Expression and distribution of penaeidin antimicrobial peptides are regulated by haemocyte reactions in microbial challenged shrimp

Expression and distribution of penaeidin antimicrobial peptidesare regulated by haemocyte reactions in microbialchallenged shrimp

Marcelo Munoz1, Franck Vandenbulcke2, Denis Saulnier3 and Evelyne Bachere1

1IFREMER/CNRS/Universite de Montpellier, �Defense et Resistance chez les Invertebres Marins�, Montpellier, France;2Laboratoire d’Endocrinologie des Annelides, Groupe de Neuroimmunite des Hirudinees, Universite des Sciences et Technologies

de Lille, France; 3IFREMER, Centre Oceanologique du Pacifique, Taravao, Tahiti, Polynesie Francaise

Penaeidins are a family of antimicrobial peptides constitu-tively produced and stored in the haemocytes of penaeidshrimp. In response to microbial stimulation, they arereleased into the blood circulation and they further attach toshrimp cuticle surfaces through a chitin-binding property. Inthe present paper, we have analysed their expression, regu-lation and distribution in shrimp tissues in response toexperimental microbial challenge. We have shown thatpenaeidin mRNA and protein are restricted to granularhaemocytes and that their expression and distribution areregulated through dramatic changes in haemocyte popula-tions, both circulating and infiltrating shrimp tissues. Twodistinct phases in the immune reactionswere evidenced: (a) amigration of haemocytes towards the infection site withinthe first 12 h following microbial injection, with a local andmassive release of peptides; (b) the appearance into theblood

circulation and tissues of a haemocyte population displayingincreased penaeidin-transcriptional activity, which maycorrespond to a systemic reaction involving haemocyteproliferation process. Finally, in vitro confrontation of hae-mocytes and bacteria revealed that penaeidins are releasedfrom granular haemocytes by a novel phenomenon ofintracellular degranulation, probably followed by the lysis ofthe cells. Furthermore, penaeidins were shown coveringbacterial surfaces suggesting that the peptides could beinvolved in opsonic activity. Penaeidin-positive bacteriawere observed to be phagocytosed mainly by hyaline cells, apopulation that does not express penaeidins.

Keywords: antimicrobial peptide; crustacea; innate immu-nity; penaeid shrimp; phagocytosis.

Antimicrobial peptides are major components of innateimmunity that have been conserved in evolution and foundin different phyla of the plant and animal kingdom.Although these immune effectors share common character-istics (small size and cationic character) and similarities instructural patterns or motifs [1], one striking feature is theirgreat diversity in terms of amino acid sequences, anti-microbial activities and modes of action. Moreover,depending on their distribution, antimicrobial peptideexpression appears to be regulated by different tissue-specific pathways [2] and these effectors may consequentlyparticipate in either a local or a systemic reaction. Antimi-crobial peptides are produced in phagocytic cells ofvertebrates [3] and invertebrates [4–6], and in various tissuessuch as epithelia of mammals and insects [7,8], or insect fatbody [9]. Peptides are produced constitutively and stored incirculating cells, where they can act intracellularly againstphagocytosed microorganisms as shown in human for

defensins [3] and in a bivalve mollusc for mytilin [6].Peptides can also be released by exocytosis upon microbialstimulation [5,10]. In various epithelia of invertebrates [2]and vertebrates [11], antimicrobial peptides are eitherproduced constitutively or induced in response to infectionor inflammation, and participate in a local antimicrobialreaction. Finally, antimicrobial peptide expression in fatbody cells is induced in response to infection and peptidesare secreted into body fluids, which characterizes the acuteor systemic reaction in insects [12].In Crustacea, penaeidins are a unique family of

antimicrobial peptides originally isolated and characterizedin the shrimp Penaeus vannamei. In previous works, threemembers of the penaeidin family, penaeidin (Pen)-1, -2and -3 were purified in their mature and active form(5.48–6.62 kDa) and cloned from the haemocytes ofexperimentally uninfected shrimp [13]. Penaeidins wereshown to be constitutively expressed in haemocytes andmature peptides were localized in the cytoplasmic granulesof the granular haemocyte population of unchallengedanimals. Regarding penaeidin gene expression and peptidedistribution, first data suggested that in response to amicrobial challenge, penaeidin transcription is notup-regulated in shrimp haemocytes, but relative penaeidinconcentration in shrimp plasma was shown to increaseupon stimulation [14]. Penaeidins, which present in theiramino acid sequences a chitin-binding motif [15] weredemonstrated to bind to shrimp cuticle surfaces inresponse to microbial challenges [14]. Thus, we speculated

Correspondence toE.Bachere,UMR5098, �Defense et Resistance chezles Invertebres Marins�, CC 80, 2 place Eugene Bataillon – 34095Montpellier, France.

Fax: +33 4 67 14 46 22, Tel.: +33 4 67 14 47 10,

E-mail: [email protected]

Abbreviations: DIG, digoxigenin; NGS, normal goat serum; ISH,

in situ hybridization; ICC, immunocytochemistry.

(Received 17 December 2001, revised 9 April 2002,

accepted 16 April 2002)

Eur. J. Biochem. 269, 2678–2689 (2002) � FEBS 2002 doi:10.1046/j.1432-1033.2002.02934.x

that penaeidin could be released from granular haemo-cytes by regulated exocytosis as demonstrated previouslyfor the antimicrobial peptide-mediated immune responsein Limulus [5].The purpose of the current study was to define the

regulation and distribution of penaeidin expression inshrimp during immune response considering Pen-3, themost abundant and representative member of the family[13,16]. We demonstrate that penaeidins are expressedexclusively in shrimp haemocytes and that experimentalmicrobial infection induces great changes in haemocytepopulations. Through in situ hybridization and immuno-histochemical analyses, haemocytic reactions were high-lighted as an important component of the immuneresponse ) involved in the distribution of the antimicro-bial peptides. Finally, to define the cellular mechanisms ofpenaeidin release, haemocytes were challenged with bac-teria in vitro, which gave new insights into haemocytefunctions and involvement of penaeidins in shrimpdefence.

M A T E R I A L S A N D M E T H O D S

Animals and immune challenge

Juvenile shrimp (8–10 g) P. vannamei (Crustacea, Deca-poda) in intermoult stage were obtained from a farm inthe province of Guayas (Ecuador) and from the FrenchPolynesia IFREMER laboratory. Shrimp microbialchallenge was performed by injecting, into the last abdo-minal segment, a suspension (50 lL; 108 cells/animal)of heat-killed (100 �C, 105 Pa, 20 min) microorganismsincluding bacteria, Aerococcus viridans, Vibrio alginolyt-icus and fungal spores of Fusarium oxysporum. Haemo-lymph and tissues were collected at different times (from0 to 72 h) post-injection as described previously [14].Unchallenged shrimps (i.e. shrimp at time 0 h) were usedas controls.

