Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/devcompimm
The role of hem
Acanthoscurria
Aline H. Fukuzawaa,
Pedro I. Silva Jrc, Re
ogia,
ulo, B
Celula
4, CEP
Aplica
logia,
2, 61 a
Received 11 October 2007; received in revised form 5 November 2007; accepted 5 November 2007
Coagulation show that 57% of the hemocytes store both gomesin and acanthoscurrin, either in the same
ARTICLE IN PRESS
Developmental and Comparative Immunology (2008) 32, 716–7250145-305X/$ - see front matter & 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dci.2007.11.002
Corresponding author. Tel.: +55 11 30917272; fax: +55 11 30917417.
E-mail address: sidaffre@icb.usp.br (S. Daffre).
1Present address: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonc-alves, 9500, CEP 91501-970 Porto
Alegre, Brazil.or in different granules. Progomesin labeling in hemocyte granules indicates that gomesin
is addressed to those organelles as a propeptide. In vivo and in vitro experiments showed
that lipopolysaccharide (LPS) and yeast caused the hemocytes to migrate. Once they have
reached the infection site, hemocytes may secrete coagulation cascade components and
AMPs to cell-free hemolymph. Furthermore, our results suggest that phagocytosis is not
the major defense mechanism activated after microbial challenge. Therefore, the main
reactions involved in the spider immune defense might be coagulation and AMP secretion.
& 2007 Elsevier Ltd. All rights reserved.Available online 3 December 2007
KEYWORDS
Spider;
Immunity;
Hemocytes;
Antimicrobial
peptides;
Secretion;
Abstract
Invertebrates protect themselves against microbial infection through cellular and humoral
immune defenses. Since the available information on the immune system of spiders is
scarce, the main goal of the present study was to investigate the role of hemocytes and
antimicrobial peptides (AMPs) in defense against microbes of spider Acanthoscurria
gomesiana. We previously described the purification and characterization of two AMPs
from the hemocytes of naı¨ve spider A. gomesiana, gomesin and acanthoscurrin. Here weaDepartamento de Parasitol
1374, CEP 05508-900 Sa˜o Pa
bDepartamento de Biologia
Av. Prof. Lineu Prestes, 152
cLaborato´rio de Toxinologia
dDepartamento de Microbio
Medicina, Rua Botucatu, 86ocytes in the immunity of the spider
gomesiana
Bruno C. Vellutinib, Daniel M. Lorenzinia,1,
nato A. Mortarad, Jose´ M.C. da Silvab, Sirlei Daffrea,
Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes,
razil
r e do Desenvolvimento, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo,
05508-900 Sa˜o Paulo, Brazil
da, Instituto Butantan, Av. Vital Brazil, 1500, CEP 05503-900 Sa˜o Paulo, Brazil
Imunologia e Parasitologia, Universidade Federal de Sa˜o Paulo—Escola Paulista de
ndar, CEP 04023-062 Sa˜o Paulo, Brazil

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Role of hemocytes in the immunity of the spider 7171. Introduction
Invertebrates are constantly exposed to microbial infec-
tions, and to protect themselves they have developed
powerful immune defense mechanisms similar to those
innate to vertebrates. These mechanisms involve cellular
and humoral responses. The former consists of encapsula-
tion, nodulation, and phagocytosis of microbes by hemo-
cytes, while humoral response comprises factors related to
the recognition of invading microorganisms, serine protease
cascades participating in melanization and coagulation, and
killing factors such as antimicrobial peptides (AMPs),
reactive oxygen species, and reactive nitrogen intermedi-
ates, including nitric oxide [1]. Over 1000 AMPs produced by
multicellular organisms (plants, invertebrates, and verte-
brates) have been isolated and characterized at the primary
structure level (http://www.bbcm.univ.trieste.it/tossi/
pag1.htm) [2–4].
Several AMPs are synthesized as large precursors. In some
cases co-translational removal of signal peptides releases the
active molecule, but in others one or more anionic propieces
are also commonly removed during proteolytic processing [5].
Depending on the organism and tissue considered, AMPs are
either constitutively stored within secretory cells, or their
synthesis is induced at the time of infection. In most insects,
AMP synthesis starts a few hours after an infection has
occurred. In other invertebrates, such as horseshoe crabs,
mussels, and shrimps, AMPs are constitutively produced and
stored in hemocyte granules [6–9].
AMPs stored in cell granules interact with and eliminate
the invading organisms. Studies on invertebrates have
demonstrated that this can be achieved by two different
processes: (i) fusion of the granules where AMPs are stored
within phagosomes [10], and/or (ii) release of these AMPs
into the plasma by exocytosis [11,12]. Similar mechanisms
are found in vertebrates [13–15].
