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

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

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

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

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

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

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-

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

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-

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

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

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

> 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-

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

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.

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,

[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

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

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

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

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
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