Northern blot analyses

Penaeidin-specific and ribosomal probes were amplified byPCR on, respectively, pen-3a cDNA clone (GenBankaccession number Y14926) and an 18 S rRNA genomicDNA clone (a gift from T. Spears, Florida State University,USA) as described previously [14]. The probes wereradiolabelled by random priming using the Ready-to-goDNA labelling kit (Amersham Pharmacia Biotech).Total RNA from shrimp haemocytes and tissues was

prepared according to the method of Trizol reagent (BRLLife technologies). Two or 10 lg total RNA were fraction-ated on denaturating 1% agarose gel containing 17%formaldehyde, and then transferred to Hybond-N filtermembranes (Amersham Pharmacia Biotech) by vacuumblotting.Membranes were hybridized at 55 �C for 12 hwith32P-labelled pen-3a cDNA fragment in a solution containing50% formamide, 5 · NaCl/Cit, 8 · Denhardt’s solution,50 mM sodium phosphate pH 6.5, 0.1% SDS and100 lgÆmL)1 denatured salmon sperm DNA. Filters werewashed twice in 2 · NaCl/Cit, 0.1% SDS at room tem-perature and twice in 1 ·NaCl/Cit, 0.1%SDS, first at roomtemperature, then at 65 �C followed by autoradiography.After stripping, the membranes were hybridized under

identical conditions with 32P-labelled 18 S ribosomal DNAprobe and subjected to further autoradiography. Penaeidintranscript and 18 S rRNA signals were quantified using the

STORMTM system (Molecular Dynamics).

Tissue preparation for histology

Tissues from juvenile shrimp were fixed in a solutioncontaining 22% formalin, 31.5% ethanol and 11.5% glacialacetic acid. After dehydration, tissues were embedded inParaplast and 8 lm sections were cut, mounted on polyL-lysin coated slides and stored at 4 �C until use.Haemolymph was collected under 1 vol. anti-aggregant

modified Alsever solution buffer [14]. Then, cells were fixedfor 10 min by addition of 1 vol. ice-cold 4% paraformal-dehyde in 100 mM NaCl/Pi containing 10% saccharose.Cells were centrifuged on slides for 5 min at 200 g in acytospin (Cyto-tek centrifuge,Miles Scientific) and stored at)20 �C until use.

Phagocytosis assay

Haemolymph was collected as described above and imme-diately centrifuged (800 g, 10 min). Supernatant was elim-inated and haemocytes were incubated at room temperaturewith bacteria (V. alginolyticus) at the ratio of 20 bacteria perhaemocyte in modified Hanks’ balanced salt solutionsupplemented with 6 mM CaCl2 and 13 mM MgCl2. Atvarious incubation times (0, 1, 3, 5, 20, 30, 45 and 60 min),cells were fixed and treated for ultrastructural analyses andimmunodetection as described below.

In situ hybridization

Probes. A plasmid containing pen-3a cDNA (GenBankaccession number Y14926) was used as template for thepreparation of the probes. Digoxigenin (DIG)-UTP-labelled and [35S]UTP-labelled antisense and sense ribo-probes were generated from linearized cDNA plasmids byin vitro transcription using RNA labelling kits, T3 RNApolymerase (Roche) and [35S]UTP (Amersham).

Hybridization. DIG-labelled riboprobes (� 40–100 ngper section) were hybridized to tissue sections as describedpreviously [17]. For cytocentrifuged cells, the protocol ofhybridization was adapted, i.e. cytocentrifuged cells wereincubated for 10 min in 100 mM glycine, 200 mM Tris/HCl pH 7.4, immersed 5 min in NaCl/Pi and fixed in100 mM phosphate buffer containing 4% paraformalde-hyde and 5 mM MgCl2. After the postfixation step, cellpreparations were washed 5 min in phosphate buffer,incubated for 10 min in 0.25% anhydride acetic preparedin 100 mM triethanolamine pH 8, and briefly washed in2 · NaCl/Cit. Samples were then rinsed in distilled water,dehydrated by graded alcohol and air dried at roomtemperature.DIG-labelled riboprobes (40–100 ng per slide) and

35S-labelled riboprobes (100 ng or 1 · 106 c.p.m. per slide)were diluted in hybridization buffer containing 50% form-amide, 10% dextran sulfate, 10 · Denhardt’s solution,0.5 mgÆmL)1 tRNA from Escherichia coli, 100 mM dithio-threitol and 0.5 mgÆmL)1 salmon sperm DNA. Hybridiza-tion was carried out overnight at 55 �C in a humid chamber.

� FEBS 2002 Antimicrobial peptide expression in shrimp (Eur. J. Biochem. 269) 2679

Slides were then washed twice (2 · 15 min) with 2 ·NaCl/Cit, treated with RNase A (20 mgÆmL)1 in 2 · NaCl/Cit)for 10 min at 37 �C and consecutively rinsed 2 · 10 min in0.1 · NaCl/Cit containing 0.07% 2-mercaptoethanol at55 �C. The probes labelled with DIG-UTP were revealedusing alkaline phosphatase-conjugated antibodies as des-cribed previously [17].

Detection and quantification of the 35S-labelled probes

After hybridization step, the slides were rinsed in 0.1 ·NaCl/Cit at 20 �C, briefly immersed in graded alcohol andair dried at room temperature. Hybridization signal wasvisualized using autoradiography. Samples were coated bydipping in LM1 Amersham liquid emulsion, immediatelydried and exposed for a 4-day period. At the end of theexposure period, the autoradiograms were developed inD19b (Kodak), fixed in 30% sodium thiosulfate (10 min atroom temperature), stained with 1% Toluidine blue andmounted with Xam (Merck). Quantification of the radio-labelling at the cellular level was performed using anAxiophot Zeiss microscope and a Biocom quantificationsystem as established [18].

Controls

Control for in situ hybridization consisted in replacingantisense riboprobe with sense riboprobe. RNase controlsections were obtained by adding a preincubation step with10 lgÆmL)1 RNase A prior to hybridization.

