Báo cáo Y học: Expression and distribution of penaeidin antimicrobial peptides are regulated by haemocyte reactions in microbial challenged shrimp pptx

Expression and distribution of penaeidin antimicrobial peptides
are regulated by haemocyte reactions in microbial
challenged shrimp
Marcelo Mun
˜
oz
1
, Franck Vandenbulcke
2
, Denis Saulnier
3
and Evelyne Bache
`
re
1
1
IFREMER/CNRS/Universite
´
de Montpellier, ÔDe
´
fense et Re
´
sistance chez les Inverte
´
bre
´
s MarinsÕ, Montpellier, France;
2
Laboratoire d’Endocrinologie des Anne
´
lides, Groupe de Neuroimmunite

´
des Hirudine
´
es, Universite
´
des Sciences et Technologies
de Lille, France;
3
IFREMER, Centre Oce
´
anologique du Pacifique, Taravao, Tahiti, Polyne
´
sie Franc¸ aise
Penaeidins are a family of antimicrobial peptides constitu-
tively produced and stored in the haemocytes of penaeid
shrimp. In response to microbial stimulation, they are
released into the blood circulation and they further attach to
shrimp cuticle surfaces through a chitin-binding property. In
the present paper, we have analysed their expression, regu-
lation and distribution in shrimp tissues in response to
experimental microbial challenge. We have shown that
penaeidinmRNAandproteinarerestrictedtogranular
haemocytes and that their expression and distribution are
regulated through dramatic changes in haemocyte popula-
tions, both circulating and infiltrating shrimp tissues. Two
distinct phases in the immune reactions were evidenced: (a) a
migration of haemocytes towards the infection site within
the first 12 h following microbial injection, with a local and
massive release of peptides; (b) the appearance into the blood
circulation and tissues of a haemocyte population displaying

increased penaeidin-transcriptional activity, which may
correspond to a systemic reaction involving haemocyte
proliferation process. Finally, in vitro confrontation of hae-
mocytes and bacteria revealed that penaeidins are released
from granular haemocytes by a novel phenomenon of
intracellular degranulation, probably followed by the lysis of
the cells. Furthermore, penaeidins were shown covering
bacterial surfaces suggesting that the peptides could be
involved in opsonic activity. Penaeidin-positive bacteria
were observed to be phagocytosed mainly by hyaline cells, a
population that does not express penaeidins.
Keywords: antimicrobial peptide; crustacea; innate immu-
nity; penaeid shrimp; phagocytosis.
Antimicrobial peptides are major components of innate
immunity that have been conserved in evolution and found
in different phyla of the plant and animal kingdom.
Although these immune effectors share common character-
istics (small size and cationic character) and similarities in
structural patterns or motifs [1], one striking feature is their
great diversity in terms of amino acid sequences, anti-
microbial activities and modes of action. Moreover,
depending on their distribution, antimicrobial peptide
expression appears to be regulated by different tissue-
specific pathways [2] and these effectors may consequently
participate in either a local or a systemic reaction. Antimi-
crobial peptides are produced in phagocytic cells of
vertebrates [3] and invertebrates [4–6], and in various tissues
such as epithelia of mammals and insects [7,8], or insect fat
body [9]. Peptides are produced constitutively and stored in
circulating cells, where they can act intracellularly against

phagocytosed microorganisms as shown in human for
defensins [3] and in a bivalve mollusc for mytilin [6].
Peptides can also be released by exocytosis upon microbial
stimulation [5,10]. In various epithelia of invertebrates [2]
and vertebrates [11], antimicrobial peptides are either
produced constitutively or induced in response to infection
or inflammation, and participate in a local antimicrobial
reaction. Finally, antimicrobial peptide expression in fat
body cells is induced in response to infection and peptides
are secreted into body fluids, which characterizes the acute
or systemic reaction in insects [12].
In Crustacea, penaeidins are a unique family of
antimicrobial peptides originally isolated and characterized
in the shrimp Penaeus vannamei. In previous works, three
members of the penaeidin family, penaeidin (Pen)-1, -2
and -3 were purified in their mature and active form
(5.48–6.62 kDa) and cloned from the haemocytes of
experimentally uninfected shrimp [13]. Penaeidins were
shown to be constitutively expressed in haemocytes and
mature peptides were localized in the cytoplasmic granules
of the granular haemocyte population of unchallenged
animals. Regarding penaeidin gene expression and peptide
distribution, first data suggested that in response to a
microbial challenge, penaeidin transcription is not
up-regulated in shrimp haemocytes, but relative penaeidin
concentrationinshrimpplasmawasshowntoincrease
upon stimulation [14]. Penaeidins, which present in their
amino acid sequences a chitin-binding motif [15] were
demonstrated to bind to shrimp cuticle surfaces in
response to microbial challenges [14]. Thus, we speculated

Correspondence to E.Bache
`
re, UMR 5098, ÔDe
´
fense et Re
´
sistance chez
les Inverte
´
bre
´
sMarinsÕ, CC 80, 2 place Euge
`
ne Bataillon – 34095
Montpellier, France.
Fax:+33467144622,Tel.:+33467144710,
E-mail:
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) Ó 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 previously
for the antimicrobial peptide-mediated immune response
in Limulus [5].
The purpose of the current study was to define the
regulation and distribution of penaeidin expression in
shrimp during immune response considering Pen-3, the
most abundant and representative member of the family