Previously we reported on the purification and character-
ization of two AMPs, acanthoscurrin and gomesin, from the
hemocytes of naı¨ve mygalomorph spider Acanthoscurria
gomesiana [16,17]. In addition, an acylpolyamine isolated
from hemocytes of the same spider species presented
antibacterial activity [18].
Acanthoscurrin is a glycine-rich peptide post-translationally
processed by the removal of the signal peptide and C-terminal
amidation. Acanthoscurrin is released into the plasma follow-
ing immune challenge [17]. Gomesin is a small-sized anti-
microbial peptide containing 18 amino acids, including
pyroglutamic acid as the N-terminus, a C-terminal arginine
a-amide, and four cysteine residues forming two disulfide
bridges [16]. This peptide presents a well-defined b-hairpin-
like structure, as determined by 2-D NMR and molecular
dynamics studies [19]. Gomesin presents marked sequence and
structural similarities to horseshoe crab tachyplesin [20,21]
and polyphemusin [22,23], and to scorpion androctonin [24].
Interestingly, gomesin also shares structural similarities with
protegrins, porcine leukocyte AMPs [25,26]. In addition,
gomesin is translated into a prepropeptide presenting
84 amino acid residues that contains a signal peptide with
23 amino acids, an 18 amino acids mature molecule, and a
C-terminal prodomain with 43 amino acid residues. Immuno-
localization studies have demonstrated that mature gomesin is
stored within hemocyte granules [27].Since the literature on the spider immune system is
limited, the main goal of the present study was to
investigate how spiders combat microbes, and the role of
hemocytes and AMPs in this process. We show that gomesin
is addressed to hemocyte granules as a propeptide and
report on the relative distribution of gomesin and acanthos-
currin in hemocytes by double-immunolocalization on
confocal microscopy. We demonstrate by means of in vivo
and in vitro experiments that hemocytes may migrate to the
microbial infection site. Once they have reached the site of
infection, hemocytes may secrete coagulation cascade
components and AMPs. Furthermore, our results suggest
that phagocytosis is not the major defense mechanism
activated after microbial challenge.
2. Material and methods
2.1. Animals and sample collection
The common spider (A. gomesiana) was obtained from
Instituto Butantan (Sa˜o Paulo, Brazil). The hemolymph was
collected from pre-chilled animals by cardiac puncture with
an apyrogenic syringe. To avoid hemocyte degranulation and
coagulation, the hemolymph was collected in the presence
of sodium citrate buffer, pH 4.6 (2:1; v:v) [28]. The
hemocytes were removed from plasma by centrifugation at
800g for 10min at 4 1C.
2.2. Production and purification of the C-terminal
prodomain of gomesin
To obtain the peptide corresponding to the C-terminal
domain, PCR was performed using the cDNA of gomesin [27]
as a template with 50 primer (AGT TTA GAT GAG ACC) and 30
primer (CTT AGT CGA AAA TAA). The PCR product was
digested with BamHI and EcoRI for subsequent cloning.
To express the recombinant C-terminal prodomain,
the PCR product was inserted into the vector pGEX-2T
(Amersham Biosciences). This vector produces recombinant
protein in fusion with glutathione S-transferase (GST). The
construct was inserted into Escherichia coli DH5a, which was
maintained in LB containing 100 mg/mL ampicillin. To induce
protein production, 0.4mM IPTG was added to the culture
medium, kept at 37 1C for 3 h, and centrifuged for 10min at
12,000g. The cells were resuspended in phosphate-buffered
saline (PBS) and disrupted by sonication using a Vibra Cell
sonicator (Branson Digital Sonifier, Model 450, USA) at 30W,
5 30 s. After that, Triton-X100 was added to a final
concentration of 1%. The cells were kept under agitation
for 30min. The homogenate was centrifuged at 12,000g for
10min at 4 1C and the recombinant protein was purified from
the supernatant.
The C-terminal prodomain was purified using a Glu-
tathione Sepharose 4B (Amersham Biosciences) column and
eluted with 50mM Tris–HCl, pH 8.0, containing 10mM
reduced glutathione. The C-terminal prodomain was cleaved
from GSTwith thrombin (Amersham Biosciences) for 16 h at
22 1C following the manufacturer’s instructions. After
cleavage, the proteins were separated by reversed-phase
chromatography on an analytical C18 (Vydac
TM, 300 Å, 5mm,
4.6mm 250mm) column equilibrated with 2% acetonitrile

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A.H. Fukuzawa et al.718(ACN) in acidified water (0.046% trifluoroacetic acid (TFA)).