Immunodetection of penaeidins

Whole animal. Eight micrometer-thick paraffin sectionswere re-hydrated and treated as follow: (a) 10 min at 20 �Cin 150 mM NaCl, 100 mM Tris/HCl pH 7.4 buffer (NaCl/Tris); (b) NaCl/Tris containing 1% normal goat serum(NGS), 1% BSA (NaCl/Tris/NGS/BSA) and 0.1% TritonX-100, 30 min at room temperature; (c) incubation withanti-penaeidin IgG (3 lgÆmL)1) diluted in NaCl/Tris/NGS/BSA, overnight at room temperature; (d) 3 · 10 min inNaCl/Tris; (e) 1 nm colloidal gold-labelled goat anti-rabbitIgG (Amersham) diluted 1 : 100 in the incubation buffer,3 h at room temperature; (f) 3 · 10 min in NaCl/Tris; (g)equilibration 2 · 5 min in 200 mM citrate buffer pH 7.4; (h)silver amplification performed with the IntensSETM kitaccording to the manufacturer’s instructions (Amersham),12 min at 20 �C; (i) 2 · 2 min in distilled water. Then,paraffin sections were mounted in XAM (Merck) andobserved using aZeissAxioskop lightmicroscope. Immuno-dection was also performed by using Texas red-tagged goatanti-rabbit serum (Jackson Immunoresearch) as describedbelow.

Circulating haemocytes. Cytocentrifuged haemocyteswere equilibrated for 10 min in NaCl/Tris before perme-abilization with 0.1% Triton X-100 in NaCl/Tris for30 min at room temperature. One hour preincubation wasperformed in the presence of 1% NGS and 1% BSA toblock nonspecific antibody binding. Rabbit anti-penaeidinpolyclonal antibody purified IgG (1.5 lgÆmL)1) [14], wasapplied for 12–16 h at room temperature in NaCl/Tris/NGS/BSA. After washing three times (10 min) in NaCl/

Tris, cells were incubated for 2 h at room temperaturewith 1 : 100 Texas red-tagged goat anti-rabbit antiserum(Jackson Immunoresearch). The slides were washed3 · 10 min in NaCl/Tris, mounted in glycerol containing25% NaCl/Tris and 0.1% p-phenylenediamine and exam-ined using a laser scanning microscope (TCS NT)equipped with a Leica (DMIRBE, Inc.) inverted micro-scope and an argon/krypton laser. Texas red signal wasdetected by exciting samples at 568 nm. Images wereacquired as single transcellular optical sections andaveraged over 16 scans per frame. Positive or negativecells were subsequently counted.Controls were incubations of anti-penaeidin IgG pread-

sorbed by purified recombinant penaeidin-3 [19].

Electron microscopy

Ultrastructural microscopy. After haemolymph collectionunder 1 vol. modified Alsever solution buffer, cells werefixed for 1 h at 4 �C in 0.1 M NaCl/Pi pH 7.4, containing2% glutaraldehyde, 4% paraformaldehyde and 10%sucrose. Cell pellets were obtained by 10 min centrifuga-tion at 800 g. The pellets were rinsed in NaCl/Pi, postfixedin 1% OsO4 for 1 h, dehydrated in graded acetonesolutions and embedded in Embed 812 Kit. Ultrathinsections (80–90 nm thick) were cut from the blocks,collected onto 200 mesh copper grids, double-stained withuranyl acetate and lead citrate and examined with a JeolJEM 100 CX.

Immunogold labelling. Immunogold detection of penaei-dins was performed on circulating cells but also on tissues.Haemocytes and dissected tissues were fixed for 1 h at 4 �Cin a mixture of 4% paraformaldehyde, 1% glutaraldehyde,10% sucrose in 100 mM NaCl/Pi, pH 7.4. Cells and tissueswere postfixed in 1% OsO4 for 3–5 min and dehydrated ingraded alcohol before embedding in LR white (TAABLaboratories).Semi-thin sections (1 lm thick) were collected on alcohol-

washed glass slides, and penaeidin immunostaining wasperformed using a gold-tagged secondary antibody andsilver amplification as described above.Ultrathin (90 nm-thick) sections from embedded pellets

or tissues were collected on nickel grids. Sections weretreated 8 min in 10% H2O2, 10 min in distilled water,30 min in NaCl/Tris/NGS/BSA and then incubated for36 h at 4 �C with 3 lgÆmL)1 rabbit anti-penaeidin IgGs inNaCl/Tris/NGS/BSA. Grids were washed three times for10 min with NaCl/Tris/NGS/BSA and incubated for 2 hat room temperature in 10 nm colloidal gold-labelled goatanti-rabbit IgGs (Amersham) diluted 1 : 100 in NaCl/Tris/NGS/BSA. Grids were then washed three times for10 min with NaCl/Tris, postfixed for 3 min in NaCl/Triscontaining 1% glutaraldehyde and washed twice for5 min with distilled water. Sections were stained for15 min with 2.5% uranyl acetate and examined with aJeol JEM 100 CX.

Statistical analyses

The data were analysed using Fisher PLSD test (P < 0.05)at 95% confidence level with STATVIEW SE + GRAPHICS

TM

program.

2680 M. Munoz et al. (Eur. J. Biochem. 269) � FEBS 2002

R E S U L T S

Tissue localization of penaeidins in nonstimulatedanimals

Penaeidins are known to be constitutively expressed inshrimp haemocytes and penaeidin transcripts were alsodetected by Northern blot in different tissues of nonstimu-lated animals [14]. In the present paper, the origin of penaei-din mRNA and peptide localization in shrimp tissues weredeterminedbyboth in situhybridizationanalyses (ISH)usingpen-3 antisense and sense RNA probes and immunocyto-chemistry (ICC) at optical and electron microscopy levels.Among the different tissues analysed, penaeidin mRNAs

were detected in circulating haemocytes in blood vessels andsinuses and in cells present within most tissues. The shape ofthe positive cells suggests that they are infiltrating haemo-cytes. A high number of cells containing penaeidin tran-scripts was detected in heart and epigastric haematopoieticnodule (also named lymphoid organ) (Fig. 1A), in bloodvessels irrigating gills and hepatopancreas (Fig. 1B and C),and to a lesser extent in all the shrimp tissues such ashaematopoietic tissue (Fig. 1D), brain, subcuticular epithe-

lia or midgut caecum (data not shown). According topenaeidin sense probe hybridization used as control, forwhich no signal was observed (Fig. 1E and F), the detectionof penaeidin transcripts with antisense riboprobewas shownto be specific for the tissues analysed. In addition, pretreat-ment of sections with RNaseA before hybridization abol-ished the positive staining providing further evidence of thesignal specificity (data not shown).Antibody used in this study was a rabbit antiserum

directed against recombinant Pen-3a [14]. The high degreeof homology between the different penaeidin forms [13]implies that the antibody recognizes different isoforms.Consequently, we qualified any immune positive signal asrelated to the presence of penaeidins.When the specific anti-penaeidin antibodies were preincubated with purifiedrecombinant penaeidins [19], penaeidin immunostainingwas no longer observed providing evidence of the specificityof the reaction (data not shown). Regarding penaeidindistribution, the peptides were shown to be localized incirculating haemocytes but also in cells located in gills,heart, brain, subcuticular epithelium, epigastric haemato-poietic nodule, midgut, midgut caecum and muscle, wherestrong labelling was observed (Fig. 2A, B and C). In order