[13,16]. We demonstrate that penaeidins are expressed
exclusively in shrimp haemocytes and that experimental
microbial infection induces great changes in haemocyte
populations. Through in situ hybridization and immuno-
histochemical analyses, haemocytic reactions were high-
lighted as an important component of the immune
response ) involved in the distribution of the antimicro-
bial peptides. Finally, to define the cellular mechanisms of
penaeidin release, haemocytes were challenged with bac-
teria in vitro, which gave new insights into haemocyte
functions and involvement of penaeidins in shrimp
defence.
MATERIALS AND METHODS
Animals and immune challenge
Juvenile shrimp (8–10 g) P. vannamei (Crustacea, Deca-
poda) in intermoult stage were obtained from a farm in
the province of Guayas (Ecuador) and from the French
Polynesia IFREMER laboratory. Shrimp microbial
challenge was performed by injecting, into the last abdo-
minal segment, a suspension (50 lL; 10
8
cells/animal)
of heat-killed (100 °C, 10
5
Pa, 20 min) microorganisms
including bacteria, Aerococcus viridans, Vibrio alginolyt-
icus and fungal spores of Fusarium oxysporum.Haemo-
lymph and tissues were collected at different times (from
0 to 72 h) post-injection as described previously [14].
Unchallenged shrimps (i.e. shrimp at time 0 h) were used

as controls.
Northern blot analyses
Penaeidin-specific and ribosomal probes were amplified by
PCR on, respectively, pen-3a cDNA clone (GenBank
accession number Y14926) and an 18 S rRNA genomic
DNA clone (a gift from T. Spears, Florida State University,
USA) as described previously [14]. The probes were
radiolabelled by random priming using the Ready-to-go
DNA labelling kit (Amersham Pharmacia Biotech).
Total RNA from shrimp haemocytes and tissues was
prepared according to the method of Trizol reagent (BRL
Life technologies). Two or 10 lg total RNA were fraction-
ated on denaturating 1% agarose gel containing 17%
formaldehyde, and then transferred to Hybond-N filter
membranes (Amersham Pharmacia Biotech) by vacuum
blotting. Membranes were hybridized at 55 °Cfor12 hwith
32
P-labelled pen-3a cDNA fragment in a solution containing
50% formamide, 5 · NaCl/Cit, 8 · Denhardt’s solution,
50 m
M
sodium phosphate pH 6.5, 0.1% SDS and
100 lgÆmL
)1
denatured salmon sperm DNA. Filters were
washed twice in 2 · NaCl/Cit, 0.1% SDS at room tem-
perature and twice in 1 · NaCl/Cit, 0.1% SDS, first at room
temperature, then at 65 °C followed by autoradiography.
After stripping, the membranes were hybridized under
identical conditions with

32
P-labelled 18 S ribosomal DNA
probe and subjected to further autoradiography. Penaeidin
transcript and 18 S rRNA signals were quantified using the
STORM
TM
system (Molecular Dynamics).
Tissue preparation for histology
Tissues from juvenile shrimp were fixed in a solution
containing 22% formalin, 31.5% ethanol and 11.5% glacial
acetic acid. After dehydration, tissues were embedded in
Paraplast and 8 lm sections were cut, mounted on poly
L
-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 fixed
for 10 min by addition of 1 vol. ice-cold 4% paraformal-
dehyde in 100 m
M
NaCl/P
i
containing 10% saccharose.
Cells were centrifuged on slides for 5 min at 200 g in a
cytospin (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 temperature
with bacteria (V. alginolyticus)attheratioof20bacteriaper

haemocyte in modified Hanks’ balanced salt solution
supplemented with 6 m
M
CaCl
2
and 13 m
M
MgCl
2
.At
various incubation times (0, 1, 3, 5, 20, 30, 45 and 60 min),
cells were fixed and treated for ultrastructural analyses and
immunodetection as described below.
In situ
hybridization
Probes. A plasmid containing pen-3a cDNA (GenBank
accession number Y14926) was used as template for the
preparation of the probes. Digoxigenin (DIG)-UTP-
labelled and [
35
S]UTP-labelled antisense and sense ribo-
probes were generated from linearized cDNA plasmids by
in vitro transcription using RNA labelling kits, T3 RNA
polymerase (Roche) and [
35
S]UTP (Amersham).
Hybridization. DIG-labelled riboprobes ( 40–100 ng
per section) were hybridized to tissue sections as described
previously [17]. For cytocentrifuged cells, the protocol of
hybridization was adapted, i.e. cytocentrifuged cells were

incubatedfor10minin100m
M
glycine, 200 m
M
Tris/
HClpH7.4,immersed5mininNaCl/P
i
and fixed in
100 m
M
phosphate buffer containing 4% paraformalde-
hyde and 5 m
M
MgCl
2
. After the postfixation step, cell
preparations were washed 5 min in phosphate buffer,
incubated for 10 min in 0.25% anhydride acetic prepared
in 100 m
M
triethanolamine pH 8, and briefly washed in
2 · NaCl/Cit. Samples were then rinsed in distilled water,
dehydrated by graded alcohol and air dried at room
temperature.
DIG-labelled riboprobes (40–100 ng per slide) and
35
S-labelled riboprobes (100 ng or 1 · 10
6
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,100m
M
dithio-
threitol and 0.5 mgÆmL
)1
salmon sperm DNA. Hybridiza-
tion was carried out overnight at 55 °C in a humid chamber.
Ó 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 in
0.1 · NaCl/Cit containing 0.07% 2-mercaptoethanol at
55 °C. The probes labelled with DIG-UTP were revealed
using alkaline phosphatase-conjugated antibodies as des-
cribed previously [17].
Detection and quantification of the
35
S-labelled probes
After hybridization step, the slides were rinsed in 0.1 ·
NaCl/Cit at 20 °C, briefly immersed in graded alcohol and
air dried at room temperature. Hybridization signal was
visualized using autoradiography. Samples were coated by
dipping in LM1 Amersham liquid emulsion, immediately
dried and exposed for a 4-day period. At the end of the
exposure period, the autoradiograms were developed in