Elution was performed with a linear 2–95% ACN gradient in
acidified water over 60min at a flow rate of 1.0mL/min.
HPLC purification was carried out at room temperature on
a Shimadzu LC-10 HPLC system with a Shimadzu diode array
detector (SPD-M10AV). The column effluent was monitored
for absorbance at 225 nm. Fractions corresponding to
absorbance peaks were hand-collected, concentrated in a
vacuum centrifuge (Savant Instruments, Inc.), and recon-
stituted in ultrapure (Milli-Q) water.
The presence of the C-terminal prodomain in the
chromatographic fractions was determined by electrospray
ionization-mass spectrometry (ESI-MS) on a Finnigan
LCQTMDuo mass spectrometer (ThermoQuest, EUA) under
the same previous conditions [17].
2.3. Antiserum production
2.3.1. Anti-acanthoscurrin
Three BALB/c mice were intraperitoneally immunized with
100 mL of PBS containing 35 mg of acanthoscurrin purified as
previously described [17] and emulsified with Lipid A from
Bordetella pertussis (Almeida, I.C.; unpublished data). The
first immunization was followed by two boosts at 2-week
intervals. Antiserum was collected 15 days after the last
injection.
2.3.2. Anti-C-terminal prodomain
The C-terminal prodomain of gomesin was coupled to bovine
serum albumin (BSA) with reagent 1-ethyl-3-(3-dimethyla-
minopropyl) carbodiimide hydrochloride (EDC, Pierce Bio-
technology) following the manufacturer’s protocol. Three
BALB/c mice were intraperitoneally immunized with 100 mL
of PBS containing 30 mg of the C-terminal prodomain
emulsified in complete Freund adjuvant. The first immuni-
zation was followed by two boosts at 2-week intervals with
aliquots emulsified in incomplete Freund adjuvant. Antiser-
um was collected 15 days after the last injection. The
C-terminal prodomain concentration was calculated with
the standard curve obtained from absorbance at 255 nm of
phenylalanine at known concentrations.
2.3.3. Anti-gomesin
The anti-gomesin antibody was obtained from a rabbit as
described in a previous study [27].
2.4. Immunocytochemistry
Hemocytes were resuspended and fixed in a sodium citrate
buffer [28] containing 3.7% formaldehyde for 15min on ice.
The hemocytes were transferred to poly-L-lysine-coated
glass slides and stored at 20 1C. The slides were incubated
for 10min in 25mM Tris–HCl, pH 7, containing 50mM
ammonium chloride, 0.2% gelatin, and 0.5% Triton X-100.
After extensive wash with PBS containing 0.1% Tween-20
(PBS-T), the slides were incubated for 1 h at room
temperature with 5% gelatin/PBS-T. Next, they were washed
again with PBS-T and incubated for 1 h at room temperature
in 5% gelatin/PBS-T with either rabbit anti-gomesin anti-
bodies (1:500), or mouse anti-acanthoscurrin antiserum
(1:200), or mouse anti-C-terminal prodomain antiserum(1:200). Control experiments were performed using either
mouse or rabbit pre-immune antiserum. Following another
washing step, the slides were incubated for 20min with
either 1:500 Alexa Fluor 488 conjugated anti-rabbit IgG
(Molecular Probes) or 1:200 Rhodamine conjugated anti-
mouse IgG (Molecular Probes), both diluted in 5% gelatin/
PBS-T. Finally, the slides were washed with PBS-T, incubated
with 10mM DAPI for 1min, and washed with PBS-T. Samples
were imaged on a BioRad 1024-UV confocal system attached
to a Zeiss Axiovert 100 microscope, using a 63 N.A. 1.4
Plan-Apochromatic (with Nomarski Differential Interference
Contrast optics) oil immersion objective.
2.5. In vivo immune challenge
For experimental immune challenge, 50 mL of fluorescein
isothiocyanate (FITC) coupled latex beads (105 particles)
were injected into the spider legs. Twenty-four hours later,
the legs were dissected and the cuticle was carefully
removed. Muscle fiber slides were analyzed under a
fluorescence microscope (Axiophot Zeiss). In other experi-
ment, 50 mL of yeast Saccharomyces cereviseae (103 cells)
resuspended in PBS were injected into the spider heart.