Fig. 2. Immunodetection of penaeidins in tissue

sections of nonstimulated shrimp. Positive cells

(arrows) are shown on semithin sections of

midgut (A), midgut caecum (B) and in muscle

(C). Ultrastructural distribution of penaeidin

immune reactivity was performed using a

10-nm gold particle-conjugated secondary

antibody. As shown in intestine, numerous

gold particles are present in electron dense

granules of two subtypes of infiltrating hae-

mocytes, namely large-granule haemocytes

(D, E) and small-granule haemocytes (F, G).

No labelling was seen throughout cytoplasm

and nucleus. bl, Basal lamina; ep, epithelia;

mf, muscular fibers; n, nucleus; star, lumen of

the intestine. Bar ¼ 10 lm (A, B, C), 1 lm(D, E, F G).

Fig. 1. Detection of penaeidin mRNA in non-

stimulated shrimp tissues by in situ hybridiza-

tion. Labelling appears in most tissues and is

particularly obvious in epigastric haemato-

poietic nodule (A), gills (B), hepatopancreas

(C) and haematopoietic tissue (D). The shape

of the positive cells evokes haemocytes

(arrows), infiltrating tissue (A, D) or free-

haemocytes in blood vessels (B, C). In a neg-

ative control consisting of sections hybridized

with pen-3a sense riboprobes, no labelling was

observed as shown for gills (E) and hepato-

pancreas (F). gf, Gill filaments; dt, digestive

tubule. Bar ¼ 10 lm.


to confirm the localization of penaeidins and to determinethe nature of the positive cells, immunogold labelling wasperformed. Penaeidin storage was confirmed to be restrictedto granular haemocytes, with large granules or smallgranules, located and infiltrating all tissues analysed suchas brain, subcuticular epithelia, epigastric haematopoieticnodule or midgut (Fig. 2D, E, F and G). The presence ofsome infiltrating haemocytes without labelling was alsofound confirming previous data about the presence ofdifferent haemocyte populations, expressing vs. not expres-sing penaeidins [14].

Microbial stimulation induces changes in the totalnumber of circulating haemocytes, and in thepopulation of haemocytes expressing penaeidins

Previous work showed that microbial challenge induces adecrease of penaeidin mRNA concentration in circulatinghaemocytes in the first hours following stimulation [14]. Inorder to define the regulation of penaeidin transcription, weanalysed time-course changes in total circulating haemocytenumber and haemocyte penaeidin mRNA levels, occurringin response to stimulation. In two independent experiments,shrimp were challenged by injection of heat-killed micro-organisms and haemolymph was collected from fiveindividual animals at different times (0, 6, 12, 48 and72 h) following injection. A strong decrease in haemocytetotal number (from 9 · 106 ± 7 · 106 cells to1.2 · 106 ± 1.4 · 106 cells) was observed in the first 12 hfollowing injection, with a significant difference (P < 0.05)at 6 h between stimulated and nonstimulated animals. Thenumber of total haemocytes returned to levels observed forunchallenged animals at 48 h and a significant increase (upto 19.8 · 106 ± 3 · 106 haemocytes; P < 0.05) of totalhaemocyte number was observed at our last time point(72 h poststimulation) (Fig. 3). In similar experiments, totalRNA was extracted from the circulating haemocytes of 10animals at the same intervals after injection, and 2 lg of

total RNA were analysed by Northern blot. The STORMTM

quantifications of penaeidin mRNA and ribosomal hybrid-ization signals were compared at each time post-injection.Analyses revealed a strong decrease in penaeidin mRNAlevels for the first 12 h and a return to nonstimulated animallevels at 48 h post-challenge. A threefold increase inpenaeidin mRNA levels was noticed at 72 h followingchallenge (Fig. 3).To better understand such a decrease in penaeidin

transcript concentration within the circulating haemocytesafter microbial challenge, Northern blot analyses wereperformed on total RNA extracted from a constant numberof haemocytes (1 · 106 cells for each individual) at everytime post-challenge, instead of constant total RNA quantity(2 lg). Hybridization signals obtained, respectively, forpen-3a transcripts and 18 S rRNA probes were quantifiedby STORM and analysed separately. Data analysis revealedan important individual variation in both pen-3a transcriptsand 18 S rRNA signals with a decrease in pen-3a transcriptlevels and constant average values with 18 S rRNA duringthe first 12 h post-challenge (data not shown). However, at48 h post-challenge, penaeidin mRNA levels appeared toincrease slightly and hybridization signals with 18 S rRNAprobes were dramatically stronger than those observed forunchallenged animals (Fig. 4).In order to determine whether changes in penaeidin

transcript and protein levels could be also associated withchanges in the composition of circulating haemocytepopulations, the percentage of circulating haemocytesexpressing and storing penaeidin was further analysed byISH and ICC, respectively. Circulating haemocytes fromfive individual shrimp were collected, counted, fixed andcytocentrifuged on slides at different times (0, 6, 12, 48 and72 h) after microbial stimulation. As shown before, signi-ficant modifications in total circulating haemocyte numberwere observed in these experiments (Fig. 5). In nonstimu-lated animals 35 ± 6% of the total haemocyte populationexpressed penaeidins. This percentage decreased to 19%(± 8%) and 13% (± 8%), respectively, 6 and 12 h aftermicrobial challenge (Fig. 5). Then, the percentage ofpenaeidin mRNA-positive haemocytes in the total circula-ting population reached 50 ± 3% at 48 h post-challenge,before returning slightly to a mean percentage (39 ± 11%)close to that observed for nonstimulated animals at 72 h

Fig. 3. Time-course analysis of total haemocyte number and penaeidin

expression (histograms) in circulating haemocytes after microbial chal-

lenge. Haemocyte counts were performed on five shrimps at different

time intervals after challenge using an haemocytometer. Vertical bars

represent mean values of haemocyte numbers at each time point (line).