D19b (Kodak), fixed in 30% sodium thiosulfate (10 min at
room temperature), stained with 1% Toluidine blue and
mounted with Xam (Merck). Quantification of the radio-
labelling at the cellular level was performed using an
Axiophot Zeiss microscope and a Biocom quantification
system as established [18].
Controls
Control for in situ hybridization consisted in replacing
antisense riboprobe with sense riboprobe. RNase control
sections were obtained by adding a preincubation step with
10 lgÆmL
)1
RNase A prior to hybridization.
Immunodetection of penaeidins
Whole animal. Eight micrometer-thick paraffin sections
were re-hydrated and treated as follow: (a) 10 min at 20 °C
in 150 m
M
NaCl, 100 m
M
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% Triton
X-100, 30 min at room temperature; (c) incubation with
anti-penaeidin IgG (3 lgÆmL
)1
) diluted in NaCl/Tris/NGS/
BSA, overnight at room temperature; (d) 3 · 10 min in
NaCl/Tris; (e) 1 nm colloidal gold-labelled goat anti-rabbit
IgG (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 m
M
citrate buffer pH 7.4; (h)
silver amplification performed with the IntensSE
TM
kit
according 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) and
observed using a Zeiss Axioskop light microscope. Immuno-
dection was also performed by using Texas red-tagged goat
anti-rabbit serum (Jackson Immunoresearch) as described
below.
Circulating haemocytes. Cytocentrifuged haemocytes
were equilibrated for 10 min in NaCl/Tris before perme-
abilization with 0.1% Triton X-100 in NaCl/Tris for
30 min at room temperature. One hour preincubation was
performed in the presence of 1% NGS and 1% BSA to
block nonspecific antibody binding. Rabbit anti-penaeidin
polyclonal antibody purified IgG (1.5 lgÆmL
)1
) [14], was
applied 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 temperature
with 1 : 100 Texas red-tagged goat anti-rabbit antiserum
(Jackson Immunoresearch). The slides were washed
3 · 10 min in NaCl/Tris, mounted in glycerol containing
25% 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 was
detected by exciting samples at 568 nm. Images were
acquired as single transcellular optical sections and
averaged over 16 scans per frame. Positive or negative
cells were subsequently counted.
Controls were incubations of anti-penaeidin IgG pread-
sorbed by purified recombinant penaeidin-3 [19].
Electron microscopy
Ultrastructural microscopy. After haemolymph collection
under 1 vol. modified Alsever solution buffer, cells were
fixed for 1 h at 4 °Cin0.1
M
NaCl/P
i
pH 7.4, containing
2% glutaraldehyde, 4% paraformaldehyde and 10%
sucrose. Cell pellets were obtained by 10 min centrifuga-
tion at 800 g. The pellets were rinsed in NaCl/P
i
, postfixed
in 1% OsO
4
for 1 h, dehydrated in graded acetone
solutions and embedded in Embed 812 Kit. Ultrathin
sections (80–90 nm thick) were cut from the blocks,
collected onto 200 mesh copper grids, double-stained with
uranyl acetate and lead citrate and examined with a Jeol
JEM 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 °C
in a mixture of 4% paraformaldehyde, 1% glutaraldehyde,
10% sucrose in 100 m
M
NaCl/P
i
, pH 7.4. Cells and tissues
werepostfixedin1%OsO
4
for 3–5 min and dehydrated in
graded alcohol before embedding in LR white (TAAB
Laboratories).
Semi-thin sections (1 lm thick) were collected on alcohol-
washed glass slides, and penaeidin immunostaining was
performed using a gold-tagged secondary antibody and
silver amplification as described above.
Ultrathin (90 nm-thick) sections from embedded pellets
or tissues were collected on nickel grids. Sections were
treated 8 min in 10% H
2
O
2
, 10 min in distilled water,
30 min in NaCl/Tris/NGS/BSA and then incubated for
36 h at 4 °Cwith3lgÆmL
)1
rabbit anti-penaeidin IgGs in
NaCl/Tris/NGS/BSA. Grids were washed three times for

10 min with NaCl/Tris/NGS/BSA and incubated for 2 h
at room temperature in 10 nm colloidal gold-labelled goat
anti-rabbit IgGs (Amersham) diluted 1 : 100 in NaCl/
Tris/NGS/BSA. Grids were then washed three times for
10 min with NaCl/Tris, postfixed for 3 min in NaCl/Tris
containing 1% glutaraldehyde and washed twice for
5 min with distilled water. Sections were stained for
15minwith2.5%uranylacetateandexaminedwitha
Jeol 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. Mun
˜
oz et al. (Eur. J. Biochem. 269) Ó FEBS 2002
RESULTS
Tissue localization of penaeidins in nonstimulated
animals
Penaeidins are known to be constitutively expressed in
shrimp haemocytes and penaeidin transcripts were also
detected 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 were
determined by both in situ hybridization analyses (ISH) using
pen-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 and
sinuses and in cells present within most tissues. The shape of
the positive cells suggests that they are infiltrating haemo-
cytes. A high number of cells containing penaeidin tran-
scripts was detected in heart and epigastric haematopoietic
nodule (also named lymphoid organ) (Fig. 1A), in blood
vessels irrigating gills and hepatopancreas (Fig. 1B and C),
and to a lesser extent in all the shrimp tissues such as
haematopoietic tissue (Fig. 1D), brain, subcuticular epithe-
lia or midgut caecum (data not shown). According to
penaeidin sense probe hybridization used as control, for
which no signal was observed (Fig. 1E and F), the detection
of penaeidin transcripts with antisense riboprobe was shown
to 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 the
signal specificity (data not shown).
Antibody used in this study was a rabbit antiserum
directed against recombinant Pen-3a [14]. The high degree
of homology between the different penaeidin forms [13]
implies that the antibody recognizes different isoforms.
Consequently, we qualified any immune positive signal as
related to the presence of penaeidins. When the specific anti-
penaeidin antibodies were preincubated with purified
recombinant penaeidins [19], penaeidin immunostaining
was no longer observed providing evidence of the specificity
of the reaction (data not shown). Regarding penaeidin
distribution, the peptides were shown to be localized in
circulating haemocytes but also in cells located in gills,
heart, brain, subcuticular epithelium, epigastric haemato-

poietic nodule, midgut, midgut caecum and muscle, where
strong labelling was observed (Fig. 2A, B and C). In order
Fig. 2. Immunodetection of penaeidins in tissue
sections of nonstimulated shrimp. Positive cells
(arrows)areshownonsemithinsectionsof
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),1lm
(D,E,FG).
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.
Ó FEBS 2002 Antimicrobial peptide expression in shrimp (Eur. J. Biochem. 269) 2681
to confirm the localization of penaeidins and to determine
the nature of the positive cells, immunogold labelling was
performed. Penaeidin storage was confirmed to be restricted
to granular haemocytes, with large granules or small
granules, located and infiltrating all tissues analysed such
as brain, subcuticular epithelia, epigastric haematopoietic
nodule or midgut (Fig. 2D, E, F and G). The presence of
some infiltrating haemocytes without labelling was also
found confirming previous data about the presence of
different haemocyte populations, expressing vs. not expres-
sing penaeidins [14].
Microbial stimulation induces changes in the total
number of circulating haemocytes, and in the
population of haemocytes expressing penaeidins
Previous work showed that microbial challenge induces a
decrease of penaeidin mRNA concentration in circulating
haemocytes in the first hours following stimulation [14]. In
order to define the regulation of penaeidin transcription, we
analysed time-course changes in total circulating haemocyte
number and haemocyte penaeidin mRNA levels, occurring
in response to stimulation. In two independent experiments,
shrimp were challenged by injection of heat-killed micro-
organisms and haemolymph was collected from five
individual animals at different times (0, 6, 12, 48 and
72 h) following injection. A strong decrease in haemocyte
total number (from 9 · 10