Three hours later, 50 mL of ice-cold 2.5% (w/v) glutaralde-
hyde in 0.1M cacodylate buffer, pH 7.4, was injected into
the heart. After 15min, the heart was dissected and
maintained in a polypropylene tube with the fixative
solution described above for 24 h at 4 1C. The same
procedure was performed with 50 mL of sterile PBS inocu-
lated into the spider heart. Fixed hearts were longitudinally
divided and processed for optical and electron microscopy
analysis.
2.6. Optical microscopy
Fixed hearts were included into a Leica resin, following the
manufacturer’s protocol. Sections of 1mm were placed onto
glass slides and stained by the May–Grunwald–Giemsa
method (modified Giemsa) [29]. Slides were examined in a
Zeiss Axiophot microscope.
2.7. Transmission electron microscopy (TEM)
Fixed hearts were post-fixed in 1.0% OsO4 in ultrapure water
and dehydrated prior to Spurr resin embedding [30].
Ultrathin sections of 70 nm were gathered onto copper grids
and stained with 0.5% uranyl acetate in ultrapure water for
5min, washed in ultrapure water, and stained again in 0.5%
lead citrate in ultrapure water. Ultrastructure was examined
on a JEOL 100 CX-II electron microscope.
2.8. Migration assay
The migration ability of hemocytes was measured in a 3-mm
Transwell filter coupled to a 24-well plate (Corning).
Hemolymph was diluted in 100 mL of serum-free modified
L15 medium [31] to a final concentration of 105 cells per
well. The hemocyte suspension was placed into the upper
chamber. Lipopolysaccharide (LPS) (1 mg/mL), yeast in a
proportion of 5:1 (yeast: hemocyte) or 10 mM gomesin was

quantified using a standard curve with different concentra-
granules [27]. Herein, the distributions of gomesin and
either rabbit or mouse pre-immune serum, respectively.
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Role of hemocytes in the immunity of the spider 719placed into the lower chamber as chemoattractant. The
preparation was kept at 37 1C for 5 h. Cells that did not
penetrate the filter were wiped out with cotton swabs, and
the cells that migrated to the filter’s lower surface were
fixed with 3.7% paraformaldehyde for 15min at 37 1C. After
washing with PBS, the cells were stained with 0.1% toluidine
blue. The filters were extensively washed with ultrapure
water and incubated with 150 mL of 1% SDS for 1 h at 37 1C to
promote cell lyses. After that, 100 mL of the SDS solution was
collected and distributed in a 96-well plate and absorbance
was determined at 630 nm. Two independent experiments
were performed (groups A and B), one in duplicate and the
other in triplicate. The experimental absorbance was
corrected through the equation: Corrected value ¼ (Experi-
mental absorbanceAverage absorbance of non-treated
samples from either group A or group B)/standard deviation
of non-treated samples from either group A or B. The
average of the corrected value and the corresponding
standard mean error (SME) were plotted. The differences
between treatments and control were statistically analyzed
by Student’s t-test for independent samples using the
software SPSS for Windows. Differences were considered
significant when Po0.05.
2.9. Phagocytic assay
Hemolymph was diluted in 100 mL of L15-modified medium
[31] supplemented with 20% bovine fetal serum to a
final concentration of 105 cells per well. The hemocyte
suspension was distributed through a 24-well plate. Either
live or heat-killed S. cereviseae (5 105 per well) were
added. After 1 h at 30 1C, the cells were fixed with PBS
containing 3.7% formaldehyde for 15min on ice and placed
onto slides. The preparation was analyzed on a Zeiss
Axiophot fluorescence microscope. Experiments were per-
formed in triplicate.
2.10. Exocytosis assay
Fresh hemolymph was diluted in 100 mL of L15-modified
medium [31] supplemented with 20% bovine fetal serum,
and distributed into a 96-well plate at a concentration of 105
cells per well. Different concentrations of LPS (1, 0.1, 0.05,
0.02, 0.01 mg/mL) were added to the medium. The
preparation was kept at 30 1C for 1 h. The supernatants
were then collected from the wells, pooled, and subjected
to solid phase extraction on Sep-Pak C18 cartridges (Waters
Associates) equilibrated with acidified water. Three step-
wise elutions were performed with 5%, 40%, and 80% ACN in
acidified water. The 40% Sep-Pak fraction was concentrated
in a vacuum centrifuge, reconstituted with 50mM sodium
bicarbonate, pH 9.6, and subjected to ELISA assay.
Three independent experiments were performed. Data are
expressed as means7SME.