Northern blot analyses were performed on 2 lg of a pool of total RNAextracted from 10 shrimps at each time point. Hybridization signals

obtained with 32P-labelled pen-3a cDNA probe were quantified by the

STORMTM system and compared to those obtainedwith the 18 S rRNA

specific probe. The penaeidin/18 S rRNA signal ratios were calculated

and the expression level in unchallenged shrimpwas normalized to 100.

Results are given as percentage expression relative to this level.

Fig. 4. Northern blot analysis of total RNAs from constant number of

haemocytes from unchallenged shrimp and shrimp 48-h post-challenge.

Total RNA was extracted from 1 · 106 haemocytes per shrimp,unchallenged (lanes 1–5) and 48 h following challenge (lanes 6–9) and

hybridized successively with 32P-labelled probes specific for pen-3a

(top) and specific for 18 S rRNA (bottom). Strong hybridization sig-

nals are observed with the 18 S rRNA probe at 48 h post-challenge

compared to those observed for unchallenged shrimp.


post-injection (Fig. 5). Regarding storage of the peptides,the percentage of penaeidin-immunoreactive haemocyteswas also established. In nonstimulated animals, the relativenumber of haemocytes storing penaeidins was similar tothat of haemocytes expressing the peptides (37 ± 4%of thetotal circulating population). At the different times post-challenge, changes similar to those observed with transcriptdetection occurred in the percentages of penaeidin-positivehaemocytes (Fig. 5). During the first 6 and 12 h post-challenge, the percentage of penaeidin-positive haemocytesdecreased, respectively, to 24 (± 4%) and 17% (± 4%) ofthe total number of circulating haemocytes, and increasedthereafter to 45 ± 6% (48 h sampling point) (Fig. 5).However, at 72 h post-stimulation, the percentage ofpenaeidin-immunoreactive haemocytes decreased dramatic-ally to 19 ± 2% of the total circulating population, apercentage inferior to that of haemocytes expressingpenaeidin observed at the same time. This last resultindicated that, at 72 h post-injection, circulating haemo-cytes display differences both in their penaeidin transcrip-tion activity and their storage ability (Fig. 5).

Microbial stimulation induces an increasein haemocyte penaeidin-transcriptional activityat 48–72 h post-challenge

To determine whether penaeidin expression could betranscriptionally regulated at the level of the haemocytesor whether changes in penaeidin transcript rates could beonly the result of changes in haemocyte populations,penaeidin mRNA content was quantified at the cellularlevel. Cytocentrifuged haemocytes, collected from shrimp

at 0, 6, 12, 48, 72 h post-injection, were probed with35S-radiolabelled penaeidin antisense riboprobes. Twenty-five haemocytes from four individual animals were analysedat each time post-injection. Quantification was expressed asArbitrary Units (AU) corresponding to the number of silvergrains counted for every haemocyte by the autoradiographyBIOCOM software. Silver grains are produced by contact of35S-emission with the autoradiographic emulsion. Thenumber of grains is proportional to the hybridizationsignal. Background level was measured and subtracted foreach slide. According to the quantification of penaeidinmRNA content in every haemocyte expressing the peptides,two groups of haemocytes were distinguished: one groupwith AU values <50 and another group with AU values>50. In nonstimulated animals (time 0) the percentage ofhaemocytes with AU values >50 constituted only 5% ofthe haemocytes analysed (Fig. 6A and B1). At 6, 12 and48 h post-stimulation, this percentage increased, respect-ively, to 19, 23 and 34% of haemocytes displaying an AUvalue >50 (Fig. 6A). At 72 h after microbial stimulation,significant differences appeared (P < 0.05) and haemocyteswith AU values >50 represented � 49% of the totalhaemocytes analysed (Fig. 6A) revealing an importantheterogeneity in penaeidin expression levels within circula-ting cell populations (Fig. 6B2). The increase of thepercentage of haemocytes with a high level of penaeidintranscriptional activity is concomitant with a decrease of therelative percentage of circulating haemocytes storingpenaeidins (Fig. 6C1 and C2).

Localization of penaeidin expression and storagein shrimp tissues after microbial challenge

In order to investigate the ability of tissues other thanhaemocytes to express penaeidins and to study the distri-bution of both transcripts and peptides in response tochallenge, shrimp tissues were analysed by Northern blot,ISH and ICC at different times post-injection.For Northern blot analyses, total RNA was extracted

from gills, midgut, cephalothorax subcuticular epitheliumand brain from 10 shrimps at 0, 3, 6, 12, 24, 48, 72 h post-stimulation. As described previously for haemocytes,STORM

TM quantified penaeidin and ribosomal hybridizationsignals were compared for every tissue at each time post-injection (Fig. 7A). Relative penaeidin mRNA levels dra-matically decreased in all the tissues analysed 6 and 12 hafter microbial challenge, and increased thereafter in gillsand midgut, at 48 or 72 h post-stimulation, up to the levelobserved in nonstimulated shrimp (Fig. 7B). However,penaeidin mRNA levels remained low in subcuticularepithelium during the 72 h after the challenge in comparisonto that observed for control shrimp (Fig. 7B).ISH analyses of the different tissues confirmed that

penaeidin transcripts were confined to haemocytes (Fig. 8).Moreover, these observations revealed that, at 6 and 12 hpost-challenge, the decrease in penaeidin mRNA levelsobserved by Northern blot in tissues could be related to adecrease in the number of haemocytes containing transcriptsthat infiltrated the tissues (Fig. 8B, E and H). At 48 and72 h, the number of haemocytes expressing penaeidin instimulated shrimp tissues appeared to be restored (Fig. 8C,F and I) and was similar to that observed in nonstimulatedanimals (Fig. 8A, D and G).

Fig. 5. Time-course analysis of percentages of haemocytes expressing

penaeidins and haemocytes storing penaeidins in the circulating popula-

tion after microbial challenge.Circulating cells were harvested from five

shrimps at different times post-injection (0, 6, 12, 48, 72 h) and fixed in

paraformaldehyde. Total haemocyte numbers were established using a

haemocytometer (line). The haemocytes were cytocentrifuged onto

slides and analysed by in situ hybridization using antisense pen-3a

riboprobes labelled with DIG-UTP and by immunocytochemistry

using an anti-penaeidin Ig detected by secondary antibody labelled

with Texas red. Immunostaining was observed by confocal micro-

scopy. The percentage of haemocytes expressing penaeidin corres-

ponds to the ratio between the number of penaeidin riboprobe-positive

cells and the total number of haemocytes (open bars). The percentage

of haemocytes storing penaeidins corresponds to the ratio between

immunopositive cells and the total number of haemocytes (black bars).

Four hundred cells per slide and three slides per shrimp were counted

and each value represents the mean of five shrimps ± SEM.