6
±7· 10
6
cells to
1.2 · 10
6
±1.4· 10
6
cells) was observed in the first 12 h
following injection, with a significant difference (P < 0.05)
at 6 h between stimulated and nonstimulated animals. The
number of total haemocytes returned to levels observed for
unchallenged animals at 48 h and a significant increase (up
to 19.8 · 10
6
±3· 10
6
haemocytes; P <0.05) of total
haemocyte number was observed at our last time point
(72 h poststimulation) (Fig. 3). In similar experiments, total
RNA was extracted from the circulating haemocytes of 10
animals at the same intervals after injection, and 2 lgof
total RNA were analysed by Northern blot. The
STORM
TM
quantifications of penaeidin mRNA and ribosomal hybrid-
ization signals were compared at each time post-injection.
Analyses revealed a strong decrease in penaeidin mRNA
levels for the first 12 h and a return to nonstimulated animal
levels at 48 h post-challenge. A threefold increase in

penaeidin mRNA levels was noticed at 72 h following
challenge (Fig. 3).
To better understand such a decrease in penaeidin
transcript concentration within the circulating haemocytes
after microbial challenge, Northern blot analyses were
performed on total RNA extracted from a constant number
of haemocytes (1 · 10
6
cells for each individual) at every
time post-challenge, instead of constant total RNA quantity
(2 lg). Hybridization signals obtained, respectively, for
pen-3a transcripts and 18 S rRNA probes were quantified
by
STORM
and analysed separately. Data analysis revealed
an important individual variation in both pen-3a transcripts
and 18 S rRNA signals with a decrease in pen-3a transcript
levels and constant average values with 18 S rRNA during
the first 12 h post-challenge (data not shown). However, at
48 h post-challenge, penaeidin mRNA levels appeared to
increase slightly and hybridization signals with 18 S rRNA
probes were dramatically stronger than those observed for
unchallenged animals (Fig. 4).
In order to determine whether changes in penaeidin
transcript and protein levels could be also associated with
changes in the composition of circulating haemocyte
populations, the percentage of circulating haemocytes
expressing and storing penaeidin was further analysed by
ISH and ICC, respectively. Circulating haemocytes from
five individual shrimp were collected, counted, fixed and

cytocentrifuged on slides at different times (0, 6, 12, 48 and
72 h) after microbial stimulation. As shown before, signi-
ficant modifications in total circulating haemocyte number
were observed in these experiments (Fig. 5). In nonstimu-
lated animals 35 ± 6% of the total haemocyte population
expressed penaeidins. This percentage decreased to 19%
(± 8%) and 13% (± 8%), respectively, 6 and 12 h after
microbial challenge (Fig. 5). Then, the percentage of
penaeidin 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 RNA
extracted from 10 shrimps at each time point. Hybridization signals
obtained with
32
P-labelled pen-3a cDNA probe were quantified by the
STORM
TM
system and compared to those obtained with the 18 S rRNA
specific probe. The penaeidin/18 S rRNA signal ratios were calculated
and the expression level in unchallenged shrimp was 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 · 10
6
haemocytes per shrimp,
unchallenged (lanes 1–5) and 48 h following challenge (lanes 6–9) and
hybridized successively with
32
P-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.
2682 M. Mun
˜
oz et al. (Eur. J. Biochem. 269) Ó FEBS 2002
post-injection (Fig. 5). Regarding storage of the peptides,
the percentage of penaeidin-immunoreactive haemocytes
was also established. In nonstimulated animals, the relative
number of haemocytes storing penaeidins was similar to
that of haemocytes expressing the peptides (37 ± 4% of the
total circulating population). At the different times post-
challenge, changes similar to those observed with transcript
detection occurred in the percentages of penaeidin-positive
haemocytes (Fig. 5). During the first 6 and 12 h post-
challenge, the percentage of penaeidin-positive haemocytes
decreased, respectively, to 24 (± 4%) and 17% (± 4%) of
the total number of circulating haemocytes, and increased
thereafter to 45 ± 6% (48 h sampling point) (Fig. 5).
However, at 72 h post-stimulation, the percentage of
penaeidin-immunoreactive haemocytes decreased dramatic-
ally to 19 ± 2% of the total circulating population, a
percentage inferior to that of haemocytes expressing

penaeidin observed at the same time. This last result
indicated 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 increase
in haemocyte penaeidin-transcriptional activity
at 48–72 h post-challenge
To determine whether penaeidin expression could be
transcriptionally regulated at the level of the haemocytes
or whether changes in penaeidin transcript rates could be
only the result of changes in haemocyte populations,
penaeidin mRNA content was quantified at the cellular
level. Cytocentrifuged haemocytes, collected from shrimp
at 0, 6, 12, 48, 72 h post-injection, were probed with
35
S-radiolabelled penaeidin antisense riboprobes. Twenty-
five haemocytes from four individual animals were analysed
at each time post-injection. Quantification was expressed as
Arbitrary Units (AU) corresponding to the number of silver
grains counted for every haemocyte by the autoradiography
BIOCOM
software. Silver grains are produced by contact of
35
S-emission with the autoradiographic emulsion. The
number of grains is proportional to the hybridization
signal. Background level was measured and subtracted for
each slide. According to the quantification of penaeidin
mRNA content in every haemocyte expressing the peptides,
two groups of haemocytes were distinguished: one group
with AU values < 50 and another group with AU values