2.11. ELISA
The quantity of gomesin in the exocytosed fluid was
determined with polyclonal anti-gomesin [27]. Microtiter
plates were coated with the pre-purified exocytosed Sep-
Pak fraction and incubated at 4 1C for 18 h. The wells wereNo fluorescence was detected in these preparations (results
not shown).
3.3. Cellular-mediated response to immune
challenge
In vivo immune challenge was performed through injection
of fluorescent beads into the spider leg. Twenty-four hours
later, numerous fluorescent beads and hemocytes exhibiting
autofluorescence in blue were visualized at 470 nm. Both
were held in a gelatinous material, which was probably
related to a coagulation response activated by non-self
particles (Figure 3).acanthoscurrin were compared in the same preparations
using Alexa Fluor 488 and rhodamine-conjugated anti-
bodies, respectively, and examined by confocal microscopy
(Figure 2). Both gomesin and acanthoscurrin labeling were
detected in hemocyte granules. Different hemocytes were
positive for only either gomesin (2%) or acanthoscurrin
(33%), but both immune reactivities often appeared within
the same cell (58%). In the latter, merged confocal
microscope images suggested that gomesin and acanthos-
currin could be packed in either different or within the same
cell granule. In addition, 7% of the cells were not labeled for
either of the AMPs. A control experiment was performed
by replacing anti-gomesin and anti-acanthoscurrin withtions of synthetic gomesin, which was produced as pre-
viously described [16].
3. Results
3.1. Localization of gomesin precursor
The translation of gomesin into a prepropeptide raised
our interest to determine the subcellular localization of
gomesin processing. To address this question, the C-terminal
prodomain localization was determined by immunofluores-
cence. The C-terminal prodomain labeling was localized in
hemocyte granules, and, most often, co-localized with anti-
gomesin labeling (Figure 1). This indicates that gomesin is
addressed to the hemocyte granules as a propeptide.
3.2. Comparative distribution of gomesin and
acanthoscurrin in hemocytes
We previously showed that gomesin is stored in hemocyteblocked with 5% of non-fat dry milk in PBS for 1 h at room
temperature. The plates were washed with PBS containing
0.1% Tween-20 and incubated with anti-gomesin (1/500)
diluted in 5% of non-fat dry milk in PBS for 1 h at room
temperature. Following another washing step, horseradish
peroxidase-conjugated goat anti-rabbit (1/1000) (Amersham
Biosciences) was added and the plates were kept at room
temperature for 1 h. After another washing step, the
enzyme activity of horseradish peroxidase was detected
with o-phenylenediamine at 490 nm. Secreted gomesin was

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Figure 1 Intracellular distribution of mature gomesin (green) an
hemocytes. Double immunofluorescence-labeling assay was perform
antibodies and Alexa Fluor 488 conjugated anti-rabbit IgG and Rho
antibodies. Nuclei were DAPI-stained. Merged confocal images indica
hemocyte granules as a propeptide. Bar, 5 mm.
Figure 2 Intracellular distribution of gomesin (green) and acanth
Hemocytes were submitted to anti-gomesin and anti-acanthoscurr
with Alexa Fluor 488 conjugated anti-rabbit IgG and Rhodamine con
Most of the cells (upper panel) presented both gomesin and acan
granules (red or green isolated granules). Bar, 5mm.
Figure 3 Clot formation. FITC-coupled latex beads (105
particles) were injected into the spider leg. The muscle fibers
of the dissected leg were observed by microscopy 24 h later. The
presence of beads (green) and hemocytes exhibiting autofluor-
escence in blue held in gelatinous material indicates the
activation of the coagulation cascade. Bar, 50 mm.
A.H. Fukuzawa et al.720d C-terminal prodomain of gomesin (red) in spider circulating
ed using anti-gomesin and anti-C-terminal prodomain as primary
damine conjugated anti-mouse IgG, respectively, as secondary
te that C-terminal prodomain and mature gomesin are stored inWe also compared heart sections from a yeast-challenged
spider with those of an unchallenged spider. In contrast to
the heart inoculated with saline buffer whose prohemocytes
predominated, the yeast-inoculated heart showed an
increased number of mature hemocytes at the inoculation
site (Figure 4A). This result suggests that these cells present
a migratory response. However, we cannot discard the
possibility that yeast challenge caused an increase in the
differentiation of prohemocytes or a new production of
the hemocytes.
The major morphological difference between prohemo-
cytes and mature hemocytes is the presence of numerous
granules in the latter. These granules present different sizes
and shapes and completely fill the cytoplasm. Non-granular
cells (prohemocytes) predominated in the heart of the
unchallenged spider, while granular cells (mature hemo-
cytes) predominated in circulating hemolymph.