Similar observations were obtained with ICC analysesrelative to the distribution of penaeidin-stained haemocyteswithin tissues and following microbial challenge (data notshown).

Haemocyte recruitment and penaeidin localizationat the site of injection

Injection of microorganisms resulted in a dramatic decreasein numbers of both circulating and tissue infiltratinghaemocytes within 3 h of injection. To study haemocytebehaviour and changes, sections of the last abdominalsegments (site of injection) were analysed at 3, 6 and 72 h byICC and ISH. Both penaeidin-producing haemocytes andreleased peptides were therefore localized. Concerningpeptide detection and distribution as studied by ICC, thelast abdominal segment of untreated animals appearedtotally devoid of immunoreactivity (Fig. 9A). Three hpost-challenge, some penaeidin-positive haemocytes wereobserved together with a slight spread of penaeidinimmunostaining near the injection site. However, 6 hpost-stimulation, an increased number of haemocytescontaining penaeidins was seen not only around the

injection sites, but also on surrounding subcuticular epithe-lia. Strong penaeidin immunoreactivity was detected aroundthe injection site revealing the presence of released peptidesand their binding to cuticular surfaces close to the injectionsite (Fig. 9B). Such reactivities were observed up to 72 hpost-injection, with an increasing number of penaeidin-positive haemocytes and an accumulation of free peptidesinto the muscle around the site of injection (Fig. 9C).Concerning penaeidin expression, an accumulation of

infiltrating haemocytes containing penaeidin transcriptsbegan also to be seen around the injection site 3 h afterstimulation (data not shown). At 6 and 72 h, a highconcentration of penaeidin-positive haemocytes wasreached around the site of injury when very few positivehaemocytes were observed in the muscle of nonstimulatedanimals or in other parts of the tail of injected shrimps(Fig. 9D and E).

Confrontation of haemocytes and bacteria

The recruitment of penaeidin-positive haemocytes aroundthe site of injection confirmed the importance of haemocyticreactions in response to microbial challenge. However, the

Fig. 6. Changes in penaeidin transcriptional

activities and penaeidin storage of circulating

haemocytes after microbial challenge.

(A) Cytocentrifuged haemocytes were

hybridized with antisense pen-3a riboprobes

labelled with [35S]UTP. The radiolabelling

appears as dark silver deposits. Individual

haemocyte titration of the level of expression

was performed using a BIOCOM system. Results

are expressed in arbitrary3 units (AU). The

level of expression was quantified in 25 cells

per slide and five slides per shrimp and each

value represents the average of four shrimps.

Histograms refer to the percentage of hae-

mocytes exhibiting more than 50 of AU

(black bars) and the percentage of haemocytes

showing less than 50 of AU (open bars). (B)

Penaeidin mRNA content in cytocentrifuged

haemocytes were visualized by silver grains

resulting from the contact of 35S-emission with

autoradiographic emulsion. Silver grains are

seen in the haemocytes of nonstimulated ani-

mals (B1); comparatively, at 72 h after

microbial challenge, stronger signals are

observed in some haemocytes (B2). (C)

Cytocentrifuged haemocytes were investigated

for penaeidin content by immunodetection

with Texas red-labelled secondary antibody.

Strong immunoreactivity is observed in hae-

mocytes from nonstimulated shrimp (C1)

whereas at 72 h post-challenge haemocytes

display weak penaeidin-immunostaining (C2).

Bars ¼ 10 lm.


strong penaeidin reactivity observed at the site of injury didnot allow investigation of the close interaction between thehaemocytes and the microorganisms and the role ofpenaeidins in these reactions. To address this question,in vitro analyses were performed. Haemocytes were incuba-ted in the presence of the bacteria V. alginolyticus, and thentreated at 1, 3, 5, 10, 20, 30, 45 and 60 min. incubation forelectron microscopy examination and penaeidin immuno-detection (immunogold labelling). Observations of controlhaemocytes (t0) confirmed penaeidin localization into

cytoplasmic granules of granular haemocytes (Fig. 10A).Haemocytes without granules or with only a few smallgranules, termed hyaline cells, presented no penaeidinimmunoreactivity as described previously [14]. In cellpreparations exposed to bacteria for 5 min, haemocyteswith granules showed slight penaeidin immunoreactivity inthe cytoplasm, when deformations of their cytoplasmicgranules began to be observed (Fig. 10B, C). At the sameincubation time with bacteria (5 min), extracellular bacteriawere not reactive to penaeidin-specific antibody

Fig. 7. Time-course analysis of penaeidin expression in shrimp tissues after microbial challenge. Ten micrograms of a pool of total RNAs, extracted

from tissues of 10 animals at different times (0, 3, 6, 12, 24, 48, 72 h) followingmicrobial injection, were hybridized successively with pen-3a and 18 S

rRNA 32P-labelled DNA probes. (A) Hybridization profiles of midgut, gills and subcuticular epithelium are shown; penaeidin mRNA levels

decrease in all the tissues within hours of challenge and increase again after 12–24 h (B) Hybridization signals were quantified STORMTM and the

penaeidin/18 S rRNA signal ratio was determined and normalized to 100 in untreated animals. Results, given as percentage expression relative to

this level, show great variations in penaeidin transcript content resulting from microbial challenge.

Fig. 8. Detection of penaeidin mRNA by in situ

hybridization in shrimp tissues after microbial

challenge. Positive haemocytes (arrows) were

detected in gills (A, B, C), epigastric haemato-

poietic nodule (D, E, F) and hepatopancreas

(G, H, I). Positive haemocytes are fairly

abundant in the tissues of untreated animals

(A, D, G), but almost undetectable in tissues

6 h after microbial injection (B, E, H). At 48 h

post-challenge, the distribution of penaeidin-

positive cells is restored and is quite similar to

that observed in unchallenged shrimp but with

more intense labelling of haemocytes (C, F, I).

Bars ¼ 10 lm.