> 50. In nonstimulated animals (time 0) the percentage of
haemocytes with AU values > 50 constituted only 5% of
the haemocytes analysed (Fig. 6A and B1). At 6, 12 and
48 h post-stimulation, this percentage increased, respect-
ively, to 19, 23 and 34% of haemocytes displaying an AU
value > 50 (Fig. 6A). At 72 h after microbial stimulation,
significant differences appeared (P < 0.05) and haemocytes
with AU values > 50 represented  49% of the total
haemocytes analysed (Fig. 6A) revealing an important
heterogeneity in penaeidin expression levels within circula-
ting cell populations (Fig. 6B2). The increase of the
percentage of haemocytes with a high level of penaeidin
transcriptional activity is concomitant with a decrease of the
relative percentage of circulating haemocytes storing
penaeidins (Fig. 6C1 and C2).
Localization of penaeidin expression and storage
in shrimp tissues after microbial challenge
In order to investigate the ability of tissues other than
haemocytes to express penaeidins and to study the distri-
bution of both transcripts and peptides in response to
challenge, 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 epithelium
and 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 hybridization
signals 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 h
after microbial challenge, and increased thereafter in gills
and midgut, at 48 or 72 h post-stimulation, up to the level
observed in nonstimulated shrimp (Fig. 7B). However,
penaeidin mRNA levels remained low in subcuticular
epithelium during the 72 h after the challenge in comparison
to 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 h
post-challenge, the decrease in penaeidin mRNA levels
observed by Northern blot in tissues could be related to a
decrease in the number of haemocytes containing transcripts
that infiltrated the tissues (Fig. 8B, E and H). At 48 and
72 h, the number of haemocytes expressing penaeidin in
stimulated shrimp tissues appeared to be restored (Fig. 8C,
F and I) and was similar to that observed in nonstimulated
animals (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.
Ó FEBS 2002 Antimicrobial peptide expression in shrimp (Eur. J. Biochem. 269) 2683
Similar observations were obtained with ICC analyses
relative to the distribution of penaeidin-stained haemocytes
within tissues and following microbial challenge (data not
shown).
Haemocyte recruitment and penaeidin localization
at the site of injection
Injection of microorganisms resulted in a dramatic decrease
in numbers of both circulating and tissue infiltrating
haemocytes within 3 h of injection. To study haemocyte
behaviour and changes, sections of the last abdominal
segments (site of injection) were analysed at 3, 6 and 72 h by
ICC and ISH. Both penaeidin-producing haemocytes and
released peptides were therefore localized. Concerning
peptide detection and distribution as studied by ICC, the
last abdominal segment of untreated animals appeared
totally devoid of immunoreactivity (Fig. 9A). Three h
post-challenge, some penaeidin-positive haemocytes were
observed together with a slight spread of penaeidin
immunostaining near the injection site. However, 6 h
post-stimulation, an increased number of haemocytes
containing penaeidins was seen not only around the
injection sites, but also on surrounding subcuticular epithe-
lia. Strong penaeidin immunoreactivity was detected around

the injection site revealing the presence of released peptides
and their binding to cuticular surfaces close to the injection
site (Fig. 9B). Such reactivities were observed up to 72 h
post-injection, with an increasing number of penaeidin-
positive haemocytes and an accumulation of free peptides
into the muscle around the site of injection (Fig. 9C).
Concerning penaeidin expression, an accumulation of
infiltrating haemocytes containing penaeidin transcripts
began also to be seen around the injection site 3 h after
stimulation (data not shown). At 6 and 72 h, a high
concentration of penaeidin-positive haemocytes was
reached around the site of injury when very few positive
haemocytes were observed in the muscle of nonstimulated
animals 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 around
the site of injection confirmed the importance of haemocytic
reactions 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 [
35
S]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 arbitrary
3
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
35
S-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.

2684 M. Mun
˜
oz et al. (Eur. J. Biochem. 269) Ó FEBS 2002
strong penaeidin reactivity observed at the site of injury did
not allow investigation of the close interaction between the
haemocytes and the microorganisms and the role of
penaeidins in these reactions. To address this question,
in vitro analyses were performed. Haemocytes were incuba-
ted in the presence of the bacteria V. alginolyticus,andthen
treated at 1, 3, 5, 10, 20, 30, 45 and 60 min. incubation for
electron microscopy examination and penaeidin immuno-
detection (immunogold labelling). Observations of control
haemocytes (t
0
) confirmed penaeidin localization into
cytoplasmic granules of granular haemocytes (Fig. 10A).
Haemocytes without granules or with only a few small
granules, termed hyaline cells, presented no penaeidin
immunoreactivity as described previously [14]. In cell
preparations exposed to bacteria for 5 min, haemocytes
with granules showed slight penaeidin immunoreactivity in
the cytoplasm, when deformations of their cytoplasmic
granules began to be observed (Fig. 10B, C). At the same
incubation time with bacteria (5 min), extracellular bacteria
were 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) following microbial injection, were hybridized successively with pen-3a and 18 S
rRNA
32
P-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
STORM
TM
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.
Ó FEBS 2002 Antimicrobial peptide expression in shrimp (Eur. J. Biochem. 269) 2685
(Fig. 10C, D). In haemocytes incubated with bacteria for
10 min, most of the granules showed gross deformation,
such as a lost of round shape and electron density, and
retraction 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 was
found in these experiments. After 20 min incubation,

extracellular penaeidin-immunoreactive bacteria were seen
in the preparation (Fig. 10G, H). Regarding phagocytosis
reactions, internalized bacteria were observed after 20 min
incubation mainly into hyaline haemocytes (Fig. 10I).
Intracellular phagocytosed bacteria observed in hyaline
cells 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 released
penaeidins before their internalization. To a lesser extent,
phagocytosed bacteria were also observed into some
granular haemocytes in which neither intracellular lysis of
their granules nor fusion of granules with phagocytic
vacuoles were seen (Fig. 10K). At the same time, a large
number of haemocytes with degenerated cytoplasm and
nuclei was observed revealing that a phenomenon of lysis
has occurred in response to Vibrio contact (Fig. 10L, M).
After 45 min incubation, degenerative haemocytes within
the preparation were predominant.
DISCUSSION
Through investigations on penaeidin expression, the aims of
the present study were to define shrimp defence mechanisms
in response to microbial infections. We applied in vivo
experimental infection model in the shrimp P. vannamei to
analyse the expression, regulation and production of
penaeidins in circulating haemocytes and tissues of the
animals.
We previously showed that penaeidins are constitutively
produced and stored in granular haemocytes of shrimps
that have not been experimentally infected, indicating
haemocytes as the main site of production of the peptides