The migratory capacity of hemocytes was confirmed
in vitro as both yeast and LPS stimulated the migration
response, which was significantly different from that of
unstimulated cells (Po0.014 and Po0.013, respectively).
Gomesin did not act as a chemoattractant at the concentra-
tion of 10 mM (Figure 4B).
oscurrin (red) in double-labeled circulating spider hemocytes.
in immunofluorescence assay. Hemocytes were then incubated
jugated anti-mouse IgG, respectively. Nuclei were DAPI-stained.
thoscurrin either in the same (yellow granules) or in different

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Role of hemocytes in the immunity of the spider 721The recruitment of mature hemocytes to the injection
site corroborated the importance of hemocytic reactions in
response to microbial challenge. However, high hemocyte
Figure 4 Migratory response of spider hemocytes. (A) Saccharomy
Three hours later, the heart was dissected and prepared for optical m
PBS-inoculated spider heart. Heart sections of yeast-challenged (lef
first image, most of the hemocytes present granules that fill the cyt
enlarged cell and parts of other two cells are shown in the inset.
prohemocytes without granules with visible nuclei (inset). Our resu
yeast injection site. Bar, 20mm (inset bar, 10 mm). (B) Hemolymph w
plate (upper chamber). LPS, yeast, and gomesin were placed in the
non-treated cells. Cells that migrated to the lower surface of the filt
treated cells was determined at 630 nm. Absorbance was corrected
values and the corresponding standard mean error (SME) were plot
(Po0.014 and Po0.013, respectively). Gomesin did not act as a he
Figure 5 Hemocyte phagocytic activity. Saccharomyces cere-
viseae (103 cells) were inoculated into the spider heart. Three
hours later, the heart was dissected and prepared for TEM
observations. The electron micrograph shows one hemocyte
containing phagocytosed yeast (P), nucleus (N), and granules
(G). Bar, 1 mm.density in the infection site made it difficult to investigate
the interaction between hemocytes and the microorgan-
isms. To address this question, sections of challenged spider
hearts were examined by transmission electron microscopy.
Only 2% of the cells contained internalized yeast (Figure 5).
Hemocyte phagocytic activity was also evaluated by in vitro
experiments. The presence of either live or heat-killed
yeasts in the culture media did not activate phagocytic
response. Some yeast was found in contact with hemocytes,
but none were internalized after 1 h. Moreover, some
hemocytes in these preparations had no cytoplasmic
granules after 30min, which suggests that they had been
secreted. In fact, the secretion of granular contents by
exocytosis was detected by TEM (Figure 6A).
3.4. Gomesin secretion after microbial challenge
Gomesin exocytosis was quantitatively assayed by ELISA
(Figure 6B). Under the assay conditions, LPS induced gomesin
secretion by the hemocytes in a concentration-dependent
ces cereviseae (103 cells) were injected into the spider heart.
icroscopy observation. The same procedure was performed with
t panel) and unchallenged spider (right panel) are shown. In the
oplasm completely, being impossible to observe the nuclei. One
The second image (right) shows one mature hemocyte (arrow)
lts suggest a migratory response from mature hemocytes to the
as distributed into 3-mm Transwell filters coupled to a 24-well
lower chamber as chemoattractants. The control corresponds to
er were stained with 0.1% toluidine blue. The absorbance of SDS-
by the equation described in Section 2.8. The average corrected
ted. Yeast and LPS attracted hemocytes to the lower chamber
mocyte chemoattractant at 10 mM (Po0.646).

concerned with the morphological characterization of
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cha
fro
y EL
ME
A.H. Fukuzawa et al.722hemocytes. Their specific functions are not known; however,
they probably are involved in blood clotting, wound healing
and fighting off infections [32]. Spider hemocytes are
originated from the heart cell wall. Prohemocytes detach
from the hematopoietic tissue and undergo mitotic divisions
until maturation [32]. It was observed that the granulocytesmanner from 0.01 to 1.0mg/mL. The highest amount of
gomesin (160nM) was observed when the hemocytes were
exposed to 1mg/mL of LPS. The hemocytes presented the
same secretory pattern in all three independent experiments
performed.