(Fig. 10C, D). In haemocytes incubated with bacteria for10 min, most of the granules showed gross deformation,such as a lost of round shape and electron density, andretraction within the granule membranes causing star-shaped contours (Fig. 10E, F). Immunoreactivity to penaei-dins was evidenced in the cytoplasm of these haemocytes,suggesting the release of granule content within the cell(Fig. 10F). No evidence of degranulation or exocytosis wasfound in these experiments. After 20 min incubation,extracellular penaeidin-immunoreactive bacteria were seenin the preparation (Fig. 10G, H). Regarding phagocytosisreactions, internalized bacteria were observed after 20 minincubation mainly into hyaline haemocytes (Fig. 10I).Intracellular phagocytosed bacteria observed in hyalinecells were shown to be immuno-positive to penaeidin(Fig. 10I, J) as well as bacteria not yet phagocytosed,suggesting that they had been covered with releasedpenaeidins before their internalization. To a lesser extent,phagocytosed bacteria were also observed into somegranular haemocytes in which neither intracellular lysis oftheir granules nor fusion of granules with phagocyticvacuoles were seen (Fig. 10K). At the same time, a largenumber of haemocytes with degenerated cytoplasm andnuclei was observed revealing that a phenomenon of lysishas occurred in response to Vibrio contact (Fig. 10L, M).After 45 min incubation, degenerative haemocytes withinthe preparation were predominant.

D I S C U S S I O N

Through investigations on penaeidin expression, the aims ofthe present study were to define shrimp defence mechanismsin response to microbial infections. We applied in vivoexperimental infection model in the shrimp P. vannamei toanalyse the expression, regulation and production of

penaeidins in circulating haemocytes and tissues of theanimals.We previously showed that penaeidins are constitutively

produced and stored in granular haemocytes of shrimpsthat have not been experimentally infected, indicatinghaemocytes as the main site of production of the peptides[14]. Here, we show that in shrimp tissues, the distribution ofpenaeidin transcripts and proteins is restricted to haemo-cytes either circulating in blood vessels irrigating tissues suchas the brain, hepatopancreas or gills, or infiltrating tissuessuch as subcuticular epithelia or midgut caecum. Penaeidinsare solely present in large-granule haemocytes and small-granule haemocytes (also called semigranular cells), and areabsent from the hyaline haemocyte population, devoid ofgranules. In the haematopoietic tissues, penaeidin tran-scripts were clearly visible in a few cells, showing thatpenaeidin expression occurs in this tissue. This result differsfrom those obtained in crayfish where the haematopoietictissue was found to be negative for prophenoloxidase [20], agene that is expressed in circulating haemocytes [21]. Thehaematopoietic tissues have been described in crustaceanspecies [22,23] but knowledge of the haematopoietic processremains limited and few data are available on the expressionof immune effectors during haemocyte differentiation andmaturation. Our observations suggest that penaeidins areexpressed either by maturating stem cells or by haemocytesearly before leaving the haematopoietic tissues. However, itcannot be excluded that circulating haemocytes expressingpenaeidins may return to infiltrate this tissue for somesignalling reaction.In invertebrates, little is known about the regulation and

expression of antimicrobial peptide encoding genes duringthe immune response, apart from insects where transcrip-tion is induced in fat body cells and surface epithelia and forwhich signalling and regulatory pathways controlling

Fig. 9. Haemocyte recruitment at the site of

microbial injection. Sections were immuno-

stained using anti-penaeidin Ig and secondary

antibody labelled with Texas red (A, B, C) and

sections were hybridized with antisense pen-3a

riboprobes labelled with DIG-UTP (D, E).

The last abdominal segment of an unchal-

lenged animal is totally devoid of immuno-

reactivity (A). Six hours post-injection,

numerous haemocytes storing penaeidin are

observed around the injury site (arrow) and an

intense immunoreactive signal is also detected

throughout the tissue (star) (B). At the same

time point, a large number of haemocytes

expressing penaeidins are present around the

injection site and near the subcuticular epi-

thelium (D). Seventy-two h after microbial

challenge, a large number of penaeidin-storing

(C) and expressing haemocytes (E) is observed

throughout surrounding tissue. Bar ¼ 20 lm(A, C, E), 10 lm (B, D).


peptide expression are particularly well characterized [2,24].In the bivalve mollusc,Mytilus galloprovincialis, antimicro-bial peptides are constitutively expressed and stored inphagocytic haemocytes where they participate in thedestruction of engulfed microorganisms [6]. In Limulus,upon microbial stimulation, antimicrobial peptides arereleased from haemocytes by regulated exocytosis [25]. Inshrimp, as previously shown [14], microbial challenge resultsin a dramatic drop of penaeidin mRNA concentration(relative to 18 S rRNA) in circulating haemocytes in theearly hours post-injection with a return to initial levels at48 h. However, at 72 h post-injection, penaeidin transcriptconcentration appears to be threefold higher than thatobserved in unchallenged shrimp. Similar kinetics (adecrease followed by a significant increase) has beenobserved in the total number of circulating haemocytes asthe result of microbial challenge, a phenomenon alreadydescribed in other crustacean species [26,27].From our results and data acquired from Northern blot,

ISH and ICC analyses, two distinct phases can be distin-guished in the immune response of shrimp to microbial

challenge. During the first phase, corresponding to the first12 h post-challenge, haemocytes constitutively producepenaeidin mRNA and protein. The decrease of penaeidindetection within total circulating populations is the result ofa decrease in penaeidin-expressing haemocytes: they leavethe blood circulation and most of the shrimp tissues andmigrate towards injured tissues.2 This is in agreement withprevious studies on other crustacean species [28]. Massiveaccumulation of penaeidin-producing haemocytes was seenaround the site of injection 6 h post-injection, as well as amassive release of penaeidin which spread into muscle tissuearound the injection site, as a local antimicrobial response.As previously demonstrated, penaeidins are released, uponstimulation, from haemocytes into haemolymphwhere theirconcentration increases; subsequently, they bind to cuticlesurfaces [14].During the second phase, at about 48–72 h post-chal-

lenge, intense penaeidin-labelling is observed in the tissuessurrounding injection site aswell as on subcuticular surfaces.Moreover, haemocytes displaying high transcriptionalactivity appear in the blood circulation as evidenced by

Fig. 10. In vitro confrontation of haemocytes with V. alginolyticus. Haemocytes were incubated with bacteria, fixed at different time intervals

(0, 1, 3, 5, 10, 20, 30 min) and embedded in resin for penaeidin immunostaining using a 10-nm gold particle-conjugated secondary antibody. (A) In

control haemocytes (t0), positive cells exhibit numerous gold particles in electron dense round granules. (B, C) After a 5-min incubation with

bacteria, the immunoreactive granules loose their round shape and retraction of the granule membranes is seen (B, arrow). All of the bacteria

observed are extracellular and are totally devoid of immunoreactivity (C, D). After 20 min of contact with bacteria, penaeidin-positive granules

have star-shaped contours (E, F, G, arrow). At the same time point, immunoreactivity is also observed in the preparation outside haemocytes and

on bacteria (H). Internalized penaeidin-positive bacteria are observed to a great extent into hyaline cells (I). In these cells that do not express

penaeidin, phagocytosed bacteria appear to be penaeidin immunopositive (J). Granular haemocytes display also phagocytic activity (K). At longer

time intervals (20 and 30 min), many cells appear as ghosts (L, M)4 , probably originating from distinct haemocyte populations. b, Bacteria; n,

nucleus; mb, plasma membrane; pg, phagosome. Bars ¼ 1 lm.