[14]. Here, we show that in shrimp tissues, the distribution of
penaeidin transcripts and proteins is restricted to haemo-
cytes either circulating in blood vessels irrigating tissues such
as the brain, hepatopancreas or gills, or infiltrating tissues
such as subcuticular epithelia or midgut caecum. Penaeidins
are solely present in large-granule haemocytes and small-
granule haemocytes (also called semigranular cells), and are
absent from the hyaline haemocyte population, devoid of
granules. In the haematopoietic tissues, penaeidin tran-
scripts were clearly visible in a few cells, showing that
penaeidin expression occurs in this tissue. This result differs
from those obtained in crayfish where the haematopoietic
tissue was found to be negative for prophenoloxidase [20], a
gene that is expressed in circulating haemocytes [21]. The
haematopoietic tissues have been described in crustacean
species [22,23] but knowledge of the haematopoietic process
remains limited and few data are available on the expression
of immune effectors during haemocyte differentiation and
maturation. Our observations suggest that penaeidins are
expressed either by maturating stem cells or by haemocytes
early before leaving the haematopoietic tissues. However, it
cannot be excluded that circulating haemocytes expressing
penaeidins may return to infiltrate this tissue for some
signalling reaction.
In invertebrates, little is known about the regulation and
expression of antimicrobial peptide encoding genes during
the immune response, apart from insects where transcrip-
tion is induced in fat body cells and surface epithelia and for
which 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).
Thelastabdominalsegmentofanunchal-
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).
2686 M. Mun
˜
oz et al. (Eur. J. Biochem. 269) Ó FEBS 2002
peptide expression are particularly well characterized [2,24].
In the bivalve mollusc, Mytilus galloprovincialis, antimicro-
bial peptides are constitutively expressed and stored in
phagocytic haemocytes where they participate in the
destruction of engulfed microorganisms [6]. In Limulus,
upon microbial stimulation, antimicrobial peptides are
released from haemocytes by regulated exocytosis [25]. In

shrimp, as previously shown [14], microbial challenge results
in a dramatic drop of penaeidin mRNA concentration
(relative to 18 S rRNA) in circulating haemocytes in the
early hours post-injection with a return to initial levels at
48 h. However, at 72 h post-injection, penaeidin transcript
concentration appears to be threefold higher than that
observed in unchallenged shrimp. Similar kinetics (a
decrease followed by a significant increase) has been
observed in the total number of circulating haemocytes as
the result of microbial challenge, a phenomenon already
described 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 first
12 h post-challenge, haemocytes constitutively produce
penaeidin mRNA and protein. The decrease of penaeidin
detection within total circulating populations is the result of
a decrease in penaeidin-expressing haemocytes: they leave
the blood circulation and most of the shrimp tissues and
migrate towards injured tissues.
2
This is in agreement with
previous studies on other crustacean species [28]. Massive
accumulation of penaeidin-producing haemocytes was seen
around the site of injection 6 h post-injection, as well as a
massive release of penaeidin which spread into muscle tissue
around the injection site, as a local antimicrobial response.
As previously demonstrated, penaeidins are released, upon
stimulation, from haemocytes into haemolymph where their

concentration increases; subsequently, they bind to cuticle
surfaces [14].
During the second phase, at about 48–72 h post-chal-
lenge, intense penaeidin-labelling is observed in the tissues
surrounding injection site as well as on subcuticular surfaces.
Moreover, haemocytes displaying high transcriptional
activity 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 (t
0
), 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.
Ó FEBS 2002 Antimicrobial peptide expression in shrimp (Eur. J. Biochem. 269) 2687
thedramaticincreaseofrRNA18Sconcentration[29]
together with an increased penaeidin expression activity.
Actually, measurement of penaeidin mRNA content using
radiolabelled probes on haemocytes collected at different
times post-infection stressed the gradual increase in penaei-
din transcriptional activity of circulating haemocytes, much
greater than that observed in unchallenged shrimp. Thus, we
assume that penaeidin up-regulation in circulating haemo-

cytes reflects an induced proliferation process, similar to
results obtained in P. japonicus. In this species, an increase
in the proliferation rate of circulating haemocytes as a result
of in vivo experimental infection with Fusarium was shown
by flow cytometry [30]. At 72 h post-stimulation, transcrip-
tionally active, young or maturating haemocyte forms, but
which are comparatively penaeidin-poor, are probably
intensively produced and released precociously from hae-
matopoietic tissues. Such a phenomenon has already been
been proposed for Syciona ingentis during the moulting
cycle and after bacterial injection [27,31]. Concomitantly, as
a result of this proliferative process, a dramatic invasion of
haemocyte producing penaeidin mRNA and protein is seen
in most of the tissues, indicating a systemic reaction. An
intense proliferation process may occur: (a) to amplify
haemocytic reactions and subsequently to increase penaei-
din representativeness within shrimp tissues, together with
other immune cellular effectors; (b) to replace into the blood
circulation and infected tissues lysed or dead haemocytes
subsequent to microbial challenge [32].
The strong immunoreactivity observed at the site of
microbial injection precluded both clarification of the
mechanisms of penaeidin release from haemocytes, and
determination of any potential involvement of penaeidin in
the elimination of microorganisms via phagocytosis. To
further address these questions haemocytes were challenged
with Vibrio in vitro. Regarding penaeidin release, there was
no indication of degranulation of granule-containing
penaeidin, or any migration of granules towards the cell
periphery, in contrast with regulated exocytosis reported in

Tachypleus [33]. Penaeidin containing haemocytes showed
striking changes in the shape and morphology of their
granules, suggesting a possible release of granule content
within the haemocyte cytoplasm. This quite original
phenomenon appears to be followed by the lysis of the
haemocytes and the release of cytoplasm content, as
suggested by the later appearance in the preparation of
numerous ghost cells and penaeidin immunoreactivity in
preparation extracellular spaces. In crustaceans, the phe-
nomenon of lysis has been reported and attributed to
hyaline cells thought to be involved in triggering the
coagulation process [22]. Such a reaction was also evidenced
here with the observation of coagulum (coagulated mater-
ial) surrounding haemocytes and suggested by the presence
of ghost cells. In this coagulum, released penaeidins could be
trapped with other immune effectors originating also from
haemocytes, such as components of the prophenoloxidase
system [34].
Regarding phagocytotic activity, in the present study
hyaline cells appear to be the most active phagocytic cells
whereas penaeidin-positive cells with large-granules are
minimally phagocytic and internalized bacteria are observed
late in this cell population. These results give new insights
into the identification of haemocyte types and their
respective function in crustaceans. Indeed, in crab and
crayfish, using haemocytes previously separated on Percoll
gradient, hyaline cells were considered to be primary
phagocytic cells [35,36]. However, in penaeid shrimp,
granular cells including large- and small-granule haemo-
cytes were described to be active in phagocytosis and to

