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EXPRESSION of ANTIMICROBIAL PEPTIDE GENES AFTER INFECTION by PARASITOID WASPS in Drosophila

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EXPRESSION of ANTIMICROBIAL PEPTIDE GENES AFTER INFECTION by PARASITOID WASPS in Drosophila 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 to be phagocytosed (4-6). The mechanism pupal parasitoids. The results show that the 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 175 176 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 25°C 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 25°C 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), 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 37°C. 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.(--) gene fused to the promoter (2.2 kb) of 8 1.1 + 1.6 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<5%). Activity was resident diptericin gene (10). In initial measured in a pool of five control or wasp- experiments the transgenic strain Dipt2.2- infected wandering larvae that were either unchallenged (-) or sacrified 3 h after bacteria! l&Z:1 was found to be totally susceptible challenge (+). The endogenous level of P-gaiac- to the parasitoids, there being no evidence tosidase was obtained by measuring the 6-galac- of a successful cellular encapsulation tosidase activity in ry50Bsibs of the Dipt2.2-fact:1 strains, devoid of Dipt2.2-facZ:l insertion. It was response against either species of Lepto- 0.91kO.2 (~8). L.h.: L. ~eferofoma; L.b.: L. bou- pi&a or Triehopria (data not shown). fardf. 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.
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