the dramatic increase of rRNA 18 S concentration [29]together with an increased penaeidin expression activity.Actually, measurement of penaeidin mRNA content usingradiolabelled probes on haemocytes collected at differenttimes post-infection stressed the gradual increase in penaei-din transcriptional activity of circulating haemocytes, muchgreater than that observed in unchallenged shrimp. Thus, weassume that penaeidin up-regulation in circulating haemo-cytes reflects an induced proliferation process, similar toresults obtained in P. japonicus. In this species, an increasein the proliferation rate of circulating haemocytes as a resultof in vivo experimental infection with Fusarium was shownby flow cytometry [30]. At 72 h post-stimulation, transcrip-tionally active, young or maturating haemocyte forms, butwhich are comparatively penaeidin-poor, are probablyintensively produced and released precociously from hae-matopoietic tissues. Such a phenomenon has already beenbeen proposed for Syciona ingentis during the moultingcycle and after bacterial injection [27,31]. Concomitantly, asa result of this proliferative process, a dramatic invasion ofhaemocyte producing penaeidin mRNA and protein is seenin most of the tissues, indicating a systemic reaction. Anintense proliferation process may occur: (a) to amplifyhaemocytic reactions and subsequently to increase penaei-din representativeness within shrimp tissues, together withother immune cellular effectors; (b) to replace into the bloodcirculation and infected tissues lysed or dead haemocytessubsequent to microbial challenge [32].The strong immunoreactivity observed at the site of

microbial injection precluded both clarification of themechanisms of penaeidin release from haemocytes, anddetermination of any potential involvement of penaeidin inthe elimination of microorganisms via phagocytosis. Tofurther address these questions haemocytes were challengedwith Vibrio in vitro. Regarding penaeidin release, there wasno indication of degranulation of granule-containingpenaeidin, or any migration of granules towards the cellperiphery, in contrast with regulated exocytosis reported inTachypleus [33]. Penaeidin containing haemocytes showedstriking changes in the shape and morphology of theirgranules, suggesting a possible release of granule contentwithin the haemocyte cytoplasm. This quite originalphenomenon appears to be followed by the lysis of thehaemocytes and the release of cytoplasm content, assuggested by the later appearance in the preparation ofnumerous ghost cells and penaeidin immunoreactivity inpreparation extracellular spaces. In crustaceans, the phe-nomenon of lysis has been reported and attributed tohyaline cells thought to be involved in triggering thecoagulation process [22]. Such a reaction was also evidencedhere with the observation of coagulum (coagulated mater-ial) surrounding haemocytes and suggested by the presenceof ghost cells. In this coagulum, released penaeidins could betrapped with other immune effectors originating also fromhaemocytes, such as components of the prophenoloxidasesystem [34].Regarding phagocytotic activity, in the present study

hyaline cells appear to be the most active phagocytic cellswhereas penaeidin-positive cells with large-granules areminimally phagocytic and internalized bacteria are observedlate in this cell population. These results give new insightsinto the identification of haemocyte types and theirrespective function in crustaceans. Indeed, in crab and

crayfish, using haemocytes previously separated on Percollgradient, hyaline cells were considered to be primaryphagocytic cells [35,36]. However, in penaeid shrimp,granular cells including large- and small-granule haemo-cytes were described to be active in phagocytosis and tocontain lysosomal enzymes and prophenoloxidase activity[22,37]. There is no model or classification scheme that isapplicable to all decapods, and different interpretationsmayalso result from the variety of experimental approaches usedin these studies. Further analyses based on expression ofimmune effectors, both transcripts and proteins, as carriedout here with penaeidins, will be of great benefit to clarifyhaemocyte lineage and identification of cell types as well astheir functions in immune response. Our data suggest thatdifferent populations of granular haemocytes may exist:(a) one population involved in a phenomenon of lysis with amassive and early release of penaeidins; and (b) anotherpopulation involved in phagocytosis of bacteria taking placelate than hyaline cell phagocytosis. No evidence fordischarge of granular penaeidin content into bacteria-containing phagosomes has been observed in shrimp, asdemonstrated in human neutrophils for defensins [38] or inmussel haemocytes for mytilins [17]. The question remainsabout the function of these intracellular penaeidins and theirpotential involvement in the elimination of internalizedmicrobes. It is attractive to assume that these two popula-tions of penaeidin-positive haemocytes can contain differentclasses of penaeidins with various functions, which areimpossible to discriminate with the tools available today.In conclusion, the expression and distribution of penaei-

dins in response to microbial challenge are regulatedthrough haemocyte reactions and haemocyte proliferationprocesses. Penaeidins may be involved in local defencereaction through their release by haemocytes and binding toshrimp cuticle surfaces. By their antimicrobial activitiesagainst Gram-positive bacteria and fungi [19], penaeidinsmay protect tissues from infections and/or participate inwound healing process. Penaeidins do not display strongantimicrobial activity against Gram-negative bacteria suchas Vibrio sp. but they can contribute to their elimination byphagocytic cells by a potential opsonic function. Indeed,extracellular bacteria as well as internalized phagocytosedbacteria were seen to be immunoreactive to penaeidins.These observations argue in favour of a coating, by releasedpenaeidin, of the bacteria before their internalization intopenaeidin-devoid hyaline cells. Finally, the diversity, thelarge distribution and abundance of penaeidins which areproduced in shrimp, together with their multiple andcomplementary properties, reveal that the penaeidin familyconstitutes a major component of the shrimp immunesystem, which should be investigated further.

A C K N O W L E D G E M E N T S

The authors thank J.C. Beauvillain and V. Mitchell for access to the

Cellular Imaging Center of the IFR 22 (Institut Federatif de Recherche

22, Faculte de Medecine Lille). This study is supported by the CNRS

(CentreNational de laRecherche Scientifique), the IFREMER (Institut

Francais de Recherche et d’Exploitation de la Mer) and the University

of Montpellier 2. It is also part of a collaborative project supported by

the European Commission, DG XII, in the program International

Cooperation with Developing Countries, INCO-DC, Contract n�IC18CT970209 (Shrimp Immunity & Disease Control).


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Expression and distribution of penaeidin antimicrobial peptides are regulated by haemocyte reactions in microbial challenged shrimp