4. Discussion
The main goal of the present work was to investigate the
involvement of hemocytes in spider immunity. In contrast to
the literature on insects [1], few reports are available on
how spiders mount an immune response. Most reports are
Figure 6 Gomesin secretion by hemocytes. Heart sections of Sac
by TEM. (A) Electron micrograph showing exocytosis of contents
released into the culture media after LPS stimulation measured b
a concentration-dependent manner. Data expressed as means7Sare the most abundant cell type in spiders. They present
numerous dense granules in cytoplasm [32,33]. Some
hemocytes called leberidiocytes have glycogen inclusions.
The increase of these cells number during the moulting
indicated that they might play a role in energy supply during
this process [34]. Cyanocytes are another type of hemocytes
that are responsible for hemocyanin production and storage
[32]. This cell type was firstly described in limulus. Different
from other types of hemocytes, the origin of cyanocytes is
not known [35].
Sherman has classified spider’s hemocytes following the
insect’s nomenclature [33]. He observed sections of a spider
heart by TEM and found at least three types of hemocytes. In
agreement with Foelix [32], granulocytes were the most
common cell type. Plasmatocytoids were quite abundant
while oenocytoids represented only 5% of the hemocytes
population. In addition, some hemocytes appeared to be in
transition from plasmatocytoids to both oenocytoids and
granular hemocytes.
During the development of this work, we observed mostly
granulocytes in circulating hemocytes from the spiderA. gomesiana. In the heart sections, we also observed cells
without cytoplasmatic granules; according to Foelix [32], we
called them prohemocytes. In addition, some hemocytes
presenting crystalline inclusions (cyanocytes) were detected
but they were rarely observed.
Recent work has shown that hemocyanin, the oxygen
carrier in spider plasma, also displays phenoloxidase activity
after limited proteolysis with either trypsin or chymotrypsin
[36]. It is well known that the final product of the
phenoloxidase system is melanin, which is deposited either
onto the surface of the microorganisms or onto a capsule of
microorganisms during the immune response of insects [37]
and crustacea [38].
In previous reports, we described the purification and
characterization of two AMPs from hemocytes of spider
A. gomesiana: gomesin and acanthoscurrin [16,17]. Here we
showed that they are often stored in the same cell (58%).
Nevertheless, they can also be stored in different cells: 33%
of the hemocytes contained only acanthoscurrin and 2% only
gomesin. Two mussel AMPs, defensins and mytilin, are stored
romyces cereviseae (103 cells)-challenged spider were observed
m hemocyte granule (G). Bar, 1mm. (B) The amount of gomesin
ISA showed that LPS induced gomesin secretion by hemocytes in
.either in the same cell or in different ones. Hemocytes that
store only mytilin seem to be involved in phagocytosis, but
the role of hemocytes that store only defensins is not clear.
Hemocytes that present both AMPs are activated later and
might be responsible for AMP secretion [7]. In spiders, the
differences in AMP distribution might be connected with
either the distinct immunity roles played by hemocytes or
differences in the cell maturation stages.
The injection of fluorescent particles into the spider leg
clearly activated a coagulation cascade. A similar response
occurs during hemolymph collection in the absence of an
anticoagulation solution. Besides, except for coagulogen, all
coagulation cascade components from horseshoe crab [39]
are found in the cDNA library of spider hemocytes [40].
Coagulation is a well-conserved response in innate
immunity and it is especially important for invertebrates,
which have open circulatory systems. Generally, in wound
repair it prevents the loss of hemolymph [41]. In horseshoe
crabs, the presence of either LPS or b-1,3 glucan triggers the
serine protease cascade that converts the soluble protein
coagulogen into the insoluble coagulin [6].

ARTICLE IN PRESS
Role of hemocytes in the immunity of the spider 723The migratory response of hemocytes to the yeast
injection site in the spider heart was confirmed by in vitro
experiments, in which LPS and yeast acted as chemoattrac-
tants, whereas 10 mM gomesin did not. In contrast to our
results, human neutrophils can be attracted by human
defensins [42]. Temporin, an AMP found on the skin of the
European frog, also promoted leukocyte chemotaxis in mice
and humans [43].
Sections of yeast-challenged spider heart revealed a very
low percentage of ingested yeast. In vitro experiments
performed with heat-killed and live S. cereviseae and E. coli
did not activate the hemocyte phagocytic response. These
results suggest that phagocytosis is not the major defense
mechanism activated upon microbial challenge. In contrast,
when S. cereviseae was injected into the cattle tick
hemocoel, approximately 20% of the hemocytes internalized
yeast [44]. Interestingly, phagocytosis in horseshoe crab was
only observed after hemocyte incubation with endotoxin-
free iron particles [45].
Exocytosis of contents of hemocytic granules was ob-
served by TEM in heart sections of yeast-challenged spiders.