contain lysosomal enzymes and prophenoloxidase activity
[22,37]. There is no model or classification scheme that is
applicable to all decapods, and different interpretations may
also result from the variety of experimental approaches used
in these studies. Further analyses based on expression of
immune effectors, both transcripts and proteins, as carried
out here with penaeidins, will be of great benefit to clarify
haemocyte lineage and identification of cell types as well as
their functions in immune response. Our data suggest that
different populations of granular haemocytes may exist:
(a) one population involved in a phenomenon of lysis with a
massive and early release of penaeidins; and (b) another
population involved in phagocytosis of bacteria taking place
late than hyaline cell phagocytosis. No evidence for
discharge of granular penaeidin content into bacteria-
containing phagosomes has been observed in shrimp, as
demonstrated in human neutrophils for defensins [38] or in
mussel haemocytes for mytilins [17]. The question remains
about the function of these intracellular penaeidins and their
potential involvement in the elimination of internalized
microbes. It is attractive to assume that these two popula-
tions of penaeidin-positive haemocytes can contain different
classes of penaeidins with various functions, which are
impossible to discriminate with the tools available today.
In conclusion, the expression and distribution of penaei-
dins in response to microbial challenge are regulated
through haemocyte reactions and haemocyte proliferation
processes. Penaeidins may be involved in local defence
reaction through their release by haemocytes and binding to
shrimp cuticle surfaces. By their antimicrobial activities

against Gram-positive bacteria and fungi [19], penaeidins
may protect tissues from infections and/or participate in
wound healing process. Penaeidins do not display strong
antimicrobial activity against Gram-negative bacteria such
as Vibrio sp. but they can contribute to their elimination by
phagocytic cells by a potential opsonic function. Indeed,
extracellular bacteria as well as internalized phagocytosed
bacteria were seen to be immunoreactive to penaeidins.
These observations argue in favour of a coating, by released
penaeidin, of the bacteria before their internalization into
penaeidin-devoid hyaline cells. Finally, the diversity, the
large distribution and abundance of penaeidins which are
produced in shrimp, together with their multiple and
complementary properties, reveal that the penaeidin family
constitutes a major component of the shrimp immune
system, which should be investigated further.
ACKNOWLEDGEMENTS
The authors thank J.C. Beauvillain and V. Mitchell for access to the
Cellular Imaging Center of the IFR 22 (Institut Fe
´
de
´
ratif de Recherche
22, Faculte
´
de Me
´
decine Lille). This study is supported by the CNRS
(Centre National de la Recherche Scientifique), the IFREMER (Institut
Franc¸ ais 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).
2688 M. Mun
˜
oz et al. (Eur. J. Biochem. 269) Ó FEBS 2002
REFERENCES
1. Bulet, P., He
´
tru, C., Dimarcq, J L. & Hoffmann, D. (1999)
Antimicrobial peptides in insects; structure and function. Dev.
Comp. Immunol. 23, 329–344.
2. Tzou, P., Ohresser, S., Ferrandon, D., Capovilla, M., Reichhart,
J.M., Lemaitre, B., Hoffmann, J.A. & Imler, J.L. (2000) Tissue-
specific inducible expression of antimicrobial peptide genes in
Drosophila surface epithelia. Immunity 13, 737–748.
3. Ganz, T. & Lehrer, R.I. (1994) Defensins. Curr. Opin. Immunol. 6,
584–589.
4. Lee,I.H.,Zhao,C.,Cho,Y.,Harwig,S.S.,Cooper,E.L.&Lehrer,
R.I. (1997) Clavanins, alpha-helical antimicrobial peptides from
tunicate hemocytes. FEBS Lett. 400, 158–162.
5. Iwanaga, S., Kawabata, S I. & Muta, T. (1998) New types of
clotting factors and defense molecules found in Horseshoe Crab
hemolymph: Their structures and functions. J. Biochem. 123, 1–15.
6. Mitta, G., Vandenbulcke, F. & Roch, P. (2000b) Original
involvement of antimicrobial peptides in mussel innate immunity.
FEBS Lett. 486, 185–190.
7. Ouellette, A. & Selsted, E. (1996) Paneth cell defensins:
endogenous peptide components of intestinal host defense.

FASEB J. 10, 1280–1289.
8. Schroder, J.M. (1999) Epithelial antimicrobial peptides: innate
local host response elements. Cell Mol. Life Sci. 56, 32–46.
9. Hoffmann, J.A. & Reichhart, J M. (1997) Drosophila immunity.
Trends Cell Biol. 7, 309–316.
10. Mitta, G., Vandenbulcke, F., Hubert, F. & Roch, P. (1999) Mussel
defensins are synthesised and processed in granulocytes then
released into the plasma after bacterial challenge. J. Cell Sci. 112,
4233–4242.
11. Hancock, R.E. & Scott, M.G. (2000) The role of antimicrobial
peptides in animal defenses. Proc. Natl Acad. Sci. USA 97, 8856–
8861.
12. Hoffmann,J.A.,Kafatos,F.C.,Janeway,C.A.&Ezekowitz,R.A.
(1999) Phylogenetic perspectives in innate immunity. Science 284,
1313–1318.
13. Destoumieux, D., Bulet, P., Loew, D., Van Dorsselaer, A.,
Rodriguez,J.&Bache
`
re, E. (1997) Penaeidins: a new family of
antimicrobial peptides in the shrimp Penaeus vannamei
(Decapoda). J. Biol. Chem. 272, 28398–28406.
14. Destoumieux, D., Munoz, M., Cosseau, C., Rodriguez, J., Bulet,
P.,Comps,M.&Bache
`
re, E. (2000) Penaeidins, antimicrobial
peptides with chitin-binding activity, are produced and stored in
shrimp granuloccytes and released after microbial challenge. J. Cell
Sci. 113, 461–469.
15. Bache
`

