; Leptopilina heterotoma and L. boulardi: strate- Bulet, P.; Lanot, R.; Reichhart, J. M.; gies to avoid cellular defense responses of Hoffmann, J. A. Characterization and transcrip Drosophila melanogaster. Exp. Parasitol. tional profiles of a Drosophila gene encoding an 70:466-475; 1990. insect defensin. A study in insect immunity. Eur. 24. Rizki, T. M.; Rizki, R. M. Selective destruction 1. Biochem. 221:201-209; 1994. of a host blood cell type by a parasitoid wasp. 12. Bulet, P.; Dimarcq, J. L.; Hetru, C.; Lagueux, Proc. Natl. Acad. Sci. U.S.A. 81:6154-6158; M.; Charlet, M.; Hegy, G.; Van Dorsselaer, A.; 1984. Hoffmann. J. A. A novel inducible antibacterial 25. Coustau, C.; Carton, Y.; Nappi, A. J.; Shot- peptide of.DrosophiZa carries an 0-glycosylated koski, F.; French-constant, R. Differential substitution. J. Biol. Chem. 268:14893-14897; induction of antibacterial traascripts in Droso- 1993. phiia susceptible and resistant to parasitism by 13. Asling, B.; Dushay, M. S.; H&mark, D. ~~to~i~~a b~uZar~j. Insect B&hem. Mol. Biol. Identification of early genes in the Drosophila (in press). immune response by PCR-based differential 26. Goving, S. Rel signal&g pathway and the display: the Attacin A gene and the evolution melaaotic tumor phenotype of Drosophila. of attacin-like proteins. Insect Biochem. Mol. Biochemical Society Transactions 24:394k Biol. 25:511-518: 1995. 1996. 14. Fehlbaum, P.; Bulet, P.; Michaut, L.; Lagueux, 27. Lemaitre, B.; Kromermetzger, E.; Michaut, L.; M.: Broekaert. W. F.: Hetru. C.: Hoffmaun. J. Nicolas, E.; Meister, M.; Georgel, P.; Reichart, A. hisect immunity: septic injury of Drosophila J. M.; Hoffmann, J. A. A recessive mutation. induces the synthesis of a potent antifungal immune deficiency (imd), defines two distinct peptide with sequence homology to plant anti- control pathways in Drosophila host defense. fungal peptides. J. Biol. Chem. 269:31159- Proc. Natl. Acad. Sci. U.S.A. 9294659469; 31 163; 1994. 1995.

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