Moreover, optical microscopy revealed that hemocytes
incubated in vitro with yeast degranulated after 30min. It
was also shown that hemocyte incubation with LPS induced
dose-dependent gomesin secretion. Furthermore, acanthos-
currin was also secreted after in vivo challenge, as
previously demonstrated [17]. In all, these results strongly
indicate that AMP secretion by spider hemocytes and
coagulation might be the major defense mechanisms
activated after infection.
Horseshoe crab hemocytes contain several defense
molecules stored in two types of secretory granules: large
granules and small granules. The former selectively accu-
mulates more than 25 defense components, such as clotting
factors, a clottable protein, coagulogen, proteinase inhibi-
tors, lectins, and antimicrobial proteins. In contrast,
small granules contain at least six AMP and several
proteins o30 kDa [6].
In the case of horseshoe crabs, LPS induces secretion of
100% of the contents of hemocyte granules in less than
20min. Exocytosis is mediated through protein G activation.
This transmembrane receptor activates phospholipase C,
which is responsible for increasing the concentration of
inositol 1,4,5-triphosphate. Consequently, intracellular
calcium is mobilized, triggering exocytosis [46]. We believe
that the secretion of the AMPs, gomesin and acanthoscurrin,
by hemocytes from challenged spiders is probably due to the
activation of G protein as shown for limulus. Ozaki et al. [47]
showed that tachyplesin, a horseshoe crab hemocyte AMP,
stimulates LPS-induced exocytosis amplification through the
same pathway, the activation of the G protein. More studies
are needed to determine if spider AMPs can amplify the
exocytosis response.
The presence of the C-terminal prodomain labeling in
hemocyte granules suggests that gomesin is addressed to the
granules as a propeptide. Progomesin processing might take
place intra- or extracellularly after propeptide secretion.
Gomesin was purified as a mature peptide using the
procedure previously described [16] through acidic homo-
genate of the hemocytes. After removing the plasma from
the hemocytes by centrifugation, the processing enzyme,
which is responsible for progomesin cleavage, must belocalized inside the hemocytes. The sequence for gomesin
shows a glycine residue followed by two basic amino acid
residues (Lys–Arg) [27]. This dibasic site is specific for
endopeptidases of the subtilisin family. These endopepti-
dases are known for processing several peptides, such as the
yeast a factor and prorenin [48]. Interestingly, sequences in
the cDNA library of spider hemocytes were similar to two
endoproteases of the subtilisin family: SPC3 from Bachios-
toma californiensis (847761) and PC5 from Rana esculenta
(23266416) [40]. Therefore, after cleavage by subtilisin, the
two remaining basic gomesin precursor residues can be
removed by carboxypeptidase and the glycine residue can
provide the amide group to form an amidated C-terminal
end through the peptidylglycine a-amidating monooxygen-
ase activity [49].
Peptides of the cathelicidin family are stored as pre-
cursors in the granules. After microbial infection, propep-
tide is secreted into the plasma, where proteases of the
elastase family cleave the cathelin domain, resulting in a
mature moiety [50]. Defensins in Paneth cells are also stored
as propeptides. Although processing occurs extracellularly,
both the processing enzyme and the propeptide are stored
within hemocyte granules. The processing enzyme, a
trypsin, is stored in the granules as a zymogen. After
reaching the plasma, it is activated and converts prodefen-
sin into the mature peptide [14].
In conclusion, our results indicate that gomesin is
addressed to the hemocyte granules as a propeptide. The
processing enzyme may be stored in hemocytes, probably in
the granules. Gomesin processing may take place in either
hemocyte granules or the plasma after secretion of both
progomesin and the processing enzyme. Moreover, our
results strongly suggest that the spider defensive mechan-
isms are very similar to those of horseshoe crab. After
microbial infection, hemocytes may migrate to the infection
site and release not only components of the coagulation
cascade but also AMPs to trap and eliminate the invading
microorganisms. Phagocytosis probably plays a secondary
role, being responsible for clearing cellular debris and
remodeling damaged tissues.
Acknowledgments
We thank Elaine Rodrigues Guadellupe for helping in
migratory assays, Sueli Daffre Carvalho for the statistical
analysis, Suzana Pessoa de Lima for the technical assistance,
and Cassiano Pereira for figure preparation. This work was
supported by Grants from Fundac- a˜o de Amparo a` Pesquisa
do Estado de Sa˜o Paulo (FAPESP) (Brazil), Conselho Nacional
de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) (Brazil).
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Role of hemocytes in the immunity of the spider 725