re, E., Destoumieux, D. & Bulet, P. (2000) Penaeidins,
antimicrobial peptides of shrimp: a comparison with other effec-
tors of innate immunity. Aquaculture 191, 71–88.
16.Gross,P.S.,Bartlett,T.C.,Browdy,C.L.,Chapman,R.W.&
Warr, G.W. (2001) Immune gene discovery by expressed sequence
tag analysis of hemocytes and hepatopancreas in the Pacific White
Shrimp, Litopenaeus vannamei, and the Atlantic White Shrimp,
L.setiferus. Dev. Comp. Immunol. 25, 565–577.
17. Mitta, G., Vandenbulcke, F., Noel, T., Romestand, B., Beauvil-
lain, J.C., Salzet, M. & Roch, P. (2000) Differential distribution
and defence involvement of antimicrobial peptides in mussel.
J. Cell Sci. 113, 2759–2769.
18. Faure-Virelizier, C., Croix, D., Bouret, S., Prevot, V., Reig, S.,
Beauvillain, J.C. & Mitchell, V. (1998) Effects of estrous cyclicity
on the expression of the galanin receptor Gal-R1 in the rat pre-
optic area: a comparison with the male. Endocrinology 139, 4127–
4139.
19. Destoumieux, D., Bulet, P., Strub, J M. & Bache
`
re, E. (1999)
Recombinant expression and range of activity of penaeidins,
antimicrobial peptides from penaeid shrimp. Eur. J. Biochem. 266,
335–346.
20. Johansson, M.W., Keyser, P., Sritunyalucksana, K. & So
¨
derha
¨
ll,
K. (2000) Haemocytes and haematopoiesis. Aquaculture 191,
45–52.

21. Sritunyalucksana, K., Cerenius, L. & Soderhall, K. (1999) Mole-
cular cloning and characterization of prophenoloxidase in the
black tiger shrimp, Penaeus monodon. Dev. Comp. Immunol. 23,
179–186.
22. Martin, G.G. & Hose, J.E. (1992) Vascular elements and blood
(hemolymph). In Microscopic Anatomy of Invertebrates,Vol.10.
F.W. Harrison & A.G. Hurnes, eds. John Wiley & Sons,
New York, 117–146.
23. Chaga,O.,Lignell,M.&So
¨
derha
¨
ll, K. (1995) The haemopoietic
cells of the freshwater crayfish, Pacifastacus leniusculus. Anim.
Biol. 4, 59–70.
24. Imler, J.L. & Hoffmann, J.A. (2000) Signaling mechanisms in the
antimicrobial host defense of Drosophila. Curr. Opin. Microbiol. 3,
16–22.
25. Iwanaga, S. & Kawabata, S I. (1998) Evolution and phylogeny of
defense molecules associated with innate immunity in horseshoe
crab. Front. Biosci. 3, D973–D984.
26. Smith, V.J. & Ratcliffe, N.A. (1980) Cellular defense reactions of
the shore crab, Carcinus maenas: in vivo hemocytic and histo-
pathological responses to injected bacteria. J. Invertebr. Pathol. 35,
65–74.
27. Martin, G.G., Poole, D., Poole, C., Hose, J.E., Arias, M.,
Reynolds, L., McKrell, N. & Whang, A. (1993) Clearance of
bacteria injected into the hemolymph of the penaeid shrimp,
Sicyonia ingentis. J. Invert. Pathol. 62, 308–315.
28. Martin, G.G., Kay, J., Poole, D. & Poole, C. (1998) In vitro nodule

formation in the ridgeback prawn, Sycionia ingentis,andthe
american lobster, Homarus americanus. J. Invert. Pathol. 117,
155–168.
29. Hansen, M.C., Nielsen, A.K., Molin, S., Hammer, K. & Kilstrup,
M. (2001) Changes in rRNA levels during stress invalidates results
from mRNA blotting: fluorescence in situ rRNA hybridization
permits renormalization for estimation of cellular mRNA levels.
J. Bacteriol. 183, 4747–4751.
30. Sequeira, T., Tavares, D. & Arala-Chaves, M. (1996) Evidence for
circulating hemocyte proliferation in the shrimp Penaeus japoni-
cus. Dev. Comp. Immunol. 20, 97–104.
31. Hose, J.E., Martin, G.G., Tiu, S. & McKrell, N. (1992) Patterns of
hemocyte production and release throughout the molt cycle in the
penaeid shrimp Sicyonia ingentis. Biol. Bull. 183, 185–199.
32. Omori, S.A., Martin, G.G. & Hose, J.E. (1989) Morphology of
hemocyte lysis and clotting in the ridgeback prawn, Sicyonia
ingentis. Cell Tissue Res. 255, 117–123.
33. Toh, Y., Mizutani, A., Tokunaga, F., Muta, T. & Iwanaga, S.
(1991) Morphology of the granular hemocytes of the Japanese
horseshoe crab Tachypleus tridentatus and immunocytochemical
localization of clotting factors and antimicrobial substances. Cell
Tissue Res. 266, 137–147.
34. Sritunyalucksana, K. & So
¨
derha
¨
ll, K. (2000) The proPO and
clotting systems in crustaceans. Aquaculture. 191, 53–69.
35. Bell, K.L. & Smith, V.J. (1993) In vitro superoxide production by
hyaline cells of the shore crab Carcinus maenas (L.). Dev. Comp.

Immunol. 17, 211–219.
36. Thornqvist, P.O., Johansson, M.W. & So
¨
derha
¨
ll, K. (1994)
Opsonic activity of cell adhesion proteins and beta-1–3-glucan
binding proteins from two crustaceans. Dev. Comp. Immunol. 18,
3–12.
37. Gargioni, R. & Barracco, M.A. (1998) Hemocytes of the palae-
monids Macrobrachium rosenbergii and M. acanthurus,andofthe
penaeid Penaeus paulensis. J. Morph. 236, 209–221.
38. Joiner, K.A., Ganz, T., Albert, J. & Rotrosen, D. (1989) The
opsonizing ligand on Salmonella typhimurium influences
incorporation of specific, but not azurophil, granule constituents
into neutrophil phagosomes. J. Cell Biol. 109, 2771–2782.
Ó FEBS 2002 Antimicrobial peptide expression in shrimp (Eur. J. Biochem. 